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 assays and battery.

This document does not create or confer legal rights or impose any legally binding requirements on the EPA or any other party. This document is distributed solely for the purpose of sharing this information with the public, consistent with the EPA transparency objectives. It is not intended to serve any other purpose, and should not be construed to represent formal dissemination of any agency determination or policy. As such, the information collection process under the agency's Information Quality Guidelines does not apply to this document.
                                       
                               Table of Contents
                                       
            SAP Review of EDSP Tier 1 Screening: Weight of Evidence
                                       
List of Acronyms	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 Guidance	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 Assays for chemical A	31
D.	Other Scientifically Relevant Information (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 Assays for Chemical S	79
D.	Other Scientifically Relevant Information (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 Available for Chemical J	128
C.	Tier 1 Screening Assays for Chemical J	128
D.	Other Scientifically Relevant Information (OSRI) for Chemical J	155
E.	Discussion	159
F.	Conclusions for Chemical J	169
9.	Case Study  --  Chemical N	170
A.	Introduction	170
B.	Data Available for Chemical N	170
C.	Tier 1 Screening Assays for Chemical N	170
D.	Other Scientifically Relevant Information (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	208


List of Tables
Table 1:  Current EDSP Tier 1 Screening Assays.	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 Assays for Chemical A.	32
Table 5.  Competitive Binding Assay of Chemical A with Estrogen Receptor from Rat Uterine Cytosol	33
Table 6:  Competitive Binding Assay of Chemical A with AR from Rat Prostate Cytosol [a]	39
Table 7:  Mean (+-SD) Hormone Concentrations 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:  Chemical 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) Body Weights and Cumulative Body 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 - Reproductive 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 Pathway for Chemical A	73
Table 19:  Thyroid Pathway for Chemical A	76
Table 20:  Summary of Tier 1 Screening Assays for Chemical S	79
Table 21:  Chemical S - Selected Group Mean (+-SD) Body Weights and Cumulative Body Weight Gains (g) in the Androgen Agonist Assay [a]	88
Table 22:  Chemical S - Selected Group Mean (+-SD) Body Weights and Cumulative Body Weight Gains (g) in the Anti-Androgen Assay [a]	89
Table 23:  Chemical S - Group Mean (+-SD) Overall (Days 1-11) Food Consumption (g/rat/day) [a]	90
Table 24:  Chemical S - Weights of Liver, Kidneys, Adrenals and Accessory Sex Organs from Androgen Agonist Assay in Rats [a]	91
Table 25:  Chemical S - Weights of Liver, Kidneys, Adrenals and Accessory Sex Organs from Anti-Androgen Assay in Rats [a]	92
Table 26:  Selected Body 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	96
Table 28: General Growth and Vaginal Opening of Rats Treated with Chemical S in the Female Pubertal Assay a	97
Table 29:  Organ Weight Data of Female Rats Treated with Chemical S in the Female Pubertal Assay a	98
Table 30:  Estrous Cyclicity Data 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 Body Weight of Male Rats Treated 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 Pubertal 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.  Thyroid Hormone Pathway for Chemical S	126
Table 43. Summary of Tier 1 Screening Assays for Chemical J	129
Table 44:  Competitive Binding Assay of Chemical J with AR from Rat Prostate Cytosol (using undefined bottom plateau)	132
Table 45: Competitive Binding Assay of Chemical J with AR from Rat Prostate Cytosol (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 Necropsy 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	152
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:  Thyroid Pathway for Chemical J	168
Table 58:  Summary of Tier 1 Screening Assays for Chemical N.	171
Table 59:  General Growth and Preputial Separation (PPS) in Rats Treated with Chemical N in the Male Pubertal Assay 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
Table 64:  Developmental and Thyroid Gross Pathology/Histopathology Endpoints1,2 in the AMA with Chemical N	186
Table 65:  Growth Endpoints[1],[2] in the AMA with Chemical N.	187
Table 66:  Estrogenic/Anti-Estrogenic Pathway for Chemical N	191
Table 67:  Androgenic Pathway for Chemical N	194
Table 68: Thyroid Pathway for Chemical N	198



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 Octyltriethoxysilane.  Run 1	34
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	35
Figure 6.  Percentage [[3]H]-Estradiol Bound to the Estrogen Receptor in the Presence of Chemical A, Unlabeled Estradiol, 19-Norethindrone or Octyltriethoxysilane.  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.  Percentage [[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.  Aromatase Inhibition 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 (Assays 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 (Assays 1  -  3), Using 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 Inhibition Response Curves for Chemical J from Each Test Run	140



