# U.S. Environmental Protection Agency **Endocrine Disruptor Screening Program SAP Review of Weight of Evidence: Evaluating Results**  **of EDSP Tier 1 Screening** Jointly developed by

the Office of Pesticide Programs, the Office of Science Coordination & Policy , and the Office of Research and Development
July 30

— August 2, 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 assay s and battery

. This document does not create or confer legal rights or impose any legally binding requirements on the EPA or an y other party . This document is distributed solely for th e purpose of sharing this information with the public, consistent with the EPA transparency objectives. I t is not intended to serve an y other purpose, and should not be construed to represent formal dissemination of any agency determination or policy . As s uch, the information collection process under the agency 's In formation Qualit y Guidelines does not appl y to this document.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **2** of **211 Table of Contents**  **SAP Review of EDSP Tier 1 Screening: Weight of Evide**  **nce** List of Acron ym s ................................ ................................ ................................ ................................ ......... 8 Preface

................................ ................................ ................................ ................................ ....................... 10 1. Background on the EPA Endocrine Disruptor Screening Program

................................ ................... 12 2. Overview of the Tier 1 Battery

................................ ................................ ................................ .......... 15 3. Overview of the Agency ’s EDSP Weight

- of

- Evidence Guidan ce ................................ .................... 18 4. Rationale for Chemical Case Study Selection

................................ ................................ ................... 23 5. Method Selection on Evaluating Endpoints

................................ ................................ ....................... 24 6. Case Study

— Chemical A

................................ ................................ ................................ ................ 31 A. Introduction

................................ ................................ ................................ ................................ . 31 B. Data Available for Chemical A

................................ ................................ ................................ .. 31 C. Tier 1 Screening Assay s for chemical A

................................ ................................ .................... 31 D. Other Scientificall y Relevant I nformation (OSRI) for Chemical A

................................ ........... 62 E. Discussion

................................ ................................ ................................ ................................ ... 67 F. Conclusions for Chemical A

................................ ................................ ................................ ....... 77 7. Case Study

— Chemical S

................................ ................................ ................................ ................. 78 A. Introduction

................................ ................................ ................................ ................................ . 78 B. Data Available for Chemical S

................................ ................................ ................................ ... 78 C. Tier 1 Screening Assay s for Chemical S

................................ ................................ .................... 79 D. Other Scientificall y Relevant I nformation (OSRI) for Chemical S

................................ .......... 114 E. Discussion

................................ ................................ ................................ ................................ . 118 F. Conclusions for Chemical S

................................ ................................ ................................ ..... 127 8. Case Study

— Chemical J

................................ ................................ ................................ ............... 128 A. Introduction

................................ ................................ ................................ ............................... 128 B. Data Avai lable for Chemical J

................................ ................................ ................................ .. 128 C. Tier 1 Screening Assay s for Chemical J

................................ ................................ ................... 128 D. Other Scientificall y Relevant I nformation (OSRI) for Chemical J

................................ .......... 155 E. Discussion

................................ ................................ ................................ ................................ . 159 F. Conclusions for Chemical J

................................ ................................ ................................ ...... 169 9. Case Study

— Chemical N

................................ ................................ ................................ .............. 170US EPA

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Page **3** of **211** A. Introduction

................................ ................................ ................................ ............................... 170 B. Data Avai lable for Chemical N

................................ ................................ ................................ 170 C. Tier 1 Screening Assay s for Chemical N

................................ ................................ ................. 170 D. Other Scientificall y Relevant I nformation (OSRI) for Chemical N

................................ ......... 187 E. Discussion

................................ ................................ ................................ ................................ . 189 F. Conclusions for Chemical N

................................ ................................ ................................ ..... 199 10. Overall Conclusions in Context of WOE Guidance

................................ ................................ ........ 199 11. Concluding Remarks

................................ ................................ ................................ ........................ 206 12. References

................................ ................................ ................................ ................................ ........ 208US EPA

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Page **4** of **211** 

### List of **Tables** Table 1: Current EDSP Tier 1 Screening Assay s. ................................ ................................ .................... 16 Table 2: Issues Associated with Case Study Chemicals.

................................ ................................ ......... 24 Table 3: Decision Logic for Interpretation of a Chemical’s Potential for Endocrine Interaction

............ 25 Table 4: Summary

of Tier 1 Screening Assay s for Chemical A.

................................ ............................. 32 Table 5. Competitive Binding Assay of Chemical A with Estrogen Receptor from Rat Uterine C yt osol

................................ ................................ ................................ ................................ ................................ ... 33 Table 6: Competitive Binding Assay of Chemical A with AR from Rat Prostate Cy tosol a

................... 39 Table 7: Mean (±SD) Hormone Concent rations Following Treatment with Chemical A for 48 Hours. a 44 Table 8: Effect of Chemical A and 4

- OH ADSN on Aromatase Activity (as Percent of Control) from Independent Runs a

................................ ................................ ................................ ................................ .... 47 Table 9: Ch emical A

- Selected Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the Androgen Agonist Component of the Hershberger Assay a

................................ ...................... 52 Table 10: Chemical A

- Accessory Sex Organ Weights (mg) from Androgen Agonist Component of the Hershberger Assay a

................................ ................................ ................................ ................................ .. 54 Table 11: Chemical A

- Selected Group Mean (±SD) Bod y Weights and Cumulative Bod y Weight Gains (g) in the Anti

- Androgen Assay a

................................ ................................ ................................ .... 55 Table 12: Chemical A

- Accessory Sex Organ Weights (mg) from Anti

- Androgen Assay in Sprague

Dawley Rats a

................................ ................................ ................................ ................................ ............ 56 Table 13: Chemical A

- Hormone Levels in Female Rats Treated with Chemical A in the Female Pubertal Assay a

................................ ................................ ................................ ................................ ......... 57 Table 14. Chemical A

- Reproductive and HPG Endpoints1 for Male Fathead Minnows

. ...................... 60 Table 15: Chemical A

- Reproducti ve and HPG Endpoints 1 for Female Fathead Minnows

. .................. 61 Table 16. Chemical A

- Growth Endpoints 1 in the FSTRA

................................ ................................ ..... 61 Table 17: Estrogenic/Anti

- Estrogenic Pathway for Chemical A

................................ ............................. 68 Table 18: Androgenic/Anti

- Androgenic Pa thway for Chemical A

................................ .......................... 73 Table 19: Th yroid Pathway for Chemical A

................................ ................................ ............................ 76 Table 20: Summary

of Tier 1 Screening Assay s for Chemical S

................................ ............................. 79 Table 21: Chemical S

- Selected Group Mean (±SD) Bod y Weights and Cumulative Bod y Weight Gains (g) in the Androgen Agonist Assay a

................................ ................................ .............................. 88 Table 22: Chemical S

- Selected Group Mean (±SD) Bod y Weights and Cumulative Bod y Weight Gains (g) in the Anti

- Androgen Assay a

................................ ................................ ................................ .... 89 Table 23: Chemical S

- Group Mean (±SD) Overall (Day s 1

- 11) Food Consumption (g/rat/day ) a

....... 90 Table 24: Chemical S

- Weights of L iver, Kidney s, Adrenals and Accessory Sex Organs from Androgen Agonist Assay

in Rats a

................................ ................................ ................................ ............................. 91 Table 25: Chemical S

- Weights of L iver, Kidney s, Adrenals and Accessory Sex Organs from Anti

Androgen Assay

in Rats a

................................ ................................ ................................ .......................... 92 Table 26: Selected Bod y Weight of Female Rats Treated with Chemical S in the Female Pubertal Assay
a

................................ ................................ ................................ ................................ ................................ .. 95 Table 27: Selected Food Consumption Data of Female Rats Treated with Chemical S in the Female Pubertal Assay a

................................ ................................ ................................ ................................ ......... 96US EPA

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Page **5** of **211** Table 28: General Growth and Vaginal Opening of Rats Treated with Chemical S in the Female Pubertal Assay a

................................ ................................ ................................ ................................ ....................... 97 Ta ble 29: Organ Weight Data of Female Rats Treated with Chemical S in the Female Pubertal Assay a

................................ ................................ ................................ ................................ ................................ ... 98 Table 30: Estrous Cy clicity D ata of Female Rats Treated with Chemical S in the Female Pubertal Assay
a

................................ ................................ ................................ ................................ ................................ .. 99 Table 31: Hormone Levels and Clinical Chemistry Data of Female Rats Treated with Chemical S in the Female Pubertal Assay a

................................ ................................ ................................ .......................... 100 Table 32: Selected Bod y Weight of Male Rats Treat ed with Chemical S in the Male Pubertal Assay a 102 Table 33: Selected Food Consumption Data of Male Rats Treated with Chemical S in the Male Pubertal Assay a

................................ ................................ ................................ ................................ ..................... 103 Table 34: General Growth and Preputial Separation of Rats Treated with Chemical S in the Male P ubertal Assay a

................................ ................................ ................................ ................................ ....... 104 Table 35: Organ Weight Data of Male Rats Treated with Chemical S in the Male Pubertal Assay a

.... 105 Table 36: Hormone Levels and Clinical Chemistry Data of Male Rats Treated with Chemical S in the Male Pubertal Assay a

................................ ................................ ................................ .............................. 108 Table 37: Chemical S

- Reproductive and HPG Endpoints 1 for Male Fathead Minnow in the FSTRA 112 Table 38: Chemical S

- Reproductive and HPG Endpoints1 for Female Fathead Minnow in the FSTRA

................................ ................................ ................................ ................................ ................................ . 112 Table 39: Chemical S

- Growth Endpoints 1 in the Fish Short

- Term Reproduction Assay (FSTRA)

.... 113 Table 40: Estrogenic/Anti

- Estrogenic Pathway for Chemical S

................................ ............................ 120 Table 41. Androgenic/Anti

- Androgenic Pathway for Chemical S

................................ ......................... 124 Table 42. Th yroid Hormone Pathway for Chemical S

................................ ................................ ........... 126 Table 43. Summary

of Tier 1 Screening Assay s for Chemical J

................................ ............................ 129 Table 44: Competitive Binding Assay of Chemical J with AR from Rat Prostate Cy tosol (using undefined bottom plateau)

................................ ................................ ................................ ....................... 132 Table 45: Competitive Binding Assay of Chemical J with AR from Rat Prostate Cy tosol (using confined bottom plateau)

................................ ................................ ................................ ................................ ........ 134 Table 46: Mean (±SD) Hormone Concentrations following Treatment with Chemical J for 48 Hours. a

................................ ................................ ................................ ................................ ................................ . 136 Table 47: Effect of Chemical J on Aromatase Activity (as Percent of Control) From Independent Runs a

................................ ................................ ................................ ................................ ................................ . 140 Table 48: Preputial Separation (PPS) in Male Rats Treated with Chemical J in the Male Pubertal Assay a

................................ ................................ ................................ ................................ ................................ . 146 Table 49: Accessory

Sex Organ Weights at Necropsy in Male Rats Treated with Chemical J in the Male Pubertal Assay a

................................ ................................ ................................ ................................ ....... 147 Table 50: Additional Organ Weights at Necrops y in Male Rats Treated with Chemical J in the Male Pubertal Assay a

................................ ................................ ................................ ................................ ....... 149 Table 51: Hormone Levels in Male Rats Treated with Chemical J in the Male Pubertal Assay a

......... 150 Table 52: Chemical J

- Reproductive and HPG Endpoints for Male Pimephales promelas in the FSTRA

................................ ................................ ................................ ................................ ................................ . 152US EPA

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Page **6** of **211** Table 53. Chemical J

- Reproductive and HPG Endpoints for Female Pimephales promelas in the FSTRA

................................ ................................ ................................ ................................ ..................... 153 Table 54: Chemical J

- Growth Endpoints in the Fish Short

- Term Reproduction Assay (FSTRA)

....... 153 Table 55: Estrogenic/Anti

- Estrogenic Pathway for Chemical J

................................ ............................. 161 Table 57: Th yroid Pathway for Chemical J

................................ ................................ ............................ 168 Table 58: Summary

of Tier 1 Screening Assay s for Chemical N.

................................ ......................... 171 Table 59: General Growth and Preputial Separation (PPS) in Rats Treated with Chemical N in the Male Pubertal Assa y a

................................ ................................ ................................ ................................ ....... 179 Table 60: Hormone Levels and Clinical Chemistry in Rats Treated with Chemical N in the Male Pubertal Assay a

................................ ................................ ................................ ................................ ....... 181 Table 61: Reproductive and HPG Endpoints for Female Fathead Minnow in the FSTRA

................... 183 Table 62: Chemical N

- Reproductive and HPG Endpoints for Male Fathead Minnow in the FSTRA. 184 Table 63: Chemical N

- Growth Endpoints in the Fish Short

- Term Reproduction Assay (FSTRA)

...... 184 Tabl e 64: Developmental and Thy roid Gross Pathology

/Histopathology

Endpoints 1,2 in the AMA with Chemical N

................................ ................................ ................................ ................................ .............. 186 Table 65: Growth Endp oints 1 , 2 in the AMA with Chemical N

. ................................ ............................. 187 Table 66: Estrogenic/Anti

- Estrogenic Pathway for Chemical N

................................ ........................... 191 T a b le 67 : Androgenic Pathway for Chemical N

................................ ................................ .................... 194 Table 68: Th yroid Pathway for Chemical N

................................ ................................ ........................... 198US EPA

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Page **7** of **211** 

### List of **Figures** U.S. Environmental Protection Agency Endocrine Disruptor Screening Program

................................ ..... 1 SAP Review of Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening

.............................. 1 Figure 1. Adverse Outcome Pathway for Estrogen Receptor (ER)

- mediated Reproductive Impairment:

................................ ................................ ................................ ................................ ................................ ... 17 Figure 2. Decision Logic for the Interpretation of Amphibian Metamorphosis Assay Results.

.............. 27 Figure 3. Decision Logic for the Interpretation of the Fish Short

- term Reproduction Assay Results.

.... 28 Figure 4. Percentage [3H]

- Estradiol Bound to the Estrogen Receptor in the Presence of Chemical A, Unlabeled Estradiol, 19

- Norethindrone or Oct yl triethoxy silane. Run 1

................................ .................. 34 Figure 5. Percentage [ 3 H]

- Estradiol Bound to the Estrogen Receptor in the Presence of Chemical A, Unlabeled Estradiol, 19

- Nor ethindrone or Oct yl triethoxy silane. Run 2

................................ ................... 35 Figure 6. Percentage [ 3 H]

- Estradiol Bound to the Estrogen Receptor in the Presence of Chemical A, Unlabeled Estradiol, 19

- Norethindrone or Oct yl triethoxy silane. Run 3

................................ .................. 36 Figure 7. Percentage [ 3 H]

- R1881 Bound to the Androgen Receptor in the Presence of Chemical A, Unlabeled R1881, or Dexamethasone. Run 1.

................................ ................................ ......................... 40 Figure 8. Percentage [ 3 H]

- R1881 Bound to the Androgen Receptor in the Presence of Chemical A, Unlabeled R1881, or Dexamethasone. Run 2.

................................ ................................ ......................... 41 Figure 9. P ercentage [ 3 H]

- R1881 Bound to the Androgen Receptor in the Presence of Chemical A, Unlabeled R1881, or Dexamethasone. Run 3.

................................ ................................ ......................... 42 Figure 10. Change in Testosterone Production Relative to Chemical A Concentration in Test Runs #1 to #3.

................................ ................................ ................................ ................................ .............................. 45 Figure 11. Aromatas e In hibition Response Curves for Chemical A.

................................ ....................... 48 Figure 12. Inhibition Response Curves for Chemical S From Each Test Run.

................................ ........ 84 Figure 13. Male plasma VTG (ng/mL) from Chemical S

................................ ................................ .... 109 Figure 14. Percentage R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, and Chemical J (Assay s 1

– 3), Using an Undefined Bottom Plateau.

........................ 133 Figure 15. Percentage R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, and Chemical J (Assay s 1

– 3), Usi ng a Constrained Bottom Plateau, for Competitive Runs 1, 2, and 3.

................................ ................................ ................................ ................................ ...... 134 Figure 16. Change in Testosterone Production Relative to Chemical J Concentration.

........................ 137 Figure 17. Change in Estradiol Production Relative to Chemical J Concentration.

.............................. 138 Figure 18. Aromatase In hibition Response Curves for Chemical J from Each Test Run

...................... 140US EPA

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Page **8** of **211** 

### List of Acronyms **Abbreviation Terminology**  **A** Androgen (hormonal pathway) **ADME** Absorption, Distribution, Metabolism, Excretion **AMA** Am

phibian Metamorphosis Assay **ANOVA** Analysis of Variance **AOP** Adverse Outcome Pathway **AR** Androgen Receptor **B max**  Binding at maximum **CG** Cowper’s Gland **ChE** Cholinesterase **CV** Coefficient of Variation **DER** Data Evaluation Record **DMF** Dimethyl

- Formamide **DMSO** Dimethyl Sulfoxide **DNA** Deoxyribonucleic Acid **DO** Dissolved Oxygen **DP** Dorsolateral Prostrate **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 Transcriptional Activation **EtOH** Ethanol **FIFRA** Federal Insecticide, Fungicide and Rodenticide Act **FQPA** Food Quality Protection Act **FSTRA** Fish Short

- Term Reproduction Assay **FT** Flutamide **GP** Glans Penis **GSI** Gonado

- Somatic Index **HLL** Hind Limb Length **HPG** Hypothalamic

- Pituitary

- Gonadal Axis **HPLC/MS/MS** High Pressure Liquid Chromatography/Mass Spectroscopy **HPT** Hypothalamic

- Pituitary

- Thyroidal Axis **IC50** Inhibitory Concentration at 50% of response **ICCVAM** Interagency Coordinating Committee on the Validation of Alternative Methods **K d**  Equilibrium Dissociation Constant **LABC** Levator Ani

- Bulbocavernosus **LC50** Lethal Concentration in 50% of test organisms **MDL** Minimum Detection Level **MIE** Molecular Initiating Event **MoA** Mode of Action **MRID** Master Record IdentifierUS EPA

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– July , 2013

Page **9** of **211 Abbreviation**  **Terminology MTC**  Maximum Tolerated Concentration **MTD** Maximum Tolerated Dose **NF stage** Nieukoop and Faber’s Staging Atlas **NIEHS** National Institute of Environmental Health Sciences **NOAEC** No Observed Adverse Effect Concentration **OCSPP** Office of Chemical Safety Pollution and Prevention **OECD** Organization 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 **PC 10**  Positive Control at 10% of response **PC 50**  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 **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) **T4** Thyroxine (tetraiodothyronine) **TP** Testosterone Propionate **TR** Thyroid Receptor **TSH** Thyroid Stimulating Hormone **VC** Vehicle Control **VO** Vaginal Opening **VP** Ventral Prostate **VTG** Vitellogenin **WoE** Weight

- of

- EvidenceUS EPA

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– July , 2013

Page **10** of **211** 

### Preface

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, fifteen were voluntar ily cancell ed or the manufacturer /importer agreed to c ease all sales and distribution of the chemical in the pesticide market. Consequently , data were submitted on 50 pesticide active ingredients and 2 inert ingredients. In support of the EDSP program, a number of Feder al Insecticide, Fungicide and Rodenticide Act ( FIFRA ) Scientific Advisory Panel (SAP) reviews were planned for 2013 (January 29

- 31; May

21

- 23, June 25

- 28, and July

30

- August 2; http:// www.epa.gov/scipol y/ sap/meetings/2013/index.html ) to address key

science issues under the EDSP program. Although these SAP reviews are separate, there is a logical sequence in the reviews that allows the results of each review to build and support EPA’s pr ogrammatic needs (see http://www.epa.gov/endo/pubs/EDSP

- comprehensive

- management
- plan.pdf ). First, under FIFRA and Safe Drinking Water Act ( SDWA ) , thousands of chemicals mu st be considered for EDSP screening. Thus, January 2013, there was a SAP review on “ Prioritizing the Universe of Endocrine Disruptor Screening Program (EDSP) Chemicals Using Computational Toxicology Tools ” to seek input on an improved process for prioritiz ing chemicals for future test orders. Second, as the agency continues to move forward in implementing the EDSP process, it is important to ensure that contributing laboratories can proficiently execute the EDSP Tier 1 assay s and that the Tier 1 battery per forms as designed to provide the necessary complementary

and redundant information to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) and thy roid (T) signaling pathway s. In May

2013, the agency

sought input from the SAP on Tier 1 assay and battery

performance based on an anal ys is of a subsample of 21 case studies on L ist 1 chemicals . EPA is still in the process of reviewing Tier 1 data submitted in response to the first set of test orders and has not reached final conclusions regarding chemical interactions with endocrine signaling pathway s. Input by the current July

- August SAP on h ow the agency will implement its 2011 EDSP weight

- of

- evidence (WoE) guidance will be helpful as EPA moves toward finalizing its evaluation of the Tier 1 data on the List 1 chemicals. T he June SAP 2013 review is important to ensure that the study designs of the wildlife Tier 2 tests are scientifically

sound and technically

practical to support Tier 2 testing decisions

. This July

- August SAP review is focused on providing comment on how the agency will employ

the 2011 EDSP weight

- of

- evidence (WoE) guidance ( at http://www.epa.gov/endo/ and in Appendix 1) toward integrating lines of evidence from the Tier 1 battery and other scientifically

relevant information (ORSI ) bey ond Tier 1 data to determine whether or not a test chemical interacts with the E, A, or T signaling pathway s. **Section 3** provides an overview of the WoE guidance which represents a hy pothesis

- based approach following the adverse outcome pathway (AOP) or mode of action ( MoA) conceptual frameworks that integ rate data along a biological continuum of key events from chemical structure through molecular initiating events, responses at the cellular and organ/tissue level, to in vivo outcomes. **Section 4** provides the justification for the four case studies presente d in this paper.US EPA

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– July , 2013

Page **11** of **211** The method for interpretation of the endpoints for each of the 11 Tier 1 assay s is discussed in **Section 5**

. The four case studies presented in **Sections 6**
**- 9**  come from the sub set of the 21 chemicals used for the May 2013 SAP review. The ca se studies are used to illustrate the basic analy tical process and concepts outlined in WoE guidance . These case studies are illustrative of the likely range of t ypical responses that will be observe d across the Tier 1 assay s and other scientificall y relevant information ( OSRI ) submitted in response to EDSP Tier 1 test orders including Part 158 data

. As discussed in **Section 10** , these case studies illustrate the varying responses along the continuum of biochemical changes, at lower levels of biological organization (earl y molecular events) as measured in vitro, as well as responses at higher levels of biological organization ( e.g ., tissue, organ, and organism) as measured through in vivo studies of intact organisms. Although there are several questions that are pertinent to the evaluation of Tier 1 screening data, the focus of the current case study anal ys es is on the interpretative process for determining whether or not there is a chemical interaction with E, A, or T signaling pathway s. This includes c haracterizing at what level of biological organization ( e.g ., molecular, cellular, tissue/organ, organism) that interaction is expressed , and describing under what conditions the chemical interacts with and perturbs the endocrine sy stem ( e.g., in an organ ism with an intact HPG axis ; in the absence of overt toxicity ; at what doses, duration and route of exposure; is metabolic activation required). Obtaining SAP input on this interpretative process is fundamental and provides the critical basis for Tier 2 t esting decisions. T his document does not focus on the Tier 2 decision

- making process itself. Because these case studies are illustrative, the analy ses presented should not be interpreted as final or complete. The agency

will fully consider the May 21

- 2 3, 2012 and July 30, 2013 SAP comments when the se reports become available in evaluating the Tier 1 and OSRI data for the 52 chemicals in L ist 1 in its final WoE determinations.US EPA

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Page **12** of **211** 

### 1. **Back ground on the EPA Endocrine Disruptor Screening Program**  The US Envi ronmental Protection Agency (EPA) “Endocrine Disruptor Screening Program” (EDSP) was initiated in response to the 1996 Food Qualit y Protection Act (FQPA), which amended the Federal Food Drug and Cosmetic Act [21 U.S.C. 346a(p)] and the Safe Drinking Water Act Amendments of 1996 (SDWA Amendments, PL 104

- 182). The EPA has interpreted these legislative provisions to include potential interactions with estrogen, androgen, and th yroid (E, A, and T) hormone signaling pathway s in humans and wildlife ( http://epa.gov/endo/)

. Based on the recommendations of the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC , 1998 ) , the EPA developed a two

- tiered screening and testing program. The purpose of EDSP Tier 1 is to identify

those chemicals that have the potential to interact with E, A or T hormone signaling pathway s, while the purpose of Tier 2 is to confirm the interaction and to evaluate the dose response for potential adverse effects involving the endocri ne sy stem identified in Tier 1 screening. A detailed history of the program can be found at the EDSP website ( http://epa.gov/endo/)

. It is important to note that unlike the assay s developed as part of Tier 1, which are considered as a battery , the Tier 2 tests do not constitute a battery . I f the Agency determines that a chemical may have the potential to interact with E, A or T pathway s, the nature and extent of testing required for Tier 2 testing will be infor

med by relevant existing data to address an y uncertainties with regard to E, A, or T related endpoints, test doses/concentrations employed, dose/concentration responses/relationships, species differences, and data deficiencies. Tier 2 testing may include one or a combination of comprehensive studies using mammals, birds, fish amphibians, invertebrates, or other special studies as appropriate

. The extended F1 mammalian reproductive toxicity test and the 2

- generation test have bee n peer reviewed and test gui delines are available. The Tier 2 ecotoxicity tests were reviewed b y the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) Scientific Advisory Panel ( SAP ) on June 25

- 28, 2013. Before finalizing Tier 2 protocols, the agency will consider the advic e of the SAP. Between October 29, 2009 and February 26, 2010, EPA issued the first test orders of the EDSP Tier 1 screening on 67 chemicals (L ist 1) ( http://www.epa.gov/endo/ ). Tier 1 test orders were issued for c hemicals based on the likelihood of exposure ; the selection was not based on the potential for endocrine activity . Of the chemicals included in the first Tier 1 test orders, there were 8 voluntary cancellations of pesticide active ingredients and manufact urers of 7 high production volume chemicals used as inert ingredients in pesticide formulations "opted out" of the pesticide market. Consequently , data were submitted on 50 pesticide active ingredients and 2 inert ingredients. The agency

selected a sub sample consisting of 21 of the 52 chemicals on L ist 1 to provide case studies to evaluate the performance of the 11 individual assay s in the EDSP Tier 1 battery and the performance of the overall battery . The subsample was based on those chemicals for which all of the EDSP Tier 1 studies had been completed or OSRI including Part 158 data had been submitted to fulfill t he test orders. The subsample reflected a range of pesticide classes and ph ys ical

- chemical properties including molecular weights, water solubilit ies , log 10 octanol

- water partition coefficient s and volatility . TheUS EPA

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– July , 2013

Page **13** of **211** subsample also reflected a range of pesti cidal modes of action. In May 2013, the agency

presented these 21 case studies to the FIFRA SAP for its input on how well each Tier 1 assay was conducted in accord with respective test guidelines and to highlight issues associated with interpretation of r esponses within each assay . In addition, the panel was as k ed to provide input on performance of the battery based on the concordance of responses within the assay s ( i.e., complementary relationships) and concordance of responses ( i.e ., redundancy ), across the Tier 1 battery (see http://www.epa.gov/scipoly /sap/meetings/2013/052113meeting.html ). When the May SAP 2013 report is made available, the agency will consider its recommen dations as it moves forward in completing evaluations of the data sets for all 52 chemicals on L ist 1. As part of the EDSP Tier 1 screening process for evaluating whether a chemical can potentiall y interact with the E, A, or T pathway , EPA considers the Tier 1 screening assay s and OSRI used to fulfill test order requirements. OSRI includes data from the open literature and te st guideline data ( e.g., the h uman 870 Guideline Series 1 and Ecological Health 850 Guideline Series 2 , 3 ) submitted to fulfill requirements to support the registration of pesticides as defined in Title 40 of the Code of Federal Regulations Part 158 4 ( s ubpar ts F and G). These test guideline data will simply be referred to as “Part 158 data” in this paper. The process used to evaluate Tier 1 screening data relies on the assessment of the quality and relevance of scientific and technical information as well as a weight

- of

- evidence (WoE) approach. In September 2011, after public comment and peer review, EPA issued its final guidance on the WoE approach (described in **Section 3** ) that will be used to evaluate the results of EDSP Tier 1 screening results and OSRI to determine whether a chemical has the potential to interact with E, A or T (USEPA, 2011 b ). The agency

has prepared four case studies from the 21 chemicals consider ed for the 2013 May SAP to illustrate the application of the decision logic, concepts, principles, and key considerations identified in the EDSP WoE guidance document (see **Section 4** for case study selection rationale).

1 USEPA. 2013. OCSPP Harmonized Test Guidelines Series 870 Health Effects Test Guidelines. http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series87 0.htm 2 USEPA. 201 2 . OCSPP Harmonized Test Guidelines Series 850 Ecological Effects Test Guidelines http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guideline s/series850.htm 3 USEPA. 1996. OCSPP Harmonized Test Guideline Series 850 Ecological Effects Test Guidelines Public Drafts http://www.epa.gov/ocspp/pubs/frs/publications/OPPTS_Harmonized/850_Ecological_Effects_Test_Guidelines/Drafts/index .html 4 CFR. 2013. Code of Federal Regulations Title 40 (Protection of Environment. Chapter I (Environmental Protection Agency) Subch apter E (Pesticide Programs) Part 158 (Data Requirements for Pesticides) http://www.ecfr.gov/cgi

- bin/text

idx?c=ecfr&tpl=/ecfrbrow se/Title40/40cfr158_m ain_02.tplUS EPA

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Page **14** of **211** The primary

purpose of the July

- Au gust 2013 SAP is to receive comment on implementation of the EDSP WoE guidance process, not on the guidance itself since the process is based on longstanding practices and principles articulated in other EPA peer

- reviewed guidance documents (USEPA 1991; 1996; 1998; 2002; 2005). The current SAP is asked to comment on the employ ment of the basic anal yt ical process, principles and concepts outlined in the 2011 EDSP WoE guidance document as illustrated with the case st udies. Because these case studies are illustrative, the anal ys es presented should not be interpreted as final or complete. The agency will consider the collective 2013 SAP report s in evaluating the Tier 1 data for the 52 chemicals in L ist 1 in its final WoE determinations for List 1 chemicals.US EPA

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Page **15** of **211** 

### 2. **Overview of the Tier 1 Battery**  As shown in **Table 1** , the Tier 1 battery is composed of 11 assay s (a combination of in vitro and relativel y short

- term in vivo screens in different taxa). A more detailed descripti on of the Tier 1 assay s can be found in the May

2013 SAP white paper entitled “ U.S. Environmental Protection Agency Endocrine Disruptor Screening Program SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance ” (at http://www.epa.gov/scipoly /sap/meetings/2013/052113meeting.html ). Briefl y, the Tier 1 battery was based on the recommendations of EDSTAC and reviewed b y the FIFRA SAP in 2008 (see http://www.epa.gov/scipoly /sap/meetings/2008/032508_mtg.htm ). The in vitro assay s are intended to provide mechanistic data for specific pathway s; whereas, the in vivo assay s evaluate a range of apical endpoints that can be associated with multiple endocrine mechanisms. The current EDSP Tier 1 screening assay s ( **Table 1** ) serve as a means to identify whether or not a chemical can interact with E, A and T along potentiall y adverse outcome pathway s (AOPs) that reflect different levels of biological organization from molecular initiating events ( e.g ., receptor binding), other key intermediate events ( e.g ., gene expression) through to adverse effects in apical endpoint s on th e hy pothalamic

- pituitary

- gonadal (HPG) and hy pothalamic

- pituitary

- thy

roid (HPT) axes. An example AOP for estrogen receptor

- mediated reproductive effects is depicted in **Figure 1** showing ty pical responses that might be observed at various levels of biological organization and the ty pes of assay s ( in vitro or in vivo ) from which observations along the pathway can be made. The molecular initiating event (MIE) in this case is chemical binding to the estrogen receptor which is measured in vitro , and veri fied using a bioassay reflecting at a higher level of biological organization, vitellogenin ( VTG ) gene expression in livers of male rainbow trout ( Oncorhynchus mykiss ) (USEPA, 2009; 2011). Also, as noted in **Figure 1** , the transition to higher levels of bio logical organization provides information, at least indirectly , o n potential compensatory processes at the individual organism level. An integrated understanding of perturbations at lower levels of biological organization in the context of effects expressed at a tissue or organ level of the intact animal provides a mean s for interpreting the degree to which apical responses may be expressed at higher levels of biological organization

.US EPA

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Page **16** of **211 Table**  **1 :**  **Current EDSP Tier 1 Screening Assays.** Screening Assay Test

Guideline Receptor Binding Steroidogenesis HP G Axis HPT Axis E Anti

- E A Anti

- A E A **In vitro** ER Binding (Rat uterine cy

tosol) OCSPP

890.1250 2 ■ ■ ERα Transcriptional Activation (Human cell line HeLa

- 9903) OCSPP 890.1300 3 OECD 455 13 ■ AR Binding (Rat prostate cy

tosol) OCSPP

890.1150 4 ■ ■ Steroidogenesis (Human Cell L ine H295R) OCSPP 890.1550 5 ■ ■ Aromatase (Human target tissue or cell line microsomes) OCSPP 890.1200 6 ■ **In vivo** Uterotrophic (Rat) OCSPP 890.1600 7 OECD 440 14 ■ Hershberger (Rat) OCSPP 890.1400 8 OECD 441 15 ■ ■ ■ 1 Pubertal Male (Rat) OCSPP 890.1500 9 ■ ■ ■ ■ ■ Pubertal Female (Rat) OCSPP 890.1450 10 ■ ■ ■ ■ ■ Fish Short

- term Reproduction OCSPP 890.1350 11 OECD 229 16 ■ ■ ■ ■ ■ ■ ■ Amphibian Metamorphosis (Frog) OCSPP 890.1100 12 OECD 231 17 ■
1 5α

- reductase inhibition only Solid black boxes indicate complementary endpoints across assays within each column. 2 http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0576

- 0005 3 http://www.regulations.gov/#!documentDetail;D=EPA

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- OPPT

- 2009

- 0576

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- OPPT

- 2009

- 0576

- 0003 5 http: //www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0576

- 0011 6 http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0576

- 0004 7 http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

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- 0576

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- 2009

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Page **17** of **211** 9 http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0576

- 0011 10 http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

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- 0009 11 http://www.regulations.gov/#!documentDetail;D=EPA

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- 0007 12 http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

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- 0002 13 http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/OECD/OECD

- TG455.pdf 14 http://iccvam.niehs.nih.gov/S uppDocs/FedDocs/OECD/OECDtg440.pdf 15 http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/OECD/OECD

- TG441.pdf 16 http://www.keepeek.com/Digital

- Asset

- Management/oecd/environment/test

- no - 229

- fish

- short

- term
- reproduction

assay_9789264185265

- en 17 http://www.oecd

ilibrary.org/docserver/download/9723101e.pdf?expires=1371412425&id=id&accname=guest& checksum=819299C4D1409C 4EEA94D4F1FDDDCBB4 **Figure 1**  **. Adverse Outcome Pathway for Estrogen Receptor (ER)**
**- med**  **iated Reproductive I**  **mpairment :**  This is a n example AOP for estrogen receptor

- mediated reproductive effects showing typical responses that might be observed at various levels of biological organization and the types of assays ( in vitro or in vivo ) from which observations along the pathw ay can be made (USEPA, 2009; 2011a, b; Schmieder et. al ., 2004).US EPA

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Page **18** of **211** 

### 3. **Overview of the Agency’s EDSP Weight** 
**- of** 
**- Evidence Guidance**  This section provides a brief description of the key aspects of the agency ’s EDSP guidance document entitled: “ Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing ”, which provides basic principles and criteria for interpreting and integrating scientific information from EDSP Tier 1 screening along with OSRI ( http://epa.gov/endo/ ; also see Appendix 1 )

. T he decision fram ework and principles articulated in the 2011 EDSP WoE guidance are not new but are based on longstanding practice and other peer

- reviewed guidance documents. Application of WoE anal ys is is an integrative and interpretive process routinely used b y EPA to evaluate human health (USEPA 1991; 1996; 2002; 2005) and ecological toxicity data (USEPA, 1998) in a manner that takes into account all relevant scientific information. The principles and criteria for weighing and integrating different lines of evidence a rticulated in existing EPA documents are considered generally applicable to evaluating data from the EDSP Tier 1 battery

. The EDSP WoE approach is characterized as a h ypothesis

- based approach generall y following the WoE framework for evaluating the conceptual framework of an AOP 5 (see SAP 2011 b ) or modes of action (MoA) as put forth in conjunction with work by the EPA (2005), the International Programme for Chemical Safet y (IPCS) (Boobis et al., 2006; 2008), and the International Life Sciences In stitute (Meek et al ., 2003; Seed et. al. , 2005). Use of the MoA/AOP conceptual framework has been a central organizing principle to more accuratel y characterize what effects may be biologicall y plausible in humans and the environment from chemical exposur es and has been endorsed by the SAP ( e.g., SAP, 2011 b ). The criteria used in MoA/AOP framework are generally applicable to the EDSP WoE evaluation and includ e considerations of biological plausibility and coherence, strength, and consistency of the bod y of evidence. Multiple lines of evidence from the EDSP Tier 1 assay s and OSRI are evaluated to address hy

potheses of chemical interactions with the E, A or T signaling pathway s. As described in **Section 2** , this analy sis is approached by considering effects at different levels of biological organization using the in vitro and in vivo Tier 1 assay s along with other available data. Expanding on the illustration presented in **Figure 1 (** and as presented in the 2011 EDSP WoE guidance) and , application of a h ypothesis

- based approach supported by corroborating evidence at different levels of biological organization follows:
 The interaction of the chemical with a molecular target, such as androgen receptor antagonism (as measured in the in vitro A R binding a ssay )

.  This leads to an altered functional cellular response, which may be indicated by increased VTG production in males (as measured in the fish short

- term reproductive assay) , and/or a decrease in serum testosterone (as measured in the male rat pubertal assay )

.

5 Ad verse outcome pathway ( AOP) and mode of action (MoA) are similar in concept and defined as the sequence of key events starting with the interaction of an agent with the molecular target through functional and anatomical changes, resulting in adverse effects (USEPA 2005; Boobis e t al . 2008; Ankley et al ., 2009).US EPA

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Page **19** of **211**  This is corroborated b y an altered structural response at the tissue or organ level, such as increase in gonado

- somatic index (GSI) an d decrease in mating tubercles in male fish , ( as evaluated in the fish short

- term reproductive assay ) , and/or de crease in androgen sensitive tissue weights (as measured in the Hershberger or male pubertal assay s)

.  Ultimately , androgen receptor antagonism may lead to an adverse outcome at the whole organism level, such as decreased fecundit y (fish) or a delay in sexual maturation (rat)

. The EDSP WoE guidance calls for consideration of the following key areas of inquiry : 1. Do the existing data provide relevant, robust, and consistent evidence , e.g., agreement among the outcomes within an indiv idual assay ( i.e ., complementarity ) and among the different assay s or studies ( i.e ., redundancy ) that the substance of interest has the potential to interact with the normal function of the E, A, or T signaling pathways?
2. If the data indicate a potential o f the chemical to interact with E, A, or T signaling pathway s, at what level of biological organization ( e.g ., receptor/target binding, cell responses, enzy me activity , organ response, males and female s ys tem integrit y, development (thyroid) and comparativ e s ys tem integrity ) is that interaction expressed , and under what conditions does the chemical perturb the endocrine s ys tem ( e.g., in an organism with an intact HPG axis , at what dose and duration, route of exposure, is metabolic activation required ) ?
3. What kind of Tier 2 testing is necessary and appropriate? The focus of this SAP review and current case study anal ys es ( **Sections 6**
**- 9**  ) is on how Questions 1 and 2 of the three preceding questions are addressed . The focus of this SAP review is on these scientific questions because they

are fundamental to the overall decision

- making process. For example, if a chemical is not determined to interact with the E, A, or T hormonal pathway s (Question 1), then no furt her testing is needed. On the other hand, if it is determined that there is a potential of the chemical to interact with E, A, or T, then further testing may be required. The WoE analy sis used to determine whether or not a chemical has potential to inter act with the endocrine s ys tem , at what level of biological organization and under what conditions will provide the critical context for what will be a highly specific case

- by

- case evaluation concerning the need for Tier 2 testing. As described in the 20 11 EDSP WoE guidance document, the WoE process involves a number of steps ranging from assembling all relevant individual studies and evaluating the scientific quality of these data to integrating the different lines of evidence to inform a judgment of a p otential endocrine interaction. The key steps are briefl y described below.US EPA

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Page **20** of **211 A**  **. Assemble and Evaluate the Scientific Quality of Individual Studies**  With respect to the sources of scientific information to be considered, it is acknowledged that the main source of scientific information considered in this WoE evaluation is from the Tier 1 assays. But the WoE guidance recognizes the importance of considering all relevant information including studies conducted b y pesticide registrants or chemical companies, as well as data from published or publically available peer

- reviewed studies. T he 2011 EDSP WoE guidance describes the relevant Part 158 health and ecological effect studies in support of pesticide registrations. In considering data bey ond EDSP Tier 1 a ssay s for the current case studies, the agency did not conduct literature searches but relied on data previously

submitted in response to 40CFR Part158 (Subparts F 6 and G 7 ) and open literature studies alread y identified in response as OSRI to the L ist 1 te st orders as OSRI. When the agency completes its final chemical

- specific WoE analy ses, the publically available literature will be more fully considered as appropriate. In addition to the principles outlined in the EDSP WoE guidance, the Office of Pestici de Programs will follow its guidance for evaluating open literature data (see : http://www.epa.gov/pesticides/science/literature

- studies.html ). The process used for evaluating e ach of the EDSP Tier 1 assay s, as well as Part 158 data submissions, is intended to be transparent. Each study is reviewed following standard evaluation procedures (SEPs) and these reviews are documented in Data Evaluation Records (DERs). In evaluating t he quality of data and relevance of scientific and technical information submitted to support agency actions, the agency

applies general assessment factors of data soundness, applicability and utility , clarity

and completeness, uncertaint y and variability , and evaluation and review. A DER is an official a gency record of review that contains a summary

of how well the study

was conducted and conforms to the guideline and the general assessment factors. The DER provides the interpretation and conclusions supp orted b y the data and identify the strengths and limitations of the study . Draft DERs were prepared for the studies used as the basis for the four chemical cases **(Sections 6**
**- 9);**  these DERs are considered draft until input from the 2013 SAPs have been considered. The May 2013 SAP white paper provided an analy sis of the Tier 1 data and generall y concluded that the contributing laboratories were able to conduct the Tier 1 assays re liably , and thus there was confidence in the measurement of the endpoints evaluated (USEPA, 2013). Based on the agency ’s evaluation and preliminary input from the May

2013 SAP, although there were minor deviations from performance criteria identified in t heir respective study guidelines, the Tier 1 data are of sufficient qualit y t o support their use in the WoE analy sis.

6 CFR. 2013. Title 40 (Protection of the Environment), Chapter 1 (Environmental Protection Agency), Subchapter E (Pesticide Programs), Part 158 (Data Requirements for Pesticides), Subpart F (Toxicology) http://www.ecfr.gov/cgi

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idx?c=ecfr&SID=435a855c4e16515181dbc82480a2a667&rgn=div6&view=text&node=40:25.0.1.1.9.6&idno=40

7 CFR. 2013. Title 40 (Protection of the Environment), Chapter 1 (Environmental Protection Agency), Subchapter E (Pesticide Programs), Part 158 (Data Requirements for Pesticides), Subpart G (Ecological Effects) http://www.ecfr.gov/cgi

bin/text

- idx?c=ecfr&SID=435a855c4e16515181dbc82480a2a667&rgn=div6&view=text&node=40:25.0.1.1.9.7&idno=40US EPA

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Page **21** of **211 B**  **. Formulate Hypotheses**  As noted in the 2011 EDSP WoE guidance document ( USEPA, 2011 b ), EPA refers to the WoE approach as “ a collect ive evaluation of all pertinent information so that the full impact of biological plausibility and coherence is adequately considered. ” The WoE is further defined as the process for characterizing the extent to which available data support a hy pothesis th at an agent causes a particular effect, which is an aspect of the WoE approach that has been routinely applied to analy ses used to support EPA regulatory

decisions ( e.g., USEPA, 2005 ). As mentioned above, with respect to evaluating the results of the EDSP Tier 1 data, the WoE analysis can be defined as the process for characterizing the extent to which the available data support a hy pothesis that an agent has the potential to interact E, A, or T signaling pathways , i.e., through estrogen agonism / antagonism , androgen agonism / antagonism , altered steroidogenesis , hy pothalamic

- pituitary

- gonadal (HPG) or

- thy roidal (HPT) axes perturbations.
**C .**  **Integrate Data at Different Levels of Biological Organization and Evaluate Extent and Nature of Complementarity and**  **Redundancy in Responses** In evaluating the potential of a chemical to disrupt endocrine signaling pathway s, the WoE anal ys is (USEPA, 2011 b ) takes into account “ considerations of biological plausibility and coherence, strength, and consistency of the body of evidence .” I n evaluating the plausibility , coherence, strength, and consistency

of the bod y of evidence, it is important to consider the extent to which observed responses are concordant within and across assay s ( i.e., complementary and redundant) as discussed in the 2008 SAP report and in the agency ’s 2013 May SAP paper. Thus, the concepts of complementarit y and redundancy

are emphasized in the current case studies in evaluating an interaction with a particular pathway consistent with the 2011 EDSP WoE guiding principles

. As presented in the agency ’s white paper for the 2013 May

SAP review:

 “complementarit y” describes the concordance of endpoints within an assay measuring multiple endpoints ; and,
 “redundancy ” describes the concordance of endpoint s across the assay s In the WoE process, the level of confidence increases with findings showing increasing complementarity of responses within an assay and redundancy

across the different assay s that span multiple levels of biological organization ( i.e

. , receptor/target binding, cell responses, enzy me activity , organ response, males and female s ys tem integrit y, development (thyroid) and comparative system integrit y ). As part of this evaluation, the magnitude, direction ( i.e ., increase or decrease), and d iagnostic specificit y of responses are important to consider . As a consequence, a more thorough understanding of E, A, or T interactions is obtained by the combined anal ys is of different endpoints and diverse t ypes of studies ( e.g ., consistency

of respons es among interrelated endpoints as opposed to isolated and discordant responses ) . For example, within the Tier 1 battery , the in vitro assay s provide data at lower levels of biological organization ( e.g ., receptor binding) for the parent compound. However, if the in vitro assay s are negative but the in vivo assay s are positive, that would not necessarily be interpreted as a discordant result because of the possibility of an active metabolite or another mechanism that may be operative inUS EPA

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Page **22** of **211** perturbing the HPG axis. On the other hand, positive in vitro assay s may indicate the potential of a chemical to initiate a molecular event/ pathway . However, if the in vivo evidence is largel y negative, that information would ty

pically be given greater consideration in the WoE evaluation because these assay s inherently

integrate responses over the different biological levels of organization and account for compensatory

processes. As indicated in the EDSP WoE guidan ce, “[i ]t is important to consider and rule out other explanations for the observed results (e.g., secondary consequences of non

- endocrine MoA or general toxicity) to the extent possible given the available data. ” I t is realized that findings in the presen ce of overt toxicity may result in uncertaint y as to whether or not the responses are mediated through an endocrine pathway. The current case studies provide examples of how endocrine responses that co

- occur with s ys temic (overt) toxicity are evaluated and addressed on a case

- by

- case basis. These anal yses consider the results from all of the Tier 1 assay s and OSRI , as well as, the nature, directionality , magnitude, and dose trends of the Tier 1 endpoint responses. Additional consideration is given to the n ature of the overt toxicity ( e.g ., mortality , reduction in body weight, behavior) and the treatment concentrations/doses at which toxicity occurred. In summary , the fundamental point made throughout the 2011 EDSP WoE guidance document is that no one study or endpoint is generall y expected to be sufficiently robust to support a decision of whether or not a chemical has the potential to interact with the E, A, or T signaling pathway s. Rather, the broad range of data provided by the Tier 1 battery of assay s and OSRI (when available) should be considered in total in the context of different levels of biological organization. **D. Develop a Short Narrati ve or Characterization of the Evidence**  T he 2011 EDSP WoE guidance document calls for a short summary narrative that characterizes the main lines of evidence and associated uncertainties and the extent to which these impact the conclusions including a des cription of confounding factors, inconsistent or conflicting data. Another important aspect of the WoE narrative is to characterize the overall strength of the evidence supporting a conclusion including whether the responses are marginally significant or c learl y statistically and biologicall y significant to discriminate among compounds that have a high to low likelihood of interacting with the E, A, or T hormonal pathway s.US EPA

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Page **23** of **211 4.**  **Rationale for Chemical Case Study Selection** As described in the White Paper in support of the 2013 May FIFRA SAP on EDSP Tier 1 Screening (Assay

and Battery

Performance) 8 , t he first set of EDSP Tier 1 test orders was issued for 67 chemicals by EPA on October 29, 2009 9 ; the selection of these chemicals for EDSP Tier 1 test orders was based on their potential for exposure and was not based on their potential to interact with endocrine

- mediated processes

. Fifteen of these 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. For the assay

and battery performance anal ys is that was prepared for the May 21

– 23 SAP Review, a sub sample consisting of 21 test chemicals that adequatel y rep resented the chemical space of the initial 52 chemicals w as selected

. This subset of chemicals represent ed a broad range of ph ys ical

- chemical properties including molecular weight (range: 138

– 1053 g/mole), water solubility (range: 0.0003

– 32,000 mg/L) , log 10 octanol

- water partition coefficient ( log K ow range: 0.8

- 6 ), and volatility (5.4 x 10 - 11

– 0.3 mm Hg) . The chemicals also represent ed a range of chemical classes ( e.g ., organophosphates, imides, azoles) and pesticidal activities ( e.g ., inerts, fungicides, herbicides, insecticides) with toxicities ranging from practicall y non toxic to mammals (acute oral LD 50 >2000 mg/kg bw) and to fish (LC 50 >100 mg/L) on an acute exposure basis to very

highl y toxic to mammals (LD 50 <10 mg/kg) and fish (L C 50 <0.1 mg/L)

. A further subsample of the 21 chemicals used in the assay /battery performance anal ys es was selected to serve as case studies for the current W oE anal ys is. The subsample consists of four chemicals which again span a range of ph ys ical

- chemical proper ties (molecular weight range: 138

– 1053 g/mole; water solubility range: 0.013

– 12,000 mg/L ; log K ow range: 1.7

– 5.2; volatility range: 10 - 7

– 0.3 mm Hg). Owing to the broad range of ph ys ical

- chemical properties, some of the test chemicals, i.e ., thos e with low solubility and high K ow values, can be difficult to maintain consistent exposure concentrations as such chemicals can also have high organic carbon partition coefficient (K oc ) values and readily sorb to aquatic toxicity testing apparatuses. Sim ilar to the range in toxicities represented in the subset of 21 chemicals, the four chemicals selected as case studies for the WoE anal ys is also range from pesticides that are very highl y toxic in mammals and fish to chemicals that are considered to be ine rt.

8 SAP 2013. Endocrine Di sruptor Screening Program (EDSP) Tier 1 Screening Assays and Battery Performance http://www.epa.gov/scipoly/sap/meetings/2013/052113meeting.html 9 Federal Register. 2009.Endoc rine Disruptor Screening Program: Tier 1 Screening Order Issuing Announcement. http://www.gpo.gov/fdsys/pkg/FR

- 2009

- 10

- 21/html/E9

- 25352.htmUS EPA

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Page **24** of **211** The subsample of four chemicals is considered illustrative of issues that are anticipated to be encountered over the broader range of test order chemicals and Tier 1 assay s potentiall y impacting the anal ys is of complementarit y within assays and redund ancy across the battery

. These issues included difficult to test substances, overt toxicity coincident with Tier 1 assay responses, outlier or discordant results with respect to the nature and directionality of changes, and weak and sometimes equivocal eff ects across assay s and taxa. **Table 2**  lists specific issues associated with each of the case studies. **Table 2:**  **Issues Associated with Case Study C hemicals.**  **Chemical Code**  **Pesticide**

**Class Issues** 

**A** Insecticide Weak/equivocal effects across assays/taxa

Tier 1 responses that may be reflective of potential interactions with perturbation of more than one endocrine modality

. Overt toxicity in fish at concentrations where potential endocrine effects were observed

. Challenging dose selection (overt toxicity) **S** Miticide Difficult

- to
- test substance ( i.e ., low solubility, high K ow , high K oc )

Overt toxicity in rat and fish assays coincident with potential endocrine effects Lack of molecular initiating event activity based on Tier 1 in vitro assays . **J** Fungicide Tie r 1 responses that may be reflective of potential interactions with

perturbation of more than one endocrine modality ; Different effects across taxa

**N** Inert Low toxicity compound

Potential endocrine effects observed only at or near limit dose in intact organisms. Lack of extensive OSRI Lack of molecular initiating event activity based on Tier 1 in vitro assays and associated adverse outcome pathways based on modified Hershberger assay.


### 5. **Method Selection on Evaluating Endpoints** As discussed in the preceding section, the determination as to whether or not a chemical has interacted with a particular hormonal pathway is based on multiple lines of evidence where EDSP Tier 1 assay

results and OSRI are considered

. Similar to the tiered EDSP process, the interpretation of data within Tier 1 involves multiple levels of refinement where in vitro assay results are considered in the context of in vivo assay s intended to provide compl e mentary information when particular hormonal pathway s are impacted. Information is integrated over multiple levels of biological organization and taxa to determine whether the hy pothesis of a potential effect on an E, A or T hormonal pathway can be supported. In reviewing the Tier 1 battery for compl e mentarity within assay s and a concordance of results across assay s given the specific endocrine modalities the assay s are designed to detect, reviewers consider the magnitude and directionality of responses in terms of their biological plausibility

.US EPA

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Page **25** of **211** Within each of the Tier 1 studies, guidance within the respective test guideline is provided for assessing whether or not a chemical is interacting with a particular hormonal pathway ; **Table 3**  provides the decision logic to evaluate evidence used for each of the Tier 1 s creening assay s. For example, for the in vitro ER Binding Assay , a chemical is classified as “interactive” with the ER if the lowest point on the fitted response curve within the range of data is less than 50% ( i.e ., more than 50% of the radiolabeled est radiol has been displaced from the receptor )

. Similarly , for the in vivo FSTRA, the test guideline states “ a statistically significant effect in one or more of the core endpoints (fecundity, secondary sex characteristics, vitellogenin, gonado

- somatic inde

x, and histopathology) may be indicative of the test material to potentially disturb the HPG axis of fishes.” **Table 3**  **: Decision Logic for Interpretation of a Chemical’s Potential for Endocrine Interaction**  **Assay Critical Endpoint(s) for Indicating**  **Potential Endocrine Interaction In vitro**  **AR Binding** Interactive : Average curve for test chemical across runs displaces 50% or more of the

radioligand ( R1881) **ER Binding** Interactive : Average curve for test chemical across runs displaces 50% or more of the

radioligand (estradiol)
**ERTA 1**  Positive : Maximum response induced by chemical (RPC ma x ) ≥ 10% of the response of the

positive control in at least two runs **Steroidogenesis** Positive : Chemical’s fold induction of estradiol and/or testosterone production is statistically

different from the solvent control (p<0.05) **Aromatase** Inhibitor: Chemical reduces aromatase activity levels by 50% or more and fit a four

parameter, one
- site competitive binding inhibition model **In vivo Hershberger**  Positive : 2 or more of the 5 measured androgen

- dependent tissue weights in the treated

animals statistically different (p ≤0.05) from the control animals **Uterotrophic** Positive : Uterine weights in the treated animals statistically increased (p ≤0.05) compared to

the control animals

**Female Pubertal** Responses in the following endpoints for potential interaction with E and/or T pathways

(usually denoted as statistically significantly different from control (p<0.05): Time to vaginal opening (VO) and/or time to onset of f irst estrus Weight of estrogen

- dependent reproductive organs Histopathology of estrogen

- dependent reproductive tissues Thyroxine (T4) Thyroid gland histology Thyroid weight Changes in other endpoints, but requires corroboration by above endpoints: Estrus c ycling Thyroid stimulating hormone (TSH)US EPA

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Page **26** of **211 Assay**  **Critical Endpoint(s) for Indicating Potential Endocrine Interaction**  **Male Pubertal** Responses in the following endpoints for potential interaction with A and/or T pathways

(usually statistically significantly different from control (p<0.05)): Age at preputial separation (PPS) Weight of androgen

- dependent reproductive organs Histopathology of androgen

- dependent reproductive tissues T4 Thyroid gland histology Thyroid weight Changes in other endpoints, but requires corroboration by above endpoints: Total serum concentrations of testoster one TSH **Fish Reproduction**  **(FSTRA)** Positive: Reported change in one of these endpoints (statistically significantly different from

control (p<0.05)): Fecundity Fertilization success Secondary sex characteristics Gonado

- som atic index (GSI) Histopathology of endocrine

- dependent reproductive tissues (gonads) Plasma concentrations of vitellogenin (VTG) Plasma concentrations of sex steroids (optional) **Amphibian Metamorphosis**  **(AMA)** Positive: Reported change in one of these endpoints (statistically significan tly different from

control (p<0.05)): Advanced development Asynchronous development Remarkable histological effects (thyroid gland) Developmental delay considered indicative of a thyroid interaction only if corroborated by treatment
- related effects on thyroid gland histology; decision logic outlined in guideline 1 ERTA

– Estrogen Receptor Transcriptional Activation
To further assist in the interpretation of results from multi

- endpoint assay s, decision trees have been developed to assist the reviewer s . The decision trees for the Amphibian Metamorphosis Assay

(AMA) and the FSTRA are depicted in **Figures 2**  and **3,** respectivel y . The decision tree for the AMA is also depicted in the test guideline (OCSPP Guideline 890.1100) itself and has been previousl y v etted

. Howerver, t he decision tree for the FSTRA is new and has not been subject to prior review; it is also more complex than that depicted for the AMA owing to the larger number of endpoints measured in the FSTRA. Additionally , it provides some guidance for reviewers in terms of assessing the relative importance of measurement endpoints and the extent of compl e mentarity exhibited in the assay

. Although the FSTRA decision tree has not been formally depicted previousl y, it is a graphic representation of w hat is discussed in the OCSPP Guideline 890.1350. Just as depicted in **Figure 3** , the decision framework is tiered. In the upper portion of the flowchart , a determination is made as to whether suitable conditions were used in the study . T he utility of the FSTRA is dependent on the extent to which the test is considered appropriate for use ( i.e ., whether performance and validity criteria are met) and whether suitable exposureUS EPA

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Page **27** of **211** concentrations had been tested . The lower portion of the framework depicts how vari ous lines of evidence from the study are assessed. As noted in **Table 3** and as depicted in **Figure 3** , critical endpoints are identified in the guideline s which are used in determining potential endocrine interac tions.

**Figure 2**  **. Decision Logic for the I nterpretation of**  **Amphibian Metamorphosis Assay R esults.** US EPA

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Page **28** of **211 Figure**  **3 . Decision Logic for the Interpretation of the Fish Short** 
**- term Reproduction Assay**  **Results.**US EPA

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Page **29** of **211** Note: Double

- ended blue arrows are intended to depict continuums of [overt] toxicity and diagnostic utility. For the AMA, the Agency is following the decision logic outlined in the test guideline (OCSPP 890.1100) and Standard Evaluation Procedure (SEP), which was informed b y the results of earlier validation studies using chemicals with known thy roidal M o As (USEPA 2008b). Although not delineated in as much detail as the FSTRA decision tree in **Figure 3**  for determining stud y quality, the decision logic for the AMA depicted in **Figure 2**  relies on high quality experiments that would include among ot her things, preliminary assessment of whether or not there is an issue with overt toxicity . Using this approach, the direction of effect is considered when evaluating whether or not treatment

- related effects on amphibian development are indicative of thy r oid activit y: developmental delay is considered indicative of thy roid interaction only

if corroborated by

treatment
- related effects on thy roid gland histology ( i.e ., “remarkable histological effects” depicted in **Figure 2**  ), whereas advanced development, as ynchronous development, or effects on thy roid gland histology

may each be considered to be diagnostic findings independentl y. For the FSTRA, the proposed logic is intended to provide i nitial context in the diagnostic ut ility of each measured endpoints in terms of an interaction with the endocrine sy stem. While the likelihood of a response being mediated through an endocrine interaction for a single endpoint is identified in **Figure 3** for the FSTRA , the assay ’s collective response across all measured endpoints need s to be considered in determining whether or not a chemical is interacting with the E and/or A pathway s. Final determinations of potential endocrine interactions need to consider and integrate all available Tier 1 data, as well as OSRI in the WoE approach described in **Section 3** and illustrated in the case studies ( **Sections 6**

- **9** ). The proposed FSTRA decision logic outlined in **Figure 3**  is comprised of basically two tiers. The first tier is to determine whether or n ot the study data are appropriate for use in evaluating potential endocrine activity by identify ing whether the study test ed doses high enough to produce a treatment

- related effect and if overt toxicity was observed in the study , and if so, what were the t ype of effects observed ( i.e ., mortality , decreases in body weight) and at what concentrations. I t is important to demonstrate that the assay was conducted at a high enough concentration to allow for the possible observation of effects ; therefore, in ac hie ving an adequate high dose, it is re cognized that some sy stemic (overt) toxicity may be observed. However, i f overt toxicity was observed at all concentrations tested, the test guideline recommends that the study be repeated using lower exposure concentrat ions. If there are an adequate number of test concentrations with no observed overt toxicity , the evaluation can proceed to the second tier where a chemical’s potential to interact with the E or A pathway is assessed. In the second tier (depicted on the left

- hand side of **Figure 3**  ), the relative degree of diagnostic utility

of specific endpoints for discerning whether or not the chemical has interacted with the endocrine sy stem is determined. If a perturbation on an endpoint has been observed, the likelih ood of that effect being mediated by an endocrine interaction is illustrated on the right

- hand side of **Figure 3** 

.US EPA

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Page **30** of **211** For example, in working through the FSTRA decision tree (after passing the toxicity assessment module) one arrives at depressed VTG in females, which according to **Figure 3** indicates a likel y endocrine interaction. However, if that response is accompanied by specific ty pes of histological changes in the ovary (depressed vitellin [y olk] deposition), decreased egg production and (when colle cted) decreased plasma testosterone and/or estradiol in the females, the chemical could, quite reliably , be labeled as a positive for the fish assay . That is, while the decision framework depict ed in **Figure 3** is intended to initially orient the reviewer i n evaluating the potential for a chemical’s interaction with the endocrine s ystem based on each measured endpoint, the responses within an assay and across assay s need to be integrated in a weight

- of

- evidence analy sis to decide whether a chemical may be en docrine

- active or not. For all in vivo Tier I assay s discussed during the May 2013 SAP, one challenge is interpreting endocrine

- related responses coincident with overt toxicity . Reviewers need to consider whether effects that may

be associated with a part icular endocrine modalit y occur at doses where cy totoxicity or other measurement endpoints ( e.g ., body weight, animal behavior) traditionally

associated with overt toxicity are observed. These data are also considered in the context of responses at lower doses with no apparent overt toxicity and , while not statistically significant , may

indicate a trend across multiple treatment levels. Although the EDSP Tier 1 assay s have been demonstrated to provide compl e mentary responses within assay s and specific patt erns of responses across assay s for known endocrine active chemicals against which the assay s were validated, similar patterns of responses may be less pronounced with chemicals that weakl y interact with endocrine pathway s. Specific patterns of responses may reflect differences between taxa and also the absorption, distribution, metabolism and excretion (ADME) of compounds encountered through in vivo studies. Additionally , t he specificit y of individual Tier 1 assays for differentiating estrogenic from anti

- androgenic or differentiating anti

- estrogenic from androgenic effects can be limited. Id entify ing which particular pathway is affected depends on whether there is sufficient concordance of information across assay s that is further informed b y the magnitu de of such responses. In summary , the reviewer must consider the totality of information as to whether or not there are sufficient information within and across assay s and OSRI data to determine if there is a concordance of information to support a pot ential interaction with the E, A, or T signaling pathway

.US EPA

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Page **31** of **211** 

### 6. **Case Study**

**— Chemical A** 

**A. Introduction**  Chemical A is a member of the organophosphate class of insecticides whose primary mode of toxic action is cholinesterase inhibition leading to neurotoxicity ( e.g ., cholinergic effects). In the case of Chemical A, the issues included: weak and/or equivocal effects across assays/taxa; potential effects on m ore than one endocrine modality ; potential endocrine

- related toxicity in the presence of overt toxicity ; c hallenges encountered in dose selection (overt toxicity ); and endpoint sensitivity ( e.g ., endocrine vs. overt toxicity ). The available information considered to determine the potential interaction of Chemical A with the E, A and/or T pathway s include all 1 1 EDSP Tier 1 studies and other scientifically relevant information (OSRI ) such as 40CFR Part 158 guideline toxicity studies. The data considered are summarized in Section s B, C and D of this chapter. A n anal ys is of the data submitted to the agency , usin g the WoE approach outlined b y the Agency (USEPA, 2011 b ), is presented in Section E

. **B. Data Available for Chemical A**  Chemical A is characterized as relativel y soluble in water with a vapor pressure indicating that the compound may

volatilize from soil and water. Although the compound has a log octanol

- water partition coefficient (log K ow ) of approximately 4, it does not bioconcentrate appreciabl y in fish, suggesting that the compound is readily metabolized and excreted. The compound is not considered pers istent in the environment since it is prone to both abiotic (hy droly sis and photoly sis) and biotic (aerobic and anaerobic) degradation/metabolism. **C. Tier 1 Screening Assays for chemical A**  The Tier 1 studies submitted in response the test order for Chemical A are shown below in **Table 4**  , and the relevant study

details and findings are summarized below

. **Table 4**  also notes whether OSRI were submitted in lieu of conducting the EDSP Tier 1 assay

.US EPA

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Page **32** of **211 Table**  **4: Summary of**  **Tier 1 Screening Assays for Chemical A. Tier 1 Assays**  **Test Guideline Satisfied by**  **OSRI (Y/N) Study Findings**  ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 No Equivocal ERα Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300; OECD 455 Yes Negative AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 No Positive 1 Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550; OECD 456 No Positive Aromatase Assay (human recombinant microsomes) OSCPP 890.1200 No Equivocal Uterotrophic Assay (Rat) OSCPP 890.1600; OECD 440 No Negative Hershberger Assay (Rat) OSCPP 890.1400; OECD 441 No Negative for Androgenic Positive for Anti

- androgenic Pubertal Female Assay (Rat) OSCPP 890.1450 No Negative for E pathway Equivocal for T pathway Pubertal Male Assay (Rat) OSCPP 890.1500 No Negative for A and T pathways Fish Short

- term Reproduction Assay OSCPP 890.1350; OECD 229 No Possible for A pathway Am phibian Metamorphosis Assay (Frog) OSCPP 890.1100; OECD 231 No Negative for T pathway
1 Classified as “Binder” according to Test Guideline OSCPP 870.1150 **ii. ER Binding Assay (Rat uterine cytosol)**  The ER binding assay

used uterine cy

tosol obtained from Sprague

- Dawley rats as the source of ER . A saturation

- bi nding assay

was conducted to demonstrate that the ER isolated from rat uterine cy tosol was present in reasonable concentrations and was functioning with appropriate affinity for the tritium ( 3 H) radio

- labeled reference estrogen prior to conducting ER compe titive

- binding assay s. The competitive

- binding assay was conducted to measure the binding of a single concentration of [ 3 H]

- 17β

- estradiol (1 nM) in the presence of increasing concentrations of Chemical A (logarithmic increase from 10 - 10 to 10 - 3 M). Dimeth yl sulfoxide (DMSO) was used as a vehicle at a final concentration of 4% (v/v). The assay

included 19

- norethindrone as a weak positive control, octy ltriethoxy silane as a negative control, and 17β

- estradiol as the natural ligand reference material. All per formance criteria were met for the saturation binding assay . The mean maximum binding (B max ) value for the three saturation binding runs was 74.6 fmol/100 μg protein and the mean saturation binding dissociation constant (K d ) was 0.331 nM.US EPA

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Page **33** of **211** In the competitive binding assay , the curves fitted to the Hill equation for the reference material showed that increasing concentrations of unlabeled 17β

- estradiol displaced [ 3 H]

- 17β

- estradiol in a manner consistent with one

- site binding, as indicated by a Hill slope of

- 1.1 and

- 1.0 for the first two runs. For the third run, the Hill slope was outside the acceptable parameter, at

- 1.6. Evidence of test compound insolubility

was reported at the highest test concentration (10 - 3 M) in the first competitive bindin g run. This test concentration was not included in the anal ys is. The results from the three competitive binding runs are summarized in **Table 5**  below and presented graphicall y in **Figures 1**
**- 3.**  The estimated mean specific binding affinity (reported as log IC 50 ) was not calculated for Chemical A as the percent specific binding did not reach 50%. The mean IC 50 for the positive 19

- norethindrone control was 5.01 × 10 - 6 M and its relative binding affinity (RBA) was 0.027%; however, this value could not be calcu lated for Chemical A. **Table 5**  **. Competitive Binding Assay of Chemical A with Estrogen Receptor from Rat Uterine**

**Cytosol Parameter**  **Run 1 b**  **Run 2 b**  **Run 3 b**  **Mean ± SE** Lo g IC 50 (M), **a** 17 β

- estradiol

- 8.9

- 9.0

- 8.7

- 8.9

19

- norethindrone

- 5.6

- 5.2

- 5.1

- 5.3 Test substance NA NA NA NA IC 50 (M), 17 β

- estradiol 1.26 × 10 - 9 1.00 × 10 - 9 2.00 × 10 - 9 1.36 × 10 - 9

19

- norethindrone 2.51 × 10 - 6 6.31 × 10 - 6 7.94 × 10 - 6 5.01 × 10 - 6 Test substance NA NA NA NA Lo g RBA(%) b , 19

- norethindrone

- 1.30

- 1.80

- 1.60

- 1.56

Test substance NA NA NA NA RBA (%) b , 19

- norethindrone 0.050 0.016 0.025 0.027

Test substance NA NA NA NA

a Data w ere obtained from the study report. b Calculated by contractor. The mean are reported for the concurrent replicates within each run. RBA (%) = relative binding affinity NA Not applicableUS EPA

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Page **34** of **211 Figure 4. Percentage [**  **3 H]**  **- Estradiol Bound to the Estrogen Receptor in the Presence of Chemical** 

**A, Unlabeled Estradiol, 19**
**- Norethindrone or**  **Octyltriethoxysilane. Run 1**US EPA

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Page **35** of **211 Figure 5. Percentage [**  **3 H]**  **- Estradiol Bound to the Estrogen Receptor in the Presence of Chemical** 

**A, Unlabeled Estradiol, 19**
**- Norethindrone or Octyltriethoxysilane. Run 2** US EPA

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Page **36** of **211 Figure 6. Percentage [**  **3 H]**  **- Estradiol Bound to the E**  **strogen Receptor in the Presence of Chemical**

**A, Unlabeled Estradiol, 19**
**- Norethindrone or Octyltriethoxysilane. Run 3** US EPA

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Page **37** of **211** In the first independent run, Chemical A showed an equivocal response at a concentration (10 - 3 M) where precipitation was observed. Th e top test concentration for the subsequent two runs was 10 - 4 M. The second run for Chemical A was classified as non

- interactive and the third independent run was classified as equivocal. When evidence of insolubility is observed, the test guideline reco mmends decreasing the concentration by half

- logs ( i.e ., run a 10 - 3.5 M concentration if precipitation is observed at 10 - 4 M); however, this was not done in these assay s. Although the binding took place at a concentration that was at or near the limit of solubility , based on the shape of the response curves and in the absence of the testing at half

- log concentrations, the overall classification for Chemical A is equivocal in this binding assay

.

**iii. ER Transcriptional Activation Assays**  The ER transcriptional assay employ s hERα

- HeLa

- 9903 cells exposed to selected , known positive and negative controls as well as Chemical A (98.7% a.i) at concentrations of 10 - 11 to 10 - 3 mg/mL in DMSO (0.1%) for approximately 24 hours. The experiments were performed using 96

- wel l plates and each Chemical A concentration was tested in six wells/plate. Cells were exposed to the test agents for 24±2 hr to induce reporter (luciferase) gene products. Luciferase expression in response to activation of the estrogen receptor b y Chemica l A was measured upon addition of a luciferase substrate and detection with a luminometer. Chemical A was tested up to the limit of cy totoxicity , which occurred at concentrations ≥10 - 4 M. The maximum level of response (RPC max ) was 8.8%, and the concentration associated with this maximum level of response, i.e ., PC max , was 10 - 5 M for the first run, and the RPC max was 5.3%, and the associated PC max was 10 - 6 M for the second run. Performance criteria were met; Lo g PC 50 and L og PC 10 for 17α

- methy ltestosterone were lower than the acceptable values, but this indicates increased sensitivity

of the cells to weak agonists. All other performance criteria were acceptable. Chemical A was negative for ER transcriptional activation in t his test sy stem
.

**iv. AR Binding Assay (Rat prostate cytosol)**  The AR binding assay

used ventral prostate cy

tosol from Sprague Dawley rats as the source of AR to measure the binding of a single concentration of [ 3 H]

- R1881 (1 nM) in the presence of increasing concentrations (10 − 10 to 10 − 3 M) of Chemical A (98.7% a.i.). DMSO was used as the solvent vehicle at a final assay

concentration of approximately 3.2%. A total of three independent runs were performed, and each run included dexamethasone as a weak positi ve control, and R1881 as the ligand reference standard.US EPA

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Page **38** of **211** Prior to conducting the competitive binding experiment, saturation binding experiments were conducted to demonstrate that AR from the rat cytosol preparations were present in adequate concentration s and functioning with the appropriate affinit y for the radiolabeled ligand. Separate cy tosolic preparations were used for each of the three competitive binding runs, so data from three saturation binding experiments were evaluated. For the cy tosolic pre paration conducted on March 30, 2011, the mean dissociation constant (K d ) for [ 3 H]

- R1881 was 0.614±0.065 nM, and the estimated B max was 1.080±0.109 fmol/100 μg protein for the single batch of prostate cy tosol that was prepared. For the cy

tosolic preparati on conducted on July 23, 2011, the K d for [ 3 H]

- R1881 was 1.080 ± 0.690 nM, and the estimated B max was 1.071 fmol/100 μg protein (±0.167) for the single batch of prostate cy tosol that was prepared. For the cy tosolic preparation conducted on September 24, 2 011, the dissociation constant (K d ) for [ 3 H]

- R1881 was 0.613 ± 0.041 nM, and the estimated B max was 0.817 fmol/100 μg protein (±0.049) for the single batch of prostate cy tosol that was prepared. The mean and individual K d values were generally below the r ange reported in the EPA validation program (0.685 to 1.57 nM), although two individual values were within the range and one value was above the range. Confidence in these numbers is generally high according to the goodness of fit (R 2 = 0.880

- 0.992) and t he small variation in R 2 and K d estimates among runs (although greater variability was observed in the July 23, 2011 Saturation Binding data). In the competitive binding experiment, p recipitation of Chemical A was visually observed at the highest tested concentration (10 − 3 M) in the first run; therefore, the data at this concentration for this run were not evaluated. No precipitation was observed at concentrations up to 10 − 3 M in Runs 2 and 3. In all three runs, specific binding of [ 3 H]

- R1881 was 93.5

- 100.5% at Chemical A concentrations of 10 − 10 to 10 − 5 M, and specific binding declined to 71.9

- 80.5% at 10 − 4 M and to 36.7

- 41.6% at 10 − 3 M (Runs 2 and 3). Based on the average binding curve for Chemical A, the estimated Log IC 50 for Chemical A was −3 .6 Log M, the relative binding affinity (RBA) was 0.00017%, and the Hill slope was ~1.00. The estimated average Log IC 50 s for R1881 ( −9.3 L og M) and the weak positive control, dexamethasone ( −4.4 Log M), were within the expected ranges, and the mean RBA f or dexamethasone was 0.0022%. The performance criteria were generally met. The results from the three c ompetitive binding experiments are summarized in **Table 6** and shown graphicall y in **Figures 7**
**- 9.**  Based on the results of three runs, Chemical A is class ified as a “b inder ” in the AR binding a ssay .US EPA

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Page **39** of **211 T**  **able 6: Competitive Binding Assay of Chemical A with AR from Rat Prostate Cytosol a**  **Parameter Run 1**  **b Run 2**  **b Run 3**  **b Mean ± SE**  **c**
r 2 (unweighted) R1881 NR NR NR NA

Dexamethasone NR NR NR NA Chemical A NR NR NR 0.9791 d Lo g IC 50 (M) R1881

− 8.9

− 10.1

− 9.0

− 9.3 ± 0.38

Dexamethasone

− 4.4

− 4.3

− 4.5

− 4.4 ± 0.06 Chemical A NA

− 3.6

− 3.9

− 3.6 ± 0.07 d IC 50 (M) c R1881 1.3 × 10 − 9 7.9 × 10 − 11 1.0 × 10 − 9 4.6 × 10 − 10 (± 3.6)

Dexamethasone 4.0 ×10 − 5 5.0 × 10 − 5 3.2 × 10 − 5 4.0 × 10 − 5 (± 0.5) Chemical A NC 2.5 × 10 − 4 1.3 × 10 − 4 2.7 × 10 − 4 (±0.5) d Lo g RBA (%) c Dexamethasone

− 2.5

− 3.8

− 2.5

− 2.9 ± 0.43

Chemical A NC

− 4.5

− 3.1

− 3.8 d RBA (%) c Dexamethasone 0.0032 0.0002 0.0032 0.0022 ± 0.0010

Chemical A NC 0.00003 0.00079 0.00017 d

a Data w ere obtained from the study report. b The mean is reported for the concurrent replicates within each run. c Calculated by reviewers and are the mathematical averages unless otherwise specified; for means expressed in scientific notation, the SE values in parentheses are presented in the same order of magnitude as the mean value. d Mean values for Chemical A were calculated by reviewers using Grap hPad PRISM; values are based on the means binding curve for Chemical A. N R Not reported NA Not applicable NC Not calculated. Although specific binding was reduced to <50% in Run 1 at 10 − 3 M. Chemical A, this data point w as not included in the analysis be cause of observed precipitation. r 2 Goodness of fit RBA Relative binding affinityUS EPA

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Page **40** of **211 Figure 7**  **. Percentage [ 3**  **H] -**  **R1881 Bound to the Androgen Receptor in the Presence of Chemical**

**A, Unlabeled R1881, or Dexamethasone. Run 1.**US EPA

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Page **41** of **211 Figure 8**  **. Percentage [**  **3 H]**  **- R1881 Bound to the Androgen Receptor in the Presence of Chemical** 

**A, Unlabeled R1881, or Dexamethasone. Run 2.**US EPA

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Page **42** of **211 Figure 9**  **. Percentage [ 3**  **H] -**  **R1881 Bound to the Androgen Receptor in the Presence of Chemical A,**

**Unlabeled R1881, or Dexamethasone. Run 3.**US EPA

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Page **43** of **211 v.**  **Steroidogenesis Assay (Human cell line H295R)** The steroidogenesis assay was conducted using H295R cells cultured in 24

- well plates in the presence of Chemical A

, (98.7% purity ) at log concentrations from 0.0001 to 100 μM in triplicate for 48 hours. The test chemical’s vehicle was DMSO and its final concentration was 0.05% (v/v). Testosterone and estradiol concentrations were measured b y high pressure liquid chromatograph y coupled with tandem mass spectroscop y (HP LC/MS

- MS) using positive ion multiple reaction monitoring. Three independent experiments were performed. A Qualit y Control (QC) plate was run concurrently

with each independent run of a test chemical plate to demonstrate that the assay responded properl y to positive control agents each at tw o concentration levels; positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production. Precipitation of the test chemical was observed in two runs and cy totoxicity in the third. As a result t he highest suitable concentration of Chemical A in Runs 1 through 3 was 10 μM

. Minimum basal hormone production was met in all blank and solvent (DMSO) control (SC) wells, except for the basal estradiol production in Run 3 (36 pg/mL), and basal hormone p roduction met the recommended fold

- increase criteria in the SC. Exposure to 10 μM forskolin induced testosterone production ≥1.5

- fold (actual 2.4

- to 2.5

- fold) and estradiol production ≥7.5

- fold (actual 8.9

- to 11.4

- fold) over the SC. Exposure to 1μM pro chloraz inhibited sy nthesis of testosterone and estradiol by ≥50% (actual 53

- 61% and 46

- 68%, respectivel y) compared to the SC. These data generall y met the performance criteria recommended by the Guideline. The within

- plate and between

- plate percent coe fficients of variation (% CVs) for the QC plates and for the test plates generally

met the performance criteria recommended by the Guideline. Although the between

- plate % CVs for estradiol were slightl y above the recommended Guideline limit of 30% for eac h set, these data are considered acceptable by the reviewers. Statistically

significant decreases in testosterone production were observed after incubation with 10 μM Chemical A in all three runs. Statistically significant increases were also observed at several lower concentrations in Runs 2 and/or 3, but these changes were not reproducible across all three test runs. Statistically

significant decreases in estradiol production were observed in Run 1 at concentrations of 0.0001 to 0.1μM, and a significant increase was observed in the same run at the 10 μM concentration. The hormone concentrations after exposure to Chemical A, and the fold

- difference change relative to SC and mean ± SEM for the three suitable assay runs (1 through 3), are presented in **Tab le 7**  **and Figure**  **10** . The fold

- changes in hormone concentration for Chemical A in the three runs are shown graphicall y in Figure 7 for testosterone. The highest suitable concentration of Chemical A in all three runs was 10 μM due to precipitation at the 10 0 μM concentration in Runs 1 and 2, and cy totoxicity greater than 20% in Run 3. Based on the hormone responses in three independent runs , Chemical was positive in the steroidogenesis assay .US EPA

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Page **44** of **211 Table 7: Mean (±SD) Hormone Concentrations Following Treatment**  **with Chemical A for 48 Hours.**  **a Nominal**  **Concentration (μM)**  **Run 1 Run 2**  **Run 3 Run 1**  **Run 2 Run 3**  **Mean b**  **± SEM b**  **Statistical Significance**  **Testosterone (pg/mL) Fold Difference**  DMSO (SC) 1850 2269 1482

— — — — — — 0.0001 2005 2527 1503 1.08 1.11* 1.01* 1.07 0.03 **Runs 2 and 3** 0.001 1810 2356 1584 0.98 1.04 1.07* 1.03 0.03 **Run 3** 0.01 1803 2359 1572 0.97 1.04 1.06* 1.02 0.03 **Run 3** 0.1 1826 2328 1484 0.99 1.03 1.00 1.01 0.01

— 1 1722 2371 1576 0.93 1.05 1.06 1.01 0.04

— 10 1413 1923 1363 0.76* 0.85* 0.92* 0.84 0.05 **Runs 1, 2, and 3** 100 732 1224 542 NA NA NA NC NC

—

Estradiol (pg/mL) Fold Difference DMSO (SC) 225 247 125

— — — — — — 0.0001 191 244 109 0.85* 0.99 0.87 0.90 0.04 **Run 1** 0.001 185 225 112 0.82* 0.91 0.89 0.87 0.03 **Run 1** 0.01 193 240 113 0.86* 0.97 0.90 0.91 0.03 **Run 1** 0.1 193 232 117 0.86* 0.94 0.94 0.91 0.03 **Run 1** 1 219 277 129 0.98 1.12 1.03 1.04 0.04

— 10 292 345 163 1.30* 1.40 1.30 1.33 0.03 **Run 1** 100 202 309 119 NA NA NA NC NC

—

a Data w ere obtained from the study report. b Calculated by the reviewers SC = Solvent control; NA = Not applicable; fold change not determined due to the presence of precipitation or cytotoxicit y. NC = Not calculated*Statistically significant (p ≤0.05)US EPA

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Page **45** of **211 Figure 10. Change in Testosterone Production Relative to Chemical A Concentration in Test** 

**Runs #1 to #3.** US EPA

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– July , 2013

Page **46** of **211 vi.**  **Aromatase Assay (Human Recombinant Microsomes)** In the in vitro aromatase (CYP 19) assay , Chemical A (purit y, 98.7% w/w,) in DMSO was incubated with human recombinant aromatase and tritiated androstenedione ( [1β

3 H(N)]

- androst

- 4

- ene - 3,17

- dione; [ 3 H]

- ASDN) at concentrations of 0, 10 − 3 , 10 − 4 , 10 − 5 , 10 − 6 , 10 − 7 , 10 − 8 , 10 − 9 , or 10 − 10 M for 15 minutes at 37°C to assess the potential of Chemical A to inhibit aromatase activity

. Aromatase activity

was determined by

measuring the amount of tritiated water produced at the end of a 15

- minute incubation period for each concentra tion of chemical. Tritiated water was quantified using liquid scintillation counting. Four independent runs were conducted, and each run included a full activity

control, a background activit y control, a positive control series (10 − 10

– 10 − 5 M) using a kno wn inhibitor (4

- hy droxy androstenedione; 4

- OH ASDN), and the test chemical series (10 − 10

– 10 − 3 M) with 3 repetitions per concentration. Aromatase activity

in the full activity

controls and background controls were acceptable. For the positive control substa nce (4

- OH ADSN), aromatase activit y averaged 0.257±0.048 nmol ∙mg protein − 1
∙ min − 1 at the lowest tested concentration (10 − 10 M) and 0.002±0.001 nmol ∙mg - protein − 1
∙ min − 1 at the highest tested concentration (10 − 5 M). These results were generall y within the rec ommended ranges for the top of the aromatase activity curve, bottom curve, Hill slope, L og IC 50 , and % CV for replicates of each concentration. For Chemical A, aromatase activity averaged 0.261±0.033 nmol ∙mg - protein − 1
∙ min − 1 at the lowest tested concentrati on of 10 - 10 M and 0.115±0.041 nmol ∙mg - protein - 1
∙ min − 1 at the highest tested concentration of 10 − 3 M. Although precipitation was noted at 10 − 3 M, the percent activity was decreased at this concentration compared to 10 – 4 M in all four runs. The goodness of fit with 4

- variable nonlinear regression improves greatl y when the 10 − 3 M concentration is included, based on increased least squares R 2 values. As shown in **Table 8,** e xcluding Run 1, which showed no inhibition for Ch emical A, the estimated Lo g IC 50 for Chemical A ranged from

– 4.93 to

– 3.90 and averaged

– 4.55 compared to 4

- OH ADSN, which averaged

- 7.22. The average slope of the concentration response curve was

– 5.29 for Chemical A and

– 1.03 for 4

- OH ADSN

.US EPA

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– July , 2013

Page **47** of **211 Table 8: Ef** 

**fect of Chemical A and 4**
**- OH ADSN on Aromatase Activity (as Percent of Control)**  **from Independent Runs a**  **Chemical Run 1**  **Run 2 Run 3**  **Run 4 Mean**  **SD %CV**  **Log IC 50**  **(μM) Chemical A** 

- 14.08

- 4.83

- 3.90

- 4.93

- 4.55 0.57 12.49 **4**
**- OH ADSN** 

- 7.29

- 7.08

- 7.19

- 7.30

- 7.22 0.10 1.42 **Slope Chemical A** 

- 3.49

- 5.01

- 5.40

- 5.46

- 5.29 0.24 4.61 **4**
**- OH ADSN** 

- 0.95

- 1.20

- 1.10

- 0.87

- 1.03 0.15 14.38 a Data obtained from the study report. Mean, SD, and %CV w ere calculated by the reviewers based on these data. Values for Chemical A we re calculated by the reviewers based on data presented on pages 42

- 48 of the study report using GraphPad Prism robust fit, 4

- variable, nonlinear regression. The highest dose, 10 – 3 M, w as excluded from the calculations due to precipitation at that dose. Run 1 for Chemical A was excluded from the calculation of mean, SD, and %CV as the results were not similar to the other three runs. Run 1 w as graphed as a line, and demonstrated l ittle (if any) inhibition. SD= Standard Deviation CV= Coefficient of Variance Aromatase activity

was inhibited at the high concentration (10 - 3 M) b y more than 50%, but the results were cofounded b y evidence of insolubility (precipitation) at this concentr ation. The next lowest concentration inhibited aromatase activity by approximately

34%. The guideline recommends that half

- log concentrations ( i.e ., 10 – 3.5 M) be tested when precipitation is observed in the test media. There is no indication in the stud y report that 1⁄2 log concentrations were evaluated for the presence of precipitate. The inhibition response curves for Chemical A from each run are shown in **Figure 11**  Based on the results of three runs, Chemical A is classified as E quivocal in the aromatase assay
.US EPA

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– July , 2013

Page **48** of **211 Figure**  **11 . Aromatase Inhibition Response Curves for Chemical A.** US EPA

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Page **49** of **211**US EPA

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– July , 2013

Page **50** of **211 vii.**  **Uterotrophic Assay (Rat)** The Uterotrophic Assay

was conducted to identify the potential estrogenic activity of Chemical A. Chemical A (98.7% a.i.) in corn oil was administered dail y via oral gavage to 56

- day old, ovariectomized female Sprague

- Dawley rats (8/group) at dose levels of 0 ( vehicle), 78 or 250 mg/kg/day . The stud y al so included a positive control group of eight ovariectomized females dosed via oral gavage with 17α

- ethy ny l estradiol (EE) at 0.1 mg/kg/day . Animals were dosed for three consecutive day s and necropsied approximately 24 hours after the final dose to determine wet and blotted uterine weights. All animals survived until scheduled terminatio n. One rat administered 78 mg/kg exhibited mild lethargy prior to dosing on Day

3. Rats administered 250 mg/kg/day exhibited the following adverse clinical effects: consistent with the cholinesterase inhibiting activity of Chemical A, two rats display ed nasal discharge, thinness and fur discoloration on Day 2; on Day

3 those same two rats plus three additional rats (total of five) display ed head and scapula fur discoloration, lethargy and hunched posture; these same five rats display ed head and scapula f ur discoloration prior to euthanasia. Bod y weights were decreased b y 15% (p<0.05) in the 250 mg/kg/day group compared to controls. Bod y weight gains were decreased (p<0.05) b y 62% in the 78 mg/kg/day group and the 250 mg/kg/day animals actuall y lost weigh t during the overall study (

- 18.1 g) compared to controls (18.5 g). Uterine weights in the Chemical A treated groups were comparable to the vehicle controls. The mean blotted uterine weight in the control group was 0.031% of the mean terminal body weight, which meets the Guideline requirement (<0.04%). Absolute wet and blotted uterus weights for the positive control group (0.1 mg/kg/day EE) were increased (p<0.05) by 254% and 162%, respectively . These increased uterine weights were in the expected range. No statistically

significant increase in uterine weight was seen in this study . Hence, Chemical A was negative in the Uterotrophic assay

.

**viii. Hershberger Assay (Rat)**  The in vivo Hershberger Assay consists of androgenic and anti

- androgenic components. To scr een for potential androgenic activity , Chemical A (98.7% a.i.,) in corn oil was administered daily via oral gavage to ten 56

- to 57

- day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 47 or 150 mg/kg/day . An androgenic positive control group consisted of 10 castrated rats exposed to 0.4 mg/kg/day

testosterone propionate (TP) b y subcutaneous (s.c.) injection for 5 days, then TP+corn oil by gavage for 5 day s.US EPA

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– July , 2013

Page **51** of **211** To screen for potential anti

- androgenic activity , Chemical A (98.7% a.i., in corn oil was administered daily via oral gavage to ten 56

- to 57

- day old, castrated male Sprague Dawley rats at dose levels of 15, 47, or 150 mg/kg/day

in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg bw/da y by subcutaneous injection. The anti

- androgen vehicle control consisted of 10 castrated rats exposed s.c. to 0.4 mg/kg bw/day

of TP for 5 day s, then TP+corn oil by gavage for 5 day s. An anti

androgenic positive control group consisted of 10 castrated r ats exposed to 0.4 mg/kg bw/day TP and 3.0 mg/kg/day

flutamide (FT) in corn oil. For both components of the Hershberger assay , the animals were dosed for 10 consecutive day s and euthanized approximately 24 hours after the final dose administration to det ermine weights of the five androgen

- dependent tissues. One animal from the TP and corn oil control ( positive control for androgen agonist, vehicle control for anti

- androgen) was found dead on day 10, and one animal from the 150 mg/kg bw/day group of the an ti

androgen assay

was found moribund prior to dosing. The remaining animals survived until scheduled termination. In the androgen agonist component of the Hershberger assay , no clinical signs of toxicity were observed in any of the dose groups. In the an ti

- androgen assay , clinical signs included one rat in the 47 mg/kg bw/day group with throat scab or lesion and shoulder scab throughout the study . For one 150 mg/kg rat, clinical signs included fur discoloration (neck/face) and soft feces on day 3, thin o n day s 5 and 6, and hunched and/or thin on day s 8

- 10. No clinical signs were observed in the 15 mg/kg bw/day group. Also in the androgen agonist assay , body weights in the 47 and 150 mg/kg bw/day groups were comparable to controls throughout the duratio n of the assay . At 150 mg/kg bw/day , overall (day s 1

- 11) bod y weights gains were decreased (p<0.05) by 64%. Animals dosed with TP had increased overall bod y weight gains (p<0.05) of 52%. There were no statistically significant increases in an y of the ac cessory

sex organs of a nimals dosed with Chemical A. Animals in the positive control TP group had statistically

significant accessory

sex organ weight increases (p<0.05) as follows: 1649% in seminal vesicles; 1210% in ventral prostate; 247% in levator an i

- bulbocavernosus (LABC) muscle; 669% in Cowper’s gland; and 88% in glans penis. The performance criteria indicated that this assay was performing as expected. Bod y weights and accessory sex organ weights for the androgen agonist component of the Hershbe rger assay are shown in **Table 9**  **and 10**  **,** respectively.US EPA

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– July , 2013

Page **52** of **211 Table 9:**  **Chemical A**
**- Selected Group Mean (±SD) Body Weights and Cumulative Body Weight** 

**Gains (g) in the Androgen Agonist Component of the Hershberger Assay a** 
**Study Day #**  **Dose**

**Vehicle Control Positive Control** 

**Vehicle + TP**  **(0.4 mg/kg/day**  **) Chemical A** 

**(47 mg/kg/day**  **) Chemical A**  **(150**

**mg/kg/day )**  N Mean SD N Mean SD N Mean SD N Mean SD 1 8 257.1 20.6 8 255.8 14.1 8 255.6 15.6 8 256.2 17. 2 4 8 270.1 23.7 8 278.5 16.4 8 263.5 17.1 8 244.8 16. 4 7 8 280.0 28.3 8 295.8 19.8 8 277.7 20.7 8 250.0 29. 2 11 8 300.7 33.4 7 321.7 25.9 8 298.3 24.2 8 271.8 24. 9
BWG (day s 1

- 11) 8 43.6 15.5 7 66.1* ( ↑52) 11.5 8 42.6 9.3 8 15.6* ( ↓64) 10. 5

a Data w ere obtained from the study report. Percent differences are presented in parentheses. b The positive control group for the androgen agonist is same as the vehicle control for the anti

- androgen. Animals were dosed with TP (s.c.) for 5 days, then TP (s.c.) + corn oil (gavage) for 5 days. This did not affect the outcome or conclusions of this study. N = Number of animals in the group SD= Standard Deviation * Significantly different from controls at p<0.05US EPA

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Page **53** of **211** In the anti

- androgen component of the Hershberger assay , there were no effects on body weights or body weight gains in the 15 and 47 mg/kg/day group, or the positive control group. At 150 mg/kg/day , overall body

weight gains were decreased (p<0.05) b y 42%. Statistically significant decreases in two or more of the target accessory sex organ weights were observed in animals dosed with Chemical A. At 15 mg/kg/day , the seminal vesicles weights were decreased 29% (p<0.05) and ventral prostate weight was decreased 26% (p<0.05). At 47 mg/kg/day, a ccessory sex organ weight decreases (p<0.05) were noted as follows: 37% in seminal vesicles; 43% in ventral prostate and 21% in LABC. At 150 mg/kg/day , accessory sex organ weight decreases (p<0.05) were noted as follows: 62% in seminal vesicles; 60% in ventral prostate; 44% in LABC; 48% in Cowper’s gland; and 13% in glans penis. Animals dosed with TP + FT had decreased (p<0.05) accessory sex organ weights as follows: 86% in seminal vesicles; 77% in ventral prostate; 45% in L ABC; 69% in Cowper’s gland; and 20% in glans penis. The %CV for mean LABC weight of the 150 mg/kg/day group (24%) exceeded the maximum allowable %CV (20%). All other %CV values were less than the maximum permissible values. Bod y weights and accessory sex organ weights for the andr ogen agonist assay

are shown in **Table 11 and 12,**  respectively. Chemical A was negative for androgenic activity , and positive for potential anti

- androgenic activity at a dose not associated with overt toxicity in the Hershberger assay

.US EPA

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– July , 2013

Page **54** of **211 Table 10:**  **Chemical A**
**- Accessory Sex Organ Weights (mg) from Androgen Agonist Component of the Hershberger Assay**  **a**
**Organ Dose** 
**Vehicle Control Chemical A**  **(47 mg/kg/day**  **) Chemical A**  **(150 mg/kg/day**  **) Positive Control**  **Vehicle + TP (0.4**  **mg/kg/day )**  **b** N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV Seminal vesicles 8 37.0 5.9 16 8 30.7 4.8 16 8 35.6 7.1 20 7 647.0* ( ↑1649) 111.6 17 Ventral prostate 8 15.7 3.5 22 8 14.5 5.0 35 8 16.1 4.8 30 7 205.6* ( ↑1210) 22.9 11
LABC 8 137.3 18.8 14 8 121.7 22.5 19 8 108.7 14.1 13 7 476.7* ( ↑247) 49.0 10 Cowper’s glands 8 6.4 1.9 29 8 5.7 1.7 29 8 4.6 0.7 16 7 49.2* ( ↑669) 3.6 7 Glans penis 8 40.6 3.7 9 8 43.1 5.5 13 8 43.2 5.0 11 7 76.4* ( ↑88) 8.4 11

a Data were obtained from the study report. Percent differences from controls were calculated by the reviewers and included in parentheses. b The positive control group for the androgen agonist is same as the vehicle control for the anti

- androgen. Animals were dosed with TP (s.c.) for 5 days, then TP (s.c.) + corn oil (gavage) for 5 days. This did not affect the outcome or conclusions of this study. N = Number of animals in the group SD= Standard Deviation CV= Coefficient of Variation * Significantly different from vehicle controls at p<0.05. No statistically

significant increases in organ weights were seen in two or more of the five androgen responsive tissues in t he andro gen agonist assayUS EPA

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– July , 2013

Page **55** of **211 Table 11:**  **Chemical A**
**- Selected Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the Anti** 
**- Androgen** 

**Assay a** 
**Study Day #**  **Dose (mg/kg/day)**

**Vehicle Control + TP (0.4**  **mg/kg/day )**  **b TP + FT**  **Positive Control Chemical A** 

**(15 mg/kg/day**  **) Chemical A** 

**(47 mg/kg/day**  **) Chemical A** 

**(150 mg/kg/day**  **)** N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 1 8 255.8 14.1 8 258.6 23.6 8 254.9 13.2 8 252.5 13.0 8 254.1 10.1 4 8 278.5 16.4 8 277.4 25.3 8 272.9 15.6 8 265.5 14.8 7 247.7 13.1 7 8 295.8 19.8 8 296.5 30.9 8 294.7 17.7 8 285.8 17.3 7 265.8 14.7 11 7 321.7 25.9 8 326.1 34.2 8 324.3 22.4 8 311.8 22.1 7 293.5 22.9 BWG (day s 1

- 11) 7 66.1 11.5 8 67.5 13.5 8 69.4 11.9 8 59.2 13.6 7 38.2* ( ↓42) 18.3

a Data w ere obtained from the study report. Percent differences from controls were calculated by the reviewers and included in parentheses. b The vehicle control for the anti

- androgen is same as the positive control group for the androgen agonist. Animals were dosed w ith TP (s.c.) for 5 days, then TP (s.c.) + corn oil (gavage) for 5 days. This did not affect the outcome or conclusions of this study. N = Number of animals in the group SD= Standard Deviation *Significantly different from controls at p<0.05US EPA

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Page **56** of **211 Table 12:**  **Chemical A**
**- Accessory Sex Organ Weights (mg) from Anti** 
**- Androgen Assay in Sprague** 
**- Dawley Rats**  **a**
**Organ Dose** 

**Vehicle Control +TP (0.4**  **mg/kg/day )**  **b Chemical A**  **(15 mg/kg/day**  **) Chemical A**  **(47 mg/kg/day**  **) Chemical A**  **(150 mg/kg/day**  **) TP + FT**  **Positive Control** N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV Seminal vesicles 8 647.0 111.6 17 8 461.1* ( ↓29) 71.9 16 8 409.6* ( ↓37) 175.3 43 7 246.3* ( ↓62) 89.9 36 8 91.6* ( ↓86) 25.0 27

Ventral prostate 8 205.6 22.9 11 8 152.2* ( ↓26) 28.1 18 8 118.2* ( ↓43) 23.0 19 7 82.6* ( ↓60) 19.5 24 8 46.6* ( ↓77) 5.1 11

LABC 8 476.7 49.0 10 8 469.0 34.1 7 8 376.2* ( ↓21) 39.7 11 7 265.6* ( ↓44) 63.6 24
c 8 262.2*

( ↓45) 47.7 18 Cowper’s glands 8 49.2 3.6 7 8 35.4 12.6 36 8 38.0 6.9 18 7 25.8* ( ↓48) 2.5 10 8 15.1* ( ↓69) 2.0 14

Glans penis 8 76.4 8.4 11 8 77.3 6.5 8 8 68.9 7.9 11 7 66.2* ( ↓13) 8.0 12 8 61.0* ( ↓20) 9.9 16

a Data w ere obtained from the study report. Percent differences from controls were calculated by the reviewers and included in parentheses. b The vehicle control for the anti

- androgen is same as the positive control group for the androgen agonist. Animals were dosed w ith TP (s.c.) for 5 days, then TP+corn oil (gavage) for 5 days. This did not affect the outcome or conclusions of this study. c The CV for mean tissue weight (150 mg/kg/day, LABC) exceeds the maximum allowable CV (20%). N = Number of animals in the group SD= Standard Deviation CV= Coefficient of Variation * Significantly different from vehicle c ontrols at p<0.05US EPA

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– July , 2013

Page **57** of **211 ix.**  **Pubertal Female Assay (Rat)** In a female pubertal assay, 16 Sprague

- Dawley (Crl:CD ® [SD] I GS) female rats/dose group were treated daily via oral gavage with Chemical A (95.9% a.i) in corn oil at doses of 0 (vehicle), 50 or 100 mg/kg bw/day from post

- natal day (PND) 22

- 42/43. Animals were examined for vaginal opening (VO) daily beginning on PND 22 and weight at day of attainment was recorded. Beginning on the day of complete VO, vaginal smears were examined for evaluation of the estrous cy cle. Following sacrifice on PND 42/43, serum thy

roxine (T 4 ), and thy

roid stimulating hormone (TSH) levels were anal yz ed b y radioimmunoassay

(RIA). Adrenal, liver, thy roid, pituitary , and urogenital organ weights were recorded, and microscopic examinations of the uterus, ovaries, thy roid, and kidney

were performed. No treatment

- related effects were observed on mortality , clinical signs, age at VO, mean age at first estrus, estrous cy cle status, clinical chemistry , or gross or microscopic patholo gy . At 100 mg/kg bw/day , the majority

of females (11/16) lost weight from PND 21 to 22, but gained weight for the remainder of the study . Body

weights ranged from 90.5

- 96.2% of control during treatment (PND 22

- 42), suggesting this dose may

be an adequate high dose. Additionally at this dose, body

weight at VO was decreased (p<0.05) b y 10%, with a decreasing (p<0.05) trend across both dose groups. At 50 mg/kg bw/day

and above, relative (to body ) liver weights were increased (p<0.05) b y 7

- 11%. Additionall y, an increasing (p<0.05) trend in unadjusted adrenal weights was observed ( ↑3

- 8%). At 100 mg/kg bw/day , relative kidney weights were increased (p<0.05) by 7%, and relative adrenal weights were increased b y 13%. These increased organ weights correlated w ith slight decreases in final body weights and slight increases in unadjusted organ weights. At 50 mg/kg bw/day

and above, serum T 4 levels were slightly decreased (p<0.05) by

17

- 23%. Serum TSH levels were similar to controls, and there were no effects obs erved on thy roid weights, or th yroid follicular cell colloid area or cell height. Hormone values are shown in **Table 1 3** 

. **Table 13: Chemical A** 
**- Hormone Levels in Female Rats Treated with Chemical A**  **in the Female**

**Pubertal Assay a** 

**Parameter Evaluated**  **Vehicle Control**  **50 mg/kg/day 100 mg/kg/day** 

**N Mean**  **SD CV (%)**  **N Mean**  **SD CV (%)**  **N Mean**  **SD CV (%)**  **Hormon es**  Serum T 4 , Total(μg/dL) 16 3.04 0.73 23.9 16 2.35* ( ↓23%) 0.5

8 24.5 16 2.51*

( ↓17%) 0.6 8 27.1 Serum TSH (ng/mL) 16 1.72 0.51 29.6 16 1.55 0.7 1 45.5 16 1.67 0.6 2 37.2 a Percent differences from controls are included in parentheses. *= p ≤ 0.05US EPA

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Page **58** of **211** At 100 mg/kg/day , mean estrous cy cle length was increased (p<0.05) from 3.7 day s to 4.6 day s, and the percent regularl y cy cling was decreased (not significant [NS]) from 92% to 69%. The mean age at first estrus and estrous cy cle status were unaffected. Because the effect on cy cle length was based on a limited period of time, the significance of this observation remains to be determined Chemical A was negative for the estrogen pathway and equivocal for the thy roid pathway

in the female pubertal assay

.

**x. Pubertal**  **Male Assay (Rat)** In a male pubertal assay , 16 Sprague

- Dawley (Crl:CD ® [SD] IGS) male rats/dose group were treated daily via gavage with Chemical A (95.9% a.i.) in corn oil at doses of 0 (vehicle), 50 or 100 mg/kg/day from post
- natal day

(PND) 23

- 53/54. Ani mals were examined for preputial separation (PPS) daily beginning on PND 30 and weight at day of attainment was recorded. Following sacrifice on PND 53/54, total serum testosterone, T 4 , and TSH levels were analy zed by RIA. Adrenal, liver, thy roid, pituit ary , and urogenital organ weights were recorded, and microscopic examinations of the testis, epididy mis, thyroid, and kidney

were performed. No treatment

- related effects were observed on mortality , clinical signs, body weights, body

weight gains, age or we ight at PPS, organ weights, hormone levels, clinical chemistry parameters, gross pathology , or microscopic examination of the thy roid, testes, epididy

mides, or kidney

. A 15% (p<0.05) decrease in T4 levels was observed at the 50 mg/kg bw/day. The minima l change observed in the T4 level at 50 mg/kg/d was not observed at 100 mg/kg/d; therefore, this change was not considered to be toxicologically significant due to the minimal magnitude and lack of dose response. Chemical A was negative for the androgen a nd thy roid pathway s in the pubertal male assay

.

**xi. Fish Short** 
**- term Reproduction Assay**  T he in vivo 21

- day sho rt
- t e r m r ep r oduc t i on as s ay of Chemical A w it h f a t he a d m i nno w s (

P im epha l e

s p ro m e l a s) w as s t u d i ed un d er f l o w - t h r ou g h cond i ti o n s. A du l t f i sh, 16 spa w n i ng g r oups ( 2 m a l es and 4 f e m a l es i n ea c h g r oup: ca . 5 m on t hs) w e r e expos e d t o Chemical A a t no m i n a l con c en t r a

ti o n s of 0 ( n eg a ti v e con t r o l ) 0.01, 0.1 and 1.0 m g a i/ L ; m ea n - m eas u r ed c o nce n t r a t i ons w e r e <L O Q ( <0

. 0008 3 ) , 0.0 1 1, 0.120, a n d 0.820 m g a i/ L. T he t e s t s y s t em w as m a i n t a i n ed a t 24 t o 25 o C a n d a pH of 6.0 t o 7.9.US EPA

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Page **59** of **211** I n t he con t r o l r ep l i c a

t e s, s pa w n i ng f

r equ e ncy occ u r r ed a t l ea s t e v e r y 4 d a y s i n t w o of t he f our r ep l i c a t e s and f ecu n d i t y i n t he co n tr o l w as 11 e gg s / f e m a l e / da y

/ r ep l i c a t e (f e cu n d it y r an g ed f

r om 6. 2

- 16 with one of t he f o u r r e p l i ca t es having ≥ 15 e gg s /f e m a l e / da y )

. F e r ti li z a t i on suc c ess i n t he c on t r ol g r oup a v e r a g ed 9 1 % ( f e r t i li z a t i on in the replicates r an g ed f r om 8 3

- 95 % )

. Fecundity was significantly reduced (p ≤0.05) by 94% a

t t he 0 .820 m g a. i

. / L l ev e l compared to the control

. Fe r t i l i z a t i on su c ce ss w as de c r e a sed 80% i n t h e 0.8 2 0 m g a. i

. / L g r oup co m pa r ed t o t he co n tr o l.
P l as m a VTG w as s i g n i f

i ca n tl y r edu c ed (p ≤0.05) r e l a t i v e t o co n tr o ls i n f e m a l e f i sh at t he 0.011 a n d 0.820 m g a. i

. / L tr e a t m ent l e v e l s, by 63 and 71 % , r esp e c t i v e l y , but was not dose

- responsive as the 37% reduction i n t he 0.1 2 0 m g a. i

. / L g r oup, compared to the controls, w a s n o t s t a t i s t i c a l l y s i g n ifi c ant. M a l e V T G l e v e l s w ere n o t s i g n ifi c a n t l y d i f f e r ent fr om t he c o n t r ol and d i d n o t exh i b i t a m ono t on i c t r end; while t he r e w as a 266 5 % i n c r e a se i n t h e m ean V T G i n t h e 0.120 m g a. i

. / L g r oup co m pa r ed t o t he c on tr o l m ean, t h i s i nc r ea s e i s d

u e t o 2 o u t of t h e 8 m easu r ed f i sh be i ng abo v e t he m ean co n tr o l V T G by 2328 and 19,01 0 % . Due to the lack of consistency across males, the spikes in two of the fish were considered artifacts. As shown in **Table 1 4**  **,** s i g n i f i c a nt reductions (p ≤0.05) i n g onado

- s o m a ti c i ndex ( G S I ) of 29 a n d 3 1 % w as obs e r v ed i n m a l e f

i sh a t t he 0

. 120 an d 0.820 m g a i/ L l e v e l s, as w e l l as a s i g n i f i ca n t 4 2% r edu c t i on f or f e m a l e f i sh at t h e 0.820 m g a i/ L l e v e l , r e l a t i v e t o c on t r o l

. N up ti a l t ub e r c l e s c o r e s in males w e r e a l so s i g n if i ca n tl y r ed

u ced (p ≤0.05) by 7 4% compared to the co n tr o l a t t he 0.820 m g a i/ L l ev e l with a decreasing trend, albeit weak, with increasing concentration ; no t u be r c l es w e r e ob se r

v ed f o r f e m a l es. E f f e c t s on g onadal h i s t o pa t ho l o g y w e r e obs e r v ed i n f e m a l e f i s h i n t he 0.820 m g a. i

. / L l e v el and i n c l u d ed an i n c r e a s e d i nc i d ence o f s t a g e 0 / 1 o v a ri es w i t h a tr o p hy of p e ri n u c l e o l a

r a nd e a r l y co r

ti c al f o l l i c l e s . Ma l e f i sh d i sp l a y ed an i nc r ea s ed i n c i den c e a n d se v e r it y (as defined as r a ng i ng fr om m i n i m al t o m ode r a t e severit y b y the pathologist) of s p e r m a t o g on i a at a l l Chemical A

- tr ea t ed l e v e l s . T he p r ese n ce o f a t e s t i s o v a w as obs e r

v ed i n one m a l e a

t 0.120 m g a. i

. / L; however, given the low incidence, it is considered an artifact. Chemical A ex p osu r e was a d d i t i on a ll y ass o cia t e d w it h a s t a t ist i c a ll y s i g n i f

i c ant r educt i on i n body

we i g ht of f e m a l e f ish a t t h e 0.120 and 0

. 820 m g a. i ./L l e v e l s, with a slight decreasing trend observed. O v e r t m o r b i d ity i n c l uding abno r

m al b e ha v ior and sw i mm i ng was a l s o obs e r v ed i n f i sh expo se d t o t he 0.820 m g a. i

. / L l e v e l

. O v e r all m ean su r v i val v a l ues w e r e 100, 9 6, 92, and 92%; however, the decreased survival with increasing exposure concentrations was not statistically s i g n ifi ca n t ( p>0.0 5 )

. M eas u r e m ent t e c hnique of t he m a l e d o r s a l nape p ad and sex s t e r o i d s we r e de s c r i b e d i n t h e p r o t o co l , but r e sul t s of t h ese obs e r v a t ions w e r e not r ep o rt e d i n t he s t u d y . As shown in **Table 1 5**  , se v e r al v a l i d i t y and p e rf or m ance c r i t e r i a w e r e n ot m et f or t h i s s t udy , where fecundit y was less than the 15 e gg s /f e m a l e / da y

/ r ep l i c a t e and s p a w n i ng f

r equ e ncy was greater than e v e r y f our d a y s for two of the four co n tr o l r ep l i c a t e s, and fertilization success was less than 9 5% in con t r ol. D i s so l v ed ox yg en also dropped be l ow t h e r equ i r e d m i n i m a percent saturation of 60% . Although some control performance measures were less than recommended, in general, the performance was reasonable.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **60** of **211** In general, there was a significant (p<0.05) decrease in fecundit y and fertility, along with a significant decrease in female GSI and VTG and male tubercle scores, reported at the highest treatment group, 0.820 mg a.i./L. Addit ionally , a significant (p<0.05) decrease in male GSI was reported at 0.120 and 0.820 mg a.i./L , along with treatment

- related effects on male gonads at all treatment groups **(Table 16).** However, signs of morbidity

(abnormal behavior and swimming patterns) wa s reported at 0.820 mg a.i./L along with a significant (p<0.05) decrease female bod y weight at 0.120 and 0.820 mg a.i./L

. The pesticidal mode of action for Chemical A is cholinesterase inhibition which can lead to cholinergic toxicity , and while cholineste rase was not measured in this study , the observation of abnormal swimming at the high concentration could be considered consistent with cholinergic toxicity . Therefore, there is uncertainty in whether the responses observed at the high concentration are r eflective of an endocrine

- mediated response or are reflective of a compromised organism with limited ability to maintain reproductive function and homeostasis. However, g iven that there were responses in two endpoints (decreases in male GSI along with gona dal histopathology ) that occurred in the absence of reported over t toxicity , they lack endocrine specificit y, and therefore, the effects may reflect an interaction with either the A pathway (A antagonism) or the E pathway

(E agonism). **Table 14 . Chemical**  **A**
**- Reproductive and HPG Endpoints1**  **for Male Fathead Minnows**  **.**

Abbreviations: GSI Gonado

- Somatic Index. NA Not applicable. VTG Vitellogenin. 1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water ) control. Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board

- certified pathologist. 2 Statistically significant [p<0.05]. LOQ=<0.00083 mg ai/L

**Treatment (mg a.i./L)**  **[measured] Tubercle**  **Score GSI**  **Gonadal Staging and**  **Histopathology. Plasma VTG** 
**Median %** 

**Diff erenc**  **e Effect? (Yes/No)**  **% Diff erence** 
Control (dilution water only ), <LOQ 29 NA NA NA
0.011 25

- 11.8 Yes 43.2 0.120 20

- 29.4 2 Yes 2665 0.820 7.8 2

- 31.3 2 Yes

- 37.0US EPA

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– July , 2013

Page **61** of **211 Table 15**  **: Chemical A** 
**- Reproductive and HPG Endpoints**  **1 for Female**  **Fa thead Minnows**  **.**

Abbreviations: GSI Gonado

- Somatic Index. NA Not applicable. VTG Vitellogenin. 1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board

- certified pathologist. 2 Statistically significant [p<0.05]. LOQ=<0.00083 mg ai/L

**Table 16 .**  **Chemical A**
**- Growth Endpoints**  **1 in the FSTRA** 
**Treatment (mg a.i./L)**  **[measured] Body Weight**  **Length Males**  **Females Males**  **Females % Diff**  **erence % Diff** 

**erence % Diff** 

**erence % Diff** 

**erence** Control (dilution water only ), <LOQ NA NA NA NA 0.011

- 5.89

- 11.1

- 1.65

- 2.47 0.120

- 16.4

- 17.9 2

- 2.32

- 2.64 0.820

- 7.2

- 22.7 2 0.69

- 1.35 Abbreviations: NA Not applicable. 1 Unless otherwise indicated, percent (%) differences are reported based on comparison to the negative (clean water) control. 2 Statistically significant [p<0.05].; LOQ=<0.00083 mg ai/L **Treatment (mg a.i./L)**  **[measured] Fecundity**  **Fertility Success**  **Tubercle**

**Score GSI**  **Gon adal Staging**  **and Histopathology**  **Plasma**

**VTG %**  **Diff erence**  **% Diff**  **erence .**  **Median %** 

**Diff erence**  **. Effect? (Yes/No)**  **% Diff**  **erence .**  Control (dilution water onl y), <LOQ NA NA 0 NA NA NA
0.011

- 20.5 6.1 0

- 17.8 No

- 62.5 2 0.120

- 21.9 5.8 0 0.44 No

- 37.1 0.820

- 93.9 2

- 79.8 2 0 42.1 2 Yes

- 71.0 2US EPA

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Page **62** of **211 xii.**  **Am phibian Metamorphosis Assay**  T h e 21

- d a y in vivo a s s a y of Chemical A on a m phibian me t amo r ph o s is of A f

ri c an c l a w e d f

rogs ( X e no p us lae v i s ) w a s s t u died un d er f lo w - throu g h c o n dition s . A m p hibian lar v ae ( 8 0 tad

p oles p er treat m ent, N F S t a g e 51) lar

v a e were e x p o s e d to con t rol and t e s t c h emical me a sured c o n c e ntratio n s of 0

. 0 0 76, 0

. 0 8 2 a n d 0

. 820 mg a.i./L . The selection of test item concentrations was determined to be adequate according to guideline recommendations. T h e t e s t s y s t em w a s maintain e d at 21 to 2 3 °C a nd a pH of 7

. 9 to 8.2. Chemical A d i d not s i gn i f i c ant l y a ff

e c t 7

- d a y n o r ma l i z e d h i nd

- li mb l eng t h ( H L L) at 0. 00 76, 0.0 8 2 a nd 0. 8 20 mg a. i ./L. R ed

u c t i o n s i n no r

m a li z e d H L L at d a y 2 1 i n t h e 0. 0 82 and 0. 8 20 m g a. i

. / L t r ea t me n t l e v e l s w e r e s i m il a r , ba s ed o n c o m pa ris on to t he ne g at i v e c o nt r o l , but t h e e ff e c t w a s o n l y s tat i s t i c a l l y s i gn i f i c ant at t he i nte r

med i a t e t r e a tm e nt l e v el ( p = 0. 0 45 )

. No s i gn i f i c ant c h an g e of m e d i an N i e u w k oop F a ber ( N F ) de v e l op m en t al s ta g e w a s ob s e r v e d at 7 d a y s or at 21 d a y s at a n y t r e a tm e nt l e v e l

. A s y n c h r on o us de v e l op m ent was not o b s e r v ed i n any of the t r ea t me n t s . B o d y w e i g ht w as significantly r e d u c ed on d a y 21 at the h i gh e s t t r ea t m e nt l e v el on l y ( p = 0.01 ) ; n o other effects o n s n ou t
- v ent l e n gt h (S V L) or b o d y we i ght w e r e ob s e r v e d. T h y r o i d g l and h y

p e r t r o p h y w a s ob s e rv ed i n the 0. 8 20 m g/ L t r ea t men t
. With respect to thy roid histopathology, m il d f o l l i c u l ar c e l l h y

pe r t r o p h y a nd h y

p e r p l a s i a w e r e r epo r t ed as c om m on in both control and treatment specimens. In c i d e n c e of i n c r e a s ed or de c r e a s ed f o lli c u l ar l umen a r ea was s p o r ad i c but a g a i n d i d n ot ap p ear t o b e treatment
- related

. C li n i c al si gns i n c l ud i ng d i s c o l o r at i on and c u r v a tu r e of t h e s p i ne (incidence values not reported) were o b s e rv ed i n s e v e r al tad p o l es a t e a c h t r ea t me n t c o n c ent r at i o n a n d i n c o nt r o l s . Un l e s s o t he r w i s e i nd i c a t ed, a l l ef f e c ts a r e r epo r ted b a s ed on c om p a ri s on to t h e n e g at i v e c ont r o l , a n d l ate s ta g e t adpo l e s ( N F S t age > 6 0) a r e e xc l u ded f

r om ana l y s e s of c ont i n uo u s e nd p o i n t s (S V L, bo d y we i ght, and no r m a li z e d H L L ) due to the effects of normal tissue resorption during metamorphosis

. The absence of significant effects on thy roid

- related endpoints in the AMA suggests that Chemical A did not interact with the amphibian HPT axis or its downstream thy roid hormonal pathway

under these study conditions.