   List of Acronyms
                                 Abbreviation
                                  Terminology
A
Androgen (hormonal pathway)
ADME
Absorption, Distribution, Metabolism, Excretion
AMA
Amphibian Metamorphosis Assay
ANOVA
Analysis of Variance
AOP
Adverse Outcome Pathway
AR
Androgen Receptor
Bmax
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 Immunosorbent 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
Kd
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 Identifier
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
PC10
Positive Control at 10% of response
PC50
Positive Control at 50% of response
PND
Post-Natal Day
QC
Quality Control
RBA
Relative Binding Affinity
RBC
Red Blood Cells
RPCmax
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-Evidence


                                     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 voluntarily cancelled or the manufacturer/importer agreed to cease 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 Federal 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/scipoly/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 programmatic 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 must 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 prioritizing 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 assays and that the Tier 1 battery performs 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 thyroid (T) signaling pathways.  In May 2013, the agency sought input from the SAP on Tier 1 assay and battery performance based on an analysis of a subsample of 21 case studies on List 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 pathways.  Input by the current July-August SAP on how 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. The 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) beyond Tier 1 data to determine whether or not a test chemical interacts with the E, A, or T signaling pathways.  Section 3 provides an overview of the WoE guidance which represents a hypothesis-based approach following the adverse outcome pathway (AOP) or mode of action (MoA) conceptual frameworks that integrate 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 presented in this paper.  
The method for interpretation of the endpoints for each of the 11 Tier 1 assays is discussed in Section 5.  The four case studies presented in Sections 6-9 come from the subset of the 21 chemicals used for the May 2013 SAP review. The case studies are used to illustrate the basic analytical process and concepts outlined in WoE guidance. These case studies are illustrative of the likely range of typical responses that will be observed across the Tier 1 assays and other scientifically 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 (early 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 analyses is on the interpretative process for determining whether or not there is a chemical interaction with E, A, or T signaling pathways. This includes characterizing 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 system  (e.g., in an organism 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 testing decisions. This document does not focus on the Tier 2 decision-making process itself.  
Because these case studies are illustrative, the analyses presented should not be interpreted as final or complete.  The agency will fully consider the May 21- 23, 2012  and July 30, 2013 SAP comments when these reports become available in evaluating the Tier 1 and OSRI data for the 52 chemicals in List 1 in its final WoE determinations.  

 
        Background on the EPA Endocrine Disruptor Screening Program

The US Environmental Protection Agency (EPA) "Endocrine Disruptor Screening Program" (EDSP) was initiated in response to the 1996 Food Quality 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 thyroid (E, A, and T) hormone signaling pathways 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 pathways, while the purpose of Tier 2 is to confirm the interaction and to evaluate the dose response for potential  adverse effects involving  the endocrine system 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 assays developed as part of Tier 1, which are considered as a battery, the Tier 2 tests do not constitute a battery. If the Agency determines that a chemical may have the potential to interact with E, A or T pathways, the nature and extent of testing required for Tier 2 testing will be informed by relevant existing data to address any 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 been peer reviewed and test guidelines are available. The Tier 2 ecotoxicity tests were reviewed by 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 advice 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 (List 1) (http://www.epa.gov/endo/).  Tier 1 test orders were issued for chemicals 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 manufacturers 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 subsample consisting of 21 of the 52 chemicals on List 1 to provide case studies to evaluate the performance of the 11 individual assays 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 the test orders. The subsample reflected a range of pesticide classes and physical-chemical properties including molecular weights, water solubilities, log10 octanol-water partition coefficients and volatility.  The subsample also reflected a range of pesticidal 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 responses within each assay. In addition, the panel was asked to provide input on performance of the battery based on the concordance of responses within the assays (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 recommendations as it moves forward in completing evaluations of the data sets for all 52 chemicals on List 1.

As part of the EDSP Tier 1 screening process for evaluating whether a chemical can potentially interact with the E, A, or T pathway, EPA considers the Tier 1 screening assays and OSRI used to fulfill test order requirements.  OSRI includes data from the open literature and test guideline data (e.g., the human 870 Guideline Series and Ecological Health 850 Guideline Series[,] ) submitted to fulfill requirements to support the registration of pesticides as defined in Title 40 of the Code of Federal Regulations Part 158 (subparts 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, 2011b).  The agency has prepared four case studies from the 21 chemicals considered 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).  