**D. Other**  **Scientifically Relevant Information (OSRI)**  **for Chemical A** This section of the case study provides a summary of the Agency review of existing data cited as OSRI

. For purposes of this case study , the information described below onl y includes data submitted by the test order recipient or the public and/or data submitted to support pesticide registration ( e.g ., Part 158 guideline studies). Existing data includes data previously submitted to the Agency in support of a registration decision believed to be relevant to one or more of the Tier 1 assay s.US EPA

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– July , 2013

Page **63** of **211 1.**  **In Vitro OSRI Data**  Kojima et al . (2005) investigated the response of an in vitro estrogen receptor

–mediated reporter gene assay following exposure to 32 chemical pesticides either alone or in combination. The E

- CALUX ® Assay (Xenobiotic Detection Sy stems, Inc, NC, USA) uses a human ovarian carcinoma cell line (BG1) transfected with a luciferase reporter gene under the control of a mouse mammary tumor virus promoter. Estrogen

- responsive concatemers ( i.e ., repetitive DNA sequences) are inserted directl y above the promoter confer estrogen responsiveness to the viral promoter and reporter gene. The transfected cells are made stable b y an antibiotic resistance gene resulting in the cell line used in the assay (BG1Luc4E 2 ). Chemicals that interact with estrogen

- responsive DNA are identified b y the luciferase gene response and quantified in terms of Relative L ig ht Units (RLU). The assay s were conducted by growing the BG1Luc4E 2 cells in medium for four day s, transferring 2 × 10 5 cells/mL into 96

- well plates and incubating at 37  ̊C with 5% CO 2 for 24 h. The medium was replaced, each test compound or an estrogen standard (E 2 ) was added, and the plates were incubated at 37  ̊C with 5% CO 2 for 24 h. The medium was replaced and the ce lls were ly sed with Promega l ys e buffer. Luciferase activity was measured via chemiluminescence with a Berthold ® Microplate Luminometer in order to compare treatment RLU to an E 2 standard. For comparative purposes, the EC 10 was used (Effective Concentrat ion at which 10% of the maximum response was observed). The data were not sufficient to allow calculation of an median effect concentration ( EC 50 ) at which 50% of the maximum response was observed

. The EC 10 of E 2 was 3.2 ×10 - 12 M, which is within the exp ected range, and the dose response curve in the paper indicates that the assay performed as expected for chemicals that have positive estrogenic activity

. Chemical A was shown to have some estrogenic activity in the BG1 Luc4E2 assay with a reported EC 10 o f 4.6 ×10 - 4 M, which is 8 orders of magnitude lower than the E 2 standard. **2. Mammalian OSRI Data**  In a subchronic dietary

toxicity study , male and female Sprague

- Dawley rats 5 months) were fed diets containing Chemical A at levels equivalent to 0, 0.03, 0.3 , 15, and 168 mg/kg/day for males and 0, 0.04, 0.4, 19 and 212 mg/kg/day for females in the diet for 13 weeks. No treatment

- related changes were seen either in the absolute or relative weights of the testes, epididy mides, prostate, uterus, ovaries, thy roid , adrenal or the pituitary

glands. No treatment

- related histopathological lesions were seen in the testes, epididy mides, seminal vesicle, prostate, uterus, ovaries, adrenal, th yroid or the pituitary glands. Clinical signs (h ypersensitivity

to sound and tou ch; hy

peractivity

and aggressiveness) were only observed at the highest dose.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **64** of **2 11**  I n a chronic dietary

toxicity study , male and female Beagle dogs were fed diets containing Chemical A at 0, 0.0032, 0.015, 4.7 and 7.7 mg/kg/day for males and 0, 0.0037, 0.020 , 4.5 and 9.1 mg/kg/day for females in the diet for 52 weeks. No treatment

- related changes were seen either in the absolute or relative weights of the testes, epididy mides, prostate, uterus, ovaries, or the adrenal and thy roid glands. No treatment
- related histopathological lesions were seen in the testes, epididymides, prostate, uterus, ovaries, vagina, mammary , adrenal, thy roid or the pituitary

glands In a combined chronic dietary toxicity /carcinogenicity

study in rats, male and female Sprague Dawley rat s were fed diets containing Chemical A at 0, 0.004, 0.06, 5 and 10 mg/kg/day for males and 0, 0.005, 0.07, 6 and 12 mg/kg/day for females for 98 weeks. No treatment

- related changes were seen either in the absolute or relative weights of the testes, epididy mides, prostate, uterus, ovaries, or the adrenal glands. No treatment

- related histopathological lesions were seen in the testes, epididy mides, seminal vesicle, prostate, uterus, ovaries, vagina, mammary , adrenal, thy roid or the pituitary

glands. In a pre

- n atal developmental toxicity

study , pregnant rats [Crl:COBS CD (SD) (BR)] received Chemical A at 0, 10, 20, and 100 mg/kg/day

in 0.2% carbox y - methy lcellulose by gavage on day s 6 through 15 of gestation. Treatment

- related findings seen only at the highest do se tested were increases in mean number of resorptions (1.8 vs 1.0 in the controls) and post

- implantation loss (13.4% vs. 6.9% in the controls). Treatment had no adverse effects on sex ratio or soft tissue abnormalities. No clinical signs related to choli nergic toxicity

were noted. In a developmental neurotoxicity study , Chemical A was administered to 27 female Crl:CD ® (SD)BR IGS rats/treatment group in the diet at concentrations of 0, 0.30, 30 or 300 ppm from gestation day (GD) 6 through postnatal (lact ation) day (PND) 21. The average dail y test article intake was 0, 0.026, 2.36, or 24.2 mg/kg/day

during gestation and 0.039, 4.06, or 39 mg/kg/day from GD 6 through PND 21. Preputial separation was delay ed at the high dose (45.0  1.88 day s) compared to c ontrols (43.1  1.41 day s). Vaginal opening was also delay ed by at the high dose (33.8  2.28 day s) compared to controls (32.5  1.10 day s). The mean bod y weight at attainment of pubert y was similar between the treated and control groups for males and females . For maternal sy stemic toxicity , the NOAEL was 24.2 mg/kg/day ), the highest dose tested. A LOAEL was not established. For maternal cholinesterase inhibition, the NOAEL was 0.026 mg/kg/day . The LOAEL was 2.36 mg/bw/day based decreases in plasma, RBC and b rain cholinesterase activities. For offspring s ys temic toxicity , the NOAE L was 2.36 mg/kg/day . The LOAEL is 24.2 mg/kg/day based reduced bod y weight, reduced bod y weight gains and delayed sexual maturation in males and females. For offspring cholinesterase inhibition, the NOAEL was 0.026 mg/kg/day . The LOAEL was 2.36 mg/kg/day based on decreased plasma and RBC cholinesterase activities both sexes In a two

- generation reproduction study , male and female Sprague

- Dawley rats were fed diets containing Chemical A at 0, 0.67, 6.69 and 35.15 mg/kg/day for males and 0, 0.77, 7.63 and 41.43 mg/kg/day for females in the diet for two successive generations. Th ere were no treatment
- related changes in testicular or ovarian weights. No treatment

- related histopathological lesions were seen in the testes, epididy mides, seminal vesicle, prostate, coagulating glands, uterus, ovaries, vagina, cervix, or the pituitary g lands. Slight decreases in male and female mating (86.7% vs. 96.7% in the control) as well as male and femaleUS EPA

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– July , 2013

Page **65** of **211** fertility (73.1% vs. 89.7%) indices were seen at the highest dose in the second generation. There was a trend towards prolonged gestation at the highest dose tested in both generations, i.e ., 36% vs. 15% in the controls in first generation and 76% vs. 40% in the controls for the second generation. None of these differences, however, reached statistical significance. The sex ratio of the pups was slightl y higher (p <0.05) for males at the highest dose tested (54.1%) compared to controls (52.7%) for the first generation. No difference in sex ratio was seen for the second generation. Some adults in the high dose group from both the first and second parental generation exhibited tremors. Sexual maturation, estrous cy cle length and periodicit y, and sperm parameters were not evaluated in this study

. For parental/sy stemic toxicity , the NOAEL is 0.67 mg/kg bw/day and the LOAEL is 6.69 mg/kg bw/day based on decreased parental weight gain. For offspring toxicity , the NOAE L is 0.67 mg/kg bw/day and the L OAEL is 6.69 mg/kg bw/day based on pup mortality

and decreased weight gain. Overall, the mammalian OSRI data do not support an interaction with the E, A o r T pathway s, with the exception of the slight delay in PPS seen in the rat developmental neurotoxicity study

.

**3. Ecotoxicity**  **OSRI Data**  I n an earl y life

- cy cle toxicity study with sheepshead minnow, Cyprinodon variegatus , based on the most sensitive endpoint s (length, dry weight, wet weight), the NOEC and LOEC were 0.0043 and 0.008 mg/L a.i., respectively

. An earl y life

- cy cle toxicity study with Chemical A was conducted using fathead minnow. The nominal concentrations of Chemical A selected for the definitive early life stage exposure were 0.094, 0.19, 0.38, 0.75, and 1.5 mg a.i./L . Statistically significant (p<0.05) decreases in larval growth were observed at all test concentrations. Egg hatching and larval survival were not affected b y any test con centration of Chemical A. The chronic effects of Chemical A on fathead minnows and brook trout ( Salvelinus fontinalis ) were determined in flow

- through s ys tems with constant toxicant concentrations. Fathead minnows exposed to the lowest concentration teste d (0.0032 mg/L) from 5 day s after hatch through spawning had a significantl y higher incidence of scoliosis ( i.e ., sideway s curvature of the spine) than the control (p = 0.05). Hatch of their progeny was reduced by

30 percent at this test concentration. Year ling brook trout exposed to 0.0048 mg/L and above began developing scoliosis and lordosis ( i.e ., inward curvature of the spine) within a few weeks. Growth of brook trout was substantially inhibited during the first 3 months at 0.0048 μg/L and above. Neurol ogical s ym ptoms were evident in brook trout at 0.0024 mg/L and above earl y in the tests, but were rarel y observed after 4 or 5 months of exposure. Exposure of mature brook trout for 6 to 8 months to concentrations ranging from 0.0096 mg/L to the lowest tes ted (0.00055 mg/L) resulted in equall y reduced growth rates for their progen y. Transfer of progen y between concentrations indicated that effects noted for progen y of both species at lower concentrations were the result of parental exposure alone and not th e exposure of progen y following fertilization.US EPA

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– July , 2013

Page **66** of **211** In a partial life

- cy cle toxicity test with fathead minnow with Chemical A, the NOAEC is 0.00092 mg/L and the LOAEC is 0.00195 mg/L based on a reduction in the number of eggs per spawn. Fish (59 days old at t est initiation) were exposed for 116 day s and reproduction was monitored with a 32

- 33 day post
hatch exposure of the F 1 generation. There was no effect on F 1 hatching success or 4

- week growth (LOAEC >0.0078 mg/L), but survival of 4

- week old larvae was affe cted at 0.0078 mg/L with a NOAEC of 0.0035mg/L.

In vivo acute toxicity

testing with freshwater fish resulted in L C 50 estimates as low as 0.090 mg/L; acute mortality data specific to fathead minnows indicate that this species is less sensitive (96

- hr LC 50 =7.8 mg/L) than other freshwater species of fish. Acute toxicity testing with aquatic

- phase amphibians ( Rana boylii ; L C 50 =7.5 mg/L ) indicates that this taxon has a sensitivity similar to that of fathead minnow. These data indicate that concentrations test ed in the FSTRA and AMA may have overlapped levels capable of eliciting overt toxicity

. In a one

- generation reproduction study with Northern bobwhite quail ( Colinus virginianus ) with Chemical A, the NOEC for the reproductive parameters was 32 mg/kg, the hi ghest concentration tested. The NOEC for cholinesterase inhibition in blood serum was less than 8.3 mg/kg, the lowest concentration tested, for both males and females.
In a one

- generation reproduction study with the mallard duck (

Anas platyrhynchos ), the NOAEC for reproductive parameters was 8.3 mg/kg diet, based on reduction in the number of eggs incubated as a percentage of eggs laid. The NOAEC for cholinesterase inhibition in blood serum was less than 4.02 mg/kg, the lowest concentration tested, for bo th males and females. Additionally , brain cholinesterase levels for females were depressed compared to the controls at all treatment levels. In a chronic life

- cy cle toxicity test with the freshwater invertebrate waterflea ( Daphnia magna) with Chemical A, there were no effects on growth and reproduction at Chemical A concentrations of ≤0.00017 mg/L. There was 100% mortality at test concentrations, ≥0.00032 mg/L . The study author reported NOAEC was 0.00017 mg/L. In a chronic toxicity

test with an estuarin e/marine invertebrate, i.e ., m ys id shrimp ( Americamysis bahia ) with Chemical A, the most sensitive endpoint was growth (dry weight) with a NOAEC and LOAEC value of 0.0.00023 and 0.00042 mg a.i./L, respectively . There was no effect on reproduction or surviv al up to 0.0016 mg a.i./L. No additional data with frog or other amphibian species have been submitted. Overall, the onl y effect observed in the OSRI ecotoxicity data that could be attributed to an interaction with the E, A or T pathway s was a reduction in the number of eggs in the freshwater fish and mallard duck. Egg reduction, however, is not an endocrine

- specific endpoint and may result from overt toxicity

.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **67** of **211 E.**  **D iscussion**  **i. Effects on Hypothalamic** 
**- Pituitary** 
**- Gonadal (HPG) Axis**  EDSP Tier 1 screening data and OSRI were evaluated together to determine whether Chemical A has potential to interact with the HPG axis or its downstream pathways related to steroidogenesis or sex steroid signaling. The Tier 1 in vitro assay s relevant to this analy sis include the A R, binding, ER binding, ERTA, aromatase, and steroidogenesis assay . Relevant in vivo assay s include the male and female pubertal rat, uterotrophic, Hershberger, and the FSTRA assay s. **Effects on Estrogen Pathway: Table 1**  **7** below summarizes the results of the available EDSP Tier 1 screening assay

result and OSRI results for determining the potential for Chemical A to interact with the estrogen pathway . The various targets of the estrogen pathway across the relevant Tier 1 a ssay s are delineated so as to facilitate determination of potential for estrogenic, anti

- estrogenic, or HPG axis effects.US EPA

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– July , 2013

Page **68** of **211 Table 1**  **7: Estrogenic/Anti** 
**- Estrogenic Pathway**  **for Chemical A Lines of Evidence Indicating Potential Interaction with**  **the Estrogenic/Anti**
**- Estrogenic Pathway**  **for Chemical A a** 
**Study Type / Literature**  **Citation E** **R B** **i n** **d i** **n g** **E R** **A c** **t i** **v a** **t i** **o n** **S t** **e r** **o i** **d o** **g e** **n e** **s i** **s U** **t e** **r i** **n e** **W e** **i g** **h t** **O v** **a r** **i a** **n W** **e i** **g h** **t O** **v a** **r i** **a n** **/ G** **o n** **a d** **S t** **a g** **i n** **g a** **n d** **H i** **s t** **o p** **a t** **h o** **l o** **g y** **P i** **t u** **i t** **a r** **y W** **e i** **g h** **t E** **s t** **r o** **u s** **C y** **c l** **i c** **i t** **y b** **A g** **e &** **W e** **i g** **h t** **a t** **V O** **F e** **r t** **i l** **i t** **y /** **F e** **c u** **n d** **i t** **y V** **i t** **e l** **l o** **g e** **n i** **n O** **v e** **r t** **T o** **x i** **c i** **t y** **O b** **s e** **r v** **e d** **C**

**EDSP Tier 1** ER Binding Equivocal. ERTA No effect Aromatase Equivocal Steroidogenesis No effect Uterotrophic No effect Female Pubertal Rat No effect No effect No effect No effect No

effect No effect No effect

FSTRA H:  follicle atrophy H:  80% ♀L :  63% ♀H:  71% XUS EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **69** of **211 Lines of Evidence Indicating Potential Interaction with**  **the Estrogenic/Anti**
**- Estrogenic Pathway**  **for Chemical A a** 
**Study Type / Literature**  **Citation E** **R B** **i n** **d i** **n g** **E R** **A c** **t i** **v a** **t i** **o n** **S t** **e r** **o i** **d o** **g e** **n e** **s i** **s U** **t e** **r i** **n e** **W e** **i g** **h t** **O v** **a r** **i a** **n W** **e i** **g h** **t O** **v a** **r i** **a n** **/ G** **o n** **a d** **S t** **a g** **i n** **g a** **n d** **H i** **s t** **o p** **a t** **h o** **l o** **g y** **P i** **t u** **i t** **a r** **y W** **e i** **g h** **t E** **s t** **r o** **u s** **C y** **c l** **i c** **i t** **y b** **A g** **e &** **W e** **i g** **h t** **a t** **V O** **F e** **r t** **i l** **i t** **y /** **F e** **c u** **n d** **i t** **y V** **i t** **e l** **l o** **g e** **n i** **n O** **v e** **r t** **T o** **x i** **c i** **t y** **O b** **s e** **r v** **e d** **C**

**OSRI** Kijoma et al. ER reporter assay Positive Subchronic toxicity (Rat) No effect No effect No effect No

effect Chronic toxicity (Rat) No effect No effect No effect Chronic toxicity (Dog) No effect No effect No effect Two

- generation reproduction (Rat) No effect No effect H: ↓37

- 59% X Developmental Neurotoxicity (Rat)  1.3 day s (p<0.05) X

a K e y to r e s pon s e s: L Low treatment, M Medium treatment, H High treatment. Arrows ( ↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. b Factors for estrous cyclicity include: age of first estrous, length of estrous, and percent of animals cycling regularly. c A n “X” in this column indicates that the potential endocrine effect(s) observed in the assay occurred in the presence of overt toxicityUS EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **70** of **211** The EDSP Tier 1 battery

of assay s measure endpoints that are considered to be complementary within an assay and/or redundant across assay s. The results for ER binding and aromatase inhibition were considered equivocal and were potentiall y confounded by the solubility limit of Chemical A. I n a published literature stud y, submitted as OSRI (Kojima, 2005), Chemical A was weakly estrogenic (an EC 10 of 4.6 x 10 - 4 M) when the ER transactivation pote ntial was evaluated in the E

- CALUX test s ys tem; however, the chemical did not transactivate the ER in the EDSP ERTA assay when tested up to a cy totoxic concentration. In the steroidogenesis assay , Chemical A did not alter estradiol production. The equivoca l in vitro responses in the ER binding and aromatase assay s were not corroborated by altered organ or tissue level responses in the mammalian in vivo assay s that would otherwise have been complementary . Chemical A did not induce a response in the uterotrophic assay and no estrogen

- related endpoints were altered in the female pubertal assay . In the FSTRA, e ffects on vitellogenin, gonadal histopathology , fertility , fecundit y, and bod yw eight were observed at concentrations where there was clear evidence of overt toxicity that included abnormal behavior (abnormal swimming) and significant body weight reductions which may be consistent with cholinergic toxic ity

. Given the directionalit y (decreases in the measured endpoints) of the FSTRA responses, the effects may not necessarily be reflective of an endocrine

- mediated response, but rather may reflect a compromised organism with limited ability

to maintain repr oductive function and homeostasis. Although control performance in this study was not entirely

consistent with performance criteria, for fecundit y and fertility, the stud y does not provide consistent evidence of estrogen pathway interaction across the var ious levels of biological organization measured in this study

. OSRI in the form of Part 158 mammalian toxicity studies do not indicate that Chemical A alters structural responses at the organ or tissue level. No organ weight or histopathological effects were seen in uterus or ovary

in subchronic (rat) or chronic (rat and dog) toxicity studies. I n a multi

- generation study in rats, the onl y potential estrogen pathway

- related effect observed was a decrease in fertility , which is a non

- specific effect and may not be an endocrine response. In a rat developmental neurotoxicity

study , a very

slight delay

in vaginal opening (1.3 day s) was observed in the offspring. I n the OSRI ecotoxicity

data , a reduction in the number of eggs was observed in freshwater fis h and mallard duck, however, this is not endocrine

- specific endpoint and the direction of the response (egg reduction) may

be reflective of overt toxicity

. An integration of data from the EDSP Tier 1 and OSRI studies do not indicate that Chemical A intera cts with the E pathway . Chemical A was not an ER agonist up to cy totoxic concentrations in the ERTA assay . The equivocal responses in the ER binding and aromatase assay s occurred at concentrations near the limit of solubility for the test compound and did not translate into effects in higher levels of biological organization in the mammalian Tier 1 in vivo studies. A slight delay

in vaginal opening was observed in a developmental neurotoxicity study in rats, but the delay

was onl y 1.3 day s and considered inconsequential . Additionally , no E pathway

- related effects were observed in the FSTRA in the absence of overt toxicity . Based upon WoE evaluation of EDSP Tier 1 data and OSRI , the absence of robust complementarity and redundancy

in responses do not sup port potential interactions with estrogen signaling pathway

.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **71** of **211 Effects on Androgen Pathway:**  **T able 1**  **8** below summarizes the results of the available EDSP Tier 1 screening assay

results and OSRI for determining the potential for Chemical A to interact with th e androgen pathway . The various targets of the androgen pathway across the relevant Tier 1 assay s are delineated so as to facilitate determination of potential for androgenic, anti

- androgenic, or HPG axis effects. Chemical A demonstrated the potential to interact with the AR and was classified as a binder in the AR binding assay . Chemical A slightly inhibited testosterone production in H295R cells in the steroidogenesis assay . In the in vivo Hershberger assay , Chemical A showed a dose

- dependent positive anti

- androgenic response in sex accessory organ weights in castrated rats at all three doses evaluated (15, 47 and 150 mg/kg/day ). However, when Chemical A was tested in the male pubertal assay using animals with an intact HPG axis, no androgen

- related ef fects were observed at doses up to 100 mg/kg/day

. A number of endocrine responses were observed ( e.g ., decrease in female VTG, fecundity , GSI , male tubercles) at the highest concentration in the FSTRA, there was also pronounced overt toxicity that include d abnormal behavior and significant body weight reductions consistent with cholinergic intoxication. Given the directionalit y of the FSTRA responses ( i.e ., decreases in the measured endpoints), the effects found at the high concentration in the FSTRA may not necessarily be reflective of an endocrine

- mediated response, but rather a reflection of a compromised organism with limited ability

to maintain reproductive function and homeostasis. Although in male fish, overt toxicity was not observed at the inter mediate concentration, possible endocrine responses were limited to two effects that lacked diagnostic specificity (i.e., altered GSI and histology ). In the Part 158 studies submitted as OSRI , no effects on organ weight or histopathology of the testes, e pididy mis or sex accessory

organs were observed with subchronic or chronic exposure of Chemical A in the rat or in the chronic toxicity study in dogs. No androgen pathway

- related effects were observed in the 2

- generation rat reproduction study . In a rat developmental neurotoxicity study in rats, a slight delay (less than 2 day s) in preputial separation was observed. There is some evidence of an interaction when evaluating the androgen pathway responses across all the considered studies. Chemical A interacts with the AR in the binding assay and decreases testosterone production in the steroidogenesis assay . In the He rshberger assay , Chemical A acts as an androgen antagonist causing dose

- related decreases in androgen

- responsive tissue weights. No effects were found in the Tier1 male pubertal assay . However, a slight delay in preputial separation was also observed in a developmental neurotoxicity study (DNT) in rats. Although these responses are consistent with the potential interaction with the A signaling pathway (via androgen antagonism and perturbation of ster oi dogenesis ), there is some uncertaint y around the observ ed responses. The in vitro effects were seen only at a high test concentration that was near the limit of solubility for the compound. In the FSTRA, there were onl y two endpoints affected in the absence of overt toxicity , and these are nonspecific respon ses and could be due to endocrine or non

- endocrine mechanisms. Effects in the Hershberger assay

were not observed in the male pubertal assay (rats with intact HPG) which was conducted at similar doses.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **72** of **211** T he in vitro responses in the AR binding and steroid ogenesis assay s (albeit at high concentrations) and the tissue responses in the Hershberger indicated that Chemical is potentially interacting with the A pathway

. In organisms with an intact HPG axis, Chemical A showed no androgen related effects in the m ale pubertal assay , and in FSTRA, several endpoints were altered that could be consistent with androgen antagonism; however, man y were in the presence of overt toxicity

. For the A signaling pathway , the in vitro assay results suggest that Chemical A has th e potential to interact with the AR and is able to alter steroidogenesis resulting in a decrease in testosterone production. Tissue level responses (decreased organ weights) consistent with AR binding and/or decreased testosterone production were observed in the Hershberger assay . However in the intact in vivo model ( e.g . pubertal male), presumabl y with compensatory mechanisms, no androgen related effects were observed. I n the FSTRA, several endpoints were altered that could be consistent with androgen a ntagonism; however, many were in the presence of overt toxicity

. The Part 158 toxicology data did not provide convincing evidence of an in vivo interaction with the A pathway

.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **73** of **211 Table 18:**  **Androgenic/Anti**
**- Androgenic Pathway**  **for Chemical A Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti** 
**- Androgenic Pathway for Chemical A**  **a**

**Study Type / Literature**  **Citation A** **R B** **i n** **d i** **n g** **S t** **e r** **o i** **d o** **g e** **n e** **s i** **s T** **e s** **t o** **s t** **e r** **o n** **e T** **e s** **t e** **s W** **e i** **g h** **t G** **o n** **a d** **S t** **a g** **i n** **g a** **n d** **H i** **s t** **o p** **a t** **h o** **l o** **g y** **E p** **i d** **i d** **y m** **i d** **e s** **W e** **i g** **h t** **E p** **i d** **i d** **y m** **i d** **e s** **H i** **s t** **o p** **a t** **h o** **l o** **g y** **P i** **t u** **i t** **a r** **y W** **e i** **g h** **t A** **c c** **e s** **s o** **r y** **S e** **x O** **r g** **a n** **W e** **i g** **h t** **s /** **2 °** **S e** **x C** **h a** **r a** **c t** **e r** **i s** **t i** **c s** **A g** **e a** **n d** **W e** **i g** **h t** **a t** **P P** **S V** **i t** **e l** **l o** **g e** **n i** **n O** **v e** **r t** **T o** **x i** **c i** **t y** **O b** **s e** **r v** **e d** **b**

**EDSP Tier 1** AR Binding Binder Steroidogenesis  Test.

Hershberger L: SV  29% VP  26% M: SV  37% VP  43% LA BC  21% H: SV  86% VP  77% LABC  45% CG  69% GP  20% Male Pubertal Rat No effect No effect No effect No

effect No effect No effect No effect No

effect

FSTRA M:  29% H:  31% L,M,H:  spermatogonia H:  74% nuptial tubercle score No effect XUS EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **74** of **211 Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti** 
**- Androgenic Pathway for Chemical A**  **a**

**Study Type / Literature**  **Citation A** **R B** **i n** **d i** **n g** **S t** **e r** **o i** **d o** **g e** **n e** **s i** **s T** **e s** **t o** **s t** **e r** **o n** **e T** **e s** **t e** **s W** **e i** **g h** **t G** **o n** **a d** **S t** **a g** **i n** **g a** **n d** **H i** **s t** **o p** **a t** **h o** **l o** **g y** **E p** **i d** **i d** **y m** **i d** **e s** **W e** **i g** **h t** **E p** **i d** **i d** **y m** **i d** **e s** **H i** **s t** **o p** **a t** **h o** **l o** **g y** **P i** **t u** **i t** **a r** **y W** **e i** **g h** **t A** **c c** **e s** **s o** **r y** **S e** **x O** **r g** **a n** **W e** **i g** **h t** **s /** **2 °** **S e** **x C** **h a** **r a** **c t** **e r** **i s** **t i** **c s** **A g** **e a** **n d** **W e** **i g** **h t** **a t** **P P** **S V** **i t** **e l** **l o** **g e** **n i** **n O** **v e** **r t** **T o** **x i** **c i** **t y** **O b** **s e** **r v** **e d** **b**

**OSRI** Subchronic toxicity (Rat) No effect No

effect No

effect No effect No effect No

effect Chronic toxicity (Rat) No effect No

effect No

effect No effect Chronic toxicity (Dog) No effect No

effect No

effect No effect No

effect Two

- generation reproduction (Rat) No effect No

effect No

effect No

effect Developmental Neurotoxicity (Rat) H:  1.9 d X

a K e y to r e s pon s e s: L Low treatment, M Medium treatment, H High treatment. Arrows ( ↓ o r ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. SV= seminal vesicle; VP= ventral prost ate; LABC= levator ani

- bulbocavernosus muscle; CG= Cowper’s gland; GP= glans penis b An “X” in this column indicates that the potential endocrine effect(s) observed in the assay occurred in the presence of over t toxicity.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **75** of **211 ii.**  **Effects on Hy pothalamic** 
**- Pituitary** 
**- Thyroidal (HPT) Axis**  In general, regulation of the HPT axis is comparable to the HPG axis, except that the feedback relationship involves thy

roid hormones ( e.g ., T 4 and T 3 ). Thy roid hormones feedback to the hy pothalamus or pituitary

to regulate thy

rotropin

- releasing hormone and TSH, respectivel y, which, in turn, regulate hormone production b y the thy roid gland. The current Tier 1 screening battery does not have a specific in vitro assay to detect chemicals with the potential to affect hy pothalamic or pituitary regulation of thy roid hormone production, but it does include three in vivo assay s that have the potential to detect these effects on the HPT axis ( i.e., the puberta l s and the AMA ). **Thyroid Hormones: Table**  **19** summarizes the results of the available EDSP Tier 1 studies and endpoints relevant for determining the potential for Chemical A to interact with thy roid regulation. The various targets of the th yroid pathway across the relevant Tier 1 assay s are delineate d so as to facilitate determination of potential for thy roid or HPT axis effects. Table 19 also includes HPT

- relevant findings from data evaluated as OSRI

. In the EDSP Tier 1 battery results, the only observed response that could be attributed to a poten tial interaction with the HPT axis was a slight decrease ( ↓23% and 17%) in serum T 4 levels observed in female pubertal rats treated with Chemical A at 50 and 100 mg/kg bw/day , respectivel y. A small decrease ( ↓15%) in T 4 was also seen in the male pubertal a ssay at 50 mg/kg bw/day , but not at 100 mg/kg bw/day and was not considered biologically significant. The depression in T4 was not associated with complementary alterations in TSH levels or changes in th yroid weights, pituitary weights, or thyroid histopa thology in the male or female pubertal assay s that might be anticipated if the compound were interacting with the HPT axis. Furthermore, the exposure period in the male pubertal assay is 10 day s longer than the female pubertal assay , y et no compelling thy r oid effects were observed in the male. Thy roid gland hy pertrophy was observed in the AMA at the highest treatment level (0.820 mg a.i./L); however, no other noteworthy effects were reported. Based on these data, there was not compelling evidence for pot ential thy roid interaction in the AMA. T here were also no changes in thy roid weights, pituitary

weights or th yroid histopathology in the mammalian Part 158 studies evaluated. These studies included a subchronic study in rats treated with Chemical A for 1 3 weeks at doses up to 168 mg/kg bw/day and 2

- ye ar rat study

and 1

- year dog stud y treated with doses up to 10 and 9 mg/kg bw/day , respectivel y.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **76** of **211 Table 19**  **: Thyroid**  **Pathway for Chemical A**  **Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Chemical A**  **a**

**Study Type / Literature Citation** **H o** **r m** **o n** **e s** **( T** **4 a** **n d** **T S** **H )** **P i** **t u** **i t** **a r** **y W** **e i** **g h** **t T** **h y** **r o** **i d** **W e** **i g** **h t** **T h** **y r** **o i** **d G** **r o** **s s** **a n** **d H** **i s** **t o** **p a** **t h** **o l** **o g** **y F** **r o** **g D** **e v** **e l** **o p** **m e** **n t** **S t** **a g** **e H** **i n** **d L** **i m** **b L** **e n** **g t** **h S** **n o** **u t** **t o** **V e** **n t** **L e** **n g** **t h** **O v** **e r** **t T** **o x** **i c** **i t** **y O** **b s** **e r** **v e** **d b** 

**EDSP Tier 1**  Male Pubertal (Rat) L: ↓T4 23% H: ↓T4 17% No

Effect No

Effect No Effect Female Pubertal (Rat) L: ↓T4 15% No Effect No

Effect No Effect AMA (Frog) Equiv ocal No Effect No

Effect No

Effect OSRI Two

- generation reproduction (Rat) No Effect No

Effect No Effect Subchronic toxicity

(Rat) No Effect No

Effect No Effect Chronic toxicity

(Rat) No Effect No Effect Chronic toxicity

(Dog) No Effect No Effect

a Key to responses: K e y to r e s pon s e s: L Low treatment, M Medium treatment, H High treatment. Arrows ( ↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. b An “X” in this column indicates that the potential endocrine effect(s) observed in the assay occurred in the presence of overt toxicity
.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **77** of **211** Evidence of slight T4 depression in the female pubertal rats was not corroborated b y complementary

changes in TSH, thy

roid weight or thy

roid histopathology . The decrease in T4 was not consistent with the magnitude of effects typically seen with thy

roid toxicants. There were also no biologically relevant changes in T4 levels in the pubertal males which are treated for 10 day s longer than female rats in the pubertal assay . The lack of thy roid pathway

- related effects in the AMA and mammalian Part 158 studies reinforce the absence of th yr oid interaction. Based upon WoE evaluation of EDSP Tier 1 data and OSRI , Chemical A does not demonstrate a potential to interact with the thy roid pathway

.
**F. Conclusions for Chemical A**  All eleven assay s compr ising the EDSP Tier 1 battery were available for Chemical A. Chemical A was evaluated for several hy pothesized endocrine modes of action for each specific E, A, and T pathway including agonism and antagonism at the estrogen and androgen receptor, altered s ter oi dogenesis , HPG and HPT perturbations. In addition to the available Tier 1 data, other relevant studies of sufficient quality were considered in this assessment. These studies included standard Part 158 toxicity data relevant to evaluating effects in endocrine tissues and functions. In determining whether Chemical A interacts with E, A, or T hormonal pathway s, the number and ty pe of effects induced, the magnitude of responses, and the pattern of responses observed across studies, taxa, and sexes wer e considered. Additionally , the conditions under which effects occur and whether overt toxicity was present were considered . This is particularly important in the case of Chemical A, given that it belongs to the organophosphate class of chemicals that res ults in cholinergic toxicity

. Integrating the in vitro and in vivo Tier 1 results, as well as consideration of relevant data bey ond Tier 1 information ( e.g ., Part 158 standard toxicology studies) did not reveal convincing evidence of an interaction with the E or T pathway s. There was reasonable compl e mentarity within assay s and redundancy

among responses across assay s in terms of a lack of clear and consistent responses for E across different levels of biological organization. For the T pathway , the AMA was negative and only

small changes in T4 were seen in the pubertal assays at doses that would be expected to cause significant cholinesterase inhibition. Although there were responses in FSTRA that could be indicative of an interaction with the estrogen signaling pathway , they were only found in the presence of overt toxicity

.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **78** of **211** With respect to the A signaling pathway , the in vitro responses in the AR binding and steroidogenesis assay s (albeit at high concentrations) and the tissue responses in the Hershb erger indicated that Chemical is potentially interacting with the A pathway

. In organisms with an intact HPG axis, Chemical A showed no androgen related effects in the male pubertal assay , and in FSTRA, several endpoints were altered that could be consist ent with androgen antagonism; however, man y were in the presence of overt toxicity

. The Part 158 toxicology data did not provide convincing evidence of an in vivo interaction with the A pathway

.


### 7. **Case Study**

**— Chemical**  **S**

**A. Introduction**  Chemical S is an organo metal compound and has a mode of action involving inhibition of oxidative phosphory lation at the site of dinitrophenol uncoupling and as such it affects the production of energy in the form of adenosine triphosphate, ATP. In the case of Chemical S, the issues included: potential interactions with more than one endocrine modality ; potential endocrine

- related effects in the presence of overt toxicity ; challenges encountered in dose selection (overt toxicity ); and endpoint sensitivity

( e.g ., endocrine v s. overt toxicity ). The available information considered to determine the potential interaction of Chemical S with the E, A and/or T pathway s include all 11 EDSP Tier 1 studies and other scientifically relevant information (OSRI) such as 40CFR Part 158 gui deline toxicity studies. The data considered are summarized in Section s B, C and D of this chapter. A n anal ys is of the data submitted to the agency , using the WoE approach outlined by the Agency

(USEPA, 2011 b ), is presented in Section E

.

**B. D**  **ata Availabl e for Chemical S**  Chemical S is a relatively insoluble compound with a high log octanol

- water partition coefficient (log K ow >5) and organic carbon partition coefficient (K oc ~321,000 L /kg). The compound is relativel y stable to abiotic routes of degradation ( i.e ., hy droly sis and photoly sis) and to aerobic metabolism; however, Chemical S can degrade slowly via anaerobic metabolism. Therefore, Chemical S is expected to persist in the environment and given its high K oc is likely to partition to sediments. Although the compound has a relatively high K ow , fish do not demonstrate appreciable bioconcentration factors suggest ing that the compound may be metabolized by

fish and/or its uptake may

be limited . The low solubili ty in water combined with the high K ow and K oc values render this chemical difficult to test in aquatic sy stems as the chemical would readily sorb to test containers.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **79** of **211 C.**  **Tier 1 Screening Assays for Chemical S**  The Tier 1 studies submitted in response the test order for Chemical S are shown below in **Table 20**  and the relevant study

details and findings are summarized below

. **Table 2 0**  **: Summary of**  **Tier 1 Screening Assays for Chemical S**

**Tier 1 Assay Test Guideline**  **Satisfied by OSRI (Y/N)**  **Study Findings** ER Binding Assay

(Rat uterine cy

tosol OSCPP 890.1250 No N egat ive 1 ERα Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300; OECD 455 No Negative AR Binding Assay

(Rat prostate cy

tosol) OSCPP 890.1150 No N egative 2 Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550; OECD 456 No Negative Aromatase Assay

(human recombinant microsomes) OSCPP 890.1200 No Equivocal Uterotrophic Assay

(Rat) OSCPP 890.1600; OECD 440 No Negative Hershberger Assay

(Rat) OSCPP 890.1400; OECD 441 No Negative for A Positive for Anti A Pubertal Female Assay

(Rat) OSCPP 890.1450 No Positive for E pathway Negative for T pathway Pubertal Male Assay

(Rat) OSCPP 890.1500 No Negative for A pathway Negative for T pathway Fish Short

- term Reproduction Assay OSCPP 890.1350; OECD 229 No Possible for A pathway 3 Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100;

OECD 231 No In adequate 4

1 Classified as “Not Interactive” according to the Test Guideline OSCPP 890.1250 2 Classified as “Non

- Binder” according to the Test Guideline OSCPP 890.1150 3 Effects coincide with decreases in survival which confounds interpretation 4 Indequate exposureUS EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **80** of **211 i.**  **ER Binding Assay (Rat uterine cytosol)** In the ER binding assay

(98.75% a.i.), uteri ne cy

tosol from Sprague Dawley rats was used as the source of ER for a Competitive Binding Experiment, which was conducted to measure the binding of a single concentration of tritiated [ 3 H]

- 17β

- estradiol (1 nM) in the presence of increasing concentrations (logarithmic increase from 10 − 12 to 10 − 5 M) of Chemical S. A solubility test showed that Chemical S was not soluble in dimethy l sulfoxide (DSMO) or ethanol at concentrations >10 - 5 M. Ethanol was used as the solvent vehicle at a final assay concentration of <3% (v/v). A total of three valid runs were performed, and each run included 19

- norethindrone as a weak positive control, octy ltriethoxy silane as a negative control, and 17

- β

- estradiol as the natural ligand reference material. A saturation binding expe riment demonstrated that the ER was present in the rat uterine cy tosol at a reasonable concentration and functioning with appropriate affinity for the 17β

- estradiol ligand. The dissociation constant (K d ) for [ 3 H]

- 17β

- estradiol was 0.1153 nM, and the estim ated average maximum binding (B max ) was 45.75 fmol/100 μg protein for the single run with the batch of uterine cy tosol that was prepared. The K d and the B max were within the expected ranges. Confidence in these numbers is high based on visual inspection of the goodness of fit. The specific binding reached a plateau, and the maximum non

- specific binding was less than 24% of total binding. In a competitive binding experiment, the estimated average specific binding affinity (reported as Lo g IC 50 ) was −8.94 Lo g M for 17β

- estradiol and −6.11 Log M for the weak positive control (19

- norethindrone). Compared to 17β

- estradiol, the mean relative binding affinity (RBA) for the weak positive control was 0.150%. All of the performance criteria for 17β

- estradiol were met across the three runs of the competitive binding assay . The guideline does not provide performance criteria for 19

- norethindrone, the weak positive control. The non

- specific binding was approximately ≤0.5%, and the curve for the reference materi al showed that increasing concentrations of unlabeled 17β

- estradiol generally displaced [ 3 H]

- 17β

- estradiol in a manner consistent with one

- site binding, as indicated b y a mean Hill slope of approximately −0.9 to −0.8. Based on the results of th r e e run s , Chemical S is classified as Not Interactive i n the ER binding a ssay .
**ii. ER Transcriptional Activation Assay**  In the ER transcriptional activation assay , hERα

- HeLa

- 9903 cells were exposed to Chemical S (97.82% a.i.) at logarithmically increasing con centrations from 10 − 13 to 10 − 6 M in 0.1% ethanol for approximately

24 hours in two independent runs. The experiments were performed using 96

- well plates and each concentration was tested in 6 wells/plate per run.US EPA

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Page **81** of **211** A preliminary

cy totoxicity test showed that Chemical S was cy totoxic in this assay s s ys tem at concentrations of 10 - 6 M (35% viability ) and above. The highest concentration tested in the ERTA assay was 10 - 6 M. Cells were exposed to the test agent for 24±2 hours at 37±1°C to induce reporter (lu ciferase) gene products. L uciferase expression in response to activation of the estrogen receptor was measured using a luciferase assay

. The performance criteria were met for the reference chemicals 17β

- estradiol, 17α

- estradiol and corticosterone, howe ver, the very weak agonist, 17α

- methy ltestosterone, did not achieve 50% transcriptional activation, therefore a PC 50 could not be calculated. Additionally, the response of the cells to 17α

- methy ltestosterone was weaker than the expected response, and the Lo g PC 10 for the two runs (

- 5.7 and

- 5.8) fell below the performance criteria range (

- 8.0 to

- 6.2) for this reference chemical. Chemical S was cy totoxic at 10 - 6 M in both runs (29

- 46.5% viability ). The highest mean induction value as expressed as the percentage of response induced by the positive control (RPC max ) for Chemical S in both runs was 2.9%, which is less than the 10% response required for a positive response. There was no evidence of ER transcriptional activation by Chemical S up to 10 - 7 M. Chemical S was negative for ER t ranscriptional activation in this test sy stem
.

**iii. AR Binding Assay (Rat prostate cytosol**  ) In the AR binding assay , ventral prostate cy tosol from Sprague Dawley rats was used as the source of AR to conduct a saturatio n binding experiment and a competitive binding experiment. The competitive binding assay was conducted to measure the binding of a single concentration of [ 3 H]

- R1881 (1 nM) in the presence of increasing concentrations (10 − 11 to 3.33 × 10 − 5 M) of Chemical S (97.85%). Ethanol was used as the solvent vehicle for Chemical S, and the reference chemicals (dexamethasone and R1881), at a final assay concentration of 4.19% (v/v). A total of three runs were performed. Each run included dexamethasone as a weak pos itive control, and R1881 as the ligand reference standard. The saturation binding experiment demonstrated that the AR was present in the rat ventral prostate cy tosol at a reasonable concentration and functioning with appropriate affinity for the R1881 lig and. The dissociation constant (K d ) for [ 3 H]

- R1881 was 1.255 nM, and the estimated B max was 15.75 fmol/100 μg protein for the single run with the batch of prostate cy tosol that was prepared. The K d and the B max were within the range for the performance c riteria. The specific binding reached a plateau, and the maximum non

- specific binding was less than 18% of total binding. The Scatchard plots for the saturation binding assay data were approximately linear. In the competitive binding experiment, the es timated average Log I C 50 was −8.79 for R1881 and −4.34 for the weak positive control (dexamethasone). Compared to R1881, the mean relativeUS EPA

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Page **82** of **211** binding affinity

(RBA) for the weak positive control was 0.0036%. No precipitation of the test compound was visuall y observed at ≤3.33 ×10 − 5 M. All of the performance criteria for R1881 and dexamethasone were met across the three runs. The non - specific binding was approximately ≤2%, and the curve for the reference material showed that increasing concentrations of un labeled R1881 generall y displaced [ 3 H]

- R1881 in a manner consistent with one

- site binding, as indicated b y a mean Hill slope of approximately −1.0. The lowest portion of the Chemical S across all runs was >75% at 3.3 × 10 − 5 M (highest concentration tested ). Based on the results of three runs, Chemical S is classified as a “ N on - binder ” in the AR binding a ssay .
**iv. Steroidogenesis Assay (Human cell line H295R)**  In a steroidogenesis assay , H295R cells cultured in vitro in 24

- well plates were incubated with Chemical S (97.82% purity ) at log concentrations from 0.00001 to 1 μM in triplicate for 48 hours. The solvent vehicle for the test chemical was ethanol, at a final assay concentration of 0.075% (v/v). Testosterone a nd estradiol levels were measured using HPL C

- MS/MS with internal standards. A Quality

Control (QC) plate was run concurrently with each independent run of a test chemical plate to demonstrate that the assay responded properl y to positive control agents at two concentration levels. Positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production. A total of 12 independent runs of the steroidogenesis assay were reportedl y conducted; however, only th e results from 5 of the test runs were reported. The remaining seven tests reportedl y failed because either the QC plates did not meet the guideline criteria or because there was excessive variability

in the test plates. Increased cy totoxicity occurred i n the Chemical S

- treated cells at 1 μM (1.9

- 21.0% viability ) in all five runs, therefore, data from the 1 μM concentration were not evaluated. Precipitation was also noted at this concentration in Run 1. No other cy totoxicity was noted. The performance criteria were generally met for the test plates and for the QC plates, with the following minor exceptions. Estradiol production in the blank and solvent control (SC) wells was less than 40 pg/mL in Runs 4 (36

- 37 pg/mL) and 5 (33

- 34 pg/mL), but the basal estradiol production was ≥2.5

- fold above the minimum detection limit (MDL) of the anal yt ical method (10 pg/mL). Inhibition of estradiol by 1 μM prochloraz slightly exceeded the 0.5

- fold performance criteria in four runs (0.51

- to 0.58

- fold), and inductio n of testosterone b y 10 μM forskolin was below the 2

- fold the performance criteria in all five runs (1.32

- to 1.68 fold). However, when the background hormone concentrations were subtracted, 10 μM forskolin induced testosterone an average of 2.56

- fold. I n addition, the between

- plate coefficient of variation (CV) for testosterone was 38.2%, which is above the recommended limit of 30%.US EPA

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Page **83** of **211** There were slight decreases (p ≤0.05) in testosterone levels (0.92

- to 0.93

- fold) at Chemical S concentrations of 0.0001

- 0.1 μM in Run 5, but the effect was not dose

- dependent and significant decreases in testosterone were not noted in the other four runs at these concentrations. The onl y other significant changes (p<0.05) in testosterone were a slight decrease (0.86

- fold) in Run 1 at and a slight increase (1.18

- fold) in Run 2, both at 0.00001 μM. For all five runs, the average fold difference in testosterone concentrations was 0.96

- to 1.02

- fold at Chemical S concentrations of 0.00001

- 0.1 μM. When hormone levels were normali zed for cell viability , there were no significant effects on testosterone levels at an y Chemical S concentration in an y run. For estradiol, the only

significant change compared to the SC was a 0.87

- fold reduction at the 0.1 μM concentration in the third run. The average fold difference in estradiol concentrations was 1.00

- to 1.05

- fold at Chemical S concentrations of 0.00001

- 0.1 μM. When estradiol levels were normalized for cell viability , there was a minor increase (p<0.05) in estradiol of 1.08

- fold in Run 1 at 0.0001 μM, and a slight decrease (p<0.05) of 0.87

- fold in Run 3 at 0.1 μM. Based on the hormone responses in three runs , Chemical S was negative in the ster oi dogenesis assay .

**v. Aromatase Assay (Human Recombinant Microsomes)**  In an in vitro aromat ase (CYP19) assay , Chemical S (97.85% a.i.) was incubated with human recombinant aromatase and tritiated androstenedione ([ 3 H]

- ASDN) in 1% ethanol at log concentrations from 10 − 10 to 10 − 5 M for 15 minutes to assess the effect of Chemical S on aromatase act ivity . The 10 − 5 M concentration of Chemical S was the highest test concentration due to solubility issues at higher concentrations. Aromatase activity

was determined by

measuring the amount of tritiated water produced at the end of a15

- minute incubation f or each concentration of test chemical. Tritiated water was quantified using liquid scintillation counting (LSC). Three runs were conducted and each run included a full activity control, a background activity control, a positive control series (10 − 10 to 10 − 5 M) using a known inhibitor ( i.e ., 4

- OH ASDN), and the Chemical S Chemical Series (10 − 10 to 10 − 5 M) with three replicates per concentration. Aromatase activity

in the full activity

controls ranged from0.309 to 0.455 nmol ∙mg - protein − 1
∙ min − 1 for the three test runs, with a mean ± standard deviation of 0.388 ± 0.048 nmol ∙mg - protein − 1
∙ min − 1

. Activity in the background controls averaged 0.0028 ± 0.001 nmol ∙mgprotein − 1
∙ min − 1 , or 0.71% of the full control activity . The response of the full activ ity controls and background controls were acceptable for each run. For the positive control substance (4

- OH ASDN), aromatase activity averaged 0.387 ± 0.018 nmol ∙mg - protein − 1
∙ min − 1 at the lowest tested concentration 10 − 10 M and 0.0026 ± 0.0003 nmol ∙mg -US EPA

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Page **84** of **211** pr otein − 1
∙ min − 1 at the highest tested concentration 10 − 5 M. The estimated Log IC 50 for 4

- OH ASDN averaged −7.23 Log M and the Hill Slope was −0.996. F or Chemical S, aromatase activity averaged 0.407 ± 0.061 nmol ∙mg - protein − 1
∙ min − 1 at the lowest tested conce ntration 10 − 10 M and 0.270 ± 0.022 nmol ∙mg - protein − 1
∙ min − 1 at the highest tested concentration 10 − 5 M. The L og IC 50 could not be calculated, as 50% inhibition was not achieved with Chemical S. However, with the exception of response seen at the 10 − 9 M (8 8.5% activity ) , the limited inhibition observed for Chemical S was generall y dose responsive, declining from 101.7% full control activity

at 10 − 7.5 M down to 68.8% activity at 10 − 5.5 M. The average lowest portion of the curves across the three runs was between 50 and 75% activity , indicating an equivocal response in this assay

( **Figure 1 2**  below). **F igure 12**  **. Inhibition Response Curves for**  **Chemical S From Each Test Run.** 

Based on the results of three runs, Chemical S is classified as E quivocal in the aromatase assay
.