The primary purpose of the July-August 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 employment of the basic analytical process, principles and concepts outlined in the 2011 EDSP WoE guidance document as illustrated with the case studies.  Because these case studies are illustrative, the analyses presented should not be interpreted as final or complete.  The agency will consider the collective 2013 SAP reports in evaluating the Tier 1 data for the 52 chemicals in List 1 in its final WoE determinations for List 1 chemicals.  

        Overview of the Tier 1 Battery

As shown in Table 1, the Tier 1 battery is composed of 11 assays (a combination of in vitro and relatively short-term in vivo screens in different taxa).  A more detailed description of the Tier 1 assays 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). 

Briefly, the Tier 1 battery was based on the recommendations of EDSTAC and reviewed by the FIFRA SAP in 2008 (see http://www.epa.gov/scipoly/sap/meetings/2008/032508_mtg.htm).  The in vitro assays are intended to provide mechanistic data for specific pathways; whereas, the in vivo assays evaluate a range of apical endpoints that can be associated with multiple endocrine mechanisms.  The current EDSP Tier 1 screening assays (Table 1) serve as a means to identify whether or not a chemical can interact with E, A and T along potentially adverse outcome pathways (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 endpoints on the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-thyroid (HPT) axes.

An example AOP for estrogen receptor-mediated reproductive effects is depicted in Figure 1 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 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 verified 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 biological organization provides information, at least indirectly, on 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 means for interpreting the degree to which apical responses may be expressed at higher levels of biological organization.
Table 1:  Current EDSP Tier 1 Screening Assays.
                                Screening Assay
                                Test Guideline
                               Receptor Binding
                                Steroidogenesis
                                   HPG Axis
                                   HPT Axis
                                       
                                       
                                       E
                                    Anti-E
                                       A
                                    Anti-A
                                       E
                                       A
                                       
                                       
In vitro
ER Binding
(Rat uterine cytosol)
OCSPP 890.1250[2]
                                      ■
                                      ■
                                       
                                       
                                       
                                       
                                       
                                       
ERα Transcriptional Activation
(Human cell line 
HeLa-9903)
OCSPP 890.1300[3]
OECD 455[13]
                                      ■
                                       
                                       
                                       
                                       
                                       
                                       
                                       
AR Binding
(Rat prostate cytosol)
OCSPP 890.1150[4]
                                       
                                       
                                      ■
                                      ■
                                       
                                       
                                       
                                       
Steroidogenesis
(Human Cell Line 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]
                                      ■
                                      ■
                                      ■
                                      ■
                                      ■
                                      ■
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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-HQ-OPPT-2009-0576-0006
[4] http://www.regulations.gov/#!documentDetail;D=EPA-HQ-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-2009-0576-0012
[8] http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0576-0008
9 http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0576-0011
[10] http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0576-0009
[11] http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0576-0007
[12] http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0576-0002
[13] http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/OECD/OECD-TG455.pdf
[14] http://iccvam.niehs.nih.gov/SuppDocs/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=819299C4D1409C4EEA94D4F1FDDDCBB4

Figure 1.  Adverse Outcome Pathway for Estrogen Receptor (ER)-mediated Reproductive Impairment:  This is an 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 pathway can be made (USEPA, 2009; 2011a, b; Schmieder et. al.,  2004).


                                       
        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).  

The decision framework 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 analysis is an integrative and interpretive process routinely used by 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 articulated 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 hypothesis-based approach generally following the WoE framework for evaluating the conceptual framework of an AOP (see SAP 2011b) or modes of action (MoA) as put forth in conjunction with work by the EPA (2005), the International Programme for Chemical Safety (IPCS) (Boobis et al., 2006; 2008), and the International Life Sciences Institute (Meek et al., 2003; Seed et. al., 2005).  Use of the MoA/AOP conceptual framework has been a central organizing principle to more accurately characterize what effects may be biologically plausible in humans and the environment from chemical exposures and has been endorsed by the SAP (e.g., SAP, 2011b). The criteria used in MoA/AOP framework are generally applicable to the EDSP WoE evaluation and include considerations of biological plausibility and coherence, strength, and consistency of the body of evidence.  Multiple lines of evidence from the EDSP Tier 1 assays and OSRI are evaluated to address hypotheses of chemical interactions with the E, A or T signaling pathways. As described in Section 2, this analysis is approached by considering effects at different levels of biological organization using the in vitro and in vivo Tier 1 assays 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 hypothesis-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 AR binding assay).
      *       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).
      *       This is corroborated by an altered structural response at the tissue or organ level, such as increase in  gonado-somatic index (GSI) and decrease in mating tubercles in male fish, (as evaluated in the fish short-term reproductive assay), and/or decrease in androgen sensitive tissue weights (as measured in the Hershberger or male pubertal assays).
      *       Ultimately, androgen receptor antagonism may lead to an adverse outcome at the whole organism level, such as decreased fecundity (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 individual assay (i.e., complementarity) and among the different assays 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 of the chemical to interact with E, A, or T signaling pathways, at what level of biological organization (e.g., receptor/target binding, cell responses, enzyme activity, organ response, males and female system integrity, development (thyroid) and comparative system integrity) is that interaction expressed, and under what conditions does the chemical perturb the endocrine system (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 analyses (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 pathways (Question 1), then no further 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 analysis used to determine whether or not a chemical has potential to interact with the endocrine system, 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 2011 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 potential endocrine interaction.  The key steps are briefly described below.