**vi. Uterotrophic Assay (Rat)**  In the in vivo Uterotrophic Assay conducted to screen for potential estrogenic activity , Chemical S (97.85% purit y) in corn oil was administered dail y via oral gavage to groups of six adult ovariectomiz ed, female Wistar (RccGab:WI ST) rats at dose levels of 0 (vehicle), 10, or 75 mg/kg bw/day for three day s from post

- natal day s (PND) 61/62 to 63/64. A positive control group was treated with a daily

dose of 17α

- eth yn yl estradiol (EE) at 0.3 μg/kg bw/day b y subcutaneous injection. Bod y weights were determined daily. All animals were euthanized and necropsied on This image cannot currently be displayed.US EPA

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Page **85** of **211** PND 64/65 approximately 24 hours after the final dose administration to determine wet and blotted uterine weights. All animals survived until scheduled termination. Clinical signs of toxicity were limited to salivation in 1/6 and 4/6 rats at 10 and 75 mg/kg bw/day , respectivel y. Terminal body weight was decreased (not significant, NS) by 5% in the 75 mg/kg bw/day group compared to vehicle cont rols. This group display ed an overall (Day s 1

- 4) bod y weight loss (p<0.05) of

- 0.68 g compared to the 10.37 g weight gain by

the controls. Body

weights in the 10 mg/kg bw/day group were comparable to the control throughout the study ; however, overall bod y weight gain was decreased (NS) b y 42% compared to controls. Uterine weights (wet and blotted) in the Chemical S treatment groups were comparable to the vehicle controls. Bod y weights and overall bod y weight gain in the positive control (EE) group were c omparable to the controls. As expected, the absolute wet and blotted uterus weights for the EE group were increased (p<0.01) b y 246 and 200%, respectivel y, over the 2 day s of necrops y. The high

- dose level was an adequate dose based on overall body weight loss during the treatment period. No statistically

significant increase in uterine weight was seen in this study . Hence, Chemical S was negative in the Uterotrophic assay

.

**vii. Hershberger Assay (Rat)** 

The dose levels for the in vivo Hershberger assay were selected based on the results from a range

finding stud y in Wistar rats (5/sex/dose). Chemical S was administered via oral gavage at doses of 0, 10, 50, or 100 mg/kg bw/day for 28 day s. At 100 mg/kg bw/day , one female was found dead on Day 19. All other animals survived to scheduled sacrifice. Salivation was observed in 2 males at 10 mg/kg bw/day

and in all animals at 50 and 100 mg/kg bw/day . Additionally , one female at 100 mg/kg bw/day

display ed dy spnea and gasping. Body weights, bod y weight gai ns, and food consumption were decreased in the 100 mg/kg bw/day males for 2

- 4 weeks of the 4 week treatment period. Although there were no significant decreases in body weights observed in the females at any dose compared to controls, body weight gains in the 100 mg/kg bw/day male group were decreased during Week 1

- 2. At 100 mg/kg bw/day, possible target organ effects based on organ weight changes were noted in the testes, levator ani

- bulbocavernosus (LABC) and Cowper’s gland, liver, and spleen. Certain biochemical disturbances such as increases in leukocy te counts, total bilirubin, AL T activity , and phosphorous levels were noted at 50 and 100 mg/kg/day ; these effects by sex were not specified in the study report. Based on these results, a high dose of 7 5 mg/kg bw/day was selected for the Hershberger Assay

.US EPA

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– July , 2013

Page **86** of **211** In a Hershberger Assay

screening for androgenic activity , Chemical S (97.85% purity ) in corn oil was administered dail y via oral gavage to groups of six 51

- day old, castrated male Wistar (RccHan:WI ST) rats at dose levels of 0 (vehicle), 10, or 75 mg/kg/day for 10 day s . The test included a vehicle control group and an androgenic positive control group consisting of six castrated rats exposed to 0.4 mg/kg bw/day of testosterone propionate (TP) by subcut aneous (s.c.) injection. In a second phase of the Hershberger Assay screening for potential anti

- androgenic activity , Chemical S (97.85% purity ) in corn oil was administered dail y via oral gavage to groups of six 51

- day old, castrated male Wistar (RccHan: WI ST) rats dose levels of 10, 40, or 75 mg/kg bw/day in conjunction with a daily

dose of reference androgen TP at 0.4 mg/kg bw/day by s.c. injection. The negative control group for anti

- androgenic activity consisted of six castrated rats dosed with TP at 0.4 mg/kg bw/day

by s.c. injection, and the antiandrogenic positive control group consisted of six castrated rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg bw/day in conjunction with a s.c. dose of TP (0.4 mg/kg bw/day ). For both assay s, bo dy

weights were determined daily and overall (Day s 1

- 11) food consumption (g/rat/day ) was determined. Animals were dosed for 10 consecutive day s and euthanized approximately

24 hours after the final dose administration to determine weights of the five and rogen - dependent accessory sex tissues ( i.e ., seminal vesicles, ventral prostate, levator ani

bulbocavernosus (LABC), Cowper's glands or glans penis), liver, kidney , and adrenal weights. All animals in both studies survived until scheduled termination. C linical signs of toxicity

in both phases of the assay were limited to salivation in 3/6 and 6/6 rats in the 10 and 75 mg/kg bw/day groups, respectivel y, in the androgen agonist study , and in 4/6 and 6/6 rats in the 40 and 75 mg/kg bw/day groups in the anti

- androgen stud y. There were no treatment

related effects on weights of liver, kidney s, adrenals or any the accessory

sex tissues of Chemical S

treated animals. In the androgen agonist assay , there were no statisticall y s ignificant (NS) differences in bod y weights at either dose of Chemical S, although overall body weight gain (Days 1

- 11) was decreased (p<0.05) by 17%, and overall food consumption was decreased (p<0.01) by 6% at 75 mg/kg bw/day . Although seminal vesicle weights were increased (p<0.05) b y 21% at 10 mg/kg bw/day , this solitary finding occurred onl y at the lowest dose. As expected, the TP positive control group had increases (p<0.01) in the weights of the seminal vesicles ( ↑1097%); ventral prostate ( ↑787%); LABC (↑114%); Cowper’s glands (↑55 1%); and glans penis ( ↑76%), indicating that the test sy stem was sensitive to an androgenic response. Because significant weight increases were not observed in two or more androgen

- dependent tissues, Chemical S is not considered to be an androgen agonist at ≤75 mg/kg/day

.US EPA

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Page **87** of **211** In the anti

- androgen assay , there were decreases (p<0.05) of 5

- 7% in body weights on Day s 711% at 40 mg/kg bw/day +TP and 7

- 10% on Day s 4

- 11 at 75 mg/kg bw/day +TP. Overall body weight gains were also decreased (p<0.01) b y 29 and 45% at 40 and 75 mg/kg bw/day , respectivel y, and food consumption was decreased (p<0.01) b y 11

- 33% in all Chemical S

- treated groups. There were no treatment
- related effects on weights of liver, kidney s or adrenals. However, significant weight decreases (p<0.05 or <0.01) were noted in the ventral prostate ( ↓19%) and glans penis (↓16%) at 10 mg/kg bw/day . At 40 mg/kg bw/day +TP, tissues weights were decreased (p<0.01) by 34% in the seminal vesicles, 40% in the ventral prostate, 16% for the LABC, 27% for the Cowper’s glands, and 20% for the glans penis. At 75 mg/kg bw/day +TP, tissues weights were decreased (p<0.01) b y 23% in the seminal vesicles, 24% in the ventral prostate, and 23% for the LABC. These decreases were not generally

dose dependent with the exception o f the ventral prostate and the glans penis. As expected, the positive FT+TP control group had decreases (p<0.01) in weights of the seminal vesicles ( ↓81%), ventral prostate (↓72%), LABC (↓47%), Cowper’s glands (↓67%), and glans penis ( ↓35%) compared to t he negative TP control group. Significant decreases in the weight of two or more androgen

- dependent tissues occurred at doses from 10 to 75 mg/kg bw/day ; however, there was overt toxicity

at the 75 mg/kg/day

dose level. Chemical S is considered to be a po tential anti

androgen at the dose level of 10 mg/kg bw/day (absence of overt toxicity ) under the conditions of this assay

. The Guideline criteria for %CV in organ weights were met in all cases for the vehicle/negative control and high dose groups. Statis tically

significant increases in organ weights were not seen in two or more of the five androgen responsive tissues in the androgen agonist assay . Chemical S was negative for androgenic activity

. Statistically

significant decreases in ventral prostate a nd glans penis weights were observed at 10 mg/kg/day

in the androgen antagonist assay in the absence of overt toxicity. Chemical S is considered positive for potential anti

- androgenic activity ; data are summarized in **Tables 2**  **1** through **2 5** 

. Chemical S was n egative for androgenic activity , and positive for potential anti

- androgenic activity at dose s not associated with overt toxicity in the Hershberger assay

.US EPA

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Page **88** of **211 Table**  **2 1: Chemical S** 
**- Selected Group Mean (±SD) Body Weights and Cumulative Body** 

**Weight Gains (g) in the Androgen Agonist Assay**  **a**
**Study Day # Dose** 

**Vehicle Control Positive Control** 

**TP (0.4 mg/kg/day**  **) Chemical S** 

**(10 mg/kg/day**  **) Chemical S** 

**(75 mg/kg/day**  **) N**  **Mean SD**  **N Mean**  **SD N**  **Mean SD**  **N Mean**  **SD** 1 6 225.9 10.2 6 226.6 8.2 6 227.5 8.0 6 224.8 7.2 7 6 255.8 12.6 6 271.1* ( ↑6) 10.5 6 258.3 9.5 6 249.8 7.5
10 6 271.9 11.2 6 292.3** ( ↑7) 11.7 6 272.4 10.8 6 260.5 8.4 11 (Terminal) 6 275.8 13.2 6 298.1** ( ↑8) 13.0 6 276.6 9.6 6 266.1 8.2 Bod y Weight Gain (Day s 1

- 11) 6 49.9 5.1 6 71.5** ( ↑43) 7.1 6 49.1 5.3 6 41.3* ( ↓17) 4.3

a Data w ere obtained from the study report. Percent differences from vehicle control are included in parentheses. N = Number of animals in the group SD = Standard Deviation * Significantly different from controls at p<0.05; ** Significantly different from controls at p<0.01US EPA

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Page **89** of **211 Table 2**  **2 : Chemical S** 
**- Selected**  **Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the Anti**
**- Androgen** 

**Assay a** 

**Study Day # Dose** 

**Negative Control TP (0.4**  **mg/kg/day )**  **Positive Control**

**FT (3 mg/kg/day**  **) + TP (0.4**  **mg/kg/day )**  **Chemical S**

**(10 mg/kg/day**  **) + TP (0.4**  **mg/kg/day )**  **Chemical S**

**(40 mg/kg/day**  **) + TP (0.4**  **mg/kg/day )**  **Chemical S (75**  **mg/kg/day ) +**  **TP (0.4 mg/kg/day**  **) N**  **Mean SD**  **N Mean**  **SD N**  **Mean SD**  **N Mean**  **SD N**  **Mean SD**  1 6 208.4 8.6 6 209.6 9.7 6 207.1 9.4 6 209.1 7.9 6 211.0 7.5 4 6 232.6 6.3 6 227.0 7.5 6 230.5 7.9 6 224.9 7.7 6 215.0* ( ↓8) 12.9
5 6 237.7 7.4 6 232.6 8.3 6 236.8 7.7 6 227.8 8.3 6 221.6* ( ↓7) 13.6
7 6 252.8 6.2 6 243.2 9.5 6 248.4 6.4 6 238.9* ( ↓5) 7.1 6 229.4** ( ↓9) 14.1
10 6 272.6 8.1 6 261.0 8.2 6 268.8 6.6 6 255.4* ( ↓6) 7.7 6 245.2** ( ↓10) 14.0 11 (Termin al) 6 277.2 9.4 6 267.2 9.0 6 273.7 6.8 6 257.9** ( ↓7) 6.5 6 248.8**

( ↓10) 15.2 Bod y Weight Gain (Day s 1

- 11) 6 68.8 12.1 6 57.5 2.1 6 66.5 6.0 6 48.7** ( ↓29) 4.2 6 37.8**

( ↓45) 11.9

a Data w ere obtained from the study report. Percent differences from negative control are included in parentheses. N = Number of animals in the group SD = Standard Deviation * Significantly different from controls at p<0.05 ** Significantly different from controls at p<0.01US EPA

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Page **90** of **211 Table**  **2 3: Chemical S** 
**- Group Mean (±SD) Overall (Days 1** 
**- 11) Food Consumption (g/rat/day)**  **a**

**Dose N**  **Mean SD** 

**Androgen Agonist Phase**  Vehicle control (corn oil; 0) 3 24.76 0.38 Positive control (TP; 0.4 mg/kg/day ) 3 27.96** ( ↑13) 0.35 Chemical S (10 mg/kg/day ) 3 24.35 0.27 Chemical S (75 mg/kg/day ) 3 23.38** ( ↓6) 0.35 **Anti**
**- Androgen**  **Phase** Negative control (TP; 0.4 mg/kg/day ) 3 23.87 0.38 Positive control (FT+TP; 3 mg/kg/day +0.4 mg /kg/day ) 3 20.69** ( ↓13) 0.58 Chemical S+TP (10 mg/kg/day +0.4 mg/kg/day ) 3 21.13** ( ↓11) 0.58 Chemical S+TP (40 mg/kg/day +0.4 mg/kg/day ) 3 18.05** ( ↓24) 0.50 Chemical S+TP (75 mg/kg/day +0.4 mg/kg/day ) 3 15.89** ( ↓33) 0.50

a Data w ere obtained from the study report. Percent differences from vehicle or negative controls are included in parentheses. N = Number of observations SD = Standard Deviation ** Significantly different from controls at p<0.01US EPA

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Page **91** of **211 Table**  **2 4**  **: Chemical S**
**- Weights of Liver, Kidneys, Adrenals and Accessory Sex Organs from Androgen Agonist Assay in Rats**  **a**

**Organ Dose** 

**Vehicle Control Positive Control**  **TP (0.4 mg/kg/day**  **) Chemical S (10**  **mg/kg/day )**  **Chemical S (75**  **mg/kg/day )**  **N Mean**  **SD CV** 

**(%) N**  **Mean SD**  **CV (%)**  **N Mean**  **SD CV**  **(%) N**  **Mean SD**  **CV (%)**  Liver (g) 6 12.24 0.47 3.8 6 13.66** ( ↑12) 0.89 6.5 6 12.11 0.60 4.9 6 12.86 0.57 4.4 Kidney s (g) 6 1.860 0.17 9.1 6 2.063 0.07 3.4 6 1.819 0.10 5.6 6 1.722 0.10 5.38 Adrenals (mg) 6 70.92 10.9 15.4 6 68.70 4.79 7.0 6 74.63 13.07 17.5 6 77.35 8.01 10.4 Seminal vesicles (mg) 6 51.7 8.3 16.1 6 618.7** ( ↑1097) 103.9 16.8 6 62.7* ( ↑21) 2.6 4.2 6 62.2 9.1 14.7 Ventral prostate (mg) 6 18.8 3.6 18.9 6 166.8** ( ↑787) 32.2 19.3 6 19.3 3.3 17.1 6 20.1 4.8 24.0 LABC (mg) 6 220.4 36.4 16.5 6 472.0** ( ↑114) 57.1 12.1 6 220.3 48.0 21.8 6 187.1 24.5 13.1 Cowper’s glands (mg) 6 6.55 2.61 39.8 6 42.65** ( ↑551) 10.4 24.4 6 8.0 2.4 30.1 6 6.2 1.6 26.1 Glans penis (mg) 6 41.5 4.4 10.5 6 73.2** ( ↑76) 5.5 7.6 6 47.3 7.2 15.3 6 45.3 7.5 16.6

a Data w ere obtained from the study report. Percent differences from vehicle controls (calculated by the reviewers) are includ ed in parentheses. N = Number of animals in the group SD = Standard Deviation CV = Coefficient of Variation * Significantly different from controls at p<0.05US EPA

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Page **92** of **211 Table 2**  **5 : Chemical S** 
**- Weights of Liver, Kidneys, Adrenals and Accessory Sex Organs from Anti** 
**- Androgen Assay in Rats**  **a**

**Organ Dose (mg/kg/day)** 

**Negative Control TP (0.4**  **mg/kg/day )**  **Positive Control**

**FT (3 mg/kg/day**  **) + TP (0.4**  **mg/kg/day )**  **Chemical S (10**  **mg/kg/day ) +**  **TP (0.4 mg/kg/day**  **) Chemical S**  **(40 mg/kg/day**  **) + TP (0.4**  **mg/kg/day )**  **Chemical S**

**(75 mg/kg/day**  **) + TP (0.4**  **mg/kg/day )**  **N Mean**  **SD CV** 

**(%) N**  **Mean SD**  **CV**

**(%) N**  **Mean SD**  **CV**

**(%) N**  **Mean SD**  **CV**

**(%) N**  **Mean SD**  **CV (%)**  Liver (g) 6 12.57 1.50 11.9 6 12.17 0.64 5.3 6 13.30 0.60 4.5 6 11.87 0.84 7.0 6 11.82 1.48 12.5 Kidney s (g) 6 1.886 0.15 8.1 6 1.741 0.13 7.6 6 1.842 0.14 7.8 6 1.698 0.15 8.9 6 1.744 0.18 10.4 Adrenals (mg) 6 73.95 4.25 5.8 6 69.60 8.94 12.9 6 63.73 6.78 10.6 6 70.13 6.85 9.8 6 70.15 8.7 12.4 Seminal vesicles (mg) 6 389.8 46.1 11.8 6 74.9** ( ↓81) 7.6 10.2 6 327.3 51.2 15.6 6 256.2** ( ↓34) 69.7 27.2 6 302.1** ( ↓23) 64.6 21.4 Ventral prostate(mg) 6 128.1 21.6 16.9 6 36.2** ( ↓72) 5.3 14.5 6 104.0*

( ↓19) 17.0 16.4 6 77.2**

( ↓40) 14.3 18.6 6 97.3** ( ↓24) 17.3 17.8 LABC (mg) 6 426.5 42.0 9.9 6 224.3** ( ↓47) 22.7 10.1 6 419.6 26.0 6.2 6 359.8** ( ↓16) 34.5 9.6 6 326.8** ( ↓23) 21.4 6.5 Cowper’s glands (mg) 6 31.7 3.6 11.3 6 10.4** ( ↓67) 2.5 23.8 6 30.1 7.0 23.4 6 23.0** ( ↓27) 4.7 20.5 6 28.2 7.7 27.3 Glans penis (mg) 6 75.27 8.9 11.8 6 49.0** ( ↓35) 7.2 14.8 6 63.4**

( ↓16) 4.2 6.7 6 60.1**

( ↓20) 3.8 6.3 6 70.5 3.3 4.6

a Data w ere obtained from the study report. Percent differences from vehicle controls are included in parentheses. N = Number of animals in the group SD = Standard Dev iation CV = Coefficient of Variation *Significantly different from controls at p<0.05 **Significantly different from controls at p<0.01US EPA

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– July , 2013

Page **93** of **211 viii.**  **Pubertal Female Assay (Rat)** In a Female Pubertal Assay , 15 Wistar rats/dose group were treated dail y via oral gavage with Chemical S (97.85% a.i) in corn oil at doses of 0, 10, or 75 mg/kg bw/day from post

- natal day

(PND) 22 to 42. An additional positive control group of 15 rats was similarly treated with 1chloro

- 2

- nitrobenzene in corn oil daily via oral gavage at a dose of 100 mg/kg bw/day. Animals were examined daily for vaginal opening (VO) beginning on PND 22; age and bod y weight at day of attainment were also recorded. Following sacrifice of the rats on PND 42, blood was collected for clinical chem istry anal ys es, including selected hormones. T otal serum t hy roxine (T 4 ) and th yroid stimulating hormone (TSH) levels were determined using the enz ym e - linked immunosorbent assay (ELISA). L iver, adrenals, pituitary , thy roid, and urogenital organs were coll ected and weighed. Histology examinations were performed on the uterus, thy roid, one ovary , one kidney , liver, pituitary , adrenals, and gross lesions. Summary d ata are presented in **Tables 2**  **6** through **3 1** 

. No treatment

- related effects were observed on mort ality . Th yroid colloid area and follicular cell height scores and the incidence of gross or microscopic lesions were similar in the treated groups and the vehicle controls. The considerable delay in onset of vaginal opening in the 75 mg/kg bw/day group ( PND 38; sacrifice at PND 42) precluded the proper evaluation of the estrous cy cle and of mean age at first vaginal estrus. At 10 mg/kg bw/day , unadjusted and adjusted ages at VO were increased (p<0.01) by 2.1

- 2.9 day s. The adjusted body

weight at VO was increased (p<0.05) by 7%. Mean cy cle length was reduced to 3.5 day s compared to 5.6 day s in the vehicle control. The number of females cy cling was increased to 80% compared to the vehicle control (67%), but the number of females regularly cy cling was 67% (p<0.05) in the treated group compared to 80% in the vehicle control. It was unclear if there was an effect on estrous cy clicit y. One animal in this group had a distended cervix; however, this finding was considered incidental. Primordial follicular ce ll counts were decreased (p<0.05) by 39% at 10 mg/kg/day , but this effect was not noted at 75 mg/kg bw/day

. At 75 mg/kg/day , increased incidences (# affected/# exposed) of slight to moderate salivation were observed post

- dose (15/15) compared to 0/15 at 0 or 10 mg/kg bw/day . Body weights were decreased (p<0.01) b y 8

- 20% from PND 24 to 42 compared to vehicle controls. Adjusted and unadjusted final body weights were decreased (p<0.01) by 13% each, and adjusted and unadjusted overall body weight gains (PND 22

- 42) were decreased (p<0.01) b y 20% each. Food consumption was decreased (p<0.01) by 27 − 37% during the measured intervals from PND 22

- 36. At 75 mg/kg bw/day , serum glucose was slightly decreased (p<0.05) by

14%, which was considered incidental. Min or decreases (p<0.05; within historical control range) were observed in serum total protein and serum albumin ( ↓8

- 10%).US EPA

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– July , 2013

Page **94** of **211** At 75 mg/kg bw/day , the following decreases (p<0.01; unless indicated otherwise) in unadjusted and adjusted organ weights were observe d compared to vehicle controls (% decrease): (i) ovaries (15

- 43%); (ii) wet uterus (24% each; not statistically significant [NS]); (iii) blotted uterus (39% each); and (iv) thy roid (14% each). Additionally , decreases (p<0.01) were noted in unadjusted, ad justed, and relative kidney weights (9

- 21%) and pituitary weights (26

- 36%). A minor increase (p<0.01) of 11% was noted in the relative liver weight. Reduced uterus size was observed (7/15) compared to 0 animals in the vehicle control. The adjusted body weight at VO was increased (p<0.05) by 12%. Unadjusted and adjusted ages at VO were increased (p<0.01) b y 6.5

- 7.3 day s. In addition, the proportion of 75 mg/kg bw/day females that did not attain VO was 12/15, compared to 0/15 vehicle controls. At 75 m g/kg bw/day , increased incidences (# affected/15) of the following microscopic findings were noted compared to the vehicle controls (0

- 1/15): absent corpus luteum in the ovaries (13), uterine luminal dilation (4), and uterine atrophy (9). C hemical S sign ificantl y altered pubertal development at 75 mg/kg, a dose that had a number of overtly toxic indications. However, there were signs of delay ed puberty in the lower dose as well in the absence of overt toxicity

. In the positive control group, slight to m oderate salivation was observed (9/15). Bod y weights were decreased (p<0.05) b y 6

- 9% during PND 24

- 33; however, bod y weights were similar to the vehicle control thereafter. Food consumption was decreased (p<0.01) b y 21% during the measured interval of PN D 22

- 29. Unadjusted and adjusted ages at incomplete VO were increased (p<0.05) b y 3.7 days, and unadjusted and adjusted ages at VO were increased (p<0.01) b y 3.0

- 3.8 day s. The following differences (p<0.05) were observed in organ weights: (i) unadjusted , adjusted, and relative liver weights increased by 51

- 54%; (ii) relative kidney weight increased by

5%; and (iii) adjusted pituitary weight decreased by

13%. The number of females regularl y c ycling was only 33% (p<0.05) compared to controls (80%). The following differences (p<0.01) were noted in clinical chemistry parameters: glucose ( ↓14%), total bilirubin ( ↑471%), and cholesterol (↑21%). Spleen enlargement (5/15) was noted compared to 0/15 in the other groups. Increased incidences (# affected/15) o f the following microscopic findings were noted compared to the vehicle controls (0

- 1/15): adrenal accessory cortical tissue (3), spleen sinusoidal congestion (11), and spleen extramedullary hematopoiesis (11). Primordial follicular cell counts were decr eased (p<0.05) b y 35%. Chemical S was positive for E pathway in the absence of overt toxicity and negative for T pathway in the pubertal female assay

.US EPA

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– July , 2013

Page **95** of **211 Table**  **2 6: Selected Body Weight of Female Rats Treated with Chemical S in the Fema**  **le Pubertal Assay a** 

**PND Vehicle Control**  **Chemical S 10 mg/kg bw/day**  **Chemical S 75 mg/kg bw/day**  **Positive Control b** 
**N Mean**  **SD CV** 

**(%) N**  **Mean SD**  **CV (%)**  **N Mean**  **SD CV**  **(%) N**  **Mean SD**  **CV (%)**  22 15 42.07 3.03 7.2 15 42.13 2.33 5.5 15 42.39 2.96 7.0 15 42.35 2.75 6.5 24 15 48.42 3.46 7.2 15 48.94 3.10 6.3 15 44.73** ( ↓8) 3.12 7.0 15 45.47* ( ↓6) 2.92 6.4 28 15 64.34 4.67 7.3 15 64.17 3.62 5.6 15 55.55** ( ↓14) 4.06 7.3 15 58.87** ( ↓9) 4.73 8.0 36 15 99.79 7.70 7 .7 15 98.51 6.11 6.2 15 80.11** ( ↓20) 8.41 10.5 15 94.96 7.69 8.1 42 15 122.78 9.33 7.6 15 119.72 7.57 6.3 15 107.08** ( ↓13) 11.27 1 0.5 15 120.68 9.21 7.6

a Data w ere obtained from the study report, and %CV was rounded by the reviewers. Percent difference from control (calculated by reviewers from unrounded cited data) is included in parentheses. b 100 mg/kg bw/day 1

- Chloro

- 2

- Nitrobenzene N Number of animals examined SD Standard Deviation CV Coeffic ient of Variation * Significantly different from controls at p<0.05. ** Significantly different from controls at p<0.01.US EPA

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– July , 2013

Page **96** of **211 Table**  **2 7: Selected Food Consumption Data of Female Rats Treated with Chemical S in the Female Pubertal Assay**  **a**

**PND Vehicle Control**  **Chemical S 10 mg/kg bw/day**  **Chemical S 75 mg/kg bw/day**  **Positive Control b** 
**N Mean**  **SD CV (%)**  **N Mean**  **SD CV (%)**  **N Mean**  **SD CV**  **(%) N**  **Mean SD**  **CV (%)** 22

- 29 8 9.27 0.48 5.2 8 8.79 0.36 4.1 8 5.82** ( ↓37) 0. 97 16.7 8 7.29** ( ↓21) 0.63 8.7 29

- 36 8 13.48 1.04 7.7 8 12.74 0.93 7.3 8 9.84** ( ↓27) 1.97 20.0 8 12.56 1.30 10.4 36

- 40 8 15.79 1.30 8.2 8 15.31 1.11 7.3 8 13.06 2.82 21.6 8 15.42 1.27 8.3 40

- 42 8 14.93 1.11 7.46 8 14.72 1.17 7.93 8 14.34 2.35 16.4 8 16.08 1.55 9.6 a Data w ere obtained from the study report, and %CV was rounded b y the reviewers. Percent difference from control (calculated by revie wers from unrounded cited data) is included in parentheses. b 100 mg/kg bw/day 1

- Chloro

- 2

- Nitrobenzene N = Number of animals examined SD = Standard Deviation CV = Coefficient of Variation **Si gnificantly different from controls at p<0.01.US EPA

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– July , 2013

Page **97** of **21 1**  **Table 2 8**  **: General Growth and Vaginal Opening of Rats Treated with Chemical S in the Female Pubertal Assay a** 

**Parameter Evaluated Vehicle Control**  **Chemical S 10 mg/kg bw/day**  **Chemical S 75 mg/kg bw/day**  **Positive Control b** 
**N Mean**  **SD CV** 

**(%) N**  **Mean SD**  **CV (%) N**  **Mean SD**  **CV (%)**  **N Mean**  **SD CV**  **(%)** Initial Body Weight (PND 22; g) U 15 42.1 3.03 7.2 15 42.1 2.33 5.5 15 42.4 2.96 7.0 15 42.4 2.75 6.5 A 15 42.2 0.63 1.5 15 42.2 0.63 1.5 15 42.3 0.63 1.5 15 42.3 0.63 1.5 Body Weight at VO (g) U 15 98.3 7.29 7.4 13 104.8 8.04 7.7 3 110.3 5.13 4.7 12 107.7 13.6 12.6 A 15 98.4 9.24 9.4 13 105.2* ( ↑7) 9.26 8.8 3 110.1* ( ↑12) 9.24 8.4 12 107.2* ( ↑9) 9.27 8.7 Final Body Weight (PND 42; g) U 15 122.8 9.33 7.6 15 119.7 7.57 6.3 15 107.1** ( ↓13) 11.27 10.5 15 120.7 9.21 7.6 A 15 122.9 4.23 3.4 15 119.9 4.23 3.5 15 106.9** ( ↓13) 4.23 4.0 15 120.6 4.23 3.5 Final Body Weight (% of control) U NA 97.5 87.2 98.3 A NA 97.5 87.0 98.1 Body Weight Gain (PND 22

- 42; g) U 15 80.7 8.25 10.2 15 77.6 6.74 8.7 15 64.7** ( ↓20) 10.75 16.6 15 78.3 7.71 9.9 A 15 80.8 4.26 5.3 15 77.7 4.26 5.5 15 64.6** ( ↓20) 4.26 6.6 15 78.3 4.26 5.4 Age at Incomplete VO (PND) U 15 32.5 2.10 6.5 14 33.4 1.16 3.5 7 38.3 3.15 8.2 13 36.2* ( ↑12) 1.92 5.3 A 15 32.5 1.91 5.9 14 33.4 1.92 5.8 6 39.1 1.93 4.9 13 36.2** ( ↑12) 1.91 5.3

A ge at Complete VO (PND) U 15 35.5 1.25 3.5 15 38.4** ( ↑8) 2.56 6.7 15 42.8** ( ↑20) 0.41 1.0 15 39.3** ( ↑11) 2.53 6.4 A 15 35.5 1.59 4.5 13 37.6** ( ↑6) 1.60 4.2 3 42.0** ( ↑18) 1.59 3.8 12 38.5** ( ↑8) 1.60 4.2 Proportion unopened (#/N) 0/15 2/15 12/15 3/15
a Data w ere obtained from the study report, and w ere sometimes rounded by the reviewers. Percent difference from control is in cluded in parentheses. b 100 mg 1

- Chloro

- 2

- Nitrobenzene U Unadjusted for body weight on PND 21 ; A Adjusted for body we ight on PND 21 N Number of animals examined ; SD Standard Deviation ; CV Coefficient of Variation ; NA Not applicable * Significantly different from controls at p<0.05 ; ** Significantly different from controls at p<0.01US EPA

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– July , 2013

Page **98** of **211 Table**  **29 :**  **Organ Weight Data of Female Rats Treated with Chemical S in the Female Pubertal Assay a** 
**Organ Vehicle Control**  **Chemical S 10 mg/kg bw/day**  **Chemical S 75 mg/kg bw/day**  **Positive Control b** 
**N Mean**  **SD CV** 

**(%) N**  **Mean SD**  **CV (%)**  **N Mean**  **SD CV**  **(%) N**  **Mean SD**  **CV (%)** 
Liver (g) U 15 5.72 0.63 11.0 15 5.49 0.58 10.5 15 5.57 0.98 17.6 15 8.65** ( ↑51) 0.96 11.1 A 15 5.73 0.40 7.0 15 5.49 0.40 7.33 15 5.56 0.40 7.25 15 8.65** ( ↑51) 0.40 4.7 R 15 4.66 0.34 7.2 15 4.58 0.31 6.7 15 5.17** ( ↑11) 0.50 9.8 15 7.16** ( ↑54) 0.40 5.5

Kidneys (g) U 15 0.95 0.12 12.6 15 0.90 0.07 7.2 15 0.75** ( ↓21) 0.09 11.6 15 0.98 0.10 10.4 A 15 0.95 0.04 4.6 15 0.91 0.04 4.9 15 0.75** ( ↓21) 0.04 5.9 15 0.98 0.04 4.5 R 15 0.77 0.05 6.8 15 0.76 0.04 5.8 15 0.70** ( ↓9) 0.03 3.65 15 0.81* ( ↑5) 0.04 5.3

Pituitary (mg) U 15 5.19 0.98 18.9 1 5 4.73 0.65 13.8 15 3.35** ( ↓35) 0.76 22.6 15 4.55 0.95 20.9 A 15 5.20 0.41 7.8 15 4.74 0.41 8.6 15 3.33** ( ↓36) 0.41 12.2 15 4.55* ( ↓13) 0.41 8.9 R 15 0.004 0.001 16.1 15 0.004 0.001 15.0 15 0.003** ( ↓26) 0.001 19.7 15 0.004 0.001 17.4 Adrenals (mg) U 15 38.17 7.15 18.7 15 38.69 6.79 17.6 15 36.29 6.00 16.5 15 37.99 7.86 20.7

A 15 38.18 3.64 9.5 15 38.70 3.64 9.4 15 3 6.29 3.64 10.0 15 37.99 3.64 9.6 R 15 0.03 0.01 21.2 15 0.03 0.01 16 .6 15 0.03 0.00 13.8 15 0.03 0.01 21.4 Ovaries (mg) U 15 1.64 0.10 6.3 15 1.63 0.13 7.7 15 1.40** ( ↓15) 0.10 6.9 15 1.58 0.13 8.0 A 15 44.67 5.10 11.4 15 44.27 5.10 11.5 15 25.36** ( ↓43) 5.10 20.1 15 39.78 5.10 12.8 Uterus, wet (mg) U 15 262.4 88.2 33.6 15 261.3 118.4 45.3 15 198.1 ( ↓24) 168.1 84.8 15 226.3 81.8 36.2 A 15 262.4 62.1 23.7 15 261.3 62.06 23.8 1 5 198.1 ( ↓24) 62.06 31.3 15 226.3 62.1 27.4US EPA

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– July , 2013

Page **99** of **211 Organ**  **Vehicle Control Chemical S**  **10 mg/kg bw/day Chemical S**  **75 mg/kg bw/day Positive Control**  **b**
**N Mean**  **SD CV** 

**(%) N**  **Mean SD**  **CV (%)**  **N Mean**  **SD CV**  **(%) N**  **Mean SD**  **CV (%)**  Uterus, blotted (mg) U 15 231.1 64.9 28.1 15 222.7 66.5 29.9 15 140.2** ( ↓39) 94.28 67.3 15 198.5 67.3 33.9 A 15 231.1 38.7 16.7 15 222.7 38.68 17.4 15 140.1** ( ↓39) 38.68 27.6 15 198.5 38.7 19.5 Thyroid, fixed (mg) U 15 11.70 1.35 11.5 15 11.69 1.84 15.7 15 10.11** ( ↓14) 1.07 10.6 15 11.57 1.71 14.8 A 15 11.69 0.79 6.7 15 11.68 0.79 6.7 15 10.11** ( ↓14) 0.79 7.8 15 11.57 0.79 6.8
a Data w ere obtained from the study report. Percent difference from control is included in parentheses. b 100 mg 1

- Chloro

- 2

- Nitrobenzene U = Unadjusted for body weight on PND 21 ; A = Adjusted for body w eight on PND 21 ; R = Organ

- to
- body weight ratio (relative to body weight) N = Number of animal s examined ; SD = Standard Deviation ; CV = Coefficient of Variation *Significantly different from controls at p<0.05.**Significantly different from controls at p<0.01. **Table 3 0**  **: Estrous Cyclicity Data of Female Rats Treated with Chemical S in the Female Puber tal Assay**  **a**
**Treatment Groups N**  **Mean Age at First Vaginal Estrus**  **(PND) Mean Cycle** 

**Length (days) Cycling (%)**  **Regularly Cycling (%)**  **Cycle Status at Necropsy (# Females)**  **Diestrus Proestrus**  **Estrus Not**  **Cycling** Vehicle 15 38 5.6 66.67 80.00 11 1 3 5 Chemical S ( 10 mg/kg/day ) 15 40 3.5 80.00 66.67* 7 2 4 1 Chemical S ( 75 mg/kg/day ) 15

--- --- 20.00 0.00* 2 1 0

--- Positive Control b 15 40 4.0 80.00 33.33* 7 0 5 0
a Data w ere obtained from the study report; b 100 mg 1

- Chloro

- 2

- Nitrobenzene

--- The considerable delay in onset of vaginal opening in the 75 mg/kg / day group (PND 38; sacrifice at PND 42) precluded the proper evaluation of the estrous cycle and mean age at first vaginal estrous. N = Number of animals *Significantly di fferent from controls at p<0.05.US EPA

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– July , 2013

Page **100** of **211 Table 3**  **1 : Hormone Levels and Clinical Chemistry Data of Female Rats Treated with Chemical S in** 

**the Female Pubertal Assay a** 

**Parameter Vehicle Control**  **Chemical S 10 mg/kg bw/day**  **Chemical S**

**75 mg/kg bw/day Positive Control**  **b**
**N Mean**  **SD CV** 

**(%) N**  **Mean SD**  **CV**

**(%) N**  **Mean SD**  **CV**

**(%) N**  **Mean SD**  **CV**

**(%) Thyroid Hormones**  Total T4 (μg/dL) 15 3.404 1.128 33.2 15 3.600 0.792 22.0 11 5.391 3.742 69.4 15 3.004 0.735 24.5 TSH (ng/mL) 9 4.098 4.507 110.0 9 4.842 3.559 73.5 15 3.215 0.849 26.4 12 2.197 1.973 89.8 Clinical Chemistry Glucose (mg/dL) 15 141.4 17.76 12.6 15 139.8 12.77 9.1 15 122.1* ( ↓14) 26.36 21.6 15 121.4*

( ↓14) 19.87 16.4 AL T (IU/L) 15 83.08 21.28 25.6 15 72.67 11.61 16.0 15 86.48 20.02 23.2 15 92.62 25.42 27.4 AST (IU/L) 15 272.0 75.95 27.9 15 260.8 58.08 22.3 15 248.8 63.17 25.4 15 232.0 26.52 11.4 Total Bilirubin (mg/dL) 15 0.07 0.10 142.6 15 0.06 0.08 151.6 15 0.12 0.12 102.6 15 0.40** ( ↑471) 0.17 43.4 Creatinine (mg/dL) 15 0.31 0.11 36.4 15 0.32 0.11 36.0 15 0.27 0.09 33.1 15 0.34 0.12 34.4 BUN (mg/dL) 15 14.87 1.72 11.6 15.11 2.02 13.4 13.65 3.34 24.5 16.52 3.22 19.5 Total Protein (g/dL) 15 5.13 0.53 10.3 15 5.25 0.37 7.0 15 4.62* ( ↓10) 0.50 10.9 15 5.36 0.46 8.7 Albumin (g/dL) 15 4.02 0.35 8.6 15 4.15 0.34 8.2 15 3.68* ( ↓8) 0.41 11.1 15 4.18 0.38 9.1 Calcium (mg/dL) 15 9.11 0.55 6.0 15 9.24 0.49 5.3 15 9.36 0.95 10.1 15 9.70 0.74 7.7 Phosphorus (mg/dL) 15 7.96 1.16 14.5 15 8.05 0.50 6.2 15 8.08 1.27 15.7 15 8.16 0.89 11.0 Cholesterol (mg/dL) 15 71.68 9.51 13.3 15 75.30 8.94 11.9 15 77.36 14.44 18.7 15 86.67** ( ↑21) 10.89 12.6 Urea (mg/dL) 15 31.85 3.69 11.6 15 32.36 4.33 13.4 15 29.22 7.16 24.5 15 35.38 6.90 19.5 AL P (lU/dL) 15 246.2 52.46 21.3 15 252.3 58.17 23.1 15 290.6 79.36 27.3 15 282.4 52.75 18.7 Globulin (g/dL) 15 1.11 0.24 21.5 15 1.10 0.15 13.6 15 0.94 0.27 29.0 15 1.18 0.17 14.5 Albumin/Globulin 15 3.72 0.60 16.1 15 3.84 0.55 14.4 15 4.15 0.97 23.3 15 3.60 0.55 15.2 a Data w ere obtained from the study report. Percent differences from controls are included in parentheses.

**ix. Pubertal Male Assay**  **(Rat)** In a Male Pubertal Assay, 15 Wistar rats/dose group were treated daily via oral gavage with Chemical S (97.85% a.i.) in corn oil at doses of 0, 10, or 75 mg/kg bw/day from post

- natal day

(PND) 23 to 53. An additional positive control group of 15 rat s was similarly treated with 1

- chloro

- 2

- nitrobenzene in corn oil daily via oral gavage at a dose of 100 mg/kg bw/day . Animals were examined for preputial separation (PPS) daily

beginning on PND 30, and age and body weight at day

of attainment were recorde d. Following sacrifice on PND 53, blood was collected for clinical chemistry anal ys es, including selected hormones. Total serum testosterone, th yroxine (T 4 ), and thy roid stimulating hormone (TSH) levels were determined using the enzy me - linked immunosorbe nt assay (ELISA). Liver, adrenals, pituitary , thy roid,US EPA

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– July , 2013

Page **101** of **211** and urogenital organs were collected and weighed. Microscopic examinations were performed on the right testes, right epidid ymis, thy roid, one kidney , liver, pituitary , adrenals, spleen, and gross les ions. Summary d ata are presented in **Tables 3 2**  through **3 6**  . No treatment

- related effects were observed on mortality or gross or microscopic pathology . No effects were noted on the thy roid of the treated groups compared to vehicle controls: thy roid colloid area and follicular cell height scores were similar; organ weights were similar; and no gross or microscopic lesion was reported. At 10 mg/kg bw/day , no adverse, treatment

- related effects were noted. An increase (p<0.01) of 22% in adjusted dorsolateral prostate weight was noted compared to the vehicle controls, but the effect was considered incidental because the effect was a slight increase in weight without effects noted on other androgen

- sensitive organs. I ncreased incidences ( number affected / numbe r exposed) of slight to moderate salivation were observed in the 10 mg/kg bw/day group (2/15) compared to the vehicle controls (0/15). This effect was considered minor and not adverse due to the lack of dose

- response ( i.e, absence of effect at the high do se). Unadjusted and adjusted age at partial PPS was increased (p<0.01) b y only 2.2 and 2.4 day s, respectively ; this effect was not corroborated with other evidence of delay ed sexual development such as changes in androgen

- dependent tissue weights or seru m testosterone concentrations

. In the high dose group (75 mg/kg bw/day ), there was an increased incidence of slight to moderate salivation observed in all animals compared to none in the vehicle control group. Bod y weights were decreased (p<0.05) b y 10

- 22% from PND 27 to 53 compared to vehicle controls. Adjusted and unadjusted final bod y weights were decreased (p<0.01) by 20% each, and adjusted and unadjusted overall bod y weight gains (PND 23

- 53) were decreased (p<0.01) b y 26% each. The adjusted bod y w eight at PPS was decreased (p<0.05) b y 11%. Corresponding to the body weight decreases, food consumption was decreased (p<0.05) b y 10

- 28% during the measured intervals from PND 23

- 30 through 51

- 53. At 75 mg/kg bw/day , the unadjusted and adjusted ages at partial PPS was delay ed (p<0.01) by 3.5 day s compared to the control, and unadjusted and adjusted ages at PPS were delay ed (p<0.01) by 5.9 days. I n addition, all of the controls completed PPS by necropsy day , as compared to only 12 of 15 of the 75 mg/kg bw/day

males

. Testosterone was decreased (p<0.05) by 56% in the high dose group. The following decreases (p<0.05) in unadjusted and adjusted androgen

- sensitive tissues organ weights were observed compared to vehicle controls (% decrease): (i) seminal ve sicle and coagulating glands (with and without fluid; 48

- 55%); (ii) ventral prostate (42% each); (iii) dorsolateral prostate (26% each); (iv) LABC (43

- 44%); (v) left and right epididy mides (24

- 25%); and (iv) left and right testes (15

- 17%). At 75 mg/kg bw/day , the following differences (p<0.05) were noted in organ weights compared to controls: (i) unadjusted and adjusted liver weights decreased b y 13% each and relative (to body ) liver weight increased b y 8%; (ii) unadjusted and adjusted kidney weight de creased by

24% each; and (iii) unadjusted and adjusted adrenal weights decreased by 4

- 12%. These effects were not considered adverse due to slight or minor magnitude without additional evidence from clinical chemistry or gross or microscopic pathology

. In addition, alanine aminotransferase (ALT) was increased (p<0.05) b y 21%,US EPA

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Page **102** of **211** and alkaline phosphatase (AL P) was decreased (p<0.01) by 34%. These slight effects on ALT and AL P were not considered adverse. In the positive control group, slight to moderate salivation was also observed (12/15) compared to 0/15 vehicle controls. An incidental decrease (p<0.05) was noted in mean bod y weight on PND 29, and a minor increase (p<0.05) was observed in adjusted body weight at PPS. An incidental decrease (p<0.01) in food consumption was noted on PND 23

- 30. Unadjusted and adjusted ages at partial PPS were increased (p<0.05) b y 2.8

- 3.0 day s, and unadjusted and adjusted ages at PPS were increased (p<0.01) by 3.0

- 3.1 day s. The following differences (p<0.05) were observ ed in organ weights: (i) unadjusted, adjusted, and relative liver weights were increased by 56

- 60%; (ii) relative kidney weight were increased by 7%; (iii) unadjusted and adjusted ventral prostate weights were decreased by 18% each; (iv) adjusted left and right epididy

mides weights were decreased by 10% each; and (v) adjusted left and right testes weights were decreased by 6

- 7%. Total T 4 was decreased (p<0.01) b y 17%. The following increases (p<0.05) were noted in clinical chemistry parameters: ALT (58% ), aspartate aminotransferase (AST; 18%), albumin (5%), and cholesterol (21%). Spleen size was increased in all animals of the positive control group compared to no animals in the other groups. All animals were observed with liver degeneration (with sing le cell necrosis) and spleen extramedullary hematopoiesis (with sinusoidal congestion) compared to none in the vehicle control group. Chemical S was negative for A and T pathway s in the pubertal male assay

. **Table 3 2**  **: Selected Body Weight of Male Rats Treated with Chemical S in the Male Pubertal Assay**  **a**

**PND Vehicle Control**  **Chemical S 10 mg/kg/day**  **Chemical S**

**75 mg/kg/day Positive Control**  **b**
**N Mean**  **SD CV** 

**(%) N**  **Mean SD**  **CV**

**(%) N**  **Mean SD**  **CV (%)**  **N Mean**  **SD CV** 

**(%)** 23 15 43.52 4.59 10.5 15 44.71 3.77 8.4 15 43.61 3.59 8.2 15 44.35 3.86 8.7 27 15 58.53 6.45 11.0 15 60.17 4.93 8.2 15 52.93* ( ↓10) 5.59 10.6 15 54.07 5.24 9.7 41 15 128.91 12.75 9.9 15 130.91 11.48 8.8 15 100.15** ( ↓22) 1 3.66 13.6 15 122.09 14.73 12.1 53 15 190.02 14.29 7.5 15 192.85 13.44 7.0 15 151.49** ( ↓20) 22.80 15.1 15 186.53 18.27 9.8

a Data w ere obtained from the study report

. Percent difference from control is included in parentheses. b 100 mg/kg/day 1

- Chloro

- 2

- Nitrobenzene N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p<0.05. ** Significantly different from controls at p<0.01.US EPA

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Page **103** of **211 Table 3**  **3 : Selected Food Consumption Data of Male Rats Treated with Chemical S in the Male** 

**Pubertal Assay a** 

**PND Vehicle Control**  **Chemical S 10 mg/kg bw/day**  **Chemical S**

**75 mg/kg b w/day**  **Positive Control b** 
**N Mea**  **n SD**  **CV (%)**  **N Mea** 

**n SD**  **CV (%)**  **N Mean**  **SD CV**  **(%) N**  **Mean SD**  **CV**

**(%)** 23

- 30 8 9.37 0.79 8.5 8 9.60 0.66 6.8 8 6.86** ( ↓2 7) 1.11 16.2 8 7.78**

( ↓17) 0.89 11.4 30

- 37 8 15.00 0.90 6.0 8 15.07 0.98 6.5 8 10.82** ( ↓28) 1.65 15.2 8 13.79 1.40 10.1 37

- 44 8 18.28 0.77 4.2 8 18.51 1.12 6.0 8 13.49** ( ↓26) 2.33 17.3 8 17.78 1.17 6.6 44

- 51 8 18.69 1.12 6.0 8 18.40 0.75 4.1 8 15.93** ( ↓15) 2.15 13.5 8 19.69 0.96 4.9 51

- 53 8 19.65 1.12 5.7 8 19.74 1.15 5.8 8 17.61* ( ↓10) 1.95 11.1 8 20.50 1.43 7.0

a Data w ere obtained from the study report

. Percent difference from control is included in parentheses. b100 m g/kg/day 1

- Chloro

- 2

- Nitrobenzene N = Number of animals examined SD = Standard Deviation CV = Coefficient of Variation *Significantly different from controls at p<0.05 **Significantly different from controls at p<0.01US EPA

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Page **104** of **211 Table 3**  **4 : General Growth and Preputial Separation of Rats Treated with Chemical S in the Male Pubertal Assay**  **a**

**Parameter Evaluated Vehicle Control**  **Chemical S 10 mg/kg bw/day**  **Chemical S 75 mg/kg bw/day**  **Posi tive Control**  **b**
**N Mean**  **SD CV** 

**(%) N**  **Mean SD**  **CV (%)**  **N Mean**  **SD CV**  **(%) N**  **Mean SD**  **CV (%)**  Initial Body Weight (PND 23; g) U 15 43.5 4.59 10 .5 15 44.7 3.77 8.4 15 43.6 3.59 8.2 15 44.4 3.86 8.7 A 15 43.9 0.65 1.5 15 44.2 0.65 1.5 15 43.9 0.65 1.5 15 44.2 0.65 1.5 Body Weight at P PS (g) U 15 162 14.3 8.8 15 169 13.1 7.8 3 149 16.2 10.9 14 174 11.2 6.4 A 15 163 12.8 7.9 15 1 69 12.8 7.6 3 145* ( ↓11) 13.2 9.1 14 174* ( ↑7) 12.8 7.3 Final Body Weigh t (PND 53; g) U 15 190 14.3 7.5 15 193 13.4 7.0 15 152** ( ↓20) 22.8 15.1 15 187 18.3 9.8 A 15 191 7.89 4.1 15 192 7.90 4.1 15 152** ( ↓20) 7.89 5.2 15 186 7.89 4.2 Final Body Weight (% of control) U NA 98.5 79.7 98.2 A NA 99.5 79.7 97.6 Body Weight Gain (PND 23

- 53; g) U 15 147 11.3 7.7 15 148 11.4 7.7 15 108** ( ↓26) 21.5 20.0 15 142 15.9 11.2 A 15 147 7.7 5.2 15 148 7.67 5.2 15 108** ( ↓26) 7.66 7.1 15 142 7.66 5.4
Age at Partial PPS (PND) U 15 34.9 2.13 6.1 15 37.1** ( ↑6) 1.92 5.2 15 38.3** ( ↑10) 1.05 2.7 15 37.7* ( ↑8) 1.33 3.5
A 15 34.8 0.79 2.3 15 37.2** ( ↑7) 0.79 2.1 15 38.3** ( ↑10) 0.79 2.1 15 37.8** ( ↑8) 0.79 2.1
Age at PPS (PND) U 15 47.5 2.50 5.3 15 48.3 2.61 5.4 15 53.4** ( ↑12) 1.24 2.3 15 50.5** ( ↑6) 2.83 5.6
A 15 47.5 1.13 2.4 15 48.5 1.13 2.3 15 53.3** ( ↑12) 1.13 2.1 15 50.6** ( ↑7) 1.12 2.2 Proportion unsep arated (#/N) 0/15 0/15 12/15 1/15
a Data w ere obtained from the study report

. Percent difference from control is included in parentheses. b 100 mg/kg/day 1

- Chloro

- 2

- Nitrobenzene U = Unadjusted for body weight on PND 21 ; A = Adjusted for body w eight on PND 21 ; N = Number of animals examined SD = Standard Deviation ; CV = Coefficient of Variation ; NA = Not applicable * Significantly different from controls at p<0.05 ; ** Significantly different from controls at p<0.01.US EPA

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Page **105** of **211 Table 3**  **5 : Organ Weight**  **Data of Male Rats Treated with Chemical S in the Male Pubertal Assay a** 
**Organ Vehicle Control**  **Chemical S 10 mg/kg bw/day**  **Chemical S 75 mg/kg bw/day**  **Positive Control b**  **N Mean**  **SD CV** 

**(%) N**  **Mean SD**  **CV (%)**  **N Mean**  **SD CV (%)**  **N Mean**  **SD CV (%)** 

Liver (g) U 15 8.46 0.82 9.6 15 8.62 0.789 9.1 15 7.33* ( ↓13) 1.37 18.7 15 13.3** ( ↑57) 1.77 13.3 A 15 8.53 0.55 6.4 15 8.53 0.548 6.4 15 7.38** ( ↓13) 0.55 7.4 15 13.3** ( ↑56) 0.55 4.1 R 15 4.45 0.22 5.0 15 4.47 0.227 5.1 15 4.82** ( ↑8) 0.38 7.9 15 7.11** ( ↑60) 0.40 5.6
Kidneys (g) U 15 1.25 0.12 9.8 15 1.21 0.104 8.6 15 0.94** ( ↓24) 0.14 14.7 15 1.30 0.16 11.9 A 15 1.25 0.06 4.9 15 1.20 0.061 5.1 15 0.95** ( ↓24) 0.06 6.5 15 1.30 0.06 4.7 R 15 0.656 0.04 6.8 15 0.628 0.036 5.8 15 0.626 0.05 8.2 15 0.699** ( ↑7) 0.65 22.7 Pituitary (mg) U 15 5.6 1.6 29.4 15 5.6 0.9 16.4 15 4.9 1.2 25.3 15 5.3 1.1 21.3 A 15 5.60 0.66 11.8 15 5.65 0.659 11.7 15 4.86 0.66 13.5 15 5.35 0.66 12.3 R 15 0.003 0.00 27.7 15 0.003 0.001 18.7 15 0.003 0.00 27.5 15 0.003 0.00 22.9 Adrenals (mg) U 15 49.1 3.8 7.7 15 48.7 6.6 13.6 15 43.1* ( ↓4) 5.8 13.4 15 48.8 8.2 16.9 A 15 49.2 3.11 6.3 15 48.4 3.11 6.4 15 43.3** ( ↓12) 3.11 7.2 15 48.7 3.11 6.4 R 15 0.026 0.00 8.8 15 0.025 0.004 15.5 15 0.029 0.00 16.6 15 0.026 0.00 16.7 Seminal vesicle + CG, with fluid (mg) U 15 246 72.9 29.7 15 252 62.8 25.0 15 112** ( ↓55) 29.7 26.6 15 217 70.5 32.5 A 15 248 29.0 11.7 15 248 29.0 11.7 15 113** ( ↓54) 28.9 25.5 15 216 28.9 13.4 Seminal vesicle + CG, without fluid (mg) U 15 198 45.3 22.9 15 209 45.0 21.5 15 103** ( ↓48) 25.0 24.3 15 185 55.6 30.1 A 15 199 20.3 10.2 15 206 20.3 9.9 15 104** ( ↓48) 20.3 19.5 15 184 20.3 11.0 Ventral prostate (mg) U 15 113 16.4 14.5 15 119 17.7 14.9 15 65.3** ( ↓42) 15.3 23.5 15 92.8* ( ↓18) 24.5 26.4 A 15 113 9.15 8.1 15 118 9.16 7.7 15 65.8** ( ↓42) 9.14 13.9 15 92.6** ( ↓18) 9.14 9.9US EPA

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Page **106** of **211 Organ**  **Vehicle Control Chemical S**  **10 mg/kg bw/day Chemical S**  **75 mg/kg bw/day Positive Control**  **b N**  **Mean SD**  **CV**