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 by pesticide registrants or chemical companies, as well as data from published or publically available peer-reviewed studies.  The 2011 EDSP WoE guidance describes the relevant Part 158 health and ecological effect studies in support of pesticide registrations.  In considering data beyond EDSP Tier 1 assays 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 and G) and open literature studies already identified in response as OSRI to the List 1 test orders as OSRI.  When the agency completes its final chemical-specific WoE analyses, 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 Pesticide 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 each of the EDSP Tier 1 assays, 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 the 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, uncertainty and variability, and evaluation and review. A DER is an official agency 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 supported by 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 analysis of the Tier 1 data and generally concluded that the contributing laboratories were able to conduct the Tier 1 assays reliably, 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 their respective study guidelines, the Tier 1 data are of sufficient quality to support their use in the WoE analysis. 

B.  	Formulate Hypotheses  
As noted in the 2011 EDSP WoE guidance document (USEPA, 2011b), EPA refers to the WoE approach as "a collective 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 hypothesis that an agent causes a particular effect, which is an aspect of the WoE approach that has been routinely applied to analyses 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 hypothesis 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, hypothalamic-pituitary-gonadal (HPG) or -thyroidal (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 pathways, the WoE analysis (USEPA, 2011b) takes into account "considerations of biological plausibility and coherence, strength, and consistency of the body of evidence."   In evaluating the plausibility, coherence, strength, and consistency of the body of evidence, it is important to consider the extent to which observed responses are concordant within and across assays (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 complementarity 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: 

   * "complementarity"  describes the concordance of endpoints within an assay measuring multiple endpoints; and, 
   * "redundancy" describes the concordance of endpoints across the assays 

In the WoE process, the level of confidence increases with findings showing increasing complementarity of responses within an  assay and redundancy across the different assays that span multiple levels of biological organization (i.e., receptor/target binding, cell responses, enzyme activity, organ response, males and female system integrity, development (thyroid) and comparative system integrity). As part of this evaluation, the magnitude, direction (i.e., increase or decrease), and diagnostic specificity of responses are important to consider.  As a consequence, a more thorough understanding of E, A, or T interactions is obtained by the combined analysis of different endpoints and diverse types of studies (e.g., consistency of responses among interrelated endpoints as opposed to isolated and discordant responses). For example, within the Tier 1 battery, the in vitro assays provide data at lower levels of biological organization (e.g., receptor binding) for the parent compound.  However, if the in vitro assays are negative but the in vivo assays 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 in perturbing the HPG axis.  On the other hand, positive in vitro assays may indicate the potential of a chemical to initiate a molecular event/pathway. However, if the in vivo evidence is largely negative, that information would typically be given greater consideration in the WoE evaluation because these assays inherently integrate responses over the different biological levels of organization and account for compensatory processes.      

As indicated in the EDSP WoE guidance, "[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." It is realized that findings in the presence of overt toxicity may result in uncertainty 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 systemic (overt) toxicity are evaluated and addressed on a case-by-case basis.  These analyses consider the results from all of the Tier 1 assays and OSRI, as well as, the nature, directionality, magnitude, and dose trends of the Tier 1 endpoint responses. Additional consideration is given to the nature 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 generally 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 pathways.  Rather, the broad range of data provided by the Tier 1 battery of assays and OSRI (when available) should be considered in total in the context of different levels of biological organization.   

      D.  Develop a Short Narrative or Characterization of the Evidence
The 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 description 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 clearly statistically and biologically significant to discriminate among compounds that have a high to low likelihood of interacting with the E, A, or T hormonal pathways.  

        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), the first set of EDSP Tier 1 test orders was issued for 67 chemicals by EPA on October 29, 2009; 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 data were submitted on a total of 52 chemicals. 
For the assay and battery performance analysis that was prepared for the May 21  -  23 SAP Review, a subsample consisting of 21 test chemicals that adequately represented the chemical space of the initial 52 chemicals was selected.  This subset of chemicals represented a broad range of physical-chemical properties including molecular weight (range: 138  -  1053 g/mole), water solubility (range: 0.0003  -  32,000 mg/L), log10 octanol-water partition coefficient (log Kow range: 0.8 - 6), and volatility (5.4 x 10[-11]  -  0.3 mm Hg).  The chemicals also represented a range of chemical classes (e.g., organophosphates, imides, azoles) and pesticidal activities (e.g., inerts, fungicides, herbicides, insecticides) with toxicities ranging from practically non-toxic to mammals (acute oral LD50>2000 mg/kg bw) and to fish (LC50>100 mg/L) on an acute exposure basis to very highly toxic to mammals (LD50 <10 mg/kg) and fish (LC50<0.1 mg/L). 
A further subsample of the 21 chemicals used in the assay/battery performance analyses was selected to serve as case studies for the current WoE analysis. The subsample consists of four chemicals which again span a range of physical-chemical properties (molecular weight range: 138  -  1053 g/mole; water solubility range:  0.013  -  12,000 mg/L; log Kow range: 1.7  -  5.2; volatility range:  10[-7]  -  0.3 mm Hg).  Owing to the broad range of physical-chemical properties, some of the test chemicals, i.e., those with low solubility and high Kow values, can be difficult to maintain consistent exposure concentrations as such chemicals can also have high organic carbon partition coefficient (Koc) values and readily sorb to aquatic toxicity testing apparatuses. 
Similar to the range in toxicities represented in the subset of 21 chemicals, the four chemicals selected as case studies for the WoE analysis also range from pesticides that are very highly toxic in mammals and fish to chemicals that are considered to be inert. 
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 assays potentially impacting the analysis of complementarity within assays and redundancy 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 effects across assays and taxa.  Table 2 lists specific issues associated with each of the case studies.
Table 2:  Issues Associated with Case Study Chemicals.
                                 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 Kow, high Koc)
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
Tier 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.

        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 assays intended to provide complementary information when particular hormonal pathways are impacted.  Information is integrated over multiple levels of biological organization and taxa to determine whether the hypothesis of a potential effect on an E, A or T hormonal pathway can be supported.  In reviewing the Tier 1 battery for complementarity within assays and a concordance of results across assays given the specific endocrine modalities the assays are designed to detect, reviewers consider the magnitude and directionality of responses in terms of their biological plausibility.  
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 screening assays.  
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 estradiol 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 index, 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 (RPCmax) >= 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 first 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 cycling
Thyroid stimulating hormone (TSH)
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 testosterone
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-somatic 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 significantly 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 assays, decision trees have been developed to assist the reviewers.  The decision trees for the Amphibian Metamorphosis Assay (AMA) and the FSTRA are depicted in Figures 2 and 3, respectively.  The decision tree for the AMA is also depicted in the test guideline (OCSPP Guideline 890.1100) itself and has been previously vetted. Howerver, the 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 complementarity exhibited in the assay.  Although the FSTRA decision tree has not been formally depicted previously, it is a graphic representation of what 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. The 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 exposure concentrations had been tested. The lower portion of the framework depicts how various 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 guidelines which are used in determining potential endocrine interactions.

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


Figure 3.  Decision Logic for the Interpretation of the Fish Short-term Reproduction Assay Results. 