**(%) N**  **Mean SD**  **CV (%)**  **N Mean**  **SD CV (%)**  **N Mean**  **SD CV (%)**  Dorsolateral prostate (mg) U 15 37.3 9.7 26.1 15 45.6 12.4 27.2 15 27.5* ( ↓26) 7.7 28.1 15 41.6 8.2 19.8 A 15 37.3 5.05 13.5 15 45.6** ( ↑22) 5.06 11.1 15 27.6** ( ↓26) 5.05 18.3 15 41.6 5.05 12.1 LABC (mg) U 15 2 96 48.1 16.2 15 285 56.1 19.7 15 167** ( ↓44) 49.3 29.5 15 277 53.4 19.3 A 15 298 24.1 8.1 15 2 82 24.1 8.6 15 168** ( ↓43) 24.1 14.3 15 277 24.1 8.7 Epididymis, left (mg) U 15 216 39.3 18.2 15 221 26.8 12.1 15 161** ( ↓25) 31.1 19.3 15 196 24.4 12.5 A 15 217 13.9 6.4 15 219 13.9 6.4 15 162** ( ↓25) 13.9 8.6 15 195* ( ↓10) 13.9 7.1 Epididymis, righ t (mg) U 15 216 38.5 17.8 15 221 25.2 11.4 15 164** ( ↓24) 29.8 18.2 15 195 22.2 11.4 A 15 217 14.4 6.6 15 2 19 14.4 6.6 15 164** ( ↓24) 14.4 8.8 15 195* ( ↓10) 14.4 7.4 Testis, left (mg ) U 15 1227 108 8.8 15 1236 90.9 7.4 15 1039** ( ↓15) 160 15.4 15 1160 92.8 8.0 A 15 1232 52.4 4.3 15 1228 52.5 4.3 15 1043** ( ↓15) 52.4 5.0 15 1158* ( ↓6) 52.4 4.5 Testis, right (m g) U 15 1224 113 9.2 15 1224 95.1 7.8 15 1019** ( ↓17) 170 16.7 15 1147 89.4 7.8 A 15 1230 54.4 4.4 15 1216 54.5 4.5 15 1023** ( ↓17) 54.4 5.3 15 1145* ( ↓7) 54.4 4.7 Thyroid, fixed ( mg) U 15 13.1 1.7 13.0 15 13.8 2.0 14.3 15 12.1 2.4 20.2 15 13.5 2.5 18.6 A 15 13.1 1.14 8.7 15 13.8 1.14 8.2 15 12.1 1.14 9.4 15 13.5 1.14 8.4US EPA

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Page **107** of **211** a Data w ere obtained from the study report

. Percent difference from control is included in parentheses. b 100 mg 1

- Chloro

- 2

- Nitrobenzene U = Unadjusted for body weight on PND 21 A = Adjusted for body weight on PND 21 R = Organ

- to
- body

w e ight ratio (relative to body weight) N = Number of animals examined S D = Standard Deviation CV = Coefficient of Variation *Significantly different from controls at p<0.05 **Significantly different from controls at p<0.01US EPA

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– July , 2013

Page **108** of **211 Table 3**  **6 : Hormone Levels and Clinical**  **Chemistry Data of Male Rats Treated with Chemical S in**

**the Male Pubertal Assay a** 

**Parameter Vehicle Control**  **Chemical S 10 mg/kg bw/day**  **Chemical S**

**75 mg/kg bw/day Positive Control**  **b**
**N Mean**  **SD CV** 

**(%) N**  **Mean SD**  **CV**

**(%) N**  **Mean SD**  **CV**

**(%) N**  **Mean SD**  **CV (%)**  **Thyroid Hormones** Total T4 (μg/dL) 15 4.178 0.543 13.0 14 4.493 0.438 9.8 15 3.971 0.486 12.3 15 3.487** ( ↓17) 0.466 13.4 TSH (ng/mL) 9 2.363 1.877 79.4 9 2.568 1.796 69.9 14 1.932 1.739 90.0 10 1.173 1.105 94.1 Testosterone (ng/mL) 14 1.836 1.059 57.7 15 2.215 1.274 57.5 14 0.814* ( ↓56) 0.593 72.8 14 1.621 1.068 65.9 **Clinical Chemistry** Glucose (mg/dL) 15 173.17 30.45 17.6 15 165.27 18.19 11.0 15 162.02 35.63 22.0 15 150.30 23.07 15.4 AL T (IU/L) 15 78.74 16.37 20.8 15 77.43 9.47 12.2 15 94.92* ( ↑21) 20.06 21.1 15 124.21** ( ↑58) 37.28 30.0 AST (IU/L) 15 162.74 29.93 18.4 15 153.18 15.51 10.1 15 187. 61 46.06 24.6 15 191.63* ( ↑18) 35.19 18.4 Total Bilirubin (mg/dL) 15 0.00 0.00 0.0 15 0.00 0.00 0.0 15 0.03 0.07 235. 4 15 0.24 0.17 72.6 Creatinine (mg/dL) 15 0.39 0.10 26.2 15 0.36 0.14 37.8 15 0.34 0.09 28.2 15 0.37 0.13 34.8 Total Protein (g/dL) 15 7.09 0.71 10.1 15 7.25 0.69 9.6 15 6.52 0.56 8.6 15 7.40 0.60 8.1 Albumin (g/dL) 15 4.50 0.18 4 .1 15 4.51 0.09 2.0 15 4.40 0.24 5.6 15 4.74** ( ↑5) 0.14 2.9 Calcium (mg/dL) 15 12.43 0.45 3.7 15 12.52 0.72 5.7 15 12.16 0.60 4.9 15 12.49 0.53 4.2 Phosphorus (mg/dL) 15 10.75 1.13 10.5 15 11.22 0.93 8.3 15 9.97 1.01 10.1 15 11.38 0.96 8.4 Cholesterol (mg/dL) 15 81.93 14.76 18.0 15 78.05 9.78 12.5 15 84.35 11.31 13.4 15 99.28** ( ↑21) 10.36 10.4 Urea (mg/dL) 15 34.88 5.89 16.9 15 30.36 4.08 13.4 15 31.99 8.19 25.6 15 39.70 5.23 13.2 AL P (lU/dL) 15 438.57 117.14 26.7 15 401.32 74.31 18.5 15 288.12** ( ↓34) 54.19 18.8 15 369.47 105.55 28.6 BUN (mg/dL) 15 16.29 2.75 16.9 15 14.18 1.90 13.4 15 14.94 3.83 25.6 15 18.54 2.44 13.2 Globulin (g/dL) 15 2.58 0.64 24.6 15 2.73 0.64 23.4 15 2.12 0.49 23.0 15 2.66 0.55 20.7 Albumin/ Globulin 15 1.84 0.44 23.7 15 1.73 0.36 20.9 15 2.1 7 0.49 22.4 15 1.86 0.40 21.7

a Data w ere obtained from the study report

. Percent difference from control is included in parentheses. S D = Standard Deviation ; CV = Coefficient of Variation *Significantly different from controls at p<0.05 **Significantly different from controls at p<0.01US EPA

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Page **109** of **211 x.**  **Fish Short**
**- term Reproduction**  **Assay (FSTRA)** The 21

- day FSTRA of Chemical S with fathead minnows ( Pimephales promelas ) was conducted under flow

- through conditions. Adult fish, 20 spawning groups (2 males and 4 females in each group: 6 months) were exposed to Chemical S at nominal conce ntrations of 0 (negative control; NC), 0 (solvent control; SC; dimethy lformamide (DMF) at 0.02mL/L), 0.16, 8.0 and 4.0 μg ai/L while mean

- measured concentrations were <LOQ (<0.010), <LOQ, 0.0834, 0.527, and 1.66 μg ai/L , respectivel y. The test sy stem was maintained at 24.8 to 25.4 o C and a pH of 7.91 to 8.35. Spawning frequency

occurred at least every

3 to 4 day s and fecundity in negative and solvent control was 26 and 34 eggs/female/day /replicate, respectively

(range: NC=21

- 36, SC=24

- 40); fertilization su ccess in the NC and SC groups averaged 93 and 90%, respectively (range: NC=89

- 98, SC=87

- 92). Fertility was significantly

reduced (p<0.05) by

13% (relative to negative control) for the 1.66 μg a.i./L treatment group **(Table 3 7**  **).** Fecundity was not significa ntly

reduced from negative control (p>0.05) at any treatment concentration; however, there was a 31% increase in fecundity in the solvent control and a 41 and 50% increase in the low and middle test concentrations with a 38% decrease at the high concentrat ion compared t o the negative control ( **Table 3 8**  **).** Male plasma vitellogenin (VTG) was significantl y increased (p=0.045) b y 90% relative to the negative control in the 1.66 μg a.i./L treatment group. There was no significant difference in VTG for females (p >0.05). The increase in male plasma VTG in this treatment group was coincident with a 38% mortality rate for males and an overall ( **i.e** ., male and female fish combined) mortality rate of 50%. Plasma vitellogenin was initially anal yz ed using a trend test ( Jonckeere

- Terpstra) and all reported values (analy zed using replicate means). The individual fish values for male VTG are depicted in **Figure 13. T**  his figure illustrates that across the negative control and all Chemical S treatment groups, there is one ind ividual fish value in each group that appears to be substantially greater than the other replicate means; additionally , all the individual values in the highest treatment group are within the range of the negative control values. A discussion of the obser ved variability

and potential outliers associated with male VTG measurements was presented in the FSTRA section of the May 2013 SAP white paper (USEPA 2013) 10 , and the results for Chemical S reflect that discussion. Furthermore, if induction of vitellogenes is in males was occurring due to Chemical S interacting with the estrogen signaling pathway , the overall change of response in the exposed fish is expected to be greater than the approximate 2

- fold increase (based on treatment means) observed in this study for males in the highest treatment. Therefore, based on a more rigorous analy sis of the male plasma VTG data, the initial statistical significance is considered an artifact of the observed variability

. This conclusion is further supported by

the relativ ely

limited extent of up

- regulation of vitellogenesis in the fish at the highest treatment group compared to the control. **Figure 13. Male plasma VTG (ng/mL) from Chemical S**  where the high treatment group was statistically

different (p ≤0.05;Jonckeere

- Terpstra using treatment replicate means and all available data)

10 http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPP

- 2013
- 0075
- 0003US EPA

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– July , 2013

Page **110** of **211** compared to the negative control. The median of the individual is represented by the solid line, with the box r epresenting the 25 th and 75 th percentile and the whiskers are the range of individual values, with potential outliers outside the whiskers (defined as greater than the median plus three times the interquartile range).

A significant 39% increase (p<0.0 5) relative to control in male gonado

- somatic index (GSI ) was observed at the 1.66 μg ai/L group. Female GSI

was not significantl y (p>0.05) different. Nuptial tubercle scores were also significantl y reduced b y 33% (p<0.05) in male fish at the 1.66 μg ai/L level; no tubercles were observed for females. The study author reported no substantial differences in gonadal staging or histopathological effects in the male testes or female ovaries for any treatment group compared to the control. Measurements of se x steroids were not reported n either was information provided on dorsal nape pads. Unless otherwise indicated, all effects are reported based on comparison to the negative (clean water) control.US EPA

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Page **111** of **211** There was a significant (p<0.05) reduction in survival (50% reduction compared to negative control) at the 1

. 66 μg ai/L treatment level. There appeared to be inadequate reporting of secondary sex characteristics and clinical signs. The stud y author reported pectoral fin hemorrhage in one male from the control gro up. There were no incidences reported in the 1.66 μg ai/L treatment group despite a significant reduction in survival. There were no significant differences in male or female body weight between the control and treatment groups at test termination; body length at test termin ation was not reported ( **Table 39**  **).**
The percent fertility

in the negative control (93%) and solvent control (90%) was slightl y less than the guideline criterion minimum of 95%. Additionally, t he dissolved oxy gen dropped below 60% in s everal solvent control treatment group replicates, with a reported minimum of 47%. Only the range was reported, so the reviewer could not determine how many or for which day s dissolved oxy gen dropped below the minimum criteria. However, once a decrease was observed, aeration was added to each test chamber on Day

10. Finally , the anal yt ical confirmation samples varied by 26

- 130%. As such the CV for the all treatments groups was greater than the 20% variance criteria. However, given the low solubility and propensity

to sorb to glass for Chemical S, deviations from the 20% may have made maintaining exposure in the water column more difficult. While these deviations to the performance criteria were observed, they did not impact the ability to interpret the r esults of the assay

. Overall, while significant changes were reported in male fish including decreased fertility and male nuptial tubercles and increased male GSI and VTG, these effects were only observed at the same test concentration in which there was a significant increase in mortalit y. Additionally , for male VTG, while a significant increase was initial ly reported, upon further evaluation, it was not determined to be statistically different from the controls based on the range of reported values and th e magnitude of change. At concentrations where no apparent overt toxicity occurred, there were no endocrine

- related responses. The pesticidal mode of action of Chemical S involves the uncoupling of mitochondrial oxidative phosphory lation resulting in the depletion of ATP, which may have affected th e overall energy reserves of the fish and may have in turn impacted energy inputs into reproduction. Given the mode of action for C hemical S along with the observed decreases in fertility and tubercles and incr eases in GSI which lack endocrine

- specificity and were coincident with effects on survival, there is uncertaint y whether the se effects were due to a possible endocrine interaction with the A (antagonism) or E pathway (agonism) or due to the fish’s re sponse to the toxic action of the chemical. Although there is uncertainty , based on the overt toxicity observed at the highest treatment level, the lack of specificity

in endpoints that were significantly affected, Chemical S is classified as “Possible” in the F STRA for impacts to E or T signaling modalities.US EPA

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Page **112** of **211 Table 37**  **: Chemical S** 
**- Reproductive and HPG Endpoints**  **1 for Male Fathead Minnow in the** 

**FSTRA**

Abbreviations: Conc. Concentration. GSI Gonado

- Somatic Index. VTG Vitellogenin. 1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. 2 Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board

- certified pathologist. 3 Statistically significant at p<0.05.

**Table 38: Chemical S**
**- Reproductive and HPG Endpoints1 for Female Fathead Minnow in the** 

**FSTRA**

Abbreviations: Conc. Concentration. Diff. Difference. Fert. Fertilization. GSI Gonado

- Somatic Index. VTG Vitellogenin. 1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. 2 Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board

- certified pathologi st. 3 Statistically significant at p<0.05.

**Treatment (μg a.i./L)**  **[mean measured] Tubercle**  **Score GSI**  **Gonadal Staging and**  **Histopathology 2**  **Plasma VTG**

**Median %**  **D iff** 

**erence Effect?** 

**(Yes/No) % Diff** 

**erence** Control (dilution water only ), if used 36.5 0 No 0 Solvent control 35.4

- 15.8 No

- 53.3 0.0834 31.0 4.44 No

- 14.4 0.527 39.0

- 4.44 No 61.9 1.66 24.0 3 39.3 3 No 89.8 3

**Treatment (μg a.i./L)**  **[mean measured]**  **Fecundity Fertility**  **Success Tubercle** 

**Score GSI**  **Gonadal Staging and**  **Histopathology 2**  **Plasma**

**VTG %**  **Diff erence**  **% Diff**  **erence Median**  **%**

**Diff erence**  **Effect?**

**(Yes/No) %** 

**Diff erence**  Control (dilution water only ), if used 0 0 0 0 No 0 Solvent control 30.8 3.2 0 7.1 No 32.8 0.0834 40.6

- 4.81 0

- 4.98 No 39.6 0.527 50.0

- 4.01 0 1.60 No 5.20 1.66

- 38.4

- 13.0 3 0 23.8 No 35.0US EPA

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– July , 2013

Page **113** of **211 Table 39**  **: Chemical S** 
**- Growth Endpoints**  **1 in the Fish Short** 
**- Term Reproduction Assay (FSTRA)** 

**Treatment (mg a.i./L)**  **[mean measured] Body Weight**  **Males Females**  **% Diff. % Diff.**  Control (dilution water only ), if used 0 0 Solvent control

- 7.9

- 4.1 0.0000834

- 1.26 2.00 0.000527

- 3.43

- 4.23 0.00166

- 3.51 2.76 Abbreviations: Diff. Difference. 1 Unless otherwise indicated, percent (%) differences are reported based on comparison to the negative (clean water) control. Termination body lengths not reported.

**xi**

. **Amphibian Metamorphosis Assay** The 21

- day

assay of Chemical S on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was studied under flow

- through conditions. Amphibian larvae (80 Nieuwkoop

- Faber [NF] Stage 51 tadpoles, 20 in each of 4 replicates) were exposed to nominal concentrations of 0 (negative and solvent controls), 0.09 , 0.300, and 1.00 μg a.i./L. Time

- weigh ted average (TWA) concentrations were <0.010 (<LOQ; controls), 0.0156, 0.0711, and 0.307 μg a.i./L

. The test sy stem was maintained at 21.5 to 22.6°C and a pH of 7.9 to 8.4. Chemical S did not significantl y increase or decrease normalized hind

- limb length (HLL) at either Day 7 or 21. Similarly , there was no significant acceleration or delay of median Day

7 and 21 NF developmental stage at any treatment level. Several individuals were excluded from the anal ys is of the Day 21 continuous endpoints (SVL , body weight, and normalized HLL) because they developed to NF Stage 61; one tadpole from the negative control and between 4

- 5 tadpoles in each of the solvent control and treated groups were excluded from the Day 21 continuous endpoint anal ys is for this reason. Unless otherwise indicated, all effects are reported based on comparison to the negative (clean water) control. No as ynchronous development was observed in any treatment group. Effects on thy roid gland histopathology

were observed in both controls and t he TWA 0.0156, 0.0711, and 0.307 μg a.i./L. Histopathological effects included mild follicular cell hy perplasia and hy

pertrophy , though there was no apparent treatment
- related effect because the incidence of these diagnoses was similar in controls and tre atments. The study author did not report clinical observations.US EPA

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– July , 2013

Page **114** of **211** There was poor recovery

(as low as 8.8% of nominal at test termination in the highest treatment level) and high variability

of measured exposure concentrations, with overall %CVs ranging fro m 33

- 69% in all treatment levels; this exceeded the performance criterion maximum CV of 20%. The very low recoveries and reported instability (likely due to low solubility

and high K oc ) of Chemical S in the test sy stem make the results of this amphibian me tamorphosis study difficult to interpret. An additional performance criterion which was not met was the decline of DO to 3.24 mg/L ( i.e ., 39% saturation), below the recommended minimum of 3.5 mg/L (40% saturation); this occurred in one replicate vessel fr om the vehicle control group on Day 13 and all test vessels were aerated upon this discovery to correct the saturation issue. Iodide in the laboratory water (13 μg/L) was higher than the guideline recommended level ( ≤10 μg/L); however, the analy sis was las t performed one y ear prior to the conduct of the study

and it is unclear if the water anal yz ed was representative of the test water, which was reported to be a combination of laboratory well water and laboratory well water that had been filtered through re verse osmosis. All other performance and validity criteria were satisfied in this study

. Overall, exposure levels in the AMA were determined to be inadequate, although there were no HPT

related effects within the range of concentrations tested.

**D. Other**  **Scientifically Relevant**  **Information (OSRI) for Chemical S**  The registrant elected to conduct the entire Tier 1 assay

. Therefore, no OSRI were submitted to the a gency . However, Chemical S has Part 158 mammalian and eco toxicity studies previously submitted to the Agency . These studies are discussed below. **1. Mammalian OSRI Data** In a subchronic toxicity

study , Chemical S was administered in the feed to 6 Carworth Farm E ( CFE ) rats/sex/group at 0, 50, 100, 300 or 600 ppm (equivalent to 2.5, 5, 15 or 30 mg/kg b w/day ) for 3 months. Testes weights and histopathology were evaluated. Bod y weight was decreased at 300 (6% in males and 7% in females and 600 ppm (13% in males and 16% in females). A slight decrease in food efficiency occurred at 600 ppm in males (27% f rom wk 0

- 4 and 12% from wk 0

- 13) and females (26% for wk 0

- 4 and 17% for Week 0

- 13). The decreases in body weights may

have been the result of the unpalatability

of the diet. No histological findings were seen in the testes. The NOAEL is 5 mg/kg bw/day a nd the LOAEL is 15 mg/kg bw/day based on elevated alkaline phosphatase in male rats. In a 2

- ye ar rat toxicity

study , Chemical S (97% a.i.) was administered in the diet to CFE rats (72/sex/group) for up to two y ears at 50, 100, 300 or 600 ppm (equivalent to 2.5, 5, 15 or 30 mg/kg bw/day

. For controls 144 rats/sex were used. Organ weights were obtained for testes. Histopathology was conducted on the testes, prostate, ovaries, uterus, mammary glands, pituitary

and thy roid glands. The diet was unpalatable to both males and females and resulted in decreases in body weights, bod y weight gains, and food consumption, especially during the earl y stages of the study . There was a significant bod y weight depression in males and females at 15 and 30 mg/kg bw/day , correlated with decreased food consumption. Only the 30 mg/kg bw/day

dose groups showed a nominal trend toUS EPA

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– July , 2013

Page **115** of **211** decreased food efficiency for weeks 0

- 52. It should be noted that the subchronic and 2

- generation studies on reproduction g enerall y show a similar nominal decrease in food efficiency at 15 and 30 mg/kg bw/day , respectively

. Mortality of females at the 30 mg/kg bw/day (33% versus 56% in controls) was less than corresponding controls. Male mortality was unchanged. Increased ab solute and relative testes weights were noted at 15 and 30 mg/kg/day , but no treatment histological correlates were noted

. No treatment

- related histopathological lesions were observed in the prostate, ovaries, uterus, mammary

glands, pituitary

and thy roid glands. T he only

effects on the intestines was a suggestion of an increased trend for soft stools, especiall y at the two highest dietary treatment levels (incidence in males was 6%, 12%, 21%, 42% and 71% from control to 30 mg/kg bw/day , respectively ). Mil d dilation of the glands in the fundic mucosa of the stomach may have shown a slight increased trend in males, but it was not considered toxicologicall y significant. For chronic toxicity , the NOAEL was 5 mg/kg bw/day and LOAEL was 15 mg/kg bw/day based on treatment
- related alkaline phosphatase increase in males and females. In a carcinogenicity

study in mice , Chemical S (97% pure) was administered in the diet to 120 control and 60 CFE mice/sex/group at 0, 50, 100, 300, or 600 ppm (0, 7.5, 15, 45 or 90 mg/k g bw/day ) for 18

- months . Testes weights were obtained. Histopathological examinations were conducted for the gonads, prostate, uterus, pituitary and thy roid glands. Body

weights were significantly (p <0.001) reduced compared with control values from week 4 (first week determined) to termination at 45 and 90 mg/kg bw/day doses for both males (3% and 17%, respectively ), and females (4% and 13%,)). At weeks 36 and 72, male bod y weights at 300 and 600 ppm were reduced b y 5% and 16%

- 18%, respectivel y, and fema le bod y weights were reduced 6%

- 3% and 16%, respectively . At termination, male body weights were reduced at the same dose levels. The NOAEL was 15 mg/kg/day and the L OAEL was 45 mg/kg/day

based on body

weight reductions. In a developmental toxicity study , 27 mated Specific Pathogen Free Wistar rats per group were administered Chemical S (98.9

- 99.1% a.i. ) b y gavage in an aqueous solution at doses of 0, 15, 30, and 60 mg/kg bw/day

on gestation day s (GDs) 6

- 15, inclusive. On GD 20, all surviving dams were euthanized and then necropsied, and all fetuses were examined externall y, weighed and sacrificed. Several high

- dose animals exhibited diarrhea at vary ing times between GDs 6 and 20 (7/27 vs. 2/27 for controls). For maternal toxicity , the NOAEL was 15 mg/k g bw/day and the LOAEL was 30 mg/kg bw/day , based on decreased body weight gain. There were no treatment

- related differences in the intrauterine parameters between the treated and control groups and there were no significant increases in litter or fetal in cidences of any individual structural abnormalities for any treated group. For developmental toxicity , the NOAEL was 60 mg/kg bw/day , the highest dose tested; a LOAEL was not established. In another developmental toxicity study , 18

- 23 pregnant New Zealand white rabbits per group were administered Chemical S (98.7% a.i) b y gelatin capsule at doses of 0, 1, 5, or 10 mg/kg bw/day on gestation day s (GD) 6

- 18, inclusive. On GD 29, all surviving does were sacrificed and the uteri were weighed and examined. Trea tment
- related deaths occurred on or after GD 19 in two mid

- dose and four high

- dose animals. The number of animals that aborted was 3, 1, 2, and 12 in the control, low

- , mid

- ,US EPA

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– July , 2013

Page **116** of **211** and high

- dose groups, respectively . Anorexia (subjective observations of not ea ting and passing few feces) was observed in 2/18, 2/18, 11/18, and 23/23 animals, respectivel y, and was often associated with abortion and/or whole litter loss. L esions of the gastric mucosa were found in the mid

- and high

dose animals that died intercurr entl y, were sacrificed following abortion, or were examined at terminal sacrifice. The overall incidence rates for lesions in the stomach were 2, 2, 6, and 8 animals in the control, low

- , mid

- , and high

- dose groups, respectively . For maternal toxicity , t he NOAEL was 1 mg/kg bw/day

and the LOAEL was 5 mg/kg bw/day based on clinical signs of toxicity and gross lesions of the stomach. No treatment

- related effects on any cesarean section parameter were observed in the low

- or mid

- dose groups. No treatment

- rel ated external, visceral, or skeletal malformations/variations were observed in this study . For developmental toxicity , the NOAEL was 5 mg/kg bw/day

and the LOAEL was 10 mg/kg bw/day based on increased numbers of resorptions/litter and post

- implantation los s/litter and decreased fetal body weights. In a two

- generation reproduction study , Chemical S (99.4% a.i.) was fed to groups of 30 male and 30 female F 0 and F 1 rats at dietary concentrations of 0, 40, 75, 250, or 500 ppm. The dietary concentrations corres ponded to doses of 2.39, 4.47, 14.8, and 30.0 mg/kg bw/day for F 0 males; 3.18, 5.92, 20.0, and 45.7 mg/kg bw/day for F 1 males; 2.76, 5.13, 16.6, and 33.6 mg/kg bw/day for F 0 females; and 3.68, 6.83, 23.9, and 54.1 mg/kg bw/day for F 1 females averaged over the premating period only . The F 0 male and female rats received treated or control diets for 72 day s prior to mating and during mating, gestation, and lactation of one litter. The F 1 pups were weaned onto the same diets as their parents and were fed thei r respective diets for 105 day s prior to mating and during mating, gestation, and lactation of one litter. The F 0 males were sacrificed after 124

- 126 day s of feeding, F 0 females after 116

- 137 day s, F 1 males after 159

- 177 day s, and F 1 females after 160

- 178 day s. Testes were weighed. Histopathological examinations were conducted for epididymides, testes, seminal vesicles, prostate, coagulating glands, ovaries, uterus, vagina, pituitary and thy roid glands

. Sexual maturation, estrous cy cle length and periodici ty , and sperm parameters were not evaluated in this study

. No treatment

- related effect were seen on survival, clinical signs, gross or microscopic findings in adult F 0 or F 1 male or female rats at an y dose, or on body weights, bod y weight gain, food con sumption, or food efficiency

at 40, 75, or 250 ppm. Body weights and overall bod y weight gain, food consumption, and food efficiency

were significantl y (p<0.05) decreased in both sexes and generations administered 500 ppm of the test material compared wit h the controls. The F 0 males weighed 8

- 19% less than controls throughout the premating/postmating period and gained 33% less weight; F 1 males weighed 23

- 25% less and gained 24% less weight. The F 0 females weighed 7

- 12% less (except for week 1) and gained 41% less weight during the premating period, and F 1 females weighed 18

- 30% less and gained 20% less weight. The F 0 and F 1 males consumed 12% less food averaged over the entire premating period, F 0 females consumed 13% less, and F 1 females consumed 7% les s. No treatment

- related effects were observed on the following parameters for F 1 or F 2 offspring: live birth, viability , lactation, or litter survival indices, the number of litter or pups born (live or stillborn), the sex ratio at birth, or the pup weig ht at birth. Pup growth was significantly (p<0.05) inhibited by

250 and 500 ppm of the test material in the diet. At 500 ppm, F 1 pups weighed 6% (N.S.), 16%, 20%, andUS EPA

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– July , 2013

Page **117** of **211** 24% less than controls on lactation day s 4 (precull), 7, 14, and 21, respectively , and gained 28% less weight between day

0 and 21. The F 2 pups at 500 ppm weighed 13%, 19%, 22%, and 29% less, respectivel y, than controls and gained 33% less weight between day 0 and 21. At 250 ppm, F 1 pups had body weights similar to those of the controls throughout the lactation period; whereas, F 2 pups weighed 9%, 12%, 9% (p<0.05), and 10% (p<0.05) less than controls on lactation day s 4 (precull), 7, 14, and 21, respectivel y, and gained 11% less weig ht. The F 2 pup weights were statisticall y significantl y decreased from control values on lactational day 14 and 21 at 250 ppm. Between lactational day

0

- 4, F 2 pups showed a weight gain decrement of 19% at 250 ppm. For parental/s ys temic toxicity , the NO AEL was14.8 mg/kg bw/day for males and 16.6 mg/kg bw/day for females and LOAEL were 30.0 mg/kg bw/day for males and 33.6 mg/kg bw/day for females based on decreased bod y weights, body weight gain, food consumption, and food efficiency in F 0 and F 1 males an d females. For reproductive toxicity , the NOAEL was 30 mg/kg bw/day in males and 33.6 mg/kg bw/day

in females; a L OAEL was not established. There were no effects on fertility at the highest dose tested. For offspring toxicity , the NOAEL was 5.13 mg/kg/day and the LOAEL was 16.6 mg/kg/day

based on decreased pup weight and weight gain in F 2 litters. Chemical S was administered in capsules to 8 control and 4 Beagle dog s /sex/group at 0, 2.5, 5, 15, 30 or 60 mg/kg/day

for 2 y ears. Organ weights were obtained for testes and thyroid

. Most dogs dosed with Chemical S were reported to have exhibited vomiting and diarrhea. It was also reported that after one - year animals dosed at 2.5

- 15 mg/kg/day , vomiting and diarrhea had subsided. I n the 30 and 60 mg/kg bw/day groups vomiting and diarrhea continued after one ye ar, but to a lesser extent. Convulsions were seen in one female from each of the 0, 2.5, 15 and 30 mg/kg bw/day dose levels. One male at 2.5 mg/kg bw/day was affected. Male (21%) and female (23%) body w eights were significantl y reduced at 60 mg/kg bw/day . Organ weights showed not effects

. The vomiting and diarrhea could be expected from a test material that irritated the gut. Possibly this vomiting and diarrhea resulted in the decreased bod y weight at 6 0 mg/kg bw/day . Vomiting and diarrhea from gut irritation is an adverse effect, but it may also reduce the dose administered, which occurred over the first y ear of the study

. The NOAEL is 5 mg/kg bw/day and LOAEL is 15 mg/kg bw/day based on excess vomiti ng and diarrhea with a bod y weight decrement at 60 mg/kg bw/day

. **2. Ecotoxicity OSRI Data**  Rainbow trout ( Onchorynchus mykiss ) were exposed to the technical grade active ingredient (TGAI) (98.6% a.i.) under flow

- through conditions in a 60

- day earl y life s tage test. DMF was used as a solvent. The study author reported that larval survival, length, and weight, were significantly less than in the solvent control at measured concentrations of 0.61 μg/L and above. The NOAEL values were 0.31 μg/L for larval s urvival and 0.61 ug/L for embry o hatching. The study was considered scientifically sound; however, raw data were not submitted and the reviewer was therefore unable to confirm the study author’s conclusions. The 96

- hour acute LC 50 value for Chemical S usi ng rainbow trout was 1.7 μg a.i./L (based on nominal concentrations) tested under static conditions.US EPA

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– July , 2013

Page **118** of **211** In a one generation avian reproduction test with bobwhite quail, nominal dietary concentrations of the TGAI at 50 and 150 ppm had no effect on bobwhite rep roductive parameters during the 21

- week exposure period. At 500 ppm there appeared to be a reduction in the number of eggs laid per hen, and an increase in the number of hens that exhibited lesions of old egg yolk peritonitis at terminal sacrifice. The NO AEC was 150 ppm. Nominal dietary

concentrations of the TGAI at 50 and 150 ppm had no effect on mallard duck reproductive parameters during a 19

- week exposure period. At 500 ppm there was a reduction in the number of eggs laid per hen. There was an incre ase in the number of females in all treatment groups that exhibited lesions of old egg yolk peritonitis at terminal sacrifice. The freshwater invertebrate waterflea, Daphnia magna, was exposed to the TGAI in a 21

- day chronic toxicity test. The NOAEL was 16 μg/L (measured) or 25 μg/L (nominal). There was a significant difference between the solvent control and the test chemical at the two highest treatment concentrations (50 and 100 μg/L, nominal) with respect to growth, percent adult survival, and total number of young. These NOAEL values are above the reported solubility of Chemical S which is about 13 μg/L. No additional data with frog or other amphibian species have been submitted.

**E. D**  **iscussion**

**i. Effects on Hypothalamic** 
**- Pituitary** 
**- Gonadal (HPG)**  **Axis** As previously

noted, the EDSP Tier 1 screening data and OSRI were evaluated together to determine whether Chemical S has potential to interact with the HPG axis or its downstream pathways related to steroidogenesis or sex steroid signaling. The Tier 1 in vitro assay s relevant to this analy sis include the AR, binding, ER binding, ERTA, aromatase, and steroidogenesis ass ay . Relevant in vivo assay s include the male and female pubertal rat, uterotrophic, Hershberger, and the FSTRA assay s. **Effects on Estrogen Pathway: Table 40**  below summarizes the results of the available EDSP Tier 1 screening assay

results and OSRI results relevant for determining the potential for Chemical S to interact with the estrogen pathway . The various targets of the estrogen pathway across the relevant Tier 1 assay s are delineated so as to facilitate determination of potential for estrogenic, anti

- estrogenic, or HPG axis effects. Chemical S was classified as “not interactive” in the in vitro estrogen receptor binding assay and negative in the ERα transcriptional activation and Uterotrophic assay s. Based on these results, Chemical S does not appea r to disrupt the estrogen signaling pathway through estrogen receptor agonism or antagonism. Chemical S showed an equivocal response in the in vitro aromatase assay and was negative in the steroidogenesis assay . However, there is some evidence that Chemi cal S may have the potential to interact with the estrogen pathway in the in vivo Female Pubertal assay . At the low dose (10 mg/kg bw/day ), significant (p ≤ 0.01) delay s ( ~ 2 day s) in vaginal opening (VO) and decreased body weight atUS EPA

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– July , 2013

Page **1 19**  of **211** VO were seen in the absence of overt toxicity . At the high dose (75 mg/kg bw/day ), estrogen

- related effects were manifested as delay ed VO (~ 7 day s; p ≤ 0.01); decreased bod y weight at VO; decreased weights of the ovaries and uterus but in the presence of overt toxicity (sal ivation, and/or decreased body weights and food consumption). I n the FSTRA assay , effects that may indicate potential interaction with the estrogen signaling pathway included alterations in fertility (13% decrease), GSI (39% increase in males), and 33% de crease in male nuptial tubercles . Additionally , while an increase in male VTG was initially reported (90% increase), upon further review of the data for this endpoint, it was concluded that this response was not different from the control and was not consi stent with the magnitude of response expected with an upregulation of VTG due to an estrogen agonism. Furthermore, effects were only observed at a concentration in which survival was markedl y affected (50% overall mortality observed). The pesticidal mod e of action of Chemical S involves the uncoupling of mitochondrial oxidative phosphory lation and resulting in the depletion of ATP. Based on the available data, another plausible mode of toxic action is related to its irritation properties including irrit ation that compromises the integrit y of the gastro

- intestinal tract in mammals leading to restricted caloric intake due to reduced food consumption. For the female pubertal assay at the high dose where overt toxicity was observed, the directionality

of th e reported responses is consistent with depletion of ATP and restricted caloric intake. As the pesticidal mode of action for Chemical S eventually leads to a depletion of ATP, there is uncertaint y in whether the responses observed in the FSTRA are potenti ally due to an interaction with the estrogen pathway

or due to the a lack of energy resources available for reproduction output. Additionally , the responses in the female rat pubertal, in the absence of overt toxicity d o not suggest an agon istic interac tion with the estrogen pathway

. The results of mammalian Part 158 studies did not reveal any potential of Chemical S to interact with the estrogen pathway

. There were reported decreases in reproduction in birds and freshwater invertebrates, but the repor ted endpoints are non

- endocrine specific. Based on WoE evaluation of EDSP Tier 1 screening assay and OSRI results, in the absence of overt toxicity , Chemical S demonstrated a potential to interact with the estrogen pathway presumably

through the perturbati on of the HPG axis in the Female Pubertal assay

. However, data from the remaining assay s are not sufficiently

compelling to support such a perturbation.US EPA

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– July , 2013

Page **120** of **211 Table 4**  **0 :**  **Estrogenic/Anti**
**- Estrogenic Pathway**  **for Chemical S Lines of Evidence**  **Indicating Potential Interaction with the Estrogenic/Anti**
**- Estrogenic Pathway**  **for Chemical S a** 
**Study Type**  **/Literature Citation** **E R** **B i** **n d** **i n** **g E** **R A** **c t** **i v** **a t** **i o** **n S** **t e** **r o** **i d** **o g** **e n** **e s** **i s** **U t** **e r** **i n** **e W** **e i** **g h** **t O** **v a** **r i** **a n** **W e** **i g** **h t** **O v** **a r** **i a** **n /** **G o** **n a** **d S** **t a** **g i** **n g** **a n** **d H** **i s** **t o** **p a** **t h** **o l** **o g** **y P** **i t** **u i** **t a** **r y** **W e** **i g** **h t** **E s** **t r** **o u** **s C** **y c** **l i** **c i** **t y** **b A** **g e** **& W** **e i** **g h** **t a** **t V** **O F** **e r** **t i** **l i** **t y** **/ F** **e c** **u n** **d i** **t y** **V i** **t e** **l l** **o g** **e n** **i n** **F i** **s h** **G o** **n a** **d a** **l W** **e i** **g h** **t (** **G S** **I )** **O v** **e r** **t T** **o x** **i c** **i t** **y O** **b s** **e r** **v e** **d c** 

**EDSP Tier 1** ER Binding Not

interactive ERTA Negative Aromatase Equivocal Steroidogenesis Negative Uterotrophic No Effect

Female Pubertal Rat ↓ 24% ↓43% No effect ↓ 35% No effect ↑ 2

- 7 day s (V0) ; ↓ 7

- 12% BW X d

FSTRA ↓ (13%) ♂ H e ↑(90%) ♂ H e ↑(39%) ♂ H e XUS EPA

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– July , 2013

Page **121** of **211 Lines of Evidence**  **Indicating Potential Interaction with the Estrogenic/Anti**
**- Estrogenic Pathway**  **for Chemical S a** 
**Study Type**  **/Literature Citation** **E R** **B i** **n d** **i n** **g E** **R A** **c t** **i v** **a t** **i o** **n S** **t e** **r o** **i d** **o g** **e n** **e s** **i s** **U t** **e r** **i n** **e W** **e i** **g h** **t O** **v a** **r i** **a n** **W e** **i g** **h t** **O v** **a r** **i a** **n /** **G o** **n a** **d S** **t a** **g i** **n g** **a n** **d H** **i s** **t o** **p a** **t h** **o l** **o g** **y P** **i t** **u i** **t a** **r y** **W e** **i g** **h t** **E s** **t r** **o u** **s C** **y c** **l i** **c i** **t y** **b A** **g e** **& W** **e i** **g h** **t a** **t V** **O F** **e r** **t i** **l i** **t y** **/ F** **e c** **u n** **d i** **t y** **V i** **t e** **l l** **o g** **e n** **i n** **F i** **s h** **G o** **n a** **d a** **l W** **e i** **g h** **t (** **G S** **I )** **O v** **e r** **t T** **o x** **i c** **i t** **y O** **b s** **e r** **v e** **d c** 

**O SRI**  Subchronic toxicity (Rat) Chronic toxicity (Rat) No effect No effect No effect No effect Chronic toxicity (Mouse) No effect Two

- generation reproduction (Rat) No effect No effect No effect No effect No

effect
a Key to responses: Positive (P), Negative (N) or Equivocal (E) observation; arrows ( ↓ or ↑) indicate the direction of the res ponse; A shaded cell i n d i ca t e s t h a t p a r a m e t e r is not routinely eva l u a t e d o r is n o t ap p li ca b l e in the assay

. b Factors for estrous cy clicity include: age of first estrous, length of estrous, and percent of animals cycling regularly. c An “X” in this column indicates that the effect(s) observed in the assay occurred in the presence of overt toxicity. d Effects at the high dose (75 mg /kg/day) occurred in the presence of overt toxicity. Effects at the low dose (10 mg/kg/day) w ere in t he absence of overt toxicity. e A 50% decrease in survival was observed at this treatment level.US EPA

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Page **122** of **211 Effects on Androgen Pathway**  **: Table**  **41** below summarizes the results of the available EDSP Tier 1 screening assay

results and OSRI results relevant for determining the potential for Chemical S to interact with the androgen pathway . The various targets of the androgen pathway across the rele vant Tier 1 assay s are delineated so as to facilitate determination of potential for androgenic, anti

- androgenic, or HPG axis effects. Chemical S was classified as a non

- binder in the in vitro Androgen Receptor Binding assay (rat prostate cy

tosol), negative in the in vitro Steroidogenesis assay (human cell line H295R) and equivocal in the in vitro Aromatase assay (human recombinant microsomes). Thus, the in vitro assay s do not provide evidence of a specific androgen mode of action. There is evidence that Chemical S has the potential to interact with the androgen pathway as shown in the in vivo Hershberger and the Male Pubertal assay s. In the anti

- androgenic phase of the Hershberger assay , si gnificant decreases in two (ventral prostate and glans penis) of the five weights examined were observed in male rats treated with 10 mg/kg/day in the absence of overt toxicity . In addition, doses of 40 and 75 mg/kg/day Chemical S induced decreases in sem inal vesicles, ventral prostate, LABC, Cowper’s gland and glans penis weights in the presence of overt toxicity that was manifested as salivation (4 out of 6 rats at 40 mg/kg bw/day and 6 out of 6 rat at 75 mg/kg bw/day ) and decreased terminal body weights and body

weight gains at 75 mg/kg bw/day . In the androgenic phase of the assay , while no treatment

- related effects were seen in androgen sensitive tissues, clinical signs of toxicity (salivation) were seen in 3 of 6 rats. In the Male Pubertal assay , res ponses relevant to interaction with the androgen pathway consistent with antagonism included delay ed preputial separation, decreases in the weights of seminal vesicles, ventral prostate, dorsolateral prostate, LABC, epididy mides, adrenals, and testes; and decreased testosterone levels, but these responses were seen only at a dose (75 mg/kg bw/day ) that was associated with overt toxicity (salivation in all treated rats; significant decreases in body weights and food consumption). As discussed in the E pathw ay discussion above, given, the decreases observed in the endpoints for the male pubertal are consistent with Chemical S’s mode of action ( i.e ., depletion of ATP and caloric intake). In the FSTRA assay , potential endocrine effects ( e.g ., alterations in fer tility , males GSI , and male tubercles) were observed only at levels with overt toxicity ( increased mortality ). However, as discussed above, there is uncertaint y in whether these responses are due to a potential endocrine

- interaction or from a response in d ecreased energy output. In creased testes weights (absolute and relative) were noted in the Part 158 chronic toxicity study in the rat; however, there were no accompan yi ng histological effects. In the Male Pubertal assay , decreased rather than increased testes weights were observed, but the decreased testes weights occurred at a dose level associated with overt toxicity

. Again, effects on reproduction were reported in the bird and invertebrate studies, but the changes reported lack endocrine

- specificity

.US EPA

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– July , 2013

Page **123** of **211** Based upon WoE evaluation of EDSP Tier 1 screening assay and OSRI results, in the absence of overt toxicity , Chemical S demonstrated a potential to interact with the androgen pathway (androgen antagonism) in only

the Hershberger assay however, this impact is not supported by information provided through the other relevant assay s .US EPA

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– July , 2013

Page **124** of **211 Table 4**  **1 . Androgenic/Anti** 
**- Androgenic Pathway**  **for Chemical S Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti** 
**- Androgenic Pathway**  **for Chemical S**  **a**

**Study Type**  **/Literature Citation** **A R** **B i** **n d** **i n** **g S** **t e** **r o** **i d** **o g** **e n** **e s** **i s** **T e** **s t** **o s** **t e** **r o** **n e** **T e** **s t** **e s** **W e** **i g** **h t** **G o** **n a** **d S** **t a** **g i** **n g** **a n** **d H** **i s** **t o** **p a** **t h** **o l** **o g** **y E** **p i** **d i** **d y** **m i** **d e** **s W** **e i** **g h** **t E** **p i** **d i** **d y** **m i** **d e** **s H** **i s** **t o** **p a** **t h** **o l** **o g** **y P** **i t** **u i** **t a** **r y** **W e** **i g** **h t** **A c** **c e** **s s** **o r** **y S** **e x** **O r** **g a** **n W** **e i** **g h** **t s** **/ 2** **° S** **e x** **C h** **a r** **a c** **t e** **r i** **s t** **i c** **s A** **g e** **a n** **d W** **e i** **g h** **t a** **t P** **P S** **V i** **t e** **l l** **o g** **e n** **i n** **F i** **s h** **G o** **n a** **d a** **l W** **e i** **g h** **t (** **G S** **I )** **O v** **e r** **t T** **o x** **i c** **i t** **y O** **b s** **e r** **v e** **d b** 

**EDSP Tier 1** AR Binding Non

binder Steroidogenesis Negative Hershberger ↓16

- 40% X c
Male Pubertal Rat ↓56% ↓17% No effect ↓24% No

effect No effect ↓25

- 55% ↓11% BW;

↑ 6 d (PPS) X d
FSTRA ↓(33%) ♂ H e ↑(39%) ♂ H e XUS EPA

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– July , 2013

Page **125** of **211 Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti** 
**- Androgenic Pathway**  **for Chemical S**  **a**

**Study Type**  **/Literature Citation** **A R** **B i** **n d** **i n** **g S** **t e** **r o** **i d** **o g** **e n** **e s** **i s** **T e** **s t** **o s** **t e** **r o** **n e** **T e** **s t** **e s** **W e** **i g** **h t** **G o** **n a** **d S** **t a** **g i** **n g** **a n** **d H** **i s** **t o** **p a** **t h** **o l** **o g** **y E** **p i** **d i** **d y** **m i** **d e** **s W** **e i** **g h** **t E** **p i** **d i** **d y** **m i** **d e** **s H** **i s** **t o** **p a** **t h** **o l** **o g** **y P** **i t** **u i** **t a** **r y** **W e** **i g** **h t** **A c** **c e** **s s** **o r** **y S** **e x** **O r** **g a** **n W** **e i** **g h** **t s** **/ 2** **° S** **e x** **C h** **a r** **a c** **t e** **r i** **s t** **i c** **s A** **g e** **a n** **d W** **e i** **g h** **t a** **t P** **P S** **V i** **t e** **l l** **o g** **e n** **i n** **F i** **s h** **G o** **n a** **d a** **l W** **e i** **g h** **t (** **G S** **I )** **O v** **e r** **t T** **o x** **i c** **i t** **y O** **b s** **e r** **v e** **d b** 

**OSRI** Subchronic toxicity (Rat) No effect No effect Chronic toxicity (Rat) ↑27% No effect No effect Chronic toxicity (Mouse) No effect No effect Chronic toxicity (Dog) No effect Two

- generation reproduction (Rat) No effect No effect No

effect No effect No effect
a Key to responses: Positive (P), Negative (N) or Equivocal (E) observation; arrows ( ↓ or ↑) indicate the direction of the res ponse; A shaded cell i n d i ca t e s t h a t p a r a m e t e r is not routinely eva l u a t e d o r is n o t ap p li ca b l e in the assay

. b An“X” in this column indicates that the effect(s) observed in the assay occurred in the presence of overt toxicity. c Effects at the mid

– and high dose (40 and 75 mg/kg/day) occurred in the presence of overt toxicity. Effects (ventral prostate and glans penis we ights) at the low dose (10 mg/kg/day) were in the absence of overt toxicity in the anti

- androgen phase of the assay. d Effects at the high dose (75 mg/kg/day) occurred in the presence of overt toxicity. There w ere no effects at the low dose (1 0 mg/kg/day). e A 50% decrease in survival was observed at this treatment level.US EPA

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– July , 2013

Page **126** of **211 ii.**  **Effects on Hyp othalamic** 
**- Pituitary** 
**- Thyroidal (HPT) Axis**  In general, regulation of the HPT axis is comparable to the HPG axis, except that the feedback relationship involves thy

roid hormones ( e.g ., T 4 and T 3 ). Thy roid hormones feedback to the hy pothalamus or pituitary

to regulate thy

rotropin

- releasing hormone and TSH, respectivel y, which, in turn, regulate hormone production by the thy roid gland. The current Tier 1 screening battery does not have a specific in vitro assay to detect chemicals with the potential to affect hy pothalamic or pituitary regulation of th yroid hormone production, but it does include three in vivo assays that have the potential to detect these effects on the HPT axis ( i.e., the pubertal s and the AMA ). **Thyroid Hormones :**  **Table 42**  below summarizes the results of the available EDSP Tier 1 screening assay and OSRI results for determining the potential for Chemical S to interact with thy roid regulation. The various targets of the thy roid pathway

across the relevant Tier 1 assay s are d elineated so as to facilitate determination of potential for th yroid or HPT axis effects.

**Table 4 2**  **. Thyroid Hormone Pathway for Chemical S**  **Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Chemical S**  **a**

**Study Type /Literature Citation** **H o** **r m** **o n** **e s** **( T** **4 a** **n d** **T S** **H )** **P i** **t u** **i t** **a r** **y W** **e i** **g h** **t T** **h y** **r o** **i d** **W e** **i g** **h t** **T h** **y r** **o i** **d H** **i s** **t o** **p a** **t h** **o l** **o g** **y F** **r o** **g D** **e v** **e l** **o p** **m e** **n t** **S t** **a g** **e H** **i n** **d L** **i m** **b L** **e n** **g t** **h S** **n o** **u t** **t o** **V e** **n t** **L e** **n g** **t h** **O v** **e r** **t T** **o x** **i c** **i t** **y O** **b s** **e r** **v e** **d b** 

**EDSP Tier 1** Male Pubertal (Rat) No effect No effect No effect No effect X Female Pubertal (Rat) No effect ↓ 35% ↓ 14% No effect X AMA (Frog) c **OSRI** Two

- generation reproduction (Rat) No effect Subchronic toxicity

(Rat) Chronic toxicity

(Rat) No effect No effect No effect Chronic toxicity

(Mouse) No effect No effect No effect Chronic toxicity

(Dog No effect a Key to responses: Positive (P), Negative (N) or Equivocal (E) observation; arrows ( ↓ or ↑) indicate the direction of the response; -- indicates that parameter was not evaluated or is not applicable. b An “X” in this column indicates that the effect(s) observed in the assay occurred in the presence of overt toxicity. c Exposure may not have been inadequate to elicit a response.US EPA

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– July , 2013

Page **127** of **211** There was no evidence that Chemical S has a potential to interact with the thy roid hormonal pathway

. The decreased thy

roid and pituitary

weights observed in the Female Pubertal assay at 75 mg/kg bw/day occurred in the presence of overt toxicity (salivati on and decreases in body weights and food consumption). There were no effects on the th yro id in the standard combined chronic toxicity /carcinogenicit y s tudies in the rat and mouse. Adequate exposure was not achieved in the Amphibian Metamorphosis assay (AMA), and therefore was not successfull y tested in this assay. Testing in the AMA assay is limited by the ph ys ico

- chemical characteristics of the test substance. With respect to Chemical S, it belongs to a class of chemicals characterized b y relatively low water solubility , high octanol

- water partition coefficients (K ow ) and high organic carbon partition coefficients (K oc ). While these compounds are relatively stable to both abiotic (hy droly sis and photol ys is) and biotic (aerobic and anaerobic) degradat ion/metabolism, their low solubility and propensity

to partition to organic matter render them difficult to test in aquatic sy stems. Based upon WoE evaluation of EDSP Tier 1 screening assay and OSRI results, Chemical S did not demonstrate a potential to in teract with the th yroid hormone pathway.

**F. Conclusions for Chemical S**  All eleven assay s comprising the EDSP Tier 1 battery were available for Chemical S. Chemical S was evaluated for several h yp othesized endocrine modes of action for each specific E, A, a nd T pathway including agonism and antagonism at the estrogen and androgen receptor, altered steroidogenesis, HPG and HPT perturbations. In addition to the available Tier 1 data, other relevant studies of sufficient quality were considered in this assessm ent. These studies included standard Part 158 toxicity data relevant to evaluating effects in endocrine tissues and functions. In determining whether Chemical S interacts with E, A, or T hormonal pathway s, the number and ty pe of effects induced, the magn itude of responses, and the pattern of responses observed across studies, taxa, and sexes were considered. Additionally , the conditions under which effects occur were considered, in particular, the dose(s) at which responses happen and whether overt toxi city was present. This is particularly

important in the case of Chemical S, given that it is a severe irritant to the gastrointestinal tract (see section 2.3.1.6. Discussion of the Toxicity Profile in the Context of Tier I Assay s). Thus, studies at doses levels at which potential irritation would occur were considered to evaluate sources of potential overt toxicity

. Additionally , as the chemical’s mode of action eventually results in a depletion of ATP, consideration was given in how this may have impact ed the responses observed in the studies. Integrating the in vitro and in vivo Tier 1 results, as well as consideration of relevant data bey ond Tier 1 information ( e.g ., Part 158 standard toxicology studies) revealed evidence of a potential interaction w ith the E or A pathway s. In the absence of overt toxicity , there was evidence of potential interaction with the androgen signaling pathway

. The compl e mentary responses in the Hershberger assay were limited to significant decreases in only two androgen

- de pendent tissues. Although androgen

- related responsesUS EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **128** of **211** were seen in the male pubertal assay and FSTRA, they

occurred in the presence of overt toxicity , and the directionality

of the reported effects was consistent with Chemical S’s mode of action

. Thus, th ere were no redundant responses across the in vivo assay s to provide confirmatory evidence. Likewise, Chemical S demonstrated a potential to interact with the estrogen signaling pathway in that only

one response (delay ed vaginal opening in the female pubertal assay ) was significant. Although there were responses in FSTRA that could be indicativ e of an interaction with the E signaling pathway , these responses were only observed in the presence of overt toxicity , which again could be due to the lack of energy reserves for input into reproductive status . The negative complementary and redundant re sponses in the rat pubertal assay

and AMA indicates that Chemical S does not interact with the thy roid hormonal pathway

.


### 8. **Case Study**

**— Chemical J** 

**A. Introduction**  C hemical J is a triazole fungicide inhibiting sterol biosynthesis which in turn disrupts membra ne function. Chemical J also acts as a hepatic microsomal enz yme inducer. In the case of Chemical J, issues included: Tier 1 responses that may be reflective of potential interactions with perturbation of more than one endocrine modalit y (i.e., EAT) and different effects across taxa which may have implications for Tier II testing. The available information considered to determine the potential interaction of Chemical J with the E, A and/or T pathway s include all 11 EDSP Tier 1 studies and other scientifi cally relevant information (OSRI ) such as 40CFR Part 158 guideline toxicity studies. The data considered are summarized in Section s B, C and D of this chapter. A n anal ys is of the data submitted to the agency , using the WoE approach outlined by the a genc y (USEPA, 2011 b ), is presented in Section E

.
**B. Data Available for Chemical J**  Chemical J is soluble in water and does not volatize appreciabl y. It is characterized as being moderately persistent as it is resistant (stable) to abiotic routes of degradation ( i.e ., hy droly sis and photoly sis) and to anaerobic biotic metabolism. The compound can slowly undergo aerobic metabolism with half

- lives >70 day s. Based on a log octanol

- water partition coefficient (log K ow ) of approximately 3 and a relatively

low propensity

to sorb to organic matter, based on its organic carbon partition coefficient (K oc ~520 mL/g oc ), Chemical J residues are not expected to bioaccumulate.

**C. Tier 1 Screening Assays**  **for Chemical J** The Tier 1 studies submitted in response the test order for Chemical J are shown below in **Table 43** and findings are summarized below

.US EPA

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– July , 2013

Page **129** of **211 Table**  **43. Summary of**  **Tier 1 Screening Assays for Chemical J Tier 1 Assays Studies**  **Test Guideline Satisfied**  **by OSRI (Y/N)**  **Study Findings** ER Binding Assay (Rat uterine cyt osol) OSCPP 890.1250 No Negative 1 ERα Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300; OECD 455 No Negative AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 No Positive 2 Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550; OECD 456 No Positive Aromatase Assay (human recombinant microsomes) OSCPP 890.1200 No Positive 3 Uterotrophic Assay (Rat) OSCPP 890.1600; OECD 440 No Negative Hershberger Assay (Rat) OSCPP 890.1400; OECD 441 No Negative for Androgenic and Anti

- androgenic Pubertal Female Assay (Rat) OSCPP 890.1450 No Negative for E and T pathways Pubertal Male Assay (Rat) OSCPP 890.1500 No Positive for A pathway Negative for T pathway Fish Short

- term Reproduction Assay OSCPP 890.1350; OECD 229 No Positive for E and A pathway Am phibian Metamorphosis Assay (Frog) OSCPP 890.1100; OECD 231 No Negative **1** Classified as “Not Interactive” according to the Test Guideline OSCPP 890.1250 2 Classified as “Binder” according to the Test Guideline OSCPP 890.1150 3 Classified as “Inhibitor” according to the Test Guideline OSCPP 890.1200US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **130** of **211 i.**  **ER Binding Assay (Rat uterine cytosol)** In the ER binding assay , uterine cy tosol from Sprague Dawley rats was used as the source of ER to conduct the binding assays . A satur ation binding experiment was conducted prior to the competitive binding experiment to demonstrate that the ER in the isolated uterine cy tosol was present in an adequate concentration and functioning with the appropriate affinity for the radiolabeled (tritium; 3 H) ligand. The c ompetitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]

- 17β

- estradiol (1 nM ) in the presence of increasing concentra tions of Chemical J (logarithmic increase from 10 − 10 to 10 − 3 M). Ethanol was used as the vehicle at a final concentration of <3% (v/v). The assay included 19

- norethindrone as a weak positive control, octy ltriethoxy silane as a negative control, and 17

- β

- e stradiol as the natural ligand reference material. In the saturation binding experiment, the maximum binding capacit y (B max ) was 59.28 fmol/100 μg protein and the dissociation constant (K d ) was 0.1032 nM. The performance criteria were met for the saturati on binding assay

. In the competitive binding experiment, Chemical J did not competitively bind to the ER at concentrations up to 10 − 3 M. Across all three runs, specific binding averaged 94.5

– 102.9% at Chemical J concentration of 10 − 10 to 10 − 3 M. The mean specific binding affinity ( IC 50 ) and relative binding affinity

(RBA) were not calculated for Chemical J because it did not achieve ≥50% displacement of [ 3 H]

- 17β

- estradiol

. The performance criteria were met for the competitive binding experiment and the reference compounds generall y performed as expected. The negative control, octy ltriethoxy silane, had no effect on binding, and there were concentration

- dependent decreases in binding for the natural ligand and the weak positive control. Based on the results of three runs , Chemical J is classified as Not Interactive in the ER binding assay .