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 by the results of earlier validation studies using chemicals with known thyroidal MoAs (USEPA 2008b). Although not delineated in as much detail as the FSTRA decision tree in Figure 3 for determining study quality, the decision logic for the AMA depicted in Figure 2 relies on high quality experiments that would include among other 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 thyroid activity: developmental delay is considered indicative of thyroid interaction only if corroborated by treatment-related effects on thyroid gland histology (i.e., "remarkable histological effects" depicted in Figure 2), whereas advanced development, asynchronous development, or effects on thyroid gland histology may each be considered to be diagnostic findings independently.  
For the FSTRA, the proposed logic is intended to provide initial context in the diagnostic utility of each measured endpoints in terms of an interaction with the endocrine system.  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 needs to be considered in determining whether or not a chemical is interacting with the E and/or A pathways. 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 not the study data are appropriate for use in evaluating potential endocrine activity by identifying whether the study tested doses high enough to produce a treatment-related effect and if overt toxicity was observed in the study, and if so, what were the type of effects observed (i.e., mortality, decreases in body weight) and at what concentrations. It is important to demonstrate that the assay was conducted at a high enough concentration to allow for the possible observation of effects; therefore, in achieving an adequate high dose, it is recognized that some systemic (overt) toxicity may be observed. However, if overt toxicity was observed at all concentrations tested, the test guideline recommends that the study be repeated using lower exposure concentrations.  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 system is determined.  If a perturbation on an endpoint has been observed, the likelihood of that effect being mediated by an endocrine interaction is illustrated on the right-hand side of Figure 3.  
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 likely endocrine interaction.  However, if that response is accompanied by specific types of histological changes in the ovary (depressed vitellin [yolk] deposition), decreased egg production and (when collected) 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 depicted in Figure 3 is intended to initially orient the reviewer in evaluating the potential for a chemical's interaction with the endocrine system based on each measured endpoint, the responses within an assay and across assays need to be integrated in a weight-of-evidence analysis to decide whether a chemical may be endocrine-active or not.
For all in vivo Tier I assays 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 particular endocrine modality occur at doses where cytotoxicity 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 assays have been demonstrated to provide complementary responses within assays and specific patterns of responses across assays for known endocrine active chemicals against which the assays were validated, similar patterns of responses may be less pronounced with chemicals that weakly interact with endocrine pathways.  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, the specificity of individual Tier 1 assays for differentiating estrogenic from anti-androgenic or differentiating anti-estrogenic from androgenic effects can be limited.  Identifying which particular pathway is affected depends on whether there is sufficient concordance of information across assays that is further informed by the magnitude 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 assays and OSRI data to determine if there is a concordance of information to support a potential interaction with the E, A, or T signaling pathway.  


        Case Study  --  Chemical 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 more than one endocrine modality; potential endocrine-related toxicity in the presence of overt toxicity; challenges 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 pathways include all 11 EDSP Tier 1 studies and other scientifically relevant information (OSRI) such as 40CFR Part 158 guideline toxicity studies.  The data considered are summarized in Sections B, C and D of this chapter.  An analysis of the data submitted to the agency, using the WoE approach outlined by the Agency (USEPA, 2011b), is presented in Section E.  
Data Available for Chemical A
Chemical A is characterized as relatively 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 Kow) of approximately 4, it does not bioconcentrate appreciably in fish, suggesting that the compound is readily metabolized and excreted.  The compound is not considered persistent in the environment since it is prone to both abiotic (hydrolysis and photolysis) and biotic (aerobic and anaerobic) degradation/metabolism.
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.



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
Amphibian 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 cytosol obtained from Sprague-Dawley rats as the source of ER.  A saturation-binding assay was conducted to demonstrate that the ER isolated from rat uterine cytosol was present in reasonable concentrations and was functioning with appropriate affinity for the tritium ([3]H) radio-labeled reference estrogen prior to conducting ER competitive-binding assays.  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).  Dimethyl 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, octyltriethoxysilane as a negative control, and 17β-estradiol as the natural ligand reference material.
All performance criteria were met for the saturation binding assay.  The mean maximum binding (Bmax) value for the three saturation binding runs was 74.6 fmol/100 μg protein and the mean saturation binding dissociation constant (Kd) was 0.331 nM.  
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 binding run.  This test concentration was not included in the analysis. 
The results from the three competitive binding runs are summarized in Table 5 below and presented graphically in Figures 1-3.  The estimated mean specific binding affinity (reported as log IC50) was not calculated for Chemical A as the percent specific binding did not reach 50%.  The mean IC50 for the positive 19-norethindrone control was 5.01 x 10[-6] M and its relative binding affinity (RBA) was 0.027%; however, this value could not be calculated 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
Log IC50 (M), a	     17β-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
IC50 (M),                           17β-estradiol
                                 1.26 x 10-9
                                 1.00 x 10-9
                                 2.00 x 10-9
                                 1.36 x 10-9
                                                               19-norethindrone
                                 2.51 x 10-6
                                 6.31 x 10-6
                                 7.94 x 10-6
                                 5.01 x 10-6
                                                                 Test substance
                                      NA
                                      NA
                                      NA
                                      NA
Log 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 were 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 applicable
Figure 4.  Percentage [3H]-Estradiol Bound to the Estrogen Receptor in the Presence of Chemical A, Unlabeled Estradiol, 19-Norethindrone or Octyltriethoxysilane.  Run 1
                                       
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 

                                       
Figure 6.  Percentage [[3]H]-Estradiol Bound to the Estrogen Receptor in the Presence of Chemical A, Unlabeled Estradiol, 19-Norethindrone or Octyltriethoxysilane.  Run 3
                                       

In the first independent run, Chemical A showed an equivocal response at a concentration (10-3 M) where precipitation was observed.  The 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 recommends 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 assays.  
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 employs 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-well 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 by Chemical A was measured upon addition of a luciferase substrate and detection with a luminometer.
Chemical A was tested up to the limit of cytotoxicity, which occurred at concentrations >=10[-4] M.  The maximum level of response (RPCmax) was 8.8%, and the concentration associated with this maximum level of response, i.e., PCmax, was 10[-5] M for the first run, and the RPCmax was 5.3%, and the associated PCmax was 10[-6] M for the second run.   Performance criteria were met; Log PC50 and Log PC10 for 17α-methyltestosterone 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 this test system.

          iv. AR Binding Assay (Rat prostate cytosol)
The AR binding assay used ventral prostate cytosol 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 positive control, and R1881 as the ligand reference standard.  
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 concentrations and functioning with the appropriate affinity for the radiolabeled ligand.  Separate cytosolic preparations were used for each of the three competitive binding runs, so data from three saturation binding experiments were evaluated.  For the cytosolic preparation conducted on March 30, 2011, the mean dissociation constant (Kd) for [[3]H]-R1881 was 0.614+-0.065 nM, and the estimated Bmax was 1.080+-0.109 fmol/100 ug protein for the single batch of prostate cytosol that was prepared.  For the cytosolic preparation conducted on July 23, 2011, the Kd for [[3]H]-R1881 was 1.080 +- 0.690 nM, and the estimated Bmax was 1.071 fmol/100 ug protein (+-0.167) for the single batch of prostate cytosol that was prepared.  For the cytosolic preparation conducted on September 24, 2011, the dissociation constant (Kd) for [[3]H]-R1881 was 0.613 +- 0.041 nM, and the estimated Bmax was 0.817 fmol/100 ug protein (+-0.049) for the single batch of prostate cytosol that was prepared.  The mean and individual Kd values were generally below the range 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 the small variation in R[2] and Kd estimates among runs (although greater variability was observed in the July 23, 2011 Saturation Binding data).
In the competitive binding experiment, precipitation 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 IC50 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 IC50s for R1881 (−9.3 Log M) and the weak positive control, dexamethasone (−4.4 Log M), were within the expected ranges, and the mean RBA for dexamethasone was 0.0022%. The performance criteria were generally met. The results from the three competitive binding experiments are summarized in Table 6 and shown graphically in Figures 7 -9. 
 Based on the results of three runs, Chemical A is classified as a "binder" in the AR binding assay.


Table 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]
Log IC50 (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]
IC50 (M) [c]	                   R1881
                                1.3 x 10[−9]
                               7.9 x 10[−11]
                                1.0 x 10[−9]
                           4.6 x 10[−10] (+- 3.6)
                                                                 	Dexamethasone
                                4.0 x10[−5]
                                5.0 x 10[−5]
                                3.2 x 10[−5]
                           4.0 x 10[−5] (+- 0.5)
                                                                   	 Chemical A
                                      NC
                                2.5 x 10[−4]
                                1.3 x 10[−4]
                          2.7 x 10[−4] (+-0.5) [d]
Log 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 were 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 GraphPad PRISM; values are based on the means binding curve for Chemical A.
    NR	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 was not included in the analysis because of observed precipitation.   
    r[2]	Goodness of fit
    RBA	Relative binding affinity

 Figure 7.  Percentage [[3]H]-R1881 Bound to the Androgen Receptor in the Presence of Chemical A, Unlabeled R1881, or Dexamethasone.  Run 1.

                                       



 Figure 8.  Percentage [[3]H]-R1881 Bound to the Androgen Receptor in the Presence of Chemical A, Unlabeled R1881, or Dexamethasone.  Run 2.
                                       

Figure 9.  Percentage [[3]H]-R1881 Bound to the Androgen Receptor in the Presence of Chemical A, Unlabeled R1881, or Dexamethasone.  Run 3.