**ii. ER Transcriptional Activation Assays**  In the ER transcriptional activation assay , hERα

- HeLa

- 9903 cells cultured in vitro were exposed to Chemical J at logarithmically increasing concentrations from 10 − 10 to 10 − 4 M in dimethy lsulfoxide (DMSO; 0.1%) for 24 hours in three independent runs. The experiments were performed using 96

- well plates and each Chemical J concentration w as tested in triplicate (3 wells/plate) in each run. Cells were exposed to the test agent for approximately 24 hours to induce reporter (luciferase) gene products. Luciferase expression in response to activation of the estrogen receptor b y Chemical J was measured upon addition of a luciferase substrate and detection with a luminometer. Chemical J was tested up to the limit of solubility , 10 − 4 M. The maximum level of response (RPC m ax ) was 6.4% for the first run, 2.8% for the second run and 7.3% for the th ird run, with the associated maximum level of response (PC m ax ) of 10 − 5 M for all three runs.US EPA

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– July , 2013

Page **131** of **211** In runs 1 and 3, all performance criteria were met for the reference chemicals 17 β

- estradiol, 17α

- estradiol, and 17 α

- methy ltestosterone. The Hill Slope values f or 17 β

- estradiol (0.4) and 17α

- estradiol (0.6) in run #2 fell outside the expected ranges (0.7 to 1.5 for 17 β

- estradiol; 0.9 to 2.0 for 17α

- estradiol). The Log PC 50 and Log PC 10 values for 17 α

- methy ltestosterone were lower than the expected values for all three runs. The Log PC 50 values for each run were

- 7.6,

- 6.6 and

- 6.1 (expected range - 6.0 to

- 5.1) and the L ogPC 10 values for the runs were

- 8.8,

- 8.9 and

- 8.8 (expected range

- 8.0 to

- 6.2). These minor deviations did not negativel y affect the inte rpretation of the study

. Chemical J was negative for ER transcriptional activation in this test sy stem.

**iii. AR Binding Assay (Rat prostate cytosol)**  In the AR binding assay , ventral prostate cy tosol homogenate from Sprague Dawley rats was used as the source of AR to conduct the binding assay s. A saturation binding experiment was conducted prior to the competitive binding experiment to demonstrate that the A R in the cy tosol preparation was present in an adequate concentration and functioning with the appropriate affinity for the radiolabeled ( 3 H) ligand. The competitive binding experiment, was conducted to measure the binding of a single concentration of the androgen ligand [ 3 H]

- R1881 (1 nM) in the presence of increasing concentrations of Chemical J (logarithmic increase from 10 − 10 to 10 − 3 M). Ethanol was used as a vehicle at a final assay concentration of <3% (v/v). The assay

included dexamethasone as a we ak positive control, and unlabeled R1881 as the ligand reference standard. In the saturation binding experiment in which two runs were conducted, the maximum binding capacity (B max ) was 3.245 fmol/100 μg protein and the dissociation constant (K d ) was 0.4 641nM. Although the B max fell slightly

below the expected range of 10 to 150 fmol/100 μg protein and the K d was also below the expected range of 0.685 to 1.57 nM, confidence in these results is high because of the goodness

- of

fit (r 2 = 0.9871

– 0.9937) an d the reproducibility between the runs. With the exception of the high concentration of 10 nM in one assay with a 24.6% nonspecific binding, non

- specific binding as a percent of total binding was <20% across the entire concentration range tested for the s aturation binding assay

. All other performance criteria and the competitive binding assay s indicated acceptable performance of the assay

. In the competitive binding experiment, Chemical J reduced the mean specific binding of the radiolabeled ligand at th e highest two concentrations in a concentration

- dependent manner; however, binding curve decreased from approximately 84% to approximately

14% over a single log unit of concentration (from 10 - 4 to 10 - 3 M) and had steep Hill slopes in runs 2 and 3. The ini tial analy sis suggested that the data for runs 2 and 3 did not fit the four

- parameter, nonlinear regression model for anal ys is of one

- site binding. However, these results may be an artifact of the data when using an undefined bottom plateau for the curve fitting. When the data were re

- analy zed by constraining the bottom of the response curves to zero, the results of all three independent runs are similar with acceptable r 2 values and standard errors.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **132** of **211** The mean estimated log IC 50 was - 3.5 M for the analy s is using a constrained bottom plateau. The range of RBA for values for Chemical J across the three runs was 0.00034% to 0.00037% indicating a weak interaction with the AR. The mean Log IC 50 values for the strong positive control R1881 and the weak positi ve control (dexamethasone) were

- 9.0 and

- 4.4 M, respectivel y, and the mean RBA for the weak positive control was 0.0028%. All performance criteria for the competitive binding assays indicated acceptable performance of the assay

. T he results from the 3 c ompetitive binding experiments, with the undefined bottom plateau, are summarized in Table **44** and shown graphically in **Figure 1 4**  **.** The results when constraining the plateau are presented in **Table 45** and **Figure 1**

. Based on the results of three runs , Chemical J is classified as a “B ind er”, albeit with a weak affinit y, in the AR binding assay . **Table 44: Competitive Binding Assay of Chemical J with AR from Rat Prostate Cytosol (using** 

**undefined bottom plateau) Parameter**  **Run 1 Run 2**  **Run 3 Mean**  r 2 (unweighted) R1881 0.9999 1.0000 0.9999 NA

Positive control 0.9999 0.9997 0.9995 NA Chemical J 0.9922 0.9969 0.9988 NA Lo g IC 50 (M) R1881

- 8.986

- 8.958

- 8.994

- 8.978

Positive control

- 4.440

- 4.382

- 4.455

- 4.426 Chemical J

- 3.419 ~ - 3.830 c ~ - 3.855 c ~ - 3.848 IC 50 (M) R1881 1.032 x 10 - 9 1.101 x 10 - 9 1.014 x 10 - 9 1.052 x 10 - 9

Positive control 3.630 x 10 - 5 4.154 x 10 - 5 3.504 x 10 - 5 3.750 x 10 - 5 Chemical J 3.810 x 10 - 4 ~1.480 x 10 - 4b ~1.395 x 10 - 4(b) ~1.420 x 10 - 4 Lo g RBA (a) Positive control

- 2.6

- 2.6

- 2.5

- 2.6

Chemical J

- 3.6 NA NA NA RBA (%) Positive control 0.0028 0.0027 0.0029 0.0028

Chemical J 0.00027 NA NA NA
a Calculated by the revie wer. b Approximate estimates as 95% CI estimates were very wide NA = Not applicable; r 2 = Goodness of fit; RBA (%) = relative binding affinityUS EPA

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– July , 2013

Page **133** of **211 Figure 1**  **4 . Percentage R1881 Bound to the Androgen Receptor in the Presence of Radioinert** 

**R1881, Dexamethasone, and Chemical J (Assays 1**

**– 3), Using an Undefined Bottom**  **Plateau. Assay #1** 

**Assay #2** 

**Assay #3**US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **134** of **211 Table 45: Competitive Binding Assay of Chemical J with AR from Rat Prostate Cytosol (using** 

**confined bottom plateau)**

**Parameter Run 1**  **Run 2 Run 3**  **Mean**

r 2 (unweighted) Chemical J 0.9819 0.9902 0.9939 NA Lo g IC 50 (M) Chemical J

- 3.552

- 3.486

- 3.493

- 3.510 IC 50 (M) Chemical J 2.805 x 10 - 4 3.266 x 10 - 4 3.214 x 10 - 4 3.112 x 10 - 4 RBA (%) Chemical J 0.00037 0.00034 0.00033 0.00035

a Calculated by the revie wer. NA = Not applicable; r 2 = Goodness of fit; RBA (%) = relative binding affinity **Figure 1 5**  **. Percentage R1881 Bound to the Androgen Receptor in the Presence of Radioinert**

**R1881, Dexamethasone, and Chemical J (Assays 1**

**– 3), Using a Constrained Bottom**  **Plateau, for Competitive Runs 1, 2, and 3.** US EPA

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– July , 2013

Page **135** of **211 iii.**  **Steroidogenesis Assay (Human cell line H295R)** In the steroidogenesis assay , H295R cells cultured in vitro in 24

- well plates were incubated with Chemical J at log concentrations from 10 − 10 to 10 − 4 M for 48 hours in triplicate in three independent experiments. The test chemical’s vehicle was DMSO, at a final concentration of 0.1% (v/v). Testosterone and estradiol levels were measured using liquid chromatography coupled with atmospheric pressure phot oionization tandem mass spectroscopy ( LC

- APPI

- MS/MS). A Quality Control (QC) plate was run concurrently

with each independent run of a test chemical plate to demonstrate that the assay responded properl y to positive control agents at two concentration lev els. The positive controls included a known inhibitor (prochloraz) and a known inducer (forskolin) of estradiol and testosterone production. Chemical J was not cy

totoxic to the H295R cells up to the test limit concentration. Viability in each run of the test plate ranged from 98

– 129% relative to solvent controls. Viability in each run of the QC plate was 84

– 127% relative to solvent controls, except for the methanol

- treated wells which were 27

– 32%. Testosterone levels were decreased (p ≤0.05) to 0.3

– 0.4

- fo ld of the solvent control values in all 3 runs at 100 μM for Chemical J. In Run 2, there were significant increases in testosterone production (1.3

- to 1.5

- fold change) at concentrations from 0.01 to 10 μM; however, the induction was not reproducible in t he other two runs. Decreased (0.8

- fold the solvent control; p ≤0.05) estradiol levels were observed at 0.1 μM for Chemical J in runs 2 and 3, and significant decreases were observed in all 3 runs at concentrations ≥1 μM (0.4

– 0.7

- fold; p ≤0.05). Decreased (p ≤0.05) estradiol (0.8

- fold the solvent control) was also noted at the lowest level tested (0.0001 μM) in Run 2. The effects of Chemical J on testosterone and estradiol production are summarized in **Table** and shown in **Figure 1** 6 and **Figure 1**  7

. Guideline acceptability

recommendations and requirements were generall y met, including lack of cy totoxicity , adequate production of testosterone and estradiol, acceptable reproducibility ( i.e ., low percent coefficients of variability (%CV), and appropriate induction and inhibition with positive controls. The exceptions included: (i) the testosterone level in the solvent control (SC) of run 2 was 470 pg/mL (< the 500 pg/mL guideline limit); (ii) the estrad iol level in the SC of run 1 was 35.5 pg/mL (< the 40 pg/mL guideline limit); and (iii) the inhibition of estradiol in run 1 with 1 μM prochloraz was 0.6

- fold (> the 0.5

- fold guideline limit). However, these exceptions were slight and did not impact the in terpretation of the results. Based on the hormone responses in three runs, Chemical J was positive in the steroidogenesis assay .US EPA

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Page **136** of **211 Table 46:**  **Mean (±SD) Hormone Concentrations following Treatment with Chemical J for 48**

**Hours .**  **a Nominal**  **Concentration (μM)**  **Run 1 Run 2**  **Run 3 Run 1**  **Run 2 Run 3**  **Mean ± SD**  **Statistical Significance**  **Testosterone (pg/mL) Fold Difference**  DMSO 616.3 461.0 631.3

— — — — — — 0.0001 512.3 497.3 540.0 0.8 1.1 0.9 0.9 0.1

— 0.001 547.3 592.3 576.7 0.9 1.3 0.9 1.0 0.2 Run #2 0.01 564.3 621.0 572.7 0.9 1.3 0.9 1.1 0.3 Run #2 0.1 613.0 640.0 603.3 1.0 1.4 1.0 1.1 0.2 Run #2 1 608.7 674.0 690.3 1.0 1.5 1.1 1.2 0.2 Run #2 10 679.7 647.0 726.7 1.1 1.4 1.2 1.2 0.2

— 100 196.7 204.7 199.0 0.3 0.4 0.3 0.4 0.1 All 3 Runs

**Estradiol (pg/mL) Fold Difference**  DMSO 42.0 49.2 57.3

— — — — — — 0.0001 38.1 41.0 55.9 0.9 0.8 1.0 0.9 0.1 Run #2 0.001 39.0 46.3 55.2 0.9 0.9 1.0 0.9 0.0

— 0.01 37.6 45.5 53.8 0.9 0.9 0.9 0.9 0.0

— 0.1 34.8 39.5 45.2 0.8 0.8 0.8 0.8 0.0 Run #2 and #3 1 22.8 35.0 29.2 0.5 0.7 0.5 0.6 0.1 All 3 Runs 10 19.1 17.6 21.3 0.5 0.4 0.4 0.4 0.1 All 3 Runs 100 28.9 28.9 33.3 0.7 0.6 0.6 0.6 0.1 All 3 Runs

a T he low er limit of quantification (LLQ) was 25 pg/mL for testosterone and 10 pg/mL for estradiol.US EPA

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Page **137** of **211 Figure 1**  **6 . Change in Testosterone Production Relative to Chemical J Concentration.** US EPA

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– July , 2013

Page **138** of **211 Figure**  **1 7**  **. Change in Estradiol Production Relative to Chemical J Concentration.** US EPA

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– July , 2013

Page **139** of **211 iv.**  **Aromatase Assay (Human Recombinant Microsomes)** In the in vitro aromatase (CYP 19) assay , Chemical J was incubated with human recombinant aromatase and tritiated androstenedione ( 1

- β [ 3 H(N)]

- androst

- 4

- ene - 3,17

- dione ( [ 3 H]ASDN)) in ethanol for 15 minutes to assess the potential of Chemical J to inhibit aromatase activit y. Chemical J was tested at logarithmic concentrations from 10 – 10 M to 10 − 3 M in 3 independent runs. Aromatase activity

was determined by

measuring the am ount of tritiated water produced at the end of a 15

- minute incubation for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting. Three runs were conducted and each run included a full activity control, a backg round activity

control, a positive control series (10 – 10 to 10 – 5 M)) with a known inhibitor (4

- OH ASDN), and the test chemical series (10 – 10 to 10 – 3 M) with 3 repetitions per concentration. Aromatase activity in the full activity

controls ranged from 0.09 0 to 0.174 nmol ∙mg - protein − 1
∙ min − 1 for the three test runs, with a mean and standard deviation of 0.136±0.016 nmol ∙mg - protein − 1
∙ min − 1 . Activity in the background controls ranged from 0.13% to 0.60% of the overall average of the background

- adjusted full ac tivity controls. The responses of the full activit y and background controls were acceptable. For the positive control substance (4

- OH ASDN), aromatase activity averaged 0.145±0.017 nmol ∙mg protein − 1
∙ min − 1 at the lowest tested concentration (10 − 10 M) and 0 .0016±0.0001 nmol ∙mg - protein − 1
∙ min − 1 at the highest tested concentration (10 − 5 M). These results were within the recommended ranges for the top of the curve, bottom of the curve, Hill slope, Log IC 50 , and CV for replicates of each concentration, with the exception of some minor deviations for Run #1. The minor deviations in Run 1 included a % top of curve of 111% (criterion: 90

- 110%) and a LogIC50 of

- 6.9 (criterion

- 7.3 to 7.0). All performance criteria were met in Run #2 and #3, with the mean performa nce criteria values within the recommended ranges. For Chemical J, aromatase activity averaged 0.127±0.013 nmol ∙mg - protein − 1
∙ min − 1 at the lowest tested concentration (10 − 10 M) and 0.00017±0.00004 nmol ∙mg - protein − 1
∙ min − 1 at the highest tested concentration (10 − 3 M). The data for Chemical J were modeled for all runs; the best fit Hill slope and Lo g IC 50 values across the three independent runs did not differ significantly. The mean goodness

- of

- fit (r 2 ) value was 0.99, and the mean log IC 50 of the test mater ial was

– 6.6. The average dose

- response curve indicated that the aromatase activity of the test material at concentrations ranging from 10 − 10 M to 10 − 8 M was essentially

equivalent to the activity observed in the full activity controls. The 10 – 6 M dose l evel reduced the activity by approximately

75%, and the 10 – 4 and 10 – 3 M dose levels reduced the activity by >99%. The mean aromatase activity data for Chemical J (expressed as % full control activity ) for each concentration tested across all 3 runs are pr esented in **Table 47** , along with the overall standard deviation and % CV. Inhibition response curves for Chemical J from each run are shown in Figure 1 **8**

. Based on the results of three, Chemical J is classified as i nhibitor in the aromatase assay

.US EPA

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Page **140** of **211 Table 47: Effect of Chemical J on Aromatase Ac**  **tivity (as Percent of Control) From Independent**

**Runs a**  **Chemical Run 1**  **Run 2 Run 3**  **Mean SEM**  **%CV Log IC**  **50 (M)**  **Chemical J**

− 6.6

− 6.8

− 6.5

− 6.6 0.09

− 2.30 **4**
**- OH ASDN** 

− 6.9

− 7.1

− 7.1

− 7.0 0.07

− 1.64 **Hill Slope Chemical J** 

− 0.92

− 0.71

− 0.87

− 0.83 0.06

− 13.16 **4**
**- OH ASDN** 

− 0.92

− 1.0

− 0.96

− 0.96 0.02

− 4.17 a = mean, SEM, and % CV estimates were calculated by the reviewer. SEM = Standard Error of the Mean. **Figure 1 8**  **. Aromatase Inhibition Response Curves for Chemical J from Each Test Run** 

.US EPA

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– July , 2013

Page **141** of **211 vi. Uterotrophic**  **Assay (Rat)** In the in vivo uterotrophic assay conducted to screen for potential estrogenic activity , Chemical J was administered dail y in corn oil via oral gavage to groups of six immature female Sprague Dawley rats at dose levels of 0 (vehicle), 50, 150, or 475 mg/kg bw/day on post
- natal day s (PND) 19

- 21. A positive control group was treated with a daily dose of 17α

- ethy

ny l estradiol (EE) at 10 μg/kg bw/day by oral gavage. Bod y weights were determined dail y. All animals were examined for vaginal opening (VO), and then euthanized and necropsied on PND 22 approximately 24 hours after the final dosing to determine wet and blotted uterine weights. All animals survived until scheduled termination. No clinical signs of toxicity were observed, and no signifi cant increase in VO was noted in the Chemical J

- treated females. At 475 mg/kg bw/day , body weights were decreased (p<0.05) b y 15% on Day 3, resulting in a 59% decrease (p<0.05) in overall (Day s 1

- 4) bod y weight gains relative to the vehicle control. Uter ine weights at this dose were comparable to the vehicle controls. Absolute wet and blotted uterus weights for the positive control (EE) group were increased (p<0.05) b y 891% and 524%, respectively. The 59% decrease in overall body weight gains in the high

- dose group indicated that these animals were dosed at adequate levels. No statistically

significant increase in uterine weight was seen in this study . Hence, Chemical J was negative in the Uterotrophic assay

.

**vii. Hershberger Assay (Rat)**  In the in vivo Hershberger assay testing for androgenic activity , Chemical J was administered dail y i n corn oil via oral gavage to groups of seven 55

- day old castrated male Sprague Dawley rats/group at dose levels of 0 (vehicle), 25, 125, or 500 mg/kg bw/day for 10 cons ecutive day s. The study included an androgenic positive control group of seven castrated male rats dosed daily with testosterone propionate (TP) at 0.4 mg/kg bw/day via subcutaneous (s.c.) injection. To screen for possible anti

- androgenic activity , addi tional groups of seven 55

- day old castrated male Sprague Dawley

rats/group were dosed dail y via oral gavage with Chemical J in corn oil at 25, 125, or 500 mg/kg bw/day

for 10 consecutive day s in conjunction with a daily s.c. injection of TP at 0.4 mg/kg bw /day . The control group consisted of seven castrated male rats dosed daily with TP (0.4 mg/kg bw/day ) by

s.c. injection, and the positive control group consisted of seven castrated male rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg bw/day f or 10 day s in conjugation with a daily s.c. dose of TP (0.4 mg/kg bw/day). Bod y weights, bod y weight gains, and food consumption were measured. Animals were euthanized approximately

24 hours after the final dose administration. The five androgen

- dependen t tissues ( i.e .,US EPA

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– July , 2013

Page **142** of **21 1**  seminal vesicles, ventral prostate, levator ani

- bulbocavernosus (LABC), Cowper's glands or glans penis) as well as adrenals, kidneys, and liver tissues were weighed and macroscopically examined. No treatment

- related effects were observed i n either the agonist or antagonist assay s on mortality , clinical signs of toxicity , body weights, bod y weight gains, food consumption, adrenals and kidney s weights, or gross pathology . The increased liver weights and microscopic liver findings at 500 mg/kg bw/day indicated that the animals were dosed to a level that showed toxicological effects, i.e ., overt toxicity

. For the androgenic portion of the study , there were no statistically significant increases in the weights of accessory sex tissue at any

dos e of Chemical J. For the TP positive control group, there were significant (p<0.05) increases in the weights of the Cowper’s glands (688%), glans penis (77%), LABC (194%), ventral prostate (917%), and seminal vesicles with coagulating glands (1105%), indi cating that the test sy stem was sensitive to an androgenic response. For the anti

- androgenic portion of the study , there was a statistically significant decrease in one of the five target accessory

sex tissues. A significant 20% decrease (p<0.05) in LABC weight was observed at 500 mg/kg bw/day

of Chemical J + TP. No significant changes in organ weights were seen in the seminal vesicle, ventral prostate, Cowper’s gland or glans penis as an y dose. The decrease in LABC weight at 500 mg/kg bw/day is not cons idered a positive result for anti

- androgenic activity in this assay as only a single organ weight was decreased. For the TP+FT positive control group, there were treatment
- related decreases in the weights of the Cowper’s glands (62%), glans penis (26%), L ABC (54%), ventral prostate (75%), and seminal vesicles with coagulating glands (81%) relative to weights in the “TP only ” treated group. These data indicate that the test sy stem was sensitive to an anti

- androgenic response. Liver weights were increased b y Chemical J treatment in both the androgenic and anti

- androgenic portions of the study . For the androgenic portion of the study , absolute liver weights were increased by 20% and 53% at 125 and 500 mg/kg bw/day Chemical J, respectively . For the anti

- andr ogenic portion of the study , there was a 59% increase in absolute liver weight in animals given 500 mg/kg bw/day with TP. These weight increases correlated with histopathologic lesions in the liver, which included hepatocellular h yp ertrophy , vacuolization , and necrosis. The performance criteria for the assay were met as the %CVs for weights of accessory sex tissues in the control and treated groups were below the maximum permissible values specified in the guideline. No statistically

significant changes w ere seen in two or more of the androgen responsive tissues. Chemical J was negative for androgenicity and anti

- androgenicit y in the Hershberger assay

.US EPA

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– July , 2013

Page **143** of **211 viii.**  **Pubertal Female Assay (Rat)** In a Female Pubertal Assay , 16 Sprague Dawley [Crl:CD(SD)] rats/dose group w ere treated daily via oral gavage with Chemical J in corn oil at doses of 0, 50, 200, or 400 mg/kg bw/day from post

- natal day (PND) 22 to 42. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and weight at day

of attainment was rec orded. Following sacrifice on PND 42, total thy roxine (T 4 ) and thy roid stimulating hormone (TSH) levels were determined using chemiluminescent immunoassay and radioimmunoassay , respectively . At termination, weights were measured for liver, kidney s, thy ro id, adrenals, pituitary , and urogenital organs, and microscopic examinations were performed on the ovaries, uterus, thy

roid, and kidney s from the control and high

- dose groups and on livers from all dose groups. At 400 mg/kg bw/day , treatment
- related incre ases (p<0.05) were observed in alanine aminotransferase (ALT; ↑24%) and gamma glutam yl transferase (GGT; ↑130%). Dose

- dependent increases (p<0.05) in absolute, relative, and adjusted liver weights were observed at 200 ( ↑18

- 19%) and 400 ( ↑36

- 41%) mg/kg bw/d ay . Animals in these dose groups also display ed treatment

- related histopathological effects consisting of very

slight to slight hepatocy

te h yp ertrophy

with altered tinctorial properties (increased cy toplasmic eosinophilia) of centrilobular and midzonal he patocy tes, very slight multifocal chronic inflammation, and very

slight to slight increased number of mitotic figures of hepatocy tes. These findings showed a dose

- dependent increase in incidence and severity , and were indicative of hepatotoxicity

at 400 m g/kg bw/day . Based on these findings in liver and on evidence of hepatic necrosis at 500 mg/kg bw/day in a concurrentl y s ubmitted Hershberger study , the high dose in the current stud y appears to be an adequate high dose. There were no significant treatme nt
- related changes or alterations on mortality , clinical signs of toxicity , body weights, bod y weight gains, age of attainment of VO, bod y weight at VO, mean age at first estrus, mean cy cle length, percent cy cling, percent regular cy cling, organ weights, s erum T 4 and TSH levels, or gross pathology . Age and weight at VO were within the acceptable range of the performance criteria. Chemical J was negative in the pubertal female assay

. No treatment

- related effects were seen for estrogen or th yroid

- related end points when tested at doses up to and including 400 mg/kg/day

.
**ix. Pubertal Male Assay (Rat)**

In the male pubertal assay 16 Sprague

- Dawley

(Crl:CD[SD]) rats/dose group were treated daily via oral gavage with Chemical J in corn oil at doses of 0, 50, 200, or 400 mg/kg bw/day from post

- natal day (PND) 23 to 53. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and weight at day

of attainment was recorded. Following sacrifice on PND 53, total serum testosterone, thy roxine (T4) , and th yro id stimulating hormone (TSH) levels were analy zed using radioimmunoassay s (TSH) or chemiluminescent assay s (testosterone and T4). L iver, kidney , pituitary , adrenals, thy roid, and urogenital organ weights were recorded, and microscopic examinati ons of the thy roid, testes, epididy mides, kidney s, and liver were performed.US EPA

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Page **144** of **211** As shown in **Tabl e 48,**  for Chemical J , at doses up to 400 mg/kg bw/day , no treatment
- related effects were observed on mortality , clinical signs, body weight, bod y weight grain, wei ght at attainment of PPS, or serum T4 levels. However, age at PPS was slightly increased (p<0.05) in the high dose group (PND 44.0 vs. PND 45.7). As shown in **Table 49** , significant, dose

- dependent decreases were also observed in the weights of the accessory sex tissues at doses of 200 mg/kg bw/day and 400 mg/kg bw/day . At 200 mg/kg bw/day , weight decreases were noted in the seminal vesicle + coagulating gland ( ↓15

- 19%) and the ventral prostate ( ↓16%). At 400 mg/kg bw/day , weight decreases were noted in the seminal vesicle + coagulating gland ( ↓33

- 41%), ventral prostate ( ↓27

- 29%), dorsolateral prostate ( ↓25

- 27%), and LABC complex ( ↓16

- 18%). Concomitant with the weight decreases in these androgen sensitive tissues, there were significant, dose

- dependent decreases in serum testosterone levels of 49% at 200 mg/kg bw/day and 85% at 400 mg/kg bw/day

. As shown in **Table 50** , other significant changes in organ weights included a 15% increase in relative adrenal weight at 400 mg/kg bw/day , and 8

- 10% decreases in pi tuitary weight at all dose levels that were not dose

- dependent. Other significant changes in clinical chemistry parameters observed at 200 and 400 mg/kg bw/day

included: 21

- 32% increase in creatinine; 27% decreased in blood urea nitrogen; and a 17

- 21% de crease in potassium. At 400 mg/kg bw/day , there were also significant changes in alkaline phosphatase ( ↓26%), total protein (↑4%) and chloride (↓3%). No significant weight changes were noted in the thy roid, but increased th yroid follicular cell heights an d decreased colloid area were noted at 400 mg/kg bw/day . No changes were noted in serum T 4 levels ( **Table 51** ). Significant (p<0.05) increases were obs erved in liver weight (unadjusted and adjusted) and relative (to body ) liver weight, which were dose

- dependent. Liver weights increased by 12

- 13% at 200 mg/kg bw/day and 27

- 28% at 400 mg/kg bw/day , and relative liver weights increased by 5%, 21% and 37% at 50, 200 and 400 mg/kg bw/day , respectivel y. Concomitant with the increases in liver weight, there were minor histological changes at 200 and 400 mg/kg bw/day, which consisted of slight to very slight hy pertrophy with altered tinctorial properties in ce ntrilobular and midzonal hepatocy tes (0/16 control, 16/16 at 200 mg/kg bw/day , and 15/15 at 400 mg/kg bw/day ) and a very slight increase the numbers of mitotic figures in hepatocy tes (2/15 control, 9/15 at 400 mg/kg bw/day ). A significant increase (53%) w as also noted in gamma glutam yl transferase (GGT) at the high dose. It appears the h igh dose (400 mg/kg bw/day ) used in this assay is adequate based on the results from the assay that showed increased liver weights and microscopic liver findings

. No treat ment related changes were seen at the low dose (50 mg/kg bw/day ).US EPA

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– July , 2013

Page **145** of **211** At 200 mg/kg bw/day , delay s in PPS, decreases in testosterone levels, changes in two androgen sensitive organs (s eminal vesicle + coagulating gland and the ventral prostate) were reported. At 400 mg/kg bw/day , the observed changes were: delay ed PPS, decreases in testosterone, decreases in four androgen sensitive tissues ( seminal vesicle + coagulating gland, ventral prostate, dorsolateral prostate, and LABC complex). Changes in the thy roid h istopathology

were also observed at 400 mg/kg bw/day along with a non - significant increasing trend in TSH across the treatment groups. Chemical J was positive for the A (anti

- A) pathway and negative for the T pathway in the pubertal male assay .US EPA

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– July , 2013

Page **146** of **211 Table 48: Prepu**  **tial Separation (PPS) in Male Rats Treated with Chemical J in the Male Pubertal Assay a** 

**Parameter Evaluated Vehicle Control**  **Chemical J 50 mg/kg bw/day**  **Chemical J 200 mg/kg bw/day**  **Chemical J 400 mg/kg bw/day**  # of Males Mean SD/

SE CV (%) # of Males Mean SD/

SE CV (%) # of Males Mean SD/

SE CV (%) # of Males Mean SD/

SE CV (%) Age at PPS (PND) U 15 44.1 1.7 3.8 16 44.1 1.4 3.1 16 44.5 1.6 3.7 15 45.6 2.9 6.5

A 15 44.0 0.44 NA 16 44.1 0.42 NA 16 44.5 0.42 NA 15 45.7* ( ↑4) 0.44 NA Proportion unseparated (#/N) 0/15 0/16 0/16 0/15

a Percent differences from controls, calculated by the reviewers, are included in parentheses

. U = Unadjusted for body weight on PND 23; A = Adjusted for body weight on PND 23; SD = Standard Deviation; SE =Standard Error; used for means adjusted for body weight on PND 23 CV =Coefficient of Variation; NA=Not applicable * Significantly different from controls at p<0.05.US EPA

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Page **147** of **211 Table 49:**  **Accessory Sex Organ Weights at Necropsy in Male Rats Treated with Chemical J in the Male Pubertal Assay a** 
**Organ Vehicle Control**  **Chemical J 50 mg/kg bw/day**  **Chemical J 200**  **m g/kg bw/day**  **Chemical J 400**  **mg/kg bw/day** # of Males Mean SD/

SE CV (%) # of Males Mean SD/

SE CV (%) # of Males Mean SD/

SE CV (%) # of Males Mean SD/

SE CV (%) Seminal vesicle + coagulating gland, with fluid (mg) U 15 522.2 120.4 23.1 b 16 500.5 115.3 23 16 424.8* ( ↓19) 116.1 27.3 15 314.5* ( ↓40) 109.2 34.7 A 15 526.9 28.0 NA 16 500.5 27.0 NA 16 425.4* ( ↓19) 27.0 NA 15 309.2* ( ↓41) 28.0 NA Seminal vesicle + coagulating gland, without fluid (mg) U 15 280.2 52.3 18.7 16 275.7 48.4 17.6 16 240.8 47.0 19.5 15 188.2* ( ↓33) 54.3 28.8
A 15 282.3 12.1 NA 16 275.6 11.7 NA 16 241.1* ( ↓15) 11.7 NA 15 185.8* ( ↓34) 12.1 NA
Ventral prostate (mg) U 15 240.8 34.2 14.2 16 227.3 35.0 15.4 16 202.6* ( ↓16) 42.4 20.9 14 175.9* ( ↓27) 38.2 21.7
A 15 243.0 8.76 NA 16 227.6 8.46 NA 16 203.2* ( ↓16) 8.46 NA 14 172.5* ( ↓29) 9.09 NA

Dorsolateral prostate (mg) U 15 156.1 43.1 27.6 16 147.2 31.3 21.3 16 143.6 27.8 19.4 14 117.3* ( ↓25) 21.1 18.0
A 15 157.8 7.53 NA 16 147.4 7.27 NA 16 144.1 7.27 NA 14 114.6* ( ↓27) 7.81 NA

LABC (mg ) U 15 467.1 90.5 19.4 16 479.7 68.2 14.2 16 449.2 68.4 15.2 15 391.2* ( ↓16) 73.2 18.7 A 15 471.2 17.1 NA 16 479.6 16.5 NA 16 449.8 16.5 NA 15 386.6* ( ↓18) 17.1 NA Epididymi s, left (mg) U 15 216 23.6 10.9 16 219 35.8 16.3 16 206.8 20.5 9.9 15 202.3 27.5 13.6

A 15 217.3 6.47 NA 16 218.9 6.25 NA 16 207.0 6.25 NA 15 200.9 6.47 NA Epididymi s, right (mg) U 15 213.4 20.7 9.7 16 221.6 25.9 11.7 16 213.4 29.4 13.8 15 207.4 26.9 13.0

A 15 214.3 6.44 NA 16 221.5 6.23 NA 16 213.5 6.23 NA 15 206.4 6.44 NA Testis, left (mg) U 15 1412 109.3 7.7 16 1429 142.7 10 16 1417 115.4 8.1 15 1440 136.3 9.5

A 15 1415 32.5 NA 16 1429 31.5 NA 16 1417 31.5 NA 15 1437 32.6 NAUS EPA

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Page **148** of **211 Organ**  **Vehicle Control Chemical J**  **50 mg/kg bw/day Chemical J**  **200 m**  **g/kg bw/day Chemical J**  **400 mg/kg bw/day**  # of Males Mean SD/

SE CV (%) # of Males Mean SD/

SE CV (%) # of Males Mean SD/

SE CV (%) # of Males Mean SD/

SE CV (%) Testis, r ight (mg) U 15 1423 119.4 8.4 16 1448 138.1 9.5 16 1428 85.3 6.0 15 1451 128.3 8.8

A 15 1426 30.3 NA 16 1448 29.3 NA 16 1428 29.3 NA 15 1447 30.3 NA
a Percent differences from controls, calculated by the reviewers, are included in parentheses. b The %CV exceeds the maximum acceptable %CV. U =Unadjusted for body weight on PND 23; A= Adjusted for body weight on PND 23; R=Organ

- to
- body

w eight ratio (relative to body weight); SD=Standard Deviation; SE =Standard Error; used for means adjusted for body weight on PND 23; CV= Coefficient of Variation *Significantly different from controls at p<0.05.US EPA

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Page **149** of **211 Table 50:**  **Additional Orga n Weights at Necropsy in Male Rats Treated with Chemical J in the Male Pubertal Assay**  **a Organ**  **Vehicle Control Chemical J**  **50 mg/kg bw/day Chemical J**  **200 m**  **g/kg bw/day Chemical J**  **400 mg/kg bw/day**  # of Males Mean SD/

SE CV (%) # of Males Mean SD/

SE CV (%) # of Males Mean SD/

SE CV (%) # of Males Mean SD/

SE CV (%)
Liver (g) U 15 13.1 1.77 13.5 16 13.2 1.22 9.25 16 14.8* ( ↑13) 1.24 8.35 15 16.8* ( ↑28) 1.64 9.79 A 15 13.2 0.36 NA 16 13.2 0.343 NA 16 14.8* ( ↑12) 0.343 NA 15 16.7* ( ↑27) 0.355 NA R 15 4.46 0.22 4.97 16 4.67* ( ↑5) 0.21 4.52 16 5.41* ( ↑21) 0.23 4.33 15 6.09* ( ↑37) 0.23 3.83 Pituitary (mg) U 15 9.6 0.9 9.5 16 8.8* ( ↓8) 0.7 8.1 16 8.6* ( ↓10) 0.9 10.0 15 8.6* ( ↓10) 1.1 12.3 A 15 9.6 0.23 NA 16 8.8* ( ↓8) 0.22 NA 16 8.6* ( ↓10) 0.22 NA 15 8.6* ( ↓10) 0.23 NA R 15 3.3 0.3 9.0 16 3.1 0.2 7.4 16 3.2 0.3 10.1 15 3.2 0.3 10.9 Adrenals (mg) U 15 43.2 7.4 17 16 44 6.2 14.1 16 43.4 7.8 17.9 15 46.8 7.2 15.3

A 15 43.5 1.69 NA 16 44.0 1.64 NA 16 43.4 1.64 NA 15 46.4 1.7 NA R 15 14.8 2.2 14.6 16 15.6 1.8 11.2 16 15.9 2.5 16 15 17* ( ↑15) 2 11.7 Thy roid, fixed (mg) U 15 11.3 b 1.5 13.6 16 11.9 1.8 15.4 15 12.5 1.3 10.6 15 12.6 1.5 11.9

A 15 11.3 0.41 NA 16 11.9 0.39 NA 15 12.5 0.41 NA 15 12.6 0.41 NA

a Percent differences from controls, calculated by the reviewers, are included in parentheses. b The m ean weight was less than the acceptable weight range. c The %CV exceeds the maximum acceptable %CV. U = Unadjusted for body weight on PND 23; A= Adjusted for body weight on PND 23; R=Organ

- to
- body

w eight ratio (relative to body weight); SD=Standard Deviation; SE = Standard Error; used for means adjusted for body weight on PND 23; CV = Coefficient of Vari ation *Significantly different from controls at p<0.05.US EPA

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Page **150** of **211 Table 51:**  **Hormone Levels in Male Rats Treated with Chemical J in the Male Pubertal Assay a** 
**Parameter Evaluated**  **Vehicle Control Chemical J**  **50 mg/kg bw/day Chemical J**  **200 mg/kg bw/day Chemical J**  **400 mg/kg bw/day**  # of Males Mean SD CV (%) # of Males Mean SD CV (%) # of Males Mean SD CV (%) # of Males Mean SD CV (%) **Hormones** Serum T 4 , Total (μg/dL) 15 4.78 0.670 14.0 16 4.69 0.481 10.3 16 5.19 0.724 14.0 15 5.15 0.458 8.89
Serum TSH (ng/mL) 15 7.07 3.23 45.7 16 8.74 6.10 69.7 16 9.06 5.14 56.8 15 11.5 6.52 58.5
Serum testosterone (ng/mL) 15 3.62 2.36 65.1 (45.7) b 16 3.46 3.24 93.7 16 1.83* ( ↓49) 1.31 71.5 15 0.561*( ↓85) 0.468 83.6

a Percent differences from controls, calculated by the reviewers, are included in parentheses b When animal #5998 is excluded from data set (in parenthe ses) the %CV is less. SD = Standard Deviation; CV= Coefficient of Variation *Significantly different from controls at p<0.05.US EPA

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Page **151** of **211 x.**  **Fish Short**
**- term Reproduction Assay (FSTRA)**  The 21

- day fish short

- term reproduction assay of Chemical J with fathead minnows ( Pimephales promelas) was studied under flow

- through conditions. Adult fish, 16 spawning groups (2 males and 4 females in each group; ca . 5.75 months old) were exposed to Chemical J at nominal concentrations of 0 (control), 0.032 0.32 , and 3.2 mg a.i./L; time

- weighted average

- measured concentrations were <LOQ (<0.00510), 0.0338, 0.327, and 3.30 mg a.i./L . The test sy stem was maintained at 24.6 to 25.4 o C and a pH of 7.2 to 7.5. All performance criteria were met in this study

. Spawnin g frequency

in the negative control occurred at least every 4 day s and fecundit y ranged from 21 to 33 eggs/female/day /replicate. Fecundity

was 25.6, 20.1, 22.6 and 0.550 eggs/female/day

/replicate in the 0 (control), 0.0338 0.327, and 3.3 mg ai/L treatment levels, respectivel y. Fecundity at the 3.3 mg a.i./L level was 98% lower than the control. No significant effect was detected for fertilization success. Fertilization rate in the control was 99%, compared to 99.5, 99.1% and 100% in the 0.0338, 0.327 and 3 .3 mg a.i./L treatment groups. Female gonadosomatic index (GSI ) exhibited a significant (p<0.05) increase of 41 and 46% at the 0.327 and 3.3 mg a.i./L levels, respectivel y, while male GSI was significantl y (p<0.05) increased 79% at the 3.3 mg a.i./L level

. Female plasma vitellogenin (VTG) was significantly reduced (p<0.05) b y 88% relative to controls at the 3.3 mg a.i./L level, while no effect was detected for male plasma VTG. Additionally , there was a significant 200% increase (p<0.05) in female testoste rone levels at the 0.327 mg a.i./L level, relative to the control. While not statistically significant (p=0.067), a 85% reduction in female plasma 17 

- estradiol concentrations at the 3.3 mg ai/L level was observed which was considered biologicall y signifi cant. Histopathology on female gonads reported severe oocy te atresia (as reported by

study pathologist using procedures described in guideline) at the 3.3 mg a.i./L level relative to the control . Male fish gonads were reported as having a increased propor tions of mature sperm (gonadal stage 3) in the 3.3 mg ai/L treatment, relative to the control. There were no significant effects on mortality (p>0.05). Minimal effects on secondary sex characteristics were observed in the study with one male fish in the 3.3 mg a.i./L and in the control were reported to have very thin fatpads while one fish each in the low and high treatment were reported to exhibit loss of territorial aggressiveness. These observations did not appear to be treatment

- related as they were r eported in both the control and treatment groups. Additionall y, a caudal fin was reported as missing in one female fish at the mid

- treatment group. In the 3.3 mg a.i./L level, a significant reduction (9%) in female body weight was observed (using a statis tical trend test); however, the impact of this magnitude of reduction, in this assay, is not considered significant. The results are presented in **Table 52**  through **Table 54 .** US EPA

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Page **152** of **211** In summary , at the highest treatment group, i.e ., 3.3 mg a.i./L, there was a significant (p<0.05) decrease in fecundit y and female plasma VTG and estradiol, along with a significant increase in male and female GSI. Additionally , alter ations in male and female gonadal histopathology were reported at the highest treatment group. Furthermore, a significant increase in female GSI and plasma testosterone was reported at 0.32 mg a.i./L. While a significant (p<0.05) decrease in female bod y weight was observed at 3.3 mg a.i./L, given the magnitude of effect (9% decrease), the impact on the interpretation of the results of this assay is not considered to be significant. Given the reported effects and the complementarity among the responses ( Vtg, GSI, fecundit y, T and E), Chemical J is considered to be positive for potential interactions with the E and A pathway s. **Table 52:**  **Chemical J**
**- Reproductive and HPG Endpoints for Male**  **Pimephales promelas in** 

**the FSTRA**

Abbreviations:. E2 17β

- estradiol. GSI Gonado

- Somatic Index. NA Not applicable. T Testosterone. VTG Vitellogenin. 1 Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board

- certified pathologist. 2 Statistically significant [p<0.05]. LOQ=<0.00510 mg ai/L **Treatment (mg a.i./L)**  **[measured] Tubercle** 

**Score GSI**  **Gonadal Staging and**  **Histopathology 1**  **Plasma**

**VTG Plasma T**  **Plasma E2**
**Median %** 

**Diff erence**  **Effect?**

**(Yes/No) %** 

**Diff erence**  **% Diff**  **erence %**  **Diff erence**  Control (<LOQ) 18.3 0 NA 0 0 0 0.032 (0.0338) 20.5 16.50 No

- 1.40

- 40.97

- 31.08 0.32 (0.327) 19.3 27.30 No

- 28.01 40.97 10.45 3.2 (3.3) 15.5 78.99 2 Yes

- 83.06

- 31.02

- 18.82US EPA

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Page **153** of **211 Table 53.**  **Chemical J**
**- Reproductive and HPG Endpoints for Female**  **Pimephales promelas**  **in the FSTRA**

Abbreviations: Diff. Difference. E2 17β

- estradiol. Fert. Fertilization. GSI Gonado

- Somatic Index. NA Not applicable. T Testosterone. VTG Vitellogenin. 1 Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board

- certified pathologist. 2 Statistically significant at p< 0.05. LOQ=<0.00510 mg ai/L
**Table 54: Chemical J** 
**- Growth Endpoints in the Fish Short** 
**- Term Reproduction Assay** 

**(FSTRA)**
**Treatment (mg a.i./L)**  **[measured] Body Weight**  **Length Males**  **Females Males**  **Females % Diff**  **erence % Diff** 

**erence % Diff** 

**erence % Diff** 

**erence** Control (<LOQ) NA NA NA NA 0.032 (0.338) 21.53

- 2.09 6.52

- 1.30 0.32 (0.327) 7.49

- 1.61 0.20

- 2.60 3.2 (3.30) 4.79

- 8.86 1 4.49

- 2.78 Abbreviations: NA Not applicable. 1 Statistically significant using a trend test (Jonckheere

- Terpstra) [p<0.05]. LOQ=<0.00510 mg ai/L **Treatment (mg a.i../L)**  **[measured] Fecundity**  **Fertility Success**  **Tubercle**

**Score GSI**  **Gonadal**

**Staging and Histo**  **patho logy**  **1 Plasma** 

**VTG Plasma** 

**T Plasma**  **E2**
**% Diff e**  **. % Diff.**  **Median %**  **Diff. Effect?** 

**(Yes/No) %** 

**Diff. %** 

**Diff. %** 

**Diff.** Control ( <LOQ) NA NA 0 NA NA NA NA NA 0.032 (0.0338) - 21.7 0.45 0 22.8 No 9.16 74.0

- 33.7 0.32 (0.327)

- 11.8 0.00 0 40.6 2 No

- 33.5 201 2 13.7 3.2 (3.30)

- 97.9 2 0.96 0 46.2 2 Yes

- 88.0 2 14.4

- 84.8 2US EPA

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Page **154** of **211 xi.**  **Amphibian Metamorphosis Assay (AMA)** The 21

- day

assay of Chemical J on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was studied under flow

- through conditions. Amphibian larvae (80 per control and treatment group, NF stage 51) were exposed to a negative control and nominal concentrations of 0.100, 0.500, and 2.50 mg a.i./L. Time

- weighted average (TWA) concentrations were <0.00474 (<LOQ; control), 0.0964, 0.438, and 2.04 mg a.i./L. The test sy stem was maintain ed at 20.1 to 22.9°C and a pH of 7.0 to 7.7. Chemical J significantly

reduced wet weight in the two highest treatment groups on Day 7 ( ≥38%, p <0.05) and in the lowest and highest treatment groups on Day 21 (≥20%, p<0.01). Similarly , snout
- vent length (SVL ) was significantly reduced at the two highest treatment groups on Day 7 ( ≥16%, p ≤ 0.05) and significant reductions were detected in the lowest and highest treatment groups on Day

21 (6%, p ≤ 0.01). There were also significant reductions in non

normalized hind limb length (HLL) at the two highest treatment groups on Day 7 ( ≥20%, p < 0.05) and at all treatment groups on Day 21 ( ≥15%, p ≤ 0.01). Statistically

significant developmental delay s (p<0.05) were observed at Chemical J concentrations associated with growth inhibition and clinical signs of toxicity . On Day 7, developmental stage was significantl y (p<0.05) delayed at the highest treatment level. Normalized HLL on Day 7 was significantly decreased at the highest treatment level (13%, p= 0.025 at 2.04 mg a.i./L) and was significantly decreased at all treatment levels on Day 21 (10%, 24%, 23% at the 0.0964, 0.438, and 2.04 mg a.i./L levels, respectively , p <0.05). There was no evidence of as ynchronous development reported on Day s 7 or 21. There were no treatment
related effects on thy

roid gland histopathology at any treatment level. Some transient abnormal behaviors (floating on the surface, l yi ng on the bottom of the tank, inverted or irregular swimming, lack of surfacing activity , and being nonresponsi ve to stimulus) were noted among control and treated tadpoles, with a greater proportion of these abnormal behaviors observed among those in the two highest treatment groups. There was no mortality in the control group and mortality did not exceed 7% in an y treatment level. All validity requirements for the amphibian metamorphosis assay were met. The performance criteria were not fully met because there were not ≥2 concentrations (non

- control) without overt toxicity available for anal ys is . A dditionally , th ere was a temperature fluctuation ( i.e ., 20.1°C) on Day 1 in the continuously

- monitored control test vessel. The presence of clinical signs of toxicity and inhibition of growth at the same concentrations as developmental delay s, in the absence of thy roid h istopathological effects, makes it difficult to interpret whether Chemical J has the potential to interact with the h ypothalamic

- pituitary

- thy roid (HPT) axis within the range of concentrations tested in the AMA. However, based on the results in the lowest treatment group which reported no observed clinical signs of toxicity , Chemical J is considered not likel y to interact with the th yroid pathway in the AMA.US EPA

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Page **155** of **211 D.**  **Other Scientifically**  **Relevant Information (OSRI) for Chemical J**  The registrant elected to conduct the 11 Tier 1 screening assay s, therefore, no OSRI were submitted to the a gency . However, Chemical J has Part 158 mammalian and eco toxicity studies previously submitted to the Agency . These studies are discussed below. **1. Mammalian OSRI Data**  In a 3

- month feeding study , groups of 10 male and 10 female COBS

- CD (SD) BR rats were administered technical Chemical J in the diet at 0, 0.25, 0.75, 2.5, 7.5, 75, 250 or 750 mg/kg bw

- day

. During weeks 3 and 4 dietary exposure levels, were increased to 0.35, 1.05, 3.5, 10.5, 35, 105, 350 or 1050 mg/kg bw

- day . During weeks 5

- 13, dietary exposure levels were again increased to 10, 30, 100, 300, 1000, 3000, 10,000 or 30,000 ppm (0.5, 1.5, 5, 15, 50, 150, 500 or 1500 mg/kg bw

- day ). Evaluati ons included , but were not limited to, clinical observations and mortality , body

weight, food consumption, hematology , clinical chemistry , organ weights, gross necrops y and histology . Complete histological examinations were performed in the 0, 150 and 500 mg/kg bw

- day

groups. All rats treated at 1500 mg/kg bw

- day died during the dosing period; no treatment
- related deaths occurred at lower doses

. At 150 mg/kg bw

- day and above, an increase incidence in small follicles was observed in the thy roid for males. A dditionally , effects on the liver and kidney s were observed ( i.e., changes in weights and histopathology ) at the 150 mg/kg bw

- day

dose.
In a 1

- ye ar feeding stud y, groups of 6 male and 6 female Beagle dogs were administered technical Chemical J at dietary concentrations of 0, 10, 100, 400 or 1600 ppm. Based on measured food consumption and body weight, the average chemical intakes were 0, 0 .34, 3.09, 14.28 and 54.22 mg/kg bw

- day in males and 0, 0.4, 3.83, 15.68 and 58.2 mg/kg bw

- day in females. Endpoints reported included body weight, food and water consumption, hematology , clinical chemistry , organ weights and histopathology . Complete histo logical examinations were not reported on the low and mid

- dose animals; however, assessment in all dietary treatment groups included testes and liver. No effects on th yroid or pituitary weights were reported. There were no histological changes in the test es, seminal vesicles, prostate, ovary , uterus, thy roid or pituitary

compared to the control group. L iver effects including, hepatocellular hy pertrophy and increased liver weight were observed 14.28 and 15.68 mg/kg bw

- day for males and females, respective ly

. In a 2

- ye ar chronic/carcinogenicity study , Chemical J was administered to 110 male and 110 female Sprague Dawley

rats in the diet. Dietary

levels for the low

- dose group were 25/35/50 ppm; for the mid

- dose group were 100/140/200 ppm and for the high

- d ose group were 400/560/800 ppm. The first dose level in each series was administered for a period of 2 weeks, the second dose level in each series was administered for a period of 2 weeks and the third dose level in each series was administered from week s 5 to termination. The overall calculated mean daily compound consumption was 0, 2.49, 9.84 or 39.21 mg/kg bw/day for males and 0, 3.23,US EPA

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Page **156** of **211** 12.86 or 52.34 mg/kg bw/day for females. Evaluations included clinical observations and mortality , body

weight, food and water consumption, hematology , clinical chemistry , gross necrops y and histopathology

. At 9.84 mg/kg bw/day in males, the mean absolute testes weights were significantly

less than controls (p<0.05; 77% of controls) at study termination. An increase i n testicular atrophy was observed at 39.21 mg/kg bw/day . Microscopicall y, the seminiferous tubules were frequently devoid of spermatid formation and germinal epithelial cells at the highest dose tested. No adverse histopathological effects were seen in th e prostate, ovaries, uterus, mammary gland, pituitary , or th yr oid at any dose tested. No changes in ovary or thy roid weights were reported.
In a 2

- ye ar supplemental carcinogenicity study , Chemical J was administered to 50 male and 50 female Sprague Dawl ey

rats at dose levels of 0 or 2500 ppm (125 mg/kg bw/day ) in the diet. Evaluations included clinical observations and mortality , body weight, food and water consumption, hematology , clinical chemistry , gross necrops y and histopathology

. At 125 mg/kg bw/d ay , Absolute and relative testes weights were significantl y lower in treated males at both the interim sacrifice (63% and 68% of controls for right testes, 58% and 63% of controls for left testes) and terminal sacrifice (81% and 85% of controls for right t estes, and 73% and 74% of controls for left testes). An increase in bilateral aspermatogenesis was also observed at this dose. The decreased spermatogenic activity was associated with an increase incidence of hy pospermia and cellular debris in the epidid ym is of treated males. Liver effects including hy pertrophy and vacuolization were also observed at 125 mg/kg bw/day

. In a 2

- ye ar feeding/oncogenicity study , groups of 110 male and 110 female Crl:CD

- 1(I CR)BR mice ( Mus musculus ) were administered technica l Chemical J at dietary concentrations of 0, 20 , 100 or 500 ppm. The overall reported mean daily compound consumption was 0, 2.7, 13.7 or 70.2 mg/kg bw

- day

for males and 0, 3.2, 16.5 or 85.2 mg/kg bw

- day for females. Of the 110 mice/sex/dose, 10/sex/dose were euthanized at 3 and 6 months and 20/sex/dose group were euthanized at 12 months. A sentinel animal program was also conducted separatel y in which 25 mice/sex were evaluated at 24 months. Evaluations included , but was not limited to, clinical observat ions and mortality , body weight, food and water consumption, clinical chemistry , gross necrops y and histopathology

. No effects on the weights of the th yroid, ovaries or pituitary were observed. There were no histological changes in the testes, seminal ves icles, prostate, ovary , uterus, mammary , thy

roid or pituitary

compared to the controls. Effects on the liver (increases in liver enzy

me (SGPT), liver weight and hepatic histopathology in one or both sexes) were observed at 500 mg/kg diet (70.2 mg/kg bw

- day for males and 85.2 mg/kg bw

- day for females).
In an 18

- month feeding/oncogenicit y study, groups of 60 female Crl:CD

- 1 (I CR)BR mice were administered technical Chemical J at dietary concentrations of 0 or 2000 ppm (393.5 mg/kg bw

day ). Ten mice/group wer e euthanized at 52 weeks. An additional 20 mice/group were added to the study as sentinels and also euthanized at 52 weeks. This study was conducted at a dietary level approaching the maximum tolerated level because it was determined that a previous studyUS EPA

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Page **157** of **211** was not conducted at sufficiently high dietary

treatment levels in females. Evaluations included clinical observations and mortality , body weight, food and water consumption, hematology , gross necrops y and histopathology . Clinical chemistry and urine anal y ses were not performed. No effects on the weights of the thy roid, ovaries or pituitary

were observed. There were no histological changes in the testes, seminal vesicles, prostate, ovary , uterus, mammary , thy roid or pituitary compared to the controls. Eff ects on body

and liver weight, and liver and adrenal histopathology

were observed at 393.5 mg/kg bw

- day , the lowest concentration tested. In a prenatal developmental toxicity study in Sprague

- Dawley

rats, Chemical J in corn oil was administered at doses o f 0, 31.3, 93.8, 312.6 or 468.9 mg/kg bw/day by gavage on gestation day s 6

- 15. For maternal toxicity , the NOAE L was 93.8 mg/kg bw/day and the LOAEL was 312.6 mg/kg bw/day

based on clinical signs (rough hair coat, salivation, alopecia, desquamation and red exudate around the mouth). The developmental toxicity NOAE L was 93.8 mg/kg bw/day and the developmental toxicity L OAEL was 312.6 mg/kg bw/day, based on increased incidences of 14 th rudimentary ribs and 7 th cervical ribs. I n a range - finding study , 100% m aternal mortality

was seen at 700 mg/kg bw/day. A prenatal developmental toxicity study was conducted in pregnant New Zealand white rabbits, in which Chemical J ( in 1% methylcellulose ) was administered by gavage at doses of 0, 20, 60, or 200 mg/kg bw/day on gestation day s 7

- 19. For maternal toxicity , the NOAEL was 60 mg/kg bw/day and the LOAEL was 200 mg/kg bw/day , based on decreased body weight and body weight gain during dosing, with a rebound weight gain effect during the post

- treatment period, and on o bservations of irregular feces, bloody urine, bloody

urogenital or anal area, and blood and/or aborted materials in the cage pan (total litter loss occurred in three does). For developmental toxicity

the NOAEL was 60 mg/kg bw/day and the LOAEL was 200 mg/ kg bw/day based on increased resorptions, decreased litter size, and decreased viability index. I n a range - findings stud y, maternal mortality was observed at doses of 464 and 700 mg/kg bw/day, and the maternal and developmental toxicity NOAEL s for that s tudy

were 100 mg/kg bw/day

. In a reproduction study , male and female CRL :CD(SD)BR rats were fed diets containing Chemical J at 0, 50, 200 or 1000 ppm (2.5, 10 or 50 mg/kg bw/day ) for two consecutive generations. At 200 ppm, centrilobular hepatocellular hy pertrophy was observed in the P 2 males. This was supported by slight but statistically

significant increases in liver weights in males. At 1000 ppm, centrilobular hepatocellular hy pertrophy was observed in both sexes in the P 1 and P 2 generations. Thes e were again supported by slight but statistically

significant increases in liver weights. For parental s ys temic toxicity , the NOAEL was 50 ppm (2.5 mg/kg bw/day ) and the LOAEL was 200 ppm (10 mg/kg bw/day ) based on hepatocellular hy pertrophy and increase s in liver weights. At 1000 ppm, an increase in the number of stillborn or percent born dead pups was observed in both generations. I n addition, multifocal or diffuse testicular atrophy was observed in males in the P 2 generation. I ncreased necrotic sper matocy tes/ spermatids or decreased spermatozoa and atroph y of the prostate were also observed in these animals. Sexual maturation, estrous cy cle length and periodicity , and sperm parameters were not evaluated in this study

. For reproductive toxicity , the NOAEL was 200 ppm (10 mg/kgUS EPA

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Page **158** of **211** bw/day ) and the LOAEL was 1000 ppm (50 mg/kg bw/day ) based on an increased incidence in the number of stillborn pups and atroph y of the testes and prostate. At 1000 ppm, there was a slight decrease in pup weight gain during la ctation. For developmental toxicity , the NOAEL was 200 ppm (10 mg/kg bw/day ) and the LOAEL was 1000 ppm (50 mg/kg bw/day ) based on a decrease in pup bod y weight gain during lactation. In evaluating the available mammalian Part 158 data for Chemical J for endpoints relevant to the E, A, or T pathway , effects on testes and prostate were observed in the two
- generation reproduction rat Part 158 study , ( multifocal or diffuse testicular atrophy , increased necrotic spermatocy

tes/spermatids or decreased spermatozo a and atroph y of the prostate )

. Additionally , d ecreased testes weight, testicular atroph y and effects on spermatogenesis were observed in the rat chronic/carcinogenicity studies.

**2. Ecotoxicity OSRI Data**  In an earl y life

- cy cle toxicity test with fathead minnow ( Pimephales promelas ) after 35 day s of exposure to Chemical J, the NOEC was reported at 0.98 mg /L , with a LOEC of 2.2 mg/L, based on reduction in standard length compared to the control. Wet weight was affected at 4 mg/L. On an acute exposure b asis, Chemical J is classified as “moderatel y toxic” to freshwater and estuarine/marine fish with a 96

- hour LC 50 of 2.4 mg a.i./L in bluegill sunfish ( Lepomis macrochirus) and 4.7 mg a.i./L in sheepshead minnow. In a one

- generation reproduction study wi th mallard ducks and Northern bobwhite quail with Chemical J, no effects on adult birds, hatchlings or reproductive parameters was observed at an y concentration tested. The NOAEC was 260 mg/kg

- diet, the highest concentration tested. In a one

- generation re production study with mallard ducks and Northern bobwhite quail with Chemical J, no effects on reproduction were observed at an y concentration tested. The NOAEC was 60 mg/kg

- diet, the highest concentration tested. In a chronic 28

- day

toxicity test with the my sid shrimp exposure to Chemical J at concentrations between 2.58 and 85.6 μg ai/L did not cause an y significant decreases on F 0 survival, F 0 male or female body length, number of y oung produced per female, survival of F 1 my sids, or F 1 male or female bod y l ength. No significant increase was observed in time to first brood. However, there was a significant decrease (p<0.050 in time to first brood at intermediate test concentrations between 5.09 and 42.0 μg a.i./L, but not at the highest concentration of 8 5.6 μg a.i./L. At each of the middle test concentrations, the mean time to first brood was approximately

one day less than the control. No additional data with frog or other amphibian species have been submitted.US EPA

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Page **159** of **211** In evaluating the available ecotoxicity Par t 158 data for Chemical J for endpoints relevant to the E, A or T pathway , reproduction did not appear to be altered (birds and m ysids), except for a significant decrease in the time to first brood compared to the control in the chronic m ys id shrimp study

.

**E. Discussion** 

**i. Effects on Hypothalamic** 
**- Pituitary** 
**- Gonadal (HPG) Axis**  As previously

noted, the EDSP Tier 1 screening data and OSRI were evaluated together to determine whether Chemical J has potential to interact with the HPG axis or its downstream pathway s related to steroidogenesis or sex steroid signaling. The Tier 1 in vitro assay s relevant to this analy sis include the AR, binding, ER binding, ERTA, aromatase, and steroidogenesis assay . Relevant in vivo assay s include the male and female pubertal rat, uterotrophic, Hershberger, and the FSTRA assay s. **Effects on Estrogen Pathway :**  **Table 5**  below summarizes the results of the EDSP Tier 1 screening assay

results and OSRI for determining the potential for Chemical J to interact with the estrogen pathway . The various targets of th e estrogen pathway across the relevant Tier 1 assay s are delineated so as to facilitate determination of potential for estrogenic, anti

- estrogenic, or HPG axis effects. Chemical J was negative for both the in vitro ER binding assay and ERTA assay

suggesti ng that Chemical J does not trigger a molecular initiating event (MIE) with signaling through the estrogen receptor. Furthermore, a negative response was observed in the uterotrophic assay , supporting this assertion. However, Chemical J was positive for alterations in enzy me activity

in both the in vitro steroidogenesis assay and aromatase assay , suggesting that Chemical J may alter hormone production along the steroidogenesis pathway . In the in vitro ster oi dogenesis assay , significant decreases (p<0.05) in estrogen production were reported. Moreover, in the in vitro aromatase assay , Chemical J was shown to strongly inhibit aromatase activity ( i.e ., conversion of androgen to estrogen). These observed alterations to ster oi dogenesis which would result in decrease estrogen levels are corroborated b y the reported results in the FSTRA in which alterations to several complementary endpoints were observed

. In the FSTRA at the highest concentration tested, a significant decrease (p<0.05) in estradiol ( ↓85% )in female fish was reported with a decrease in VTG ( ↓88%) along with an significant increase in GSI (↑46%) and significant decrease in the apical endpoint of fecundit y ( ↓98%). An increase in gonadal oocy te atresia in females was also observed at this high c oncentration. Additionally , a significant increase in male GSI ( ↑80%) along with alterations in gonadal histopathology was reported.US EPA

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Page **160** of **211** S i gnificant increase s in female testosterone ( ↑200%) a nd GSI ( ↑41%) were reported at the next lower concentration which may suggest an inhibition of aromatase activit y. While significant changes or alterations in estrogen related responses were not reported in the female rat pubertal assay , differences in sensitivities between fish and rats regarding chemicals that appear to alter steroidogenesis have been reported in the open literature (Ankley and Gray , 2013). For the uterotrophic assay , as these test animals have limited ability to produce sex steroids under the test conditions, the negative response in this assay could be anticipated for a chemical whose primary mode of action is altering sterol sy nthesis. Therefore, based upon WoE evaluation of EDSP Tier 1 data, Chemical J may alter ster oi dogenesis which then interferes with the estrogen signaling pathway and the HPG a xis.US EPA

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Page **161** of **211 Table**  **55: Estrogenic/Anti** 
**- Estrogenic Pathway**  **for Chemical J Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti** 
**- Estrogenic Pathway for Chemical J**  **a**

**Study Type / Literature Citation** **E R** **B i** **n d** **i n** **g E** **R A** **c t** **i v** **a t** **i o** **n S** **t e** **r o** **i d** **o g** **e n** **e s** **i s** **U t** **e r** **i n** **e W** **e i** **g h** **t O** **v a** **r i** **a n** **W e** **i g** **h t** **o r** **F i** **s h** **G S** **I O** **v a** **r i** **a n** **/ G** **o n** **a d** **S t** **a g** **i n** **g a** **n d** **H i** **s t** **o p** **a t** **h o** **l o** **g y** **P i** **t u** **i t** **a r** **y W** **e i** **g h** **t E** **s t** **r o** **u s** **C y** **c l** **i c** **i t** **y b** **A g** **e &** **W e** **i g** **h t** **a t** **V O** **F e** **r t** **i l** **i t** **y /** **F e** **c u** **n d** **i t** **y V** **i t** **e l** **l o** **g e** **n i** **n E** **s t** **r a** **d i** **o l** **O v** **e r** **t T** **o x** **i c** **i t** **y O** **b s** **e r** **v e** **d c** 

**EDSP Tier 1** ER Binding N ERTA N Aromatase P Steroidogenesis P Uterotrophic N Female Pubertal Rat N N N N N N N

FSTRA ↑(41%) M; ↑(46%) H ♀ P ↓ (98%) H ↓

(88%) H ♀ ↓

(85%) H ♀US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **162** of **211 Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti** 
**- Estrogenic Pathway for Chemical J**  **a**

**Study Type / Literature Citation** **E R** **B i** **n d** **i n** **g E** **R A** **c t** **i v** **a t** **i o** **n S** **t e** **r o** **i d** **o g** **e n** **e s** **i s** **U t** **e r** **i n** **e W** **e i** **g h** **t O** **v a** **r i** **a n** **W e** **i g** **h t** **o r** **F i** **s h** **G S** **I O** **v a** **r i** **a n** **/ G** **o n** **a d** **S t** **a g** **i n** **g a** **n d** **H i** **s t** **o p** **a t** **h o** **l o** **g y** **P i** **t u** **i t** **a r** **y W** **e i** **g h** **t E** **s t** **r o** **u s** **C y** **c l** **i c** **i t** **y b** **A g** **e &** **W e** **i g** **h t** **a t** **V O** **F e** **r t** **i l** **i t** **y /** **F e** **c u** **n d** **i t** **y V** **i t** **e l** **l o** **g e** **n i** **n E** **s t** **r a** **d i** **o l** **O v** **e r** **t T** **o x** **i c** **i t** **y O** **b s** **e r** **v e** **d c** 

**OSRI** Subchronic toxicity (Rat) N N Chronic toxicity

(Dog) N N N Chronic toxicity

(Rat) N N N Chronic toxicity

(Dog) N N N Carcinogenicit y (Mouse) N N Two

- generation reproduction (Rat) N N N Avian Reproduction N Aquatic Invertebrate Reproduction N

a Key to responses: Positive (P), negative (N) or equivocal (E) observation; arrow s ( ↓ or ↑) indicate the direction of the res ponse; A shaded cell indicates that parameter wa s not evaluated or is not applicable. b Factors for estrous cyclicity include: age of first estrous, length of estrous, and percent of animals cycling regularly. c An “X” in this column indicates that the effect(s) observed in the assay occurred in the presence of overt toxicity.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **163** of **211 Effects on Androgen Pathway**  **: Table 56**  below summarizes the results of the EDSP Tier 1 screening assay

results and OSRI for determining the potential for Chemical J to interact with the androgen pathway . The various targets of the androgen pathway across the relevant Tier 1 assay s are delineated so as to facilitate determination of potential for androgenic, anti

androgeni c, or HPG axis effects. As discussed for the estrogen pathway , the results for Chemical J demonstrate that it may interfere with steroidogenesis, which is further supported b y in the in vitro steroidogenesis assay which reported a significant decrease in t estosterone production. Several complementary responses along different levels of biological organization were reported in the male pubertal assay including a significant decrease in testosterone ( ↓49

- 85%) correlated with a decrease in multiple androgen

- d ependent tissue weights ( ↓15

- 41%) and finall y a delay in PPS ( ↓1.7

- d). Additionally , while not dose

- dependent, a decrease in pituitary weight was observed in this assay at all test doses ( ↓8

- 10%). While testosterone was reported to not be affected in mal es in the FSTRA, the study

did report a significant increase in male GSI ( ↑80%) along with alterations in the male gonads reported as increased proportions of mature sperm relative to spermatogenic precursor cells. Th us , there was redundancy between the m ale pubertal and FSTRA in that alterations in androgen

- dependent tissues were reported in both assay s. Furthermore, as discussed in the estrogen pathway section, alterations in several levels of biological organization for the females were reported which included alterations to enzy me activit y up to the apical endpoint of fecundit y. Chemical J was classified as a “binder” in the AR binding assay ; based on the anal ys is of the response curves, Chemical J has a weak affinity for the androgen

- receptor. This weak in vitro binding with the AR did not manifest in a response in the Hershberger assay suggesting that androgen

- related effects observed in the male pubertal and FSTRA are not mediated through AR signaling in vivo . Furthermore, given that the Hershberg er uses castrated rats and have limited ability to produce sex steroids, the negative results in this assay would be consistent with a chemical that alters steroidogenesis by a means other than the inhibition of 5α

- reductase. Additionally , in a two

- gener ation reproduction rat Part 158 study , multifocal or diffuse testicular atrophy , increased necrotic spermatocy tes/spermatids or decreased spermatozoa and atrophy of the prostate were observed in these animals. Decreased testes weight, testicular atroph y a nd effects on spermatogenesis were observed in the rat chronic/carcinogenicity studies. Based upon WoE evaluation of EDSP Tier 1 data and OSRI , Chemical J appears to alter ster oi dogenesis which then interferes with the androgen signaling pathway and the HP G axis.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **164** of **211 Table 56**  **: Androgenic/Anti** 
**- Androgenic Pathway**  **for Chemical J**  **Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti**
**- Androgenic Pathway for Chemical J**  **a**

**Study Type / Literature**  **Citation A** **R B** **i n** **d i** **n g** **S t** **e r** **o i** **d o** **g e** **n e** **s i** **s T** **e s** **t o** **s t** **e r** **o n** **e T** **e s** **t e** **s W** **e i** **g h** **t o** **r M** **a l** **e F** **i s** **h G** **S I** **G o** **n a** **d S** **t a** **g i** **n g** **a n** **d H** **i s** **t o** **p a** **t h** **o l** **o g** **y E** **p i** **d i** **d y** **m i** **d e** **s W** **e i** **g h** **t E** **p i** **d i** **d y** **m i** **d e** **s H** **i s** **t o** **p a** **t h** **o l** **o g** **y P** **i t** **u i** **t a** **r y** **W e** **i g** **h t** **A c** **c e** **s s** **o r** **y S** **e x** **O r** **g a** **n W** **e i** **g h** **t s** **/ 2** **° S** **e x** **C h** **a r** **a c** **t e** **r i** **s t** **i c** **s A** **g e** **a n** **d W** **e i** **g h** **t a** **t P** **P S** **F e** **c u** **n d** **i t** **y /** **f e** **r t** **i l** **i t** **y V** **i t** **e l** **l o** **g e** **n i** **n O** **v e** **r t** **T o** **x i** **c i** **t y** **O b** **s e** **r v** **e d** **b**

**EDSP Tier 1** AR Binding P Aromatase P Steroidogenesis P Hershberger N
Male Pubertal Rat ↓(49%)M ↓(85%)H N o

effect N o effect N o

effect N o effect ↓(8%)L; ↓(10%) M ↓(10%)H ↓(15

-

19%) M; ↓(16

- 41%) H ↑(1.7d ) H

FSTRA ↑ (200%) M ♀ ↑

(80%) P resent ↓ (98%) H ♀ ↓ (88%) H ♀US EPA

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– July , 2013

Page **165** of **211 Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti** 
**- Androgenic Pathway for Chemical J**  **a**

**Study Type / Literature**  **Citation A** **R B** **i n** **d i** **n g** **S t** **e r** **o i** **d o** **g e** **n e** **s i** **s T** **e s** **t o** **s t** **e r** **o n** **e T** **e s** **t e** **s W** **e i** **g h** **t o** **r M** **a l** **e F** **i s** **h G** **S I** **G o** **n a** **d S** **t a** **g i** **n g** **a n** **d H** **i s** **t o** **p a** **t h** **o l** **o g** **y E** **p i** **d i** **d y** **m i** **d e** **s W** **e i** **g h** **t E** **p i** **d i** **d y** **m i** **d e** **s H** **i s** **t o** **p a** **t h** **o l** **o g** **y P** **i t** **u i** **t a** **r y** **W e** **i g** **h t** **A c** **c e** **s s** **o r** **y S** **e x** **O r** **g a** **n W** **e i** **g h** **t s** **/ 2** **° S** **e x** **C h** **a r** **a c** **t e** **r i** **s t** **i c** **s A** **g e** **a n** **d W** **e i** **g h** **t a** **t P** **P S** **F e** **c u** **n d** **i t** **y /** **f e** **r t** **i l** **i t** **y V** **i t** **e l** **l o** **g e** **n i** **n O** **v e** **r t** **T o** **x i** **c i** **t y** **O b** **s e** **r v** **e d** **b**

**OSRI** Subchronic toxicity (Rat) N o effect N o effect N o

effect N o effect N o

effect N o

effect Chronic toxicity (Dog) N o effect N o effect N o

effect N o effect

Chronic toxicity (Rat) (  23% ) M; (  19

- 42%); H b Testes atrophy Present

Carcinogenicit y (Mouse) N o effect N o effect N o

effect Two

generation reproduction (Rat) N o effect Testes atrophy N o

effect P resent P resentUS EPA

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– July , 2013

Page **166** of **211 Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti** 
**- Androgenic Pathway for Chemical J**  **a**

**Study Type / Literature**  **Citation A** **R B** **i n** **d i** **n g** **S t** **e r** **o i** **d o** **g e** **n e** **s i** **s T** **e s** **t o** **s t** **e r** **o n** **e T** **e s** **t e** **s W** **e i** **g h** **t o** **r M** **a l** **e F** **i s** **h G** **S I** **G o** **n a** **d S** **t a** **g i** **n g** **a n** **d H** **i s** **t o** **p a** **t h** **o l** **o g** **y E** **p i** **d i** **d y** **m i** **d e** **s W** **e i** **g h** **t E** **p i** **d i** **d y** **m i** **d e** **s H** **i s** **t o** **p a** **t h** **o l** **o g** **y P** **i t** **u i** **t a** **r y** **W e** **i g** **h t** **A c** **c e** **s s** **o r** **y S** **e x** **O r** **g a** **n W** **e i** **g h** **t s** **/ 2** **° S** **e x** **C h** **a r** **a c** **t e** **r i** **s t** **i c** **s A** **g e** **a n** **d W** **e i** **g h** **t a** **t P** **P S** **F e** **c u** **n d** **i t** **y /** **f e** **r t** **i l** **i t** **y V** **i t** **e l** **l o** **g e** **n i** **n O** **v e** **r t** **T o** **x i** **c i** **t y** **O b** **s e** **r v** **e d** **b**

Avian Reproduction N o effect Aquatic Invertebrate Reproduction N o effect

a Key to responses: Positive (P), negative (N) or equivocal (E) observation; arrows ( ↓ or ↑) indicate the direction of the res ponse; A shaded cell indicates that parameter was not evaluated or is not applicable; L, M, and H refer to low , m iddle and high test concentration, respectively; ♂ = male; ♀ = female b 125 mg/kg bw/day dose in supplemental chronic/carcinogenicity study.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **167** of **211 ii.**  **Effec ts on Hypothalamic** 
**- Pituitary** 
**- Thyroidal (HPT) Axis**  In general, regulation of the HPT axis is comparable to the HPG axis, except that the feedback relationship involves thy

roid hormones ( e.g ., T 4 and T 3 ). Thy roid hormones feedback to the hy pothalamus or pituitary

to regulate thy

rotropin

- releasing hormone and TSH, respectivel y, which, in turn, regulate hormone production by the thy roid gland. The current Tier 1 screening battery does not have a specific in vitro assay to detect chemicals with the potential t o affect hy pothalamic or pituitary regulation of th yroid hormone production, but it does include three in vivo assays that have the potential to detect these effects on the HPT axis ( i.e., the pubertal s and the AMA ). **Thyroid Hormones :**  **Table 57**  below summarizes the results of the EDSP Tier 1 screening assay results and OSRI for determining the potential for Chemical J to interact with thy roid regulation. The various targets of the th yroid pathway across the relevant Tier 1 assay s are delineated so as to facilitate determination of potential for thy roid or HPT axis effects. There were no changes to the thy roid pathway

endpoints in the f

emale pubertal study . In the male pubertal assay , alterations in thy roid histopathology

were reported and included increased th yroid follicular cell heights and decreased colloid area. There was also a significant decrease in pituitary weights. Additiona lly , while not significant (p>0.05), dose

- dependent increases in serum TSH levels ranging from 23

- 63% were observed. However, there were no significant changes in thy roid weight or serum T 4 levels. A significant decrease (8

- 10%) in pituitary weight in th e male pubertal assay was reported at all test concentrations; however the observed decrease was not dose

- dependent. For the AMA, while the observation of overt toxicity in two of the three test concentrations limited the ability to interpret the results i n those treatments, the absence of potential thyroid

- mediated effects in the remaining low test concentration allowed for the overall conclusion that Chemical J was not likely affecting t he thy

roid pathway

in the AMA. In a three

- month feeding Part 158 stu dy with male and female COBS

- CD (SD) BR rats, increases in the number of small follicles of thy roid in males were reported; however, incidences for this effect were not reported. This finding is not an adverse response and only indicates less colloid in th e follicle which is a normal response to maintain homeostasis of the thyroid hormone s ys tem. Overall, while there were changes in th yroid histology in the male rat pubertal assay as well as in one Part 158 rat feeding study, which are likel y normal homeost atic responses of the thy roid hormone sy stem There was no evidence of thy roid interactions in the female pubertal, and the overall conclusion for the AMA was that Chemical J was likely inactive. Therefore, based upon WoE evaluation of EDSP Tier 1 data an d OSRI, there is no evidence that Chemical J results in significant changes in thy roid hormones through interaction with the HPT axis.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **168** of **211 Table**  **57: Thyroid Pathway**  **for Chemical J Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway f**  **or Chemical J a** 

**Study Type / Literature Citation** **H o** **r m** **o n** **e s** **( T** **4 a** **n d** **T S** **H )** **P i** **t u** **i t** **a r** **y W** **e i** **g h** **t T** **h y** **r o** **i d** **W e** **i g** **h t** **T h** **y r** **o i** **d G** **r o** **s s** **a n** **d H** **i s** **t o** **p a** **t h** **o l** **o g** **y F** **r o** **g D** **e v** **e l** **o p** **m e** **n t** **S t** **a g** **e H** **i n** **d L** **i m** **b L** **e n** **g t** **h S** **n o** **u t** **t o** **V e** **n t** **L e** **n g** **t h** **O v** **e r** **t T** **o x** **i c** **i t** **y O** **b s** **e r** **v e** **d b** 

**EDSP Tier 1** Male Pubertal (Rat) N o effect ↓ (8%) L; ↓ (10%) M. H N o

effect P Female Pubertal (Rat) N o effect N o effect No effect No effect AMA (Frog) No effect No effect No effect No effect x c
OSRI Two

- generation reproduction (Rat) No effect No effect No effect Subchronic toxicity (Rat) No effect No effect No effect Chronic toxicity

(Dog) No effect No effect Chronic toxicity

(Rat) No effect No effect Carcinogenicit y (Mouse) No effect No effect

a Key to responses: Positive (P), negative (N); arrow s ( ↓ or ↑) indicate the direction of the response; A shaded cell indicates that parameter was not evaluated or is not applicable. b An “X” in this column indicates that the potential endocrine effect(s) observed in the assay occurred in the presence of overt toxicity c While overt toxicity was observed in this assay in the tw o of the three treatment groups, at the lowest treatment groups, at the lowest treatment groups, where there was an no overt toxicity observed, there w ere no indicati ons that the effects observed (delayed development and growth) were due the chemical’s interaction with the thyroid pathway.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **169** of **211 F.**  **Conclusion s for Chemical J**  All eleven assay s comprising the EDSP Tier 1 battery were available for Chemical J. Chemical J was evaluated for several h yp othesized endocrine modes of action for each specific E, A, and T pathway including agonism and antagonism at the estrogen and andro gen receptor, altered ster oi dogenesis , HPG and HPT perturbations. In addition to the available Tier 1 data, other relevant studies of sufficient quality were considered in this assessment. These studies included standard Part 158 toxicity data relevant to evaluating effects in endocrine tissues and functions. In determining whether Chemical J interacts with E, A, or T hormonal pathway s, the number and ty pe of effects induced, the magnitude of responses, and the pattern of responses observed across stud ies, taxa, and sexes were considered. Additionally , the conditions under which effects occur were considered, in particular, the dose(s) at which responses happen and whether overt toxicity was present. Integrating the in vitro and in vivo Tier 1 result s, as well as consideration of relevant data bey ond Tier 1 information ( e.g ., Part 158 standard toxicology studies) demonstrated that Chemical J appears to alter steroidogenesis which affected both the estrogen and androgen hormonal signaling pathway s and the HPG axis. For the E pathway , the result in the FSTRA provided complimentary responses within that assay along with the positive results in the steroidogenesis aromatase assay providing redundancy across the assay s. Additionally , the negative response s in the ERB, ERTA and uterotrophic assay s provide evidence that the effects observed for Chemical J are not estrogen receptor

- mediated. While responses were not observed in the female pubertal, differential sensitivities among the fish and rat have been reported for chemicals that alter s teroidogenesis (Ankley and Gray , 2013)

. With respect to the A signaling pathway , the in vitro responses in the AR binding assay suggest that Chemical J has a weak affinity for AR binding; whereas, the negative results in the Hershberger assay suggest that Chemical J does not directl y interact on the androgen receptor. However, there were complementary

responses within the male pubertal and FSTRA assay s and redundancy across biolog ical levels of organization between those two assay s. The results in the in vivo assay s, along with the positive results in the steroidogenesis assay demonstrate that Chemical J may have the potential to interact with the androgen s ys tem via a perturbatio n to steroidogenesis. For the T pathway , the AMA and female pubertal assay s were negative. The only finding was a change in thy roid histology

in male rats which is likel y reflective of a normal response to maintain thyroid homeostasis. There were no changes in T4 and non

- significant increases in TSH found in the male rat; all other thy roid

- mediated endpoints were not affected. Based on this evidence, Chemical J does not appear to interact with the HPT axis.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **170** of **211** 

### 9. **Ca se Study** 

**— Chemical**  **N**

**A. Introduction**  Chem ical N, a cy clic unsaturated ketone which has irritant properties, is one of four chemicals selected from the 21 chemicals used in the EDSP assay and battery

performance analy sis to serve as case studies for the current WoE analysis for Tier 1 screening. T he available information considered to determine the potential interaction of Chemical N with the E, A and/or T pathway s included all 11 EDSP Tier 1 studies and OSRI , such as the Part 158 mammalian and ecotoxicity data, when available. In the case of Che mical N, issues included potential endocrine effects observed in a mammalian Tier 1 assay ( m ale p ubertal) that occurred only near the limit dose (1000 mg/kg/bw/day ), an effect on fish vitellogenin that was not corroborated b y other effects at that test con centration, and a generally smaller dataset of OSRI from which additional lines of evidence could be drawn for interpretation

. The data considered are summarized below in Sections **B, C** and **D** of this chapter

. A n anal ys is of the data submitted to the agency , using the WoE approach outlined by the Agency

(USEPA, 2011 b ), is presented in Section **E**

.
**B. Data**  **A vailable**  **for Chemical N** Chemical N is relativel y soluble in water with a low log octanol

- water partition coefficient (log K ow ~2). The chemical can persist in the environment although it undergoes aerobic degradation. Based on the chemical

- phy sical characteristics of the compound, it is considered moderately mobile and can move to surface water via runoff/erosion. With its relatively low K ow , the compound i s not expected to bioconcentrate in aquatic organism.

**C. Tier 1 Screening Assays**  **for Chemical N** The Tier 1 studies submitted in response the test order for Chemical N are shown below in **Table 58**  , and the relevant study

details and findings are summarized below.US EPA

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– July , 2013

Page **171** of **211 Table**  **58 :**  **Summary of Tier 1 Screening Assays for Chemical N.** 

**Tier 1 Assays Test Guideline**  **Satisfied by OSRI (Y/N)**  **Study Findings** ER Binding Assay

(Rat uterine cy

tosol) OSCPP 890.1250 No Negative 1 ERα Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300; OECD 455 No Negative AR Binding Assay

(Rat prostate cy

tosol) OSCPP 890.1150 No Negative Steroidogenesis Assay

(Human cell line H295R) OSCPP 890.1550; OECD 456 No Negative Aromatase Assay

(human recombinant microsomes) OSCPP 890.1200 No Negative Uterotrophic Assay

(Rat) OSCPP 890.1600;

OECD 440 No Negative Hershberger Assay

(Rat) OSCPP 890.1400;

OECD 441 No Negative for A and Anti

- A Pubertal Female Assay (Rat) OSCPP 890.1450 No Negative

- E and T pathway s Pubertal Male Assay

(Rat) OSCPP 890.1500 No Positive

- A pathway Negative

- T pathway Fish Short

- term Reproduction Assay OSCPP 890.1350; OECD 229 No Possible for A pathway Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; OECD 231 No Negative

- T pathway

1 Classified as “Not Interactive” according to the Test Guidelines 890.1250

. 2 Negative for both androgenic and anti

- androgenic effectsUS EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **172** of **211 i.**  **ER Binding Assay (Rat uterine cytosol)** In the ER binding assay for Chemical N, uterine cy tosol from Sprague Dawley rats was used as the source of ER for a Competitive Binding Experiment, which was conducted to measure the binding of a single concentration of [ 3 H]

- 17β

- estradiol (1 nM) in the presence of increasing con centrations (logarithmic increase from 10 − 10 to 10 − 3 M) of Chemical N. Ethanol was used as the solvent vehicle at a final concentration of <3%. Saturation binding experiments were conducted to demonstrate that the ER was present in a reasonable concentra tion in the cy tosol preparation and functioning with the appropriate affinit y for the radiolabeled ligand. In the competitive binding experiment , a total of three valid runs were performed and each run included 19

- norethindrone as a weak positive control, octy ltriethoxy silane as a negative control, and 17

- β

- estradiol as the natural ligand reference material. In the Saturation Binding Experiment, the maximum binding capacit y (B max ) was 59.28 fmol/100 μg protein and the dissociation constant (K d ) was 0.1032 n M. I n the Competitive Binding Experiment, the estimated mean Log IC 50 was −8.917 Log M for 17 β

- estradiol and −5.436 Log M for the weak positive control. The mean Relative Binding Affinity (RBA) was 0.033% for the weak positive control. All performance cr iteria were met. Chemical N had no effect on specific binding of the [ 3 H]

- ligand; therefore, a Log IC 50 and RBA could not be calculated for Chemical N. Based on the results of t hree runs, Chemical N is classified as Not Interactive in the ER binding a ssa y.

**ii. ER Transcriptional Activation Assay**  In the ER transcriptional activation assay , hERα

- HeLa

- 9903 cells were exposed to Chemical N (98.7% a.i.) at logarithmicall y increasing concentrations from 10 − 9 M to 10 − 3 M in dimethy lsulfoxide (DMSO; 0.1%) in three i ndependent runs. The experiments were performed with 96

- well plates and each Chemical N concentration was tested in triplicate (3 wells/plate) in each run. Cells were exposed to the test agent for approximately 24 hrs to induce reporter (luciferase) gene p roducts. L uciferase expression in response to activation of the estrogen receptor by Chemical N was measured upon addition of a luciferase substrate and detection with a luminometer with acceptable sensitivity

. Chemical N was tested up to the guideline li mit of 10 − 3 M. I n addition, reference chemicals were included for each run to confirm the adequacy of the experimental conditions for the detection of ER agonism. The reference chemicals were: 17β

- estradiol (strong agonist), 17α

- estradiol (weak agonist), 17α

- methy ltestosterone (very weak agonist), and corticosterone (negative). The reference chemical results adequatel y met the test guideline performance criteria. . For test chemical N, the RPC max was 5.9% for the first run, 7.8% for the second run and 9.9% for the third run. Because the RPC max < PC 10 in all three runs, Chemical N was negative for ER transcriptional activation in this test sy stem.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **173** of **211 iii.**  **AR Binding Assay (Rat prostate cytosol)** In the in vitro AR binding assay , ventral prostate cy tosol from Spr ague Dawley rats was used as the source of AR to conduct a Competitive Binding Experiment, which was conducted to measure the binding of a single concentration of [ 3 H]

- R1881 (1 nM) in the presence of increasing concentrations of Chemical N (logarithmic inc rease from 10 − 10 to 10 − 3 M). Ethanol was used as a vehicle at a final assay concentration of <3%. The assay included dexamethasone as a weak positive control, and R1881 as the ligand reference standard. The s aturation b inding e xperiment was conducted in duplicate and summarized in a submitted study profile. I n the S a t u ra tion B indi n g E x p er im e nt, the m a x i m u m binding ca

p ac i t y ( B m a x ) wa s 3.245 f m o l/ 100 μ g p r o t e i n a nd t he d i ss o ci a ti on c ons t a nt (K d ) wa s 0.4641 nM. T h e se v a l u e s w er e b e l ow t h e ra n ge of v a l u e s fr om t he v a li d ati on s t ud i e s (0.685

- 1.57 nM). T h e S ca t c h ar d plot i nd i ca t e d a li n e a r re s pon s e a cro s s t he con c en tr a ti o n s of li g a nd a dd e d. N onsp e c i f i c b i n d i ng a s a p e r ce nt of t o t a l b i nd i ng wa s l e ss t h a n 20 % acro s s t h e en t i r e c o n c en t ra ti o n r a n g e i n t he s a t u ra ti on b i nd i ng a s s a y s (ra n g e 6. 2

- 19.8 % ), w it h t he e x ce p ti on of t he h i g h c o n ce n t ra ti on ( 10 n M) i n one a ss a y , w h i c h wa s 24.6 %

. In the c ompetitive b inding e xperiment, the estimated mean Log IC 50 s for R1881 ( −8.978 Log M) and the weak positive control, dexamethasone ( −4.412 Log M), were within the expected ranges. The mean relative binding affinit y (RBA) for the weak positive control was 0.0027%. All performance criteria were met. Chemical N h ad no effect on specific binding of the [ 3 H]

- ligand; therefore, a L og I C 50 and RBA could not be calculated for Chemical N as it did not result in 50% inhibition at any concentration. Based on the results of three assay s, Chemical N is classi fied as a “No n - Binder” in the AR binding assay .

**iv. Steroidogenesis Assay (Human cell line H295R)**  In the steroidogenesis assay , H295R cells cultured in vitro in 24

- well plates were incubated with Chemical N (98.7±1.3% purity , 1136041301) at concentrations of 0.0001 to 10 0 μM for 48 hours in triplicate for three independent experiments. The solvent vehicle for Chemical N was DMSO and its final concentration was 0.1% (v/v). Testosterone and estradiol levels were measured by using LC

- APPI

- MS/MS. A Quality Control (QC) plat e was run concurrently with each independent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels. The positive controls included a known inhibitor (prochloraz) and a known in ducer (forskolin) of estradiol and testosterone production.US EPA

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Page **174** of **211** The g uideline minimum basal hormone production levels for testosterone (500 pg/mL) were generally met in both blank and solvent control (SC) wells for testosterone. Although the mean basal estradiol production was less than the recommended lower limit (40 pg/mL ) for experimental runs 1 and 2 (29.2 and 32.6 pg/mL, respectively ), the values were more than 2.5

- fold greater than the minimum detection level MDL. Testosterone and estradiol produc tion in the presence of 10 μM forskolin met induction requirements. Although testosterone inhibition was greater than the required 50% in the presence of 1 μM prochloraz, inhibition of estradiol only reached 40% in Runs #2 and #3. Because all other guide line requirements were met, these results were deemed acceptable. No solubility

or cy totoxicity issues were reported for Chemical N at any of the test concentrations. The within

- plate coefficients of variability (expressed as a percent; %CV) of the vehicl e controls in the test assay s ranged between 3.3

– 13.9%, and the between

- plate CV was 30.2% for testosterone and 22.1% for estradiol. Although the % CV estimate for testosterone is slightly higher than the Guideline

recommended limit of ≤ 30%, the reviewer s consider these data acceptable. No effect on testosterone production was observed at an y test concentration. Slightly decreased (0.9

- , 0.8

- fold) estradiol levels were observed at two mid

- level Chemical N concentrations in a single run only , and a sli ght increase (1.2

- fold) was noted at the highest concentration in a separate run. These effects were not reproducible or concentration

- dependent. Based on hormone responses in three runs, Chemical N was negative in the ster oi dogenesis assay .

**v. Aromatase**  **Assay (Human Recombinant Microsomes)** In the aromatase (CYP19) assay , Chemical N (98.7% a.i.) in ethanol (1%) was incubated with human recombinant aromatase and tritiated androstenedione (1

- β [ 3 H(N)]

- Androst

- 4

- ene - 3,17

- dione; [ 3 H]ASDN) for 15 minutes at 37 °C to assess the effect of Chemical N on aromatase activity

. Chemical N was tested at logarithmic concentrations from 10 – 10 M to 10 − 3 M in three independent runs. Aromatase activity

was determined by

measuring the amount of tritiated water produced at the end of a15

- minute incubation for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (L SC). Three independent runs were conducted and each run included a full activity control, a background activity control , a positive control series (10 − 10 to 10 − 5 M) using a known inhibitor (4

- OH ASDN), and the test chemical series (10 − 10 to 10 − 3 M) with 3 repetitions per concentration. Results for the positive control were within the recommended ranges for the top of the c urve, bottom curve, Hill Slope, and coefficient of variation (CV) for replicates of each concentration within runs with the exception of the top % of the curve for one run was 112% exceeding the recommended range of 90

- 110%. The mean Log IC 50 values for 4

- OH ASDN for the three runs (

- 7.45,

- 7.35 and

- 7.35) exceeded the recommended range of

- 7.3 to

- 7.0 M.US EPA

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Page **175** of **211** For Chemical N, aromatase activity averaged 0.224±0.018 nmol ∙mg - protein − 1
∙ min − 1 at the lowest tested concentration (10 − 10 M) and 0.187±0.027 nmol ∙mg - pro tein − 1
∙ min − 1 at the highest tested concentration (10 − 3 M). The average dose

- response curve indicated that aromatase activity at concentrations of Chemical N ranging from 10 − 10 M to 10 − 3 M was essentially equivalent to the activity observed in the full act ivity

controls. The 10 – 3 M dose level reduced the aromatase activity b y approximately 20%. Based on the results of three runs , Chemical N is classified as a Non

- Inhibitor in the aromatase assay.

**vi. Uterotrophic Assay (Rat)**  In the uterotrophic assay

conducte d to screen for potential estrogenic activity , Chemical N (98% a.i.) in corn oil was administered daily via subcutaneous (s.c.) injection to groups of 6 ovariectomized female, Sprague

- Dawley

rats at dose levels of 0 (untreated and vehicle controls), 5, 15, or 50 mg/kg/day on postnatal day s (PND) 56

- 58. A positive control group was treated with 17α

- ethy ny l estradiol (EE) in corn oil by

s.c. injection at a dose level of 0.27 μg/kg/day . : The high dose (50 mg/kg/day ) was selected based on the results of a r ange - finding study in which 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. Therefore, dosing was considered adequate in this study

. All animals were euthanize d and then necropsied approximately 24 hours after the final dose on PND 59 to determine wet and blotted uterine weights. All animals survived until scheduled termination. No clinical signs of toxicity were observed in animals for an y Chemical N treated groups. No effects on bod y weights were observed for the Chemical N treated groups; however, overall body weight gains for the 50 mg/kg/day group were decreased (p<0.05) b y 32% compared to the vehicle control. In the positive control group (EE), overal l body weight gains were decreased (p<0.05) by 28% on Day s 1

- 4. Uterine weights in the Chemical N treated groups were comparable to the untreated and vehicle controls. As expected, absolute wet and blotted uterus weights for the positive control (EE) gro up were increased (p<0.05) b y 86% each. No statistically

significant increase in uterine weight was seen in this study . Hence, Chemical N was negative in the Uterotrophic assay .US EPA

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Page **176** of **211 vii.**  **Hershberger Assay (Rat)** In the in vivo Hershberger assay testing for andro genic activity , Chemical N ( ≥98.7±0.1%) was administered dail y in corn oil via oral gavage to groups of seven 55

- day old castrated male Sprague Dawley rats/group at dose levels of 0 (vehicle), 100, 400, or 800 mg/kg/day for 10 consecutive day s. The study included an androgenic positive control group of seven castrated male rats dosed dail y with testosterone propionate (TP) in corn oil at 0.4 mg/kg/day via subcutaneous (s.c.) injection. To screen for possible anti

- androgenic activity , additional groups of seven 55

- day old castrated male Sprague Dawley

rats/group were dosed dail y via oral gavage with Chemical N in corn oil at 100, 400, or 800 mg/kg/day

for 10 consecutive day s in conjunction with a daily s.c. injection of TP in corn oil at 0.4 mg/kg/day . Th e control group for anti

- androgenic activity consisted of seven castrated male rats dosed daily

with TP (0.4 mg/kg/day ) b y s.c. injection, and the positive anti

- androgen control group consisted of seven castrated male rats dosed orall y with flutamide (FT) in corn oil at 3 mg/kg/day for 10 day s in conjugation with a daily s.c. dose of TP (0.4 mg/kg/day ). Bod y weights, bod y weight gains and food consumption were measured. Animals were euthanized approximately

24 hours after the final dose administration. The five androgen

- dependent tissues (seminal vesicles, ventral prostate, levator ani

- bulbocavernosus (L ABC), Cowper's glands or glans penis), adrenals, kidney s, and liver were weighed and macroscopicall y examined. No adverse treatment

- related effects were observed on mortality , clinical signs of toxicity , body weights, bod y weight gains, food consumption, or gross pathology in animals dosed with Chemical N. No treatment

- related effects were observed at any dose on the weights of any of the accessory androgen

- dependent tissues, or on the adrenals or kidney s in the androgenic and anti

- androgenic studies. L i ver weights were increased (p ≤0.05) b y Chemical N treatment in both the androgenic and anti

- androgenic portions of the study by 27

– 28% at 400 mg/kg/day and b y 32

– 34% at 800 mg/kg/day. The high dose was selected based on the results of a range

- finding stud y in which at a 1000 mg/kg/day dose, there was excessive toxicity as evidenced b y significant bod y weight losses, marked food consumption decrease, and clinical signs of distress. Therefore, dosing was considered adequate in this study

. For the androgenic portion of the study , the TP positive control group demonstrated treatment

- related increases (p ≤0.05) in the weights of the seminal vesicles with coagulating glands (↑1079%), ventral prostate ( ↑995%), LABC (↑96%), Cowper’s glands (↑603%), and glans penis ( ↑77%), indicating that the test sy stem was sensitive to an androgenic response. TP administration also resulted in increased food consumption and body weight gains, consistent with an androgenic response. For the anti

androgenic portion of the study , the TP + flutamide group demonstrated treatment

- related decreases (p ≤0.05) in weights of the seminal vesicles with coagulating glands (↓78%), ventral prostate (↓68%), LABC ( ↓38%), Cowper’s glands (↓54%), and glans penis (↓17%) compared to weights in the TP

tr eated group. These data indicate that the test s ystem was sensitive to an anti

- androgenic response. Flutamide administration did not affect food consumption or body weight gains.US EPA

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Page **177** of **211** In the androgen agonist assay , the guideline specified maximum %CV levels were exceeded in the ventral prostate at 800 mg/kg/day (55.2% vs 45% criterion) and LABC at 400 mg/kg/day (35.5% vs 30% criterion). The anti

- androgen assay did not meet the following Guideline criteria for maximum %CV levels: (i) LABC at 400 mg/kg/day w ith TP (23.5% vs 20% criterion); (ii) LABC in the flutamide positive control (26.2% vs 20% criterion); and (iii) Cowper's gland in the flutamide positive control (35.1% vs 30% criterion). The report also compared two control groups: one control group was a dministered the vehicle by oral gavage onl y while the other group was administered the vehicle b y oral gavage and s.c. injection. Bod y weights in animals given corn oil by both the oral and s.c. routes were similar to the body weights in animals given cor

n oil by oral gavage only . However, a minor decrease (p ≤0.05) in overall (Day s 1

– 11) body weight gain of 11% was noted in the animals given corn oil by both the oral and s.c. routes compared to animals given corn oil b y gavage only . Minor decreases (p ≤0. 05) of 6

– 8% of food consumption during the intervals of Day s 1

– 4 and 7

– 11 were observed in animals given corn oil by both the oral and s.c. routes compared to the food consumption in animals given corn oil by oral gavage only . This effect corresponded to the minor decrease noted in overall (Days 1

– 11) body weight gain. No differences (p>0.05) were detected in an y organ weight between the two control groups. Chemical N was negative for androgenicit y and anti

- androgenicit y in the Hershberger assay

.
**viii. Pubert**  **al Female Assay (Rat)** In the Female Pubertal Assay , 16 Sprague Dawley rats/dose group were treated dail y via oral gavage (4 mL /kg) with Chemical N (98.8 ± 0.1% purity ) in corn oil at doses of 0 (corn oil), 50, 200, or 800 mg/kg/day

from post

- natal day

(PN D) 22 to 42. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainment was recorded. Following sacrifice on PND 42, total thy

roxine (T 4 ) levels were determined using a chemiluminescent immunoassay a nd thy roid stimulating hormone (TSH) levels were determined using a radioimmunoassay ; serum was also anal yz ed for levels of selected clinical chemistry parameters. Adrenal, liver, pituitary , thy roid, and urogenital organ weights were recorded and microsco pic examinations of the ovaries, uterus, thy roid, and kidney s were performed. One animal each in the control and 200 mg/kg/day groups died prior to study termination, and one animal in the 50 mg/kg/day group was removed from the study due to accidental in

jury . All other rats survived until scheduled sacrifice. No treatment

- related effects were observed on clinical signs, body weight, body

weight gains, or gross or microscopic pathology. There were no effects of treatment on age or body weight at attain ment of VO, mean age at first vaginal estrus, mean cy cle length, percent cy cling, or percent regularl y cy cling. There were no dose

- related effects on serum T 4 or TSH.US EPA

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Page **178** of **211** At 800 mg/kg/day , adjusted, absolute, and relative liver weights were increased (p<0.05 ) b y 16

- 18% compared to controls. There were no effects on liver weights at doses ≤200 mg/kg/day . There were no treatment
- related effects on the weights of kidneys, pituitary , adrenal, ovaries, uterus, or thy roid. Serum urea nitrogen levels were decrea sed (p<0.05) in the 50, 200, and 800 mg/kg/day dose groups ( ↓17%, ↓25%, and ↓25%, respectivel y). Levels of creatinine were increased (p<0.05) in rats dosed at 200 and 800 mg/kg/day

( ↑200% and ↑500%, respectively ). At 800 mg/kg/day , levels of chloride and alkaline phosphatase were decreased (p<0.05) b y 2% and 22%, respectively, and levels of cholesterol were increased (p<0.05) by 14%. There were no dose

- related effects on the other clinical chemistry parameters. Organ weights in the control group were wi thin the acceptable range of the guideline performance criteria, with the exception of %CV for liver (13.29%; maximum 13.13%). Chemical N was negative for the estrogen and th yroid pathway s in the female pubertal assay

.
**ix. Pubertal Male Assay (Rat)**  In the Mal e Pubertal Assay , 16 Sprague Dawley rats/dose group were treated daily via oral gavage (4 mL /kg) with Chemical N (98.8 ± 0.1% a.i.) in corn oil at doses of 0 (corn oil), 50, 200, or 800 mg/kg/day

from post

- natal day

(PND) 23 to 53. Animals were examined f or preputial separation (PPS) daily beginning on PND 30 and weight at day of attainment was recorded. Following sacrifice on PND 53, total T 4 and testosterone levels were determined using chemiluminescent immunoassay s and TSH levels were determined using a radioimmunoassay. Serum was also analy zed for levels of selected clinical chemistry

parameters. Adrenal, liver, pituitary , thy roid, and urogenital organ weights were recorded, and microscopic examinations of the testes, epididy mides, thy

roid, and kidne ys were performed. One animal each in the 200 and 800 mg/kg/day groups died prior to study termination. All other rats survived until scheduled sacrifice. No clinical signs of toxicity were observed in any

of the surviving rats. The results for one rat in the 800 mg/kg/day group were excluded from the study due to a vascular abnormality

which was considered to be unassociated with treatment. No treatment

- related effects were observed on mortality , clinical signs, body weights, body

weight gains, or gross or microscopic pathology

. As shown in **Table 5**  **9** , at 800 mg/kg/day , a statistically significant (p<0.05) delay in the age of attainment of PPS (1.8 day s, adjusted for body weight on PND 23) was observed compared to the controls. Although the unadjusted a ge at attainment of PPS was increased in the 800 mg/kg/day group (+1.7 day s compared to the unadjusted mean in the control group), the difference was not statistically significant. There were no effects on the age at attainment of PPS at 50 and 200 mg/kg/ day groups. Bod y weights, bod y weight gains, and bod y weight at attainment of PPS were similar across all dose groups.US EPA

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– July , 2013

Page **179** of **211 Table 5**  **9 : General Growth and Preputial Separation (PPS) in Rats Treated with Chemical N in the Male Pubertal Assay**  **a**

**Parameter Evaluated Vehicle Control**  **Chemical N (50 mg/kg/day)**  **Chemical N (200 mg/kg/day)**  **Chemical N (800 mg/kg/day)**  **N Mean**  **SD/**

**SE b**  **CV (%)**  **N Mean**  **SD/**

**SE b**  **CV (%)**  **N Mean**  **SD/**

**SE b**  **CV (%)**  **N Mean**  **SD/**

**SE b**  **CV (%)**  Initial bod y weight (PND 23; g) U 16 54.3 4.1 7.6 16 54.6 4.7 8.6 16 54.8 4.0 7.3 15 54.9 5.5 10.0 A NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Bod y weight at PPS (g) U 16 209.9 17.5 8.34 16 211.3 19.5 9.23 15 215.8 17.9 8.30 13 218.5 16.6 7.59 A 16 210.0 4.47 NA 16 211.2 4.47 NA 15 215.8 4.61 NA 13 218.6 4.95 NA
Final bod y weight (g) U 16 275.0 28.4 10.3 16 275.5 25.4 9.2 15 283.4 26.7 9.4 14 273.4 25.6 9.4 A 16 275.5 5.13 NA 16 274.9 5.13 NA 15 283.0 5.30 NA 14 273.8 5.49 NA Final bod y weight (% of control) U NA NA NA NA 16 100.2 9.2 9.2 15 103.1 9.7 9.4 14 99.4 9.3 9.4 A NA NA NA NA NR NR NR NR NR NR NR NR NR NR NR NR Bod y weight gain (final

– initial; g) U 16 220.6 25.5 11.6 16 220.8 22.3 10.1 15 228.8 25.4 11.1 14 219.0 22.0 10.0 A NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR Age at PPS (PND) U 16 44.8 2.1 4.72 16 44.6 1.7 3.91 15 45.1 2.1 4.65 13 46.5 2.2 4.69 A 16 44.7 0.38 NA 16 44.7 0.38 NA 15 45.2 0.40 NA 13 46.5* 0.43 NA Proportion unseparated #/N) 0/16 NA 0/16 NA 0/15 NA 0/13 NA

a Data w ere o btained from the study report. b Standard deviation (SD) reported for unadjusted values and standard error of the mean (SE) reported for adjusted values. U = Unadjusted for body weight on PND 23 A = Adjusted for body weight on PND 23 N = Number of animals examined CV = Coefficient of Variation NA = N ot applicable ; Not reported ; *Significantlydifferent from controls at (p<0.05)US EPA

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Page **180** of **211** At 800 mg/kg/day , the adjusted, absolute, and relative weight of seminal vesicles + coagulating glands (with and without fluid) was decreased (p<0.05) by 24

- 26%. Seminal vesicl es weights in the 50 and 200 mg/kg/day

groups were similar to controls. There were no dose

- related effects on the weights of kidney s, pituitary

glands, adrenal glands, ventral prostate, dorsolateral prostate, levator ani

bulbocavernosus muscle (LABC), epi didy mides, testes, or thy

roid. Relative liver weight was increased (p<0.05) at 50 mg/kg/day by 7%. Adjusted, absolute, and relative liver weights were increased (p<0.05) at 200 mg/kg/day b y 16

- 20%, and at 800 mg/kg/day by 32%

- 33% compared to controls. As shown in **Table 6**  **0** , at 800 mg/kg/day , the level of serum testosterone was decreased (p<0.05) b y 58% compared to the control. L evels were also decreased at 50 and 200 mg/kg/day ; however, the decreases were not statistically significant. Serum urea nitro gen levels were decreased (p<0.05) in the 200 and 800 mg/kg/day

dose groups ( ↓25%, and ↓42%, respectively ). Levels of creatinine were increased (p<0.05) in rats dosed at 800 mg/kg/day ( ↑400%). Levels of alkaline phosphatase were decreased (p<0.05) b y 15% at 50 mg/kg/day , by 18% at 200 mg/kg/day , and by 25% at 800 mg/kg/day

. There were no dose

- related effects on serum T 4 , serum TSH, or the other evaluated clinical chemistry parameters. Chemical N was positive for the A pathway and negative for the T path way in the pubertal male assay

.US EPA

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Page **181** of **211 Table 6**  **0 : Hormone Levels and Clinical Chemistry in Rats Treated with Chemical N in the Male Pubertal Assay**  **a**
**Parameter Evaluated**  **Vehicle Control Chemical N**  **(50 mg/kg/day) Chemical N**  **(200 mg/kg/day) Chemical N**  **(800 mg/kg/day) N**  **Mean SD**  **CV**

**(%) N**  **Mean SD**  **CV (%)**  **N Mean**  **SD CV**  **(%) N**  **Mean SD**  **CV (%)**  **Hormones** Serum T 4 , Total (μg/dL) 16 5.02 0.508 10.13 16 5.15 0.621 12.06 15 5.13 0.616 11.99 14 4.67 0.610 13.08 Serum TSH (ng/mL) 16 6.29 4.045 64.27 16 8.40 6.038 71.88 15 8.07 5.673 70.33 14 5.43 4.920 90.64 Serum testosterone (ng/mL) 16 2.96 2.277 76.87 16 2.14 ( ↓25) 1.547 72.19 15 1.76 ( ↓41) 0.988 56.09 14 1.245* ( ↓58) 0.856 68.79
Creat inine (μmol/L ) 16 0.1 0.0 NA 16 0.1 0.0 NA 15 0.2 0.1 41.74 14 0.5* ( ↑400) 0.1 30.49 Serum urea nitrogen (mg/dL) 16 12 2 15.01 16 11 2 21.08 15 9* ( ↓25) 2 23.50 14 7* ( ↓42) 2 28.51 Alkal ine phosphatase (μ/L ) 16 343 69 20.20 16 292* ( ↓15) 51 17.51 15 282* ( ↓18) 46 16.35 14 258* ( ↓25) 46 18.03

a Dat a w ere obtained from the study report. Percent differences from controls, are included in parentheses N = Number of animals examined SD = Standard Deviation CV = Coefficient of Variation *Significantly different from controls at p<0.05.US EPA

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Page **182** of **211 x.**  **Fish S hort**  **- term Reproduction Assay**  The 21

- day

short

- term reproduction assay of Chemical N with fathead minnows ( Pimephales promelas ) was studied under flow

- through conditions. Adult fish, 16 spawning groups (2 males and 4 females in each group; 6 months old) we re exposed to Chemical N at nominal concentrations of 0 (control), 0.8, 8.0, and 80 mg ai/L; mean

- measured concentrations were < LOQ ( 0.199), 0.748, 7.62, and 79.8 mg ai/L , respectivel y. The test sy stem was maintained at 24.6 to 25.4 o C and a pH of 7.0 to 7.5. Adult survival in both sexes at all treatment levels was 100%. Male and female body weight were both significantl y (p<0.05) reduced 24% of control at the 79.8 mg ai/L treatment level **(Table 63**  **).** There were no notable observations in secondary sex characteristics for an y treatment group compared to the controls. However, the study author reported reduced feeding and an observable decrease in bod y weight in high

- dose males compared to the control s. Spawning frequency

occurred at least every

4 days and fecundit y in controls was 31.6 eggs/female/day

/replicate (range 19.1

- 36.5); fertilization success in the control group averaged 98.4%. Fecundity

was significantly

(p<0.05) reduced 94% at the 79.8 mg ai/L treatment **(Table 61**  **).** There was also a significant reduction in fertility of 70%, (p<0.05) at the 79.8 mg ai/L treatment level compared to the control **(Table 62)**  **.** Female vitellogenin (VTG) was significantl y (p<0.05) reduced 44 and 77% of control a t the 7.62 and 79.8 mg ai/L treatment levels, respectivel y, and was dose

- responsive. There were no significant differences in male VTG (p>0.05) between the treatment groups. There were no significant differences (p>0.05) in gonado

- somatic index (GSI ) for males or females compared to the control. Tubercle scores in males in the treatment groups were not significantl y different (p>0.05) from the control; tubercles were not observed in an y female. Although not anal yz ed statisticall y, there were no reported t reatment
- related histopathology observations in males, but there was a reported increase in incidence of mild to moderate oocy te atresia at the 79.8 mg ai/L treatment level compared to the female control fish. Sex

- steroids were not measured. Observation o f dorsal fat (nape) pads was reported but further analy sis of these data was not reported. Validity

criteria were met in this study

. Overall, several effects were observed in females and males at the highest treatment concentration of 79.8 mg ai/L ; howe ver, these effects (significantly reduced fecundity

( ↓94%), fertility

(↓70%), and body weight ( ↓24)) may

be explained by reduced feeding . Visual observations of reduced feeding and emaciation of male fish was reported , and significant body weight reductio ns were observed in both males and females at the highest treatment
. Significant decreases in female VTG were measured in the two highest treatment groups; however, there is uncertainty whether the decrease in the 79.8 mg ai/L group was due to reduced feeding

. A reduction in female caloric intake, reflected in reduced bod y weight at the high treatment level in this study , implies that less energy is available for vitellogenesis and for reproduction in general (Kime et al. 1999). The 44% reduction in VT G inUS EPA

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Page **183** of **211** females at 7.62 mg ai/L relative to controls occurred in the absence of effects on body weight but was not manifested in changes in GSI or fecundity or reported alterations in gonadal histopathology which might be expected to complement a decrease in VTG . T herefore, while the reported VTG decrease could be due to an interaction of Chemical N with endocrine function, the veracit y or biological significance of this is uncertain. Conceptually , a decrease in female VTG could be reflective of direct estr ogen antagonism or androgen agonism or antagonism, since either an increase in circulating androgens or a decrease in testosterone substrate (as a pre

- cursor to estradiol) could result in lower levels of circulating estrogens (USEPA 2007). **Table 61: Re productive and HPG Endpoints**  **for Female Fa**  **thead M**  **innow in the FSTRA** 

Abbreviations: Conc. Concentration. GSI Gonado

- Som atic Index. VTG Vitellogenin. 1 Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board

- certified pathologist. 2 Statistically significant at p<0.05. LOQ=<0.199 mg ai/L. **Mean**   ** Measured** **Treatment (mg a.i./L)**  **[measured] Fecundity**  **Fertility Success**  **Tubercle**

**Score GSI**  **Gonadal Staging and**  **Histopathology Plasma** 

**VTG**
**% Diff**  **erence %**  **Diff erence**  **Median %** 

**Diff erence**  **Effect?**

**(Yes/No) %** 

**Diff erence**  Negative Control (<LOQ) 0 0 0 0 No 0
0.748 1.03

- 0.66 0 6.76 No

- 4.33 7.62

- 5.46

- 0.41 0 10.61 No
**- 43.89**  **2** 79.8
**- 94.30**  **2**

**- 70.26**  **2** 0

- 28.32 **Yes -**  **77.18 2** US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **184** of **211 Table**  **62 :**  **Chemical N**
**- Reproductive and HPG Endpoints for Male**  **Fa thead**  **M innow**  **in the**

**FSTRA.**

Abbreviations: Conc. Concentration. Diff. Difference. GS I Gonado

- Som atic Index. VTG Vitellogenin. 1 Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board

- certified pathologist. LOQ = <0.199 mg ai/L.

**Table 63**  **: Chemical N** 
**- Growth Endpoints**  **in the Fish Short**
**- Term Reproduction Assay** 

**(FSTRA)**
**Mean -**  **Measured Treatment**  **(mg a.i./L) [measured]**  **Body Weight Length** 

**Males Females**  **Males Females**  **% Diff erence**  **% Diff**

**erence % Diff** 

**erence % Diff** 

**erence** Control (dilution water onl y), if used 0 0 0 0 0.748 13.34

- 7.96 3.48

- 2.66 7.62 4.84

- 3.35 3.53

- 0.74 79.8
**- 23.57**  **1**

**- 24.30**  **1**

- 0.10

- 3.00

1 Statisticallysignificant at p<0.05. L OQ=<0.199 mg ai/L. **Mean -**  **Measured Treatment**  **(mg a.i./L) [measured]**  **Tubercle Score GSI**  **Gonadal Staging and**  **Histopathology 1**  **Plasma VTG**
**Median %** 

**Diff erence**  **Effect? (Yes/No) % Diff**  **erence** Negative Control (<LOQ) 30.1 0 No 0

0.748 31.5

- 0.20 No 46.24
7.62 31.9 4.20 No - 48.79
79.8 23.6

- 4.40 No - 88.63US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **185** of **211 xi.**  **Am phibian Metamorphosis Assay**  The 21

- day

assay of Chemical N on amphibian metamorphosis of Xenopus laevis was studied under flow

- through conditions. African clawed frog ( Xenopus laevis ) larvae (80/treatment, [Nieuwkoop

Faber (NF), 51] were exposed to 0 (negative control), 0.600, 6.00, and 60.0 mg a.i./L. Mean

measured concentrations were <0.199 (<LOQ; control), 0.584, 5.63, and 59.0 mg a.i./L. The test sy stem was maintained at 21.7 to 22.8°C and a pH of 7.0 to 7.6. As shown in **Table 64**  **,** Chemical N ha d no significant effect (p>0.05) on normalized hind

- limb length (HLL) at an y treatment level (up to including 59.0 mg a.i./L) on Day 7 or 21. Non

normalized HLL was also anal yz ed and showed no effects at either time point. Further, there was no significa nt acceleration or delay of median NF developmental stage on Day 7 or 21. No asy nchronous development was observed in any treatment group. Mild follicular cell hy pertrophy was observed in the control and all treatment groups and did not appear to be treatm ent
- related. The only treatment
- related effects observed in this study were for the significant (p<0.05) 15 and 5% reductions, relative to the negative control, in Day 21 wet weight and Day

21 SVL at the 59.0 mg a.i./L level **(Table 65**  **).** All effects are re ported based on comparison to the negative (clean water) control. Anal ys is of Day 21 wet weight, snout

- to
- vent length (SVL), HLL, and normalized HLL endpoints excluded performance of late

- stage tadpoles ( i.e ., 21 day NF >60). There were several individua ls from the control and Chemical N

- treated groups which matured bey ond NF stage 60 by Day 21 (range of NF=61

- 62, with no clear pattern in occurrence). Late stage individuals did not comprise more than 20% of an y treatment level and there was no evidence to suggest a dose

- dependent relationship for late stage individuals ( i.e ., 13% of individuals were NF>60 in the negative control group, compared to 8, 10, and 18% in the 0.584, 5.63, and 59.0 mg a.i./L treatment groups, respectivel y). At test termination, ta dpoles with bent tails were observed in the control (17%) and all treatment groups (20, 29, and 17%) and, like thy roid gland observations, did not appear to be related to Chemical N exposure. No apparent cause of this common malformation was reported. Po tentially relevant nutritional and water quality variables in the study are described here, for context. Sera Micron feed was administered at one

- half the guideline recommended rate. Animals were maintained in an in

house culture; the original source of the animals was not reported. Iodide in dilution water was measured, but the measurement and the guideline

- recommended iodide range (0.5 to 10 ug/L) were below the limit of detection (<50 ug/L). It was not reported whether an y iodide supplementation was provided bey ond what was available in the diet (54 ug/g). Continuous flow diluters provided complete turnover every

2.7 hours. The dilution water was natural surface water treated by a municipal drinking water plant and was then sand

- and carbon

- filtered , buffered, and radiated with ultraviolet prior to use. The dilution water source was previously anal yz ed for pesticides, heav y metals, and other contaminants. Ammonia (<10 mg/L), perchlorate (<0.2 mg/L), and chlorate (<10 mg/L) were below the limits of detection. Water hardness (mean 69.5 mg/L as CaCO 2 ) was slightl y higher than recommended in the guideline (40

- 48 mg/L as CaCO 2 ). Ultimately , the presence of bent tails in these tadpoles was not considered to have impacted the stud y conclusions in this case.US EPA

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– July , 2013

Page **186** of **211** The absence of significant effects on thy roid

- related endpoints in the AMA suggests that Chemical N did not interact with the amphibian HPT axis or its downstream thy roid hormonal pathway

under these study

conditions. **Table 64**  **: Developmental and Thy**  **roid Gross Pathology/Histopathology Endpoints 1,2**  **in the AMA with Chemical**  N

.

**Treatment (mg a.i./L)**  **[mean**   ** measured]** **NF Developmental Stage Hind Limb Length**  **3 Asynchronus**  **Development Thyroid Gross**  **and Histopathology**  **Day 7 Day 21**  **Day 7 Day 21**  **Day 7 Day 21**  **Day 21 Median**  **p Median**  **p %**  **Diff. P**  **%**

**Diff. p**  **%**

**Diff. P**  **%**

**Diff. p**  **Treatment**  

** Related Effects** **(Yes/No)** Negative Control 54

-- 59

-- -- -- -- -- -- -- -- -- No 0.584 54 0.16 60 0.44 0.12 0.99 0.37 0.28

-- -- -- -- No 5.63 54 0.18 60 0.28 2.25 0.87 3.30 0.21

-- -- -- -- No 59.0 54 0.12 60 0.10 2.60 0.82 4.03 0.16

-- -- -- -- No Statistical Test Jonckheere

Terpstra Jonckheere

Terpstra Dunnett Jonckheere

Terpstra N/A N/A N/A
A b b r e v i a t i on s : Di ff

. D i ff e r e n c e. NA Not a p p li c a b l e. 1 U n l e s s othe r w i s e i nd i c ated, effe c ts a r e r epo r ted ba s ed on c om pa r i s on to the cl ean w ater c ont r o l

. Con cl u s i ons r ega r d i ng h i s t o patho l ogy may be hea v i l y w e i ghted by the e x pe r t op i n i on of a boa r d

- c e r t i f i ed patho l og i s t. 2 U n l e s s ot h e r w is e s p e c i f i e d, ef f e c ts a r e c on si d e r e d s t at i s t i c a l l y si g n i f i c a nt at p < 0.05. 3 H i nd

- l i m

b l e ng t h i s no r m a l i z ed to s n ou t
- v ent l e ng t h ( S V L )

. H S e v e r al t ad

p o l es w e r e e x c l ud e d f

r om the a n a l y s i s of Day 2 1 e n dp o i nts f o r w et we i g ht, S V L, HLL, a nd n o r m HLL. E x cl ud e d i n d i v i du a l s c o m p ri s e d 13, 8, 10, a nd 1 8% of t he c o nt r o l , 0.5 8 4, 5

. 63, a nd 59.0 m g a i / L t r eat m ent g r o u p to t al s a m p l e si z e s , r e s p e c t i v e ly

. La t e s tage i n d i v id ua l s r a ng e d f r om N F 6 1 to 6 2 w i th no ap p a r ent pat t e r n of o cc u r r en c e o r do s e - d e p en d en c y .US EPA

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– July , 2013

Page **187** of **211 Table**  **65 :**  **Growth Endpoints 1**  **, 2**  **in the AMA with Chemical N**  **. Treatment (mg**  **a.i./L) [mean**  **** **measured] Snout** 
**- Vent Length**  **Body Weight**

**Day 7 Day 21**  **Day 7 Day 21**  **% Diff**  **erence p**  **%**

**Diff erence**  **p %** 

**Diff erence**  **P %** 

**Diff erence**  **p** Negative Control

-- -- -- -- -- -- -- -- 0.584

- 0.78 0.99

- 0.90 0.39 0 0.99

- 2.68 0.28 5.63 2.54 0.93

- 0.58 0.33 11.48 0.80

- 2.73 0.33 59.0

- 2.96 0.90

- 4.86 0.013

- 3.93 0.99

- 15.1 0.016 Statistical Test Dunnett Jonckheere

Terpstra Dunnett Jonckheere

Terpstra
1 U n l e s s o th e r w i s e i n d i c a t ed , e f f e c ts a r e r e po r ted b a s ed on c o m p a ri so n to t

h e n eg a t i v e ( c l e an w a t e r ) c o n t r o l

. 2 U n l e s s o t

he r w i s e s p e c i f

i ed , e ffe c ts a r e c o n s i d e r e d s t a t i s t i c a l l y s i gn i f i c an t a t p < 0 .0 5. H S e v e r a l t a d po l e s we r e e x c l ud ed f r om t h e a na l y s i s o f D a y 2 1 en d p o i n ts f o r w et we i g h t, S V L , H LL , and no r m HL L

. E x c l ud ed i nd i v i d ua l s c o m p ris e d 13 , 8 , 10 , a n d 18 % o f t h e c on t r o l , 0 .58 4 , 5 .6 3 , a n d 59

. 0 m g a i /L t r ea t m e n t g r o up t o t a l s a m p l e s i z e s , r e s pe c t i v e ly

. La te s t a ge i n d i v i d ua l s r an g ed f r om NF 61 t o 62 w i th no a pp a r en t p a tt e r n o f o c c u rr en c e o r d o s e - d e p en d e n c y . **D. Other**  **Scientifically Relevant**  **Information (OSRI) for Chemical N** 

**1. Mammalian OSRI Data**  The registrant elected to conduct the entire Tier 1 assay . Howev er, limited in vitro and mammalian in vivo data were submitted as OSRI

. These studies are discussed below Chemical N was evaluated in a ER α ye ast two
- hy brid (Y2H) s ys tem. The sy stem is based on a ligand

- dependent interaction of two proteins, a hormone receptor and a coactivator, and hormonal activity is detected by

β

- galactoside activity . Chemical N had a 10% relative effective concentratio n (REC10; the concentration showing 10% of the agonist activity of 10 - 7 M 17β

- estradiol) of > 10 - 4 M. Chemical N was also evaluated in ER α chloramphenicol acetyltransferase transcriptional activation assay . Chemical N was negative in this assay up to the highest concentration tested (10 - 5 M). In a 90

- day

subchronic oral toxicity

study , groups of 10 male and 10 female Sprague

- Dawley rats were given Chemical N (unspecified purit y) administered at 0, 750, 1500 or 3000 ppm in the diet (equivalent to 0, 57, 102 .5 or 233.8 mg/kg bw/day in males and 0, 78.9, 163.8 or 311.8 mg/kg bw/day in females) resulted in decrease in body weight of onl y males rats in high dose group. No effect on testes or th yroid weight was observed. No histopathological findings were seen in the testes, prostate, ovaries, uterus, pituitary or thy roid. For females, the NOAEL was 311.8 mg/kg bw/day ; a LOAEL was not established. For males, the NOAEL was 233.8 mg/kg bw/day and LOAEL was 102.5 mg/kg/day based on the decrease in body weight.US EPA

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– July , 2013

Page **188** of **211** In a 90

- day

subchronic oral toxicity

study , groups of beagle dogs (4 dogs/sex/dose) were given Chemical N (unspecified purity ) in gelatin capsules at 0, 35, 75 or 150 mg/kg bw/day

. No effect on thy roid weight was observed. No histopathological findings were seen in the testes, prostate, seminal vesicle, ovaries, uterus, pituitary or thy roid. The NOAEL was 150 mg/kg bw/day (highest dose tested); a LOAEL was not established In a carcinogenicity

study , Chemical N (94% and 97%) was administered via gavage to 5 0 F344/N rats/sex/dose in corn oil at dose levels of 0, 250 or 500 mg/kg bw/day , 5 days/week for 103 weeks. Carcinomas of the preputial gland were increased in high dose male rats. Under the conditions of this study , there was some evidence of carcinogeni city in male F344/N rats as shown by the occurrence of renal tubular cell adenomas and adenocarcinomas and carcinomas of the preputial gland. There were no effects on the weights of the testes, ovaries, or thy roids. No histopathological effects were seen in the testes, prostate, ovaries, uterus, mammary , or th yro id. There was no evidence of carcinogenicity in female F344/N rats. In a carcinogenicity

study , Chemical N (94% and 97%) in corn oil was administered via gavage to 50 B6C3Fl albino mice/sex/dose a t dose levels of 0, 250 or 500 mg/kg bw/day , 5 day s per week for 103 weeks. In the high dose male mice, Chemical N exposure was associated with increased incidences of hepatocellular adenomas and carcinomas) and of mesench ym al tumors of the integumentary

s ys tem (fibroma, fibro sarcoma, neuro fibrosarcoma, or sarcoma). No compound

related neoplastic or non

- neoplastic lesions associated with Chemical N exposure were seen in female mice. No effects on the weights of the testes, ovaries, mammary , or thy roids w ere observed. No histopathological effects were seen in the testes, prostate, ovaries, uterus mammary , or thy roid. There was no evidence of carcinogenicit y of Chemical N in female B6C3Fl mice. In a developmental toxicity study , Chemical N [96.8% purity ] was administered b y the whole

- body inhalation route, as a vapor, to groups of 22 mated female CD

- 1 mice and 22 mated female Fischer 344 rats at dose levels of 0, 25, 50, or 115 ppm [0,0.14, 0.28, or 0.64 mg/L ] from day s 6 through 15 of gestation. For mice, the maternal NOAEL was 50 ppm and the LOAEL was 115 ppm, based on decreased gestation day

18 body weight, corrected for uterine weight. A developmental LOAEL was not established. The developmental NOAEL was 115 ppm, the highest dose tested. For rats, the maternal NOAEL was 25 ppm and the LOAEL was 50 ppm, based on an increased incidence of clinical signs. The developmental NOAEL was 50 p pm and the LOAEL was 115 ppm, based on the significantl y decreased fetal crown

- rump distance in female fetuses.US EPA

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– July , 2013

Page **189** of **211 2.**  **Ecotox icity OSRI Data**  No ecotoxicity OSRI were available for review.

**E. Discussion**  **i. Effects**  **on H**  **y pothal**  **a m**  **i c**  **- Pituit**  **a r**  **y -**  **Gonad a**  **l (HPG)**  **A x**  **is** EDSP Tier 1 screening data and OSRI were evaluated together to determine whether Chemical N has potential to interact with the HPG axis or its downstream pathways related to steroidogenesis or sex steroid signaling. The Tier 1 in vitro assay s relevant to this analy sis include the AR, binding, ER binding, ERTA, aromatase, and steroidogenesis assay s. Relevant in vivo assay s include the male and female pubertal rat, uterotrophic, Hershberger, and the FSTRA assay s. **E f**  **f ect**  **s on E**  **s tr**  **og e**  **n P**  **at h**  **w ay**  **: T**  **a ble**  **66** b e low summ ar i z e s the re sults of the a v a il a ble ED S P studi e s a nd e ndpoints re l e v a nt f

or d e t er mining the po t e nti a l f

or Chemical N to int erac t with the e st r og e n signaling p a th w a y . Effects on body weight or size are noted; such effects are ty picall y in response to generalized toxicity

but could be endocrine mediated. Any other signs of overt toxicity or cy totoxicity in relevant in vitro and in vivo assay s are identified to help dist inguish responses due to generalize toxicity

versus endocrine activity and to provide a characterization of the observed responses and an y uncertainties. Chemical N did not produce responses in the in vitro assay s, the in vivo Uterotrophic assay , or the fe male pubertal rat assays indicative of estrogen agonism or antagonism, or disruption of ster oi dogenesis . There were, however, some responses that may potentially

be related to interaction with the estrogen or androgen pathway s reported in FSTRA. Plasma V TG was significantly lower in female fish exposed to an intermediate concentration (7.62 mg/L) of Chemical N than in control females ( ↓44%, p<0.05) . N o overt toxicity , such as reduced feeding or reduced body weight, was observed at this treatment level. Reduced feeding was observed in male fish and body weights were significantl y reduced in both male and female fish at the high treatment level (79.8 mg/L) , where reproduction was almost completely suppressed and multiple other endpoints were affected: fecu ndity ( ↓ 94% ) , fertilization success ( ↓ 70% ) , female plasma VTG ( ↓ 7 7 % ) , and a slight increase in oocy te atresia which could be consistent with impaired reproduction and reduced VTG

. While reduced feeding and lower bod y weights might account for effects at the high treatment level because less energy was available for reproduction, this does not explain the 44% decrease in female VTG at the intermediate treatment level. However, no other complementary responses consistent with an effect on the E (or A) hormonal pathway s were observed at the intermediate treatment level. When considered together, the absence of effects on estrogen receptor binding, estrogen receptor transactivation, and uterine weight in the uterotrophic assay suggest that Chemical N does not interact with the estrogen signaling pathway as an estrogen receptor agonist or antagonist. There was also no response detected in the in vitro steroidogenesis assay . The reduct ion in plasma VTG ,US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **190** of **211** increase in oocy

te atresia, and effects on other non

- endocrine

- specific endpoints (fecundity and fertilization success) may be consistent with an effect on the estrogen signaling pathway through perturbation of the HPG axis. However, th e onl y effect observed in the absence of overt toxicity was at an intermediate test concentration in the FSTRA in which there was a 44% reduction in female VTG which did not manifest into higher level effects . All other effects in this study were observed at the high concentration, which approaches the historical limit concentration for testing with pesticide active ingredients.

.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **191** of **211 Table**  **66 :**  **Estrogenic/Anti**
**- Estrogenic Pathway for Chemical N** 
**L in**  **e s**  **of E**  **vidence I**  **n dicating**  **Potential for**  **I nteraction**  **w ith**  **t he**  **Es trogen/Anti** 
**- Estrogenic**  **Path w**  **a y for Chemical N**  **a**
**.**

**S t**  **udy T**  **y p**  **e/Literature Citation** **E R** **B i** **n d** **i n** **g E** **R A** **c t** **i v** **a t** **i o** **n A** **r o** **m a** **t a** **s e** **S t** **e r** **o i** **d o** **g e** **n e** **s i** **s U** **t e** **r i** **n e** **W e** **i g** **h t** **O v** **a r** **i a** **n W** **e i** **g h** **t o** **r G** **o n** **a d** **o -** **S o** **m a** **t i** **c I** **n d** **e x** **( G** **S I** **) G** **o n** **a d** **a l** **S t** **a g** **i n** **g a** **n d** **H i** **s t** **o p** **a t** **h o** **l o** **g y** **P i** **t u** **i t** **a r** **y W** **e i** **g h** **t E** **s t** **r o** **u s** **C y** **c l** **i c** **i t** **y b** **A g** **e &** **W i** **g h** **t a** **t V** **O F** **e c** **u n** **d i** **t y** **F e** **r t** **i l** **i z** **a t** **i o** **n s** **u c** **c e** **s s** **V i** **t e** **l l** **o g** **e n** **i n** **O v** **e r** **t T** **o x** **i c** **i t** **y o** **b s** **e r** **v e** **d b** 

**EDSP Tier 1** ER B i nd i ng Not

Interactive E R T A Negative
A ro m atase Non

Inhibitor S t e r o i do g en e s i s Negative
U ter o tr o ph i c No effect Fe m a l e Pube r t al R at No effect No effect No effect No

effect No effect No effect
FS T R A No effect ↑ Oocy

te atresia H ↓94% H ↓70% H ↓44% ♀M ↓77% ♀HUS EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **192** of **211 OSRI**  Yeast 2

- hy

brid assay ER α Negative ER α transcriptional activation Negative
Subchronic toxicity (Rat) No effect
Subchronic toxicity (Dog) No effect Carcinogenicit y (Rat) No effect No effect Carcinogenicit y (Mouse) No effect No effect Developmental toxicity (Rat) Developmental toxicity (Mouse)
a K e y to r e s pon s e s: L Low treatment, M Medium treatment, H High treatment. A rr o w s ( ↓ o r ↑ ) i nd i ca te t h e d ir ec ti o n o f t h e r e s pon s e. A shaded cell i n d i ca t e s t h a t p a r a m e t e r is not routinely eva l u a t e d o r is n o t ap p li ca b l e in the assay

. b An “X” in this column indicates that the effect(s) observed in this assay occurred in the presence of overt toxicity

.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **193** of **211 E**  **f f**  **ects on**  **A nd**  **rogen P**  **a t**  **h w**  **ay :**  **T a**  **ble 67**  summ ar i z e s the re sults of the a v a il a ble ED S P studi e s a nd e ndpoints re l e v a nt f

or d e t er mining the po t e nti a l f

or Chemical N to int erac t with the androgen signaling p a th w a y . Effects on body weight or size are noted; such effects are ty picall y in response to generalized toxicity

but could be endocrine mediated as a decrease in T could is often associated with a decrease in bod y weight. An y other signs of overt toxicity or cy to toxicity in relevant in vitro and in vivo assay s are identified to provide a characterization of the observed responses and any uncertainties, e.g. , to help distinguish effects due to generalized toxicity from potential endocrine activity , when possible. Chemical N caused no effects consistent with an androgenic or anti

- androgenic response in the in vitro assay s or the in vivo Hershberger assay . However, in the male pubertal rat assay , there was a statistically

significant decrease in serum testosterone (58% lower than controls) at a dose near the limit dose (800 mg/kg bw / day ). Other effects found at 800 mg/kg bw / day included a ~24% reduction in the weight of seminal vesicles and a 1.8 day delay in PPS. No effects were seen at lower doses (50 and 200 mg/ kg bw/day ). Effects in fish described in the previous section may also be relevant to the androgen signaling pathway ; however, the onl y effect unaccompanied by reduced body weight (males and females) was a 44% reduction in female plasma VTG at the medium treatment level (7.62 mg/L). When considered together, the absence of effects in mechanistic studies but presence of some in vivo effects in intact rats and fish suggest that Chemical N may alter the androgen signaling pathway through HPG axis perturbati on or another unspecified mechanism

.US EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **194** of **211 T**  **a b**  **le 67**  **: Androgenic Pathway for Chemical**  **N L**  **in e**  **s o**  **f E**  **v idence**  **I n**  **dic at**  **ing Pot**  **en t**  **i a**  **l for**  **I n**  **t er**  **a c**  **t i**  **o n**  **w i**  **t h**  **t he**  **Androgenic/An t**  **i**
**- Androgenic**  **Activity for Chemical N a** 
**.**

**S t**  **ud y**  **T y**  **p e**  **/ Literature Citation** **A R** **B i** **n d** **i n** **g S** **t e** **r o** **i d** **o g** **e n** **e s** **i s** **T e** **s t** **o s** **t e** **r o** **n e** **T e** **s t** **e s** **W e** **i g** **h t** **o r** **G o** **n a** **d o** **- S** **o m** **a t** **i c** **I n** **d e** **x (** **G S** **I )** **G o** **n a** **d a** **l S** **t a** **g i** **n g** **a n** **d H** **i s** **t o** **p a** **t h** **o l** **o g** **y E** **p i** **d i** **d y** **m i** **d e** **s W** **e i** **g h** **t E** **p i** **d i** **d y** **m i** **d e** **s H** **i s** **t o** **p a** **t h** **o l** **o g** **y P** **i t** **u i** **t a** **r y** **W e** **i g** **h t** **A c** **c e** **s s** **o r** **y S** **e x** **O r** **g a** **n W** **e i** **g h** **t s** **A g** **e a** **n d** **W e** **i g** **h t** **a t** **P P** **S S** **e c** **o n** **d a** **r y** **S e** **x C** **h a** **r a** **c t** **e r** **i s** **t i** **c s** **V i** **t e** **l l** **o g** **e n** **i n** **O v** **e r** **t T** **o x** **i c** **i t** **y O** **b s** **e r** **v e** **d b** 

AR B i nd i ng Not

Interactive S t e r o i do g en e s i s Negative Hershberger No effect No effect No effect No effect
Ma l e Pube r t al R at ↓58% H No effect No effect No effect No effect No effect ↓24% H S V ↑1.8 H

FS T R A Not measured No

effect ↑ Oocy

te atresia H No effect ↓44% ♀M ↓77% ♀HUS EPA

– SAP Review of EDSP Tier 1 Screening: Weight of Evidence

– July , 2013

Page **195** of **211** OSRI Subchronic toxicity (Rat) No effect No effect Subchronic toxicity (Dog) No effect No effect Carcinogenicit y (Rat) No effect No effect Carcinogenicit y (Mouse) No effect No effect

a K e y to r e s pon s e s: L Low treatment, M Medium treatment, H High treatment; d Days; Sem ves Seminal vesicles + coagulating glands. A rr o w s ( ↓ o r ↑ ) i nd i ca te t h e d ir ec ti o n o f t h e r e s pon s e. A shaded cell i n d i ca t e s t h a t p a r a m e t e r is not routinely eva l u a t e d o r is n o t ap p li ca b l e in the assay

. b An “X” in this column indicates that the effect(s) observed in this assay occurred in the presence of overt toxicity

. SV= Seminal vesicleUS EPA

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Page **196** of **211 Summary**  **HPG Axis :**  There were limited complementary and redundant responses in vivo with respect to potential interaction of Chemical N with the estrogen signaling pathway . There were some responses in FSTRA that may be suggestive of an effect on the estrogen (antagonist) or androgen (antagonist or agonist) signaling pathway , but most of these responses were observed at aquatic concentrations approaching the historical limit concentration for testing with pesticides (100 mg/L)

. At the high treatment level, the impairment o f reproduction and associated endpoints may be reflective of a non - endocrine MoA , given the observations of reduced feeding in male fish and reduced bod y weight in both males and females. For example, the Agency ’s Assessment Tool for Ecological Risk (ASTE R) classifies Chemical N, a ketone, as a nonpolar narcotic, meaning that baseline toxicity

data for this chemical structure fit the mathematical model for a substance with no specific, receptor

- mediated MoA (USEPA 2013)

. However, there was no reduction in feeding or body weight reported at the intermediate treatment level where a significant, 44% reduction in female VTG was still observed. For the androgen signaling pathway (anti

- A) there was some complementarit y found in the male pubertal assay and some redundancy in response when the effects observed in FSTRA are viewed together with those in the male pubertal assay . Lower testosterone and effects on androgen

- sensitive tissues (seminal vesicles) and developmental timing (age at PPS) were observed in mal e pubertal rats near a limit dose of 800 mg/kg/day

. Given that testosterone (T) and estrogen (E) were not measured in the FSTRA, the hy pothesis of potential E or A antagonism as a cause for the decrease in female fish VTG was not confirmed. . However, a decrease in VTG would be consistent with a decrease in E given that testosterone (T) was decreased in the male pubertal and is a precursor to E. A decrease in E may lead to a decrease in VTG. The absence of effects consistent with anti

- A MoA in the HPG

- interrupted Hershberger assay may suggest an effect higher in the HPG axis.

**ii. Effects on HPT Axis and**  **Th y**  **r oid**  **Hormone Pathway**  In general, regulation of the HPT axis is comparable to the HPG axis, except that the feedback relationship involves thy

roid hormo nes ( e.g ., T 4 and T 3 ). Thy roid hormones feedback to the hy pothalamus or pituitary

to regulate thy

rotropin

- releasing hormone and TSH, respectivel y, which, in turn, regulate hormone production by the thy roid gland. The current Tier 1 screening battery does not have a specific in vitro assay to detect chemicals with the potential to affect h ypothalamic or pituitary

regulation of thy

roid hormone production, but it does include three in vivo assay s that have the potential to detect these effects on the HPT axis ( i.e., the puberta l s and the AMA ).
**Thyroid Hormones: T**  **a ble**  **68** s u mm ar i z e s the re sults of the a v a il a ble ED S P studi e s a nd e ndpoints re l e v a nt f or d e t er mining the po t e nti a l f

or Chemical N to int erac t with the HPT axis or thy roid hormone signaling p a th w a y . Effects on bod y weight or size are noted; such effects may be endocrine

- mediated or in response to generalized toxicity . Other signs of overt toxicity are identified to provide context for the observed responses. There are currently no in vitro assay s in the Tier 1 battery

relevant to this modality

.US EPA

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Page **197** of **211** The Tier 1 assay s showed no evidence of interaction with the HPT axis or thy roid hormonal pathway in pubertal rats or metamorphosing amphibians. The slight reductions in amphibian body weight and size and eff

ects on some mammalian clinical chemistry parameters at the high treatment levels demonstrate that the exposure was sufficiently high to challenge the test organisms. No OSRI was available relevant to the thy roid hormonal pathway

. Over all, the Tier 1 screening data suggest that Chemical N does not interact with the HPT axis or thy roid hormonal pathway

.US EPA

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– July , 2013

Page **198** of **211 Table 68: Thyroid Pathway for Chemical N**  **Lines of Evidence Indicating Potential for Interaction with the Thyroid Pathway for Chemical N**  **a**

**S t**  **udy T**  **y pe**  **/Literature Ciation** **D e** **v e** **l o** **p m** **e n** **t a** **l S** **t a** **g e** **( A** **m p** **h i** **b i** **a n** **) A** **s y** **n c** **h r** **o n** **o u** **s D** **e v** **e l** **o p** **m e** **n t** **( A** **m p** **h i** **b i** **a n** **) H** **i n** **d L** **i m** **b L** **e n** **g t** **h b** **( A** **m p** **h i** **b i** **a n** **) T** **h y** **r o** **i d** **W e** **i g** **h t** **T h** **y r** **o i** **d H** **i s** **t o** **p a** **t h** **o l** **o g** **y T** **4 T** **S H** **P i** **t u** **i t** **a r** **y W** **e i** **g h** **t B** **o d** **y W** **e i** **g h** **t (** **B W** **) o** **r A** **m p** **h i** **b i** **a n** **S n** **o u** **t -** **V e** **n t** **L e** **n g** **t h** **( S** **V L** **) O** **v e** **r t** **T o** **x i** **c i** **t y** **O b** **s e** **r v** **e d** **b**

M ale P ub e r tal ( R at) No effect No effect No effect No effect No effect No effect F e m ale P ub e r tal ( R at) No effect No effect No effect No effect No effect No effect Amphibian Metamorphosis No effect No effect No effect No effect ↓15% BW H ↓5% SVL H Subchronic toxicity

(Rat) No effect No effect
Subchronic toxicity

(Dog) No effect No effect
Carcinogenicit y (Rat) No effect No effect
Carcinogenicit y (Mouse) No effect No effect

a K e y to r e s pon s e s: L Low treatment, M Medium treatment, H High treatement. A rr o w s ( ↓ o r ↑ ) i nd i ca te t h e d ir ec ti o n o f t h e r e s pon s e ; A shaded cell i n d i ca t e s t h a t p a r a m e t e r is not routinely eva l u a t e d o r is n o t ap p li ca b l e in the assay

. b Hind

- limb length is norm alized to snout

- vent length prior to analysis.US EPA

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Page **199** of **211 F.**  **Conclusions for Chemical N**  The absence of effects in the mechanistic in vitro , uterotrophic, and Hershberger assay s suggests that Chemical N does not affect estrogen or androgen receptor

- mediated signaling. The in vivo results of the Tier 1 screen may

indicate, however, that Chemical N may alter the estrogen or androgen signaling pathway s through perturbation of the HPG axis. For the estrogen pathway there were no redundant responses across assay s , and limited complementary responses within the FSTRA , at treatment levels without reduced feeding

. For the androgen pathway there was some redundancy across two in vivo assay s (fish and male pubertal rat) , and complementary responses within the in vivo male pubertal assay suggestive of an anti

- A effect . Although several responses were found even in the absence of overt toxicity , most re sponses occurred near limit doses of Chemical N. Given the high doses or aquatic concentrations needed to elicit these responses and the reduced feeding and bod y weights observed at high test level in fish, there is some uncertaint y as to whether the obse rved responses may be secondary

to to a non
- endocrine MoA , e.g. , nonpolar narcosis

. The Tier 1 data showed no evidence of potential interaction with the HPT axis or thy roid hormonal pathway

.

**10. Overall Conclusions in Context of WOE Guidance**  The purpose of this section is to come back to the decision framework articulated in the 2011 EDSP WoE guidance (as summarized in Section 3) and illustrate with four case studies how the agency plans to implement the process to determine the potential of a chemical to interact with the E, A, or T signaling pathway s using Tier 1 data OSRI. Because the EDSP WoE guidance is based on concepts routinely employ ed in the agency ’s WoE evaluations, the purpose of this analy sis is not to evaluate the guidance itself but rather its proposed implementation. As discussed in Section 3, there are several questions that are pertinent to the evaluation of Tier 1 screening data. However, the focus of the current anal ys is was on the WoE process for determining (1) whether or not there is a chemical interaction with E, A, or T signaling pathway s,(2) at what level of biological organization is that interaction expressed ( e.g ., receptor/target binding, cell responses, enz yme activity, organ response etc) , and under what condit ions ( e.g., in an organism with an intact HPG ; in the absence of overt toxicity ; at what doses, duration and route of exposure ; is metabolic activation required) does the chemical interact with and perturb the endocrine s ys tem. Determining responses to qu estions 1 and 2 is the fundamental anal ys is that provides the critical basis for Tier 2 testing decisions, and thus the current review focuses on data interpretation as it relates to these scientific questions.
As discussed in Section 4, the selection o f the first set of chemicals for EDSP Tier 1 test orders was based on their potential for exposure and was not based on their potential to interact with endocrine

mediated processes

. The subset of 21 chemicals used for the assay and battery

performance an al ys is presented to the May

2013 SAP represented a broad range of ph ys ical

- chemical properties, chemical classes and pesticidal modes of action and associated toxicities. The further subsample used as caseUS EPA

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– July , 2013

Page **200** of **211** studies for this WoE analysis also span s a broad range of phy sical

- chemical properties and toxicities; however, they

were also selected to be illustrative of issues that are anticipated to be encountered over the broader range of test order chemicals and Tier 1 assay s potentially impacting the anal ys is o f complementarit y within assays and redundancy across the battery of assays. The four case studies presented in **Sections 6**
**- 9**  are illustrative of the vary ing degrees of complementarit y and redundancy observed within and across the Tier 1 assay s responses. The case studies also demonstrate the vary ing responses along the continuum of biochemical changes, at lower levels of biological organization as measured in vitro, as well as responses at higher levels of biological organization ( e.g ., tissue, organ, and organism) as measured through in vivo studies of intact organisms. A pivotal aspect of the EDSP WoE guidance is use of a h ypothesis

- based approach. This hy pothesis

based anal ys is is supported by the AOP/MoA conceptual framework that promotes a s ys tematic and transparent approach to integrate all relevant t ypes of data in characterizing responses at different levels of biological organization ( e.g ., receptor/target binding, cell responses, enzy me activity , organ response, males and fe male sy stem integrit y, development (thyroid) and comparative system integrit y ) (see SAP 2010; SAP 2011 b ; SAP 2012 a, b ). Inherent within the AOP/MoA concept is the importance of understanding the adaptive or homeostatic capacity of biological s ys tems and t heir limits, relative to concentration/dose and duration of exposure. Thus, a biological process may be perturbed at lower levels of biological organization but the biological sy stem may compensate, or depending on the degree of perturbation may

become un stable, and therefore, dy sfunctional. The Tier 1 in vivo data along with other available in vivo data ( e.g ., from Part 158) provide the means to evaluate perturbations at lower levels of biological organization that may or may not progress to apical endp oints in vivo

. The evaluation of the experimental data concerning E, A, or T h ypotheses on chemical interactions is based on the WoE principles of biological plausibility , coherence, strength, and consistency of the body of evidence, as evaluated through observations of the degree of complementarity and redundancy

among responses. These principles are not unique to the EDSP WoE but are common to the Agency ’s overall process for evaluating data ( e.g ., EPA, 2005). This conceptual framework, as considered in the context of AOP/ M o A , enables a robust characterization of the degree of confidence in conclusions regarding E, A, or T interactions. As discussed below and as alluded to in the preceding discussions on Chemicals A, S , J and N, all four case studies i llustrate this hy pothesis

- based decision framework within the EDSP WoE guidance and emphasize the importance of understanding the responses along a continuum consisting of different levels of biological organization.US EPA

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– July , 2013

Page **201** of **211** The determination whether a chemical is interacting with a particular pathway is based on an anal ysis of endpoints that are identified in each of the EDSP Tier 1 assay s ( **Table 3** ). While the in vitro studies contain comparatively

few endpoints, the in vivo Tier 1 studies have multiple endpoin ts and in some cases have decision logic trees 11 to assist reviewers in determining the extent to which complementarity exists and the degree of diagnostic utility in discerning an endocrine interaction. This decision logic is intended to provide a consiste nt and transparent process for evaluating studies across stud y reviewers and reduces potential reviewer bias. As discussed in Section 5, it is important to demonstrate that the assay was conducted at a high enough concentration to allow for the possible ob servation of effects; the EDSP Tier 1 assay s provide guidance on selecting maximum tolerated dose (MTD) or the limit dose used. I n achieving an adequate high dose, it is recognized that some sy stemic (overt) toxicity may be observed or conversel y that rela tively high exposure levels may

be utilized in limit testing that can potentially challenge the ability

of the test organism to clear the test material. As indicated in the previous white paper (USEPA 2013) on test assay and battery

performance, the major ity of chemicals for which the first set of Tier 1 test orders was issued has pesticidal modes of action and are designed to be toxic. That white paper also discussed the extent to which overt toxicity was observed across many of the in vivo Tier 1 assay s and that in many cases this overt toxicity

appeared to be consistent with the pesticidal mode of action for the test chemical. Even for inert chemicals, sufficiently high test concentrations can result in more general forms of toxicity

such as nonpolar narcosis that can interfere with ADME of the test material. An understanding of the mode of toxicity informs the weight that is placed on certain Tier 1 responses in the presence of overt toxicity . For example, in the case of Chemical A, the effects found at the high concentration in FSTRA may be a reflection of cholinergic intoxication and a compromised organism with limited ability

to maintain reproductive function and homeostasis. L ikewise, in the case of Chemical S, the majority of Tier 1 in vivo responses were decreases in the measured endpoints and were largel y expressed in the presence of overt toxicity . This could be consistent with its pesticidal mode of action as an uncoupler of mitochondrial oxidative phosphory lation resulting in de pletion of ATP. 

### Chem **ical A** In apply ing the WoE guidance framework, the c ase s tudy with Chemical A is an example of how a chemical appears to initiate a molecular event along a pathway via binding to the androgen receptor and by altered steroidogenesis a s measured the in vitro Tier 1 assay s. These molecular initiating events appear to translate into in vivo responses indicative of anti

- androgenic effects in the Hershberger assay ( using the castrated rat) with an increasing dose trend in regards to the ma gnitude of organ weight decreases and number of tissues affected. Chemical A, however, did not produce an y organ weight or histological effects in androgen

- dependent tissues when evaluated in the Tier 1 male rat pubertal assay

11 The FSTRA decision tree is presented in Section 4 which is new proposal for SAP comment, while the AMA decision tree is well established and included in both EPA and OECD test guidelines.US EPA

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Page **202** of **211** and other mammalian toxicolo gy studies (including the 2

- generation reproductive, subchronic and chronic studies) available through Part 158. Although there were no effects on sexual development in the male pubertal Tier 1 assay up to 100 mg/kg bw / day , there was a slight delay in the age of onset for male pubertal development at 24 mg/kg bw / day reported in a developmental neurotoxicity test (DNT) which assesses an animal with an intact brain

- pituitary

- gonadal axis that may provide adequate compensatory

changes . Given the lack of effec ts in the Tier 1 male pubertal assay , the slight delay in PPS observed in the DNT might be spurious. In the FSTRA, Chemical A produced an increasing dose trend in the number of endpoints that could be consistent with an anti

- androgen response; however, sev eral of these measurement endpoints occurred at test concentrations exhibiting overt toxicity

. In the absence of overt toxicity at lower concentrations, two endpoints were affected which lack specificit y as to their diagnostic ability ( i.e ., GSI and hist ology ). Using the EDSP WoE hy pothesis

- based approach in evaluating responses along a biological continuum, based on in vitro Tier 1 data, Chemical A has the potential to initiate the androgen AOP at a molecular level (via AR binding or altered steroidogene sis). With a gonadectomized in vivo model (Hershberger assay), anti

- androgenic responses at the tissue level occurred. Within the context of an intact mammalian organism, the Tier 1 male pubertal data and the Part 158 subchronic, chronic, and 2

- generation reproductive data did not support an anti

- androgen AOP. Thus, within the in vitro and the mammalian database, Chemical A has the potential to initiate an anti

- androgen AOP via androgen receptor binding or altered steroidogenesis; however, it appears that the perturbation at this level of biological organization is not expressed in the intact mammalian organism presumably due to compensatory

mechanisms. Assuming that the MIEs observed ( i.e., AR binding and altered steroidogenesis) are conserved across taxa , the Tier 1 f ish assay also indicates that an anti

- androgenic effect may be expressed, but at doses that are overtly toxic. From the Tier 1 observations, it could be that fish are more responsive than the rat to these MIEs/AOPs (particularl y altered ster oidogenesis).


### Chem **ical S** The case study

with Chemical S is an example of a difficult to test substance given its phy sical

- chemical properties. The low water solubility combined with the high log K ow (>5) and K oc (~321,000 L /kg) values makes this chemical difficult to test in aquatic sy stems. Chemical S is also a severe irritant to the gastrointestinal tract, which is a potential source of overt toxicity in mammalian assay

s ys tems. The low palatability

of or a gut irritation induc ed by Chemical S can limit the absorption and sy stemic toxicity of the compound following oral doses. At sufficiently low doses, the gastrointestinal tract is able to tolerate Chemical S but at higher doses, the integrity of the gastrointestinal tract is likely compromised.US EPA

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– July , 2013

Page **203** of **211** Although the in vitro Tier 1 assay s were negative for Chemical S and the MI E is uncertain, there were Tier 1 in vivo responses that could be indicative of interaction with the androgen signaling pathway

. Chemical S resulted in an ant i

- androgen response in that there was with a dose trend in the Hershberger assay with respect to the number of androgen sensitive tissues (decreased weights) affected. In the absence of overt toxicity , two tissues were affected. There were also a number o f responses in the male pubertal assay

consistent with an anti

- androgenic interaction, but these effects only occurred near or at higher overtl y toxic doses. The minor increase in PPS at the lower dose was not corroborated with other evidence of delay ed sexual development and was not considered adverse. The Part 158 data, which included subchronic, chronic, and multi

- generation reproductive studies, did not show any androgen

- related responses at similar mg/kg bw doses, except for an increase in testis we ight after chronic exposure. This response is unclear given the in vivo observations in EDSP Tier 1 and might be a spurious result. Using the EDSP WoE hypothesis

- based approach in evaluating responses along a biological continuum, based on the in vivo m ammalian Tier 1 data (Hershberger and male pubertal assay s), Chemical S has the potential to interact with the androgen pathway . The negative in vitro data may indicate that a different MI E may be occurring or that an activated metabolite is responsible f or the in vivo effects. There is a greater degree of complementarity and redundancy

in responses but at doses that are likel y c ompromising the integrity of the gastro

- intestinal tract. At lower doses, fewer responses are observed. Thus, when the Tier 1 da ta are integrated with the Part 158 mammalian data (including longer exposure durations), it appears that the organism can compensate at lower doses. For the androgen signaling pathway , there were no corroborative responses in FSTRA in the absence of over t toxicity , i.e ., e ffects on male GSI and secondary sex characteristics (tubercles) were onl y observed at concentration with e ffects on survival. The situation is similar to the rat in the sense that fewer responses are found at lower doses that are not o vertly toxic. With respect to the estrogen pathway , there was limited complementarit y and redundancy found among the available data in that only one estrogen related response was observed in the female pubertal assay (delay ed vaginal opening). There were no responses found in the Part 158 studies indicative of an interaction with the estrogen pathway . For the E pathway , again, while there were observed complementary responses observed in the male fish in the FSTRA ( i.e., increases in GSI along with decreases in fertility and secondary

sex characteristics ) , these responses were only observed in the presence of overt toxicity ( i.e. , e ffects on survival). The absence of complementary and redundant responses in the rat pube rtal assay s and AMA provides some support for a lack of interaction with the thy roid hormonal pathway . Although Chemical S was difficult to test in the AMA, as evidenced by high CVs in measured concentrations that were consistently

well below nominal conc entrations, there is uncertaint y whether results would have been substantially

different had the test been repeated to achieve exposure concentrations similar to what were achieved in the FSTRA. The Part 158 studies are in agreement with the EDSP Tier 1 d ata in that there were no effects found relevant the T hormonal pathway

.US EPA

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Page **204** of **211** Consistent with the low solubility of Chemical S, a solvent (dimethy lformamide) was used in an attempt to achieve test concentrations sufficient to challenge the test species in bo th the FSTRA and AMA. I n the FSTRA, an effort was made to increase the flow

- through rate of the exposure solutions from the recommended 45 mL/min to 70 mL /min and even then, mean coefficients of variation for measured concentrations ranged between 28

- 49% relative to the recommended value of ≤20% while test substance recovery

ranged between 42 to 59%. In the AMA, mean coefficients of variation for measured concentrations ranged between 33 to 69% while test substance recovery was as low as 9% of nominal. In both aquatic studies, the solubility limit of the compound and propensity to absorb rendered it difficult to maintain consistent test concentrations at the desired levels. These data illustrate that for some compounds, phy sical

- chemical characteristics will limit the extent to which exposure concentrations can be consistently maintained

. However, the fact that exposure concentrations are measured enables reviewers to determine responses relative to those measured concentrations even if the level of vari ability

deviates from performance standards. 

### Chem **ical J** The case study

with Chemical J is another example of how a chemical may initiate a molecular event, i.e ., binding to the androgen receptor (albeit with low binding affinity ), but this initiating event did not translate to higher level responses in in vivo, as measured in the Hershberger assay which is sensitive to androgen agonists/antagonists. However, when diverse t ypes of data are integrated along the biological continuum ( i.e., in vitro and i n vivo Tier I assay s), Chemical J appears to interfere with steroidogenesis as the biochemical key

event which translates into apical responses at higher levels of biological organization across taxa. Consistent with AR binding and altered steroidogenesi s, several complementary

and redundant responses are observed in vivo in the male pubertal assay ( e.g., decrease in testosterone correlated with decrease in several androgen

- dependent tissue weights and delay in male sexual development) and in fish (decrea se in female VTG along with an increase in female and male GSI with a decrease in fecundity correlated with alterations in the female and male gonads). Part 158 data provide additional support for this hy pothesis in that other mammalian studies reported g onado

histology and organ weight changes in androgen

- sensitive tissues and altered spermatogenesis. It appears that the in vivo mammalian responses are generally occurring within a similar dose range (in terms of mg/kg bw per day ) in the EDSP Tier 1 and Part 158 studies, although the Part 158 studies treatments are dietary

and the Tier 1 doses are given by gavage and may

reflect differences in absorption, distribution, metabolism and excretion of the compound as a result of different dosing regimens (gava ge vs. dietary )

. As discussed earlier, an important aspect of the AOP/MoA concept is the consideration of whether the molecular perturbation is significant enough to disrupt normal functioning at the organism level. Case Study J is a good example of this consideration with respect to chemical perturbation of the hy pothalamic

- pituitary

- thy

roid axis. The only

noted changes in vivo included, a trend that was not statistically

significant toward increased TSH in the male rat pubertal assay and changes in thy

r oid histology ( increased th yr oid follicular cell height and decreased colloid area) in the male pubertal assay ,US EPA

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Page **205** of **211** as well as, in one Part 158 rat feeding stud y (increase in number of small follicles). Given that Chemical J is known to induce microsomal enzy m es, these changes are likel y reflective of a normal adaptive biological response to maintain homeostasis as the liver clears the test chemical. There were no changes in T4 levels

. There appears to be an absence of potential thy roid

- mediated developmental effects in the amphibian Tier 1 assay . The lack of th yroid

- related responses across taxa demonstrates that Chemical J does not a lter HPT axis. 

### Chem **ical N** In the case of Chemical N, the mechanistic in vitro studies, as well as, the in vivo Uterotrophic and Hershberger assay s were all negative. Nonetheless, t here were limited complementary and redundant responses in the rat pubertal and fish Tier 1 assay s with respect to potential interaction of Chemical N with the HPG axis. These response s, however, were only observed at or near limit doses in the rat studies and aquatic concentrations approaching the historical limit concentration for aquatic testing ( i.e ., 100 mg/L). However, a response at the tissue level was observed in female fish in that a decrease plasma vitellogenin occurred at a test concentration approximately 10 times lower than the limit concentration. This decrease in VTG did not manifest into alterations in other measured endpoints that would be consistent with a decrease in VTG ( i.e., GSI , fecundit y, or gonadal histopathology ). Although the molecular initiating event is unclear, testosterone is a precursor to estrogen and decreased testosterone may

lead to decreased estrogen which would be consistent with the decreased VTG observed in the female fish. Furthermore, lower testosterone levels were observed in the rat pubertal assay s as well as effects on androgen

- sensitive tissues (seminal vesicles) and delay ed sexual developmental (age at PPS), but only near a limit dose (800 mg/kg bw/day). Thus, Chemical N may alter the HPG axis under conditions where normal compensatory processes are likel y exceeded or overwhelmed given that effects only occurred near limit doses.US EPA

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Page **206** of **211** 

### 11. **Concluding Remarks** Because of the complexity of some of the in vivo assay s where measurement endpoints can span multiple levels of biological organization and degrees of specificit y as direct measures of interactions with endocrine

- mediated processes, decision frameworks hav e evolved to assist reviewers in making determinations. These decision frame work s are informed by the results of earlier validation studies using chemicals with relatively well defined E, A or T AOPs/MoAs ( e.g ., the decision tree for the AMA depicted in **F igure 2**  ). A tiered decision logic tree ( **Figure 3** ) has been developed for the fish assay depicting what is contained in the FSTRA guidance and provide s a means of consistently and transparentl y identifying potential interactions with the E or A pathway s. The extent of complementarity among measurement endpoints with any assay though must be considered in the context of the direction and magnitude of chan ge as well as whether there are confounding effects such as overt toxicity or whether effects are observed at levels that are environmental ly relevant. Also, measurement endpoints can represent vary ing degrees of specificit y in terms of being considered “ diagnostic” for an interaction with the E, A or T pathway

.
In summary , the chemicals selected as case studies in these WoE analy ses are drawn from the subset of chemicals that received the first set of Tier 1 test orders based on their potential for expos ure and not endocrine disrupting potential. Similar to the 21 chemicals that were used in the assay and battery performance anal ys is, the WoE case studies represent a range of ph ys ical

- chemical properties and pesticidal modes of action and associated toxic ities. The case studies are intended to represent issues considered reflective of those that will be encountered across the broad chemical and toxicological domain likely

to receive EDSP test orders. Toward e nsuring that an adequate range of exposure lev els are tested in the Tier 1 screen, upper test concentrations/dosages frequently reflect the toxic mode of action of the pesticide or more generalized patterns of toxicity ( e.g ., nonpolar narcosis) that may obscure/confound efforts to distinguish effects specificall y related to an endocrine

- mediated AOP. T he approach articulated within the 2011 EDSP WoE guidance document relies on an hy pothesis

- based framework that integrates diverse data at different levels of biological organization to provide a robust and transparent approach to determinations of potential chemical interactions with E, A, or T signaling pathway s. This conceptual framework promotes the sy stematic use of a battery of assay s and other relevant information that are designed to evaluate par ticular responses along specific endocrine pathway s . The scientific determination of an endocrine interaction focuses on comparing responses to what is known about the underly ing biology as described in the AOP/MoA as an aid in the decision

making process. As illustrated by the case studies, the degree of complementarity and redundancy

in observed responses, as well as the conditions under which this responses occur , provides an important basis in judging the biological plausibility , coherence, strength, an d consistency of the bod y of evidence in characterizing the confidence in conclusions regarding potential chemical interactions with E, A, or T. The chemical endocrine interactions that were observed for the case studies were context specific withUS EPA

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Page **207** of **211** respect to the level of biological organization and the conditions in which these interactions occurred. The decision logic used as part of the analy s e s provides a means of en su r ing clear and consistent interpretations of potential interactions based on individua l endpoints and combinations of endpoints within and across assay s.US EPA

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Page **208** of **211** 

### 12. **References** Ankley GT, Bennett RS, Erickson RJ, Hoff DJ, Hornung,MW, Johnson RD, Mount DR, Nichols JW, Russom CL , and PK Schmieder (2009) Adverse Outcome Pathway s: A Conceptual Framework to Support Ecotoxicology

Research and Risk Assessment. Environmental Toxicology and Chemistry

29:1

- 12. ASTER . Agenc y’s Assessment Tool for Ecological Risk (ASTER) http://www.epa.gov/med/Prods_Pubs/aster.htm Boobis AR , Cohen SM , Dellarco V , McGregor D , Meek ME , Vickers C , Willcocks D , Farland W

. (2006)

. IPCS framework for anal yz ing the relevance of a cancer mode of action for humans. Cr it Rev Toxicol. 36(10):781

- 92. Boobis AR , Doe JE , Heinrich

- Hirsch B , Meek ME , Munn S , Ruchirawat M , Schlatter J , Seed J , Vickers C

. (2008)

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### Appendix
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