          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 uM 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 by high pressure liquid chromatography coupled with tandem mass spectroscopy (HPLC/MS-MS) using positive ion multiple reaction monitoring.  Three independent experiments were performed.  A Quality Control (QC) plate was run concurrently with each independent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents each at two 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 cytotoxicity in the third.  As a result the highest suitable concentration of Chemical A in Runs 1 through 3 was 10 uM.  
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 production met the recommended fold-increase criteria in the SC.  Exposure to 10 uM 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 1uM prochloraz inhibited synthesis of testosterone and estradiol by >=50% (actual 53-61% and 46-68%, respectively) compared to the SC.  These data generally met the performance criteria recommended by the Guideline.  
The within-plate and between-plate percent coefficients 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 slightly above the recommended Guideline limit of 30% for each set, these data are considered acceptable by the reviewers.
Statistically significant decreases in testosterone production were observed after incubation with 10 uM 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 0.1uM, and a significant increase was observed in the same run at the 10 uM 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 Table 7 and Figure 10.  The fold-changes in hormone concentration for Chemical A in the three runs are shown graphically in Figure 7 for testosterone.  The highest suitable concentration of Chemical A in all three runs was 10 uM due to precipitation at the 100 uM concentration in Runs 1 and 2, and cytotoxicity greater than 20% in Run 3.  
Based on the hormone responses in three independent runs, Chemical was positive in the steroidogenesis assay.
Table 7:  Mean (+-SD) Hormone Concentrations Following Treatment with Chemical A for 48 Hours.[a]
                          Nominal Concentration (uM)
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 were 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 cytotoxicity.
NC = Not calculated*Statistically significant (p<=0.05)
Figure 10.  Change in Testosterone Production Relative to Chemical A Concentration in Test Runs #1 to #3.
                                       
                                       
                                       
          vi. Aromatase Assay (Human Recombinant Microsomes)
In the in vitro aromatase (CYP 19) assay, Chemical A (purity, 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 concentration 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 activity control, a positive control series (10[−10] - 10[−5] M) using a known inhibitor (4-hydroxyandrostenedione; 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 substance (4-OH ADSN), aromatase activity 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 generally within the recommended ranges for the top of the aromatase activity curve, bottom curve, Hill slope, Log IC50, 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 concentration 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 greatly when the 10[−3] M concentration is included, based on increased least squares R[2] values.  
As shown in Table 8, excluding Run 1, which showed no inhibition for Chemical A, the estimated Log IC50 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. 
Table 8: Effect 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 IC50 (uM)
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 were calculated by the reviewers based on these data.  Values for  Chemical A were 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, was 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 was graphed as a line, and demonstrated little (if any) inhibition.
SD= Standard Deviation
CV= Coefficient of Variance

Aromatase activity was inhibited at the high concentration (10[-3] M) by more than 50%, but the results were cofounded by evidence of insolubility (precipitation) at this concentration.  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 study 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 Equivocal in the aromatase assay.

Figure 11.  Aromatase Inhibition Response Curves for Chemical A.
                                       

                                       


                                       

                                       

          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 daily 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 study also included a positive control group of eight ovariectomized females dosed via oral gavage with 17α-ethynyl estradiol (EE) at 0.1 mg/kg/day.  Animals were dosed for three consecutive days and necropsied approximately 24 hours after the final dose to determine wet and blotted uterine weights.
All animals survived until scheduled termination.  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 displayed nasal discharge, thinness and fur discoloration on Day 2; on Day 3 those same two rats plus three additional rats (total of five) displayed head and scapula fur discoloration, lethargy and hunched posture; these same five rats displayed head and scapula fur discoloration prior to euthanasia.
Body weights were decreased by 15% (p<0.05) in the 250 mg/kg/day group compared to controls.  Body weight gains were decreased (p<0.05) by 62% in the 78 mg/kg/day group and the 250 mg/kg/day animals actually lost weight 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 screen 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) by subcutaneous (s.c.) injection for 5 days, then TP+corn oil by gavage for 5 days.
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/day 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 days, then TP+corn oil by gavage for 5 days.  An anti-androgenic positive control group consisted of 10 castrated rats 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 days and euthanized approximately 24 hours after the final dose administration to determine 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 anti-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 anti-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 on days 5 and 6, and hunched and/or thin on days 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 duration of the assay.  At 150 mg/kg bw/day, overall (days 1-11) body weights gains were decreased (p<0.05) by 64%.  Animals dosed with TP had increased overall body weight gains (p<0.05) of 52%.  There were no statistically significant increases in any of the accessory sex organs of animals 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 ani-bulbocavernosus (LABC) muscle; 669% in Cowper's gland; and 88% in glans penis.  The performance criteria indicated that this assay was performing as expected.  Body weights and accessory sex organ weights for the androgen agonist component of the Hershberger assay are shown in Table 9 and10, respectively. 
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
(days 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 were 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<0.05

  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) by 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, accessory 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 LABC; 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.  Body weights and accessory sex organ weights for the androgen 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. 

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 the androgen agonist assay
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
(days 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 were 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 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<0.05



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 (47mg/kg/day)
 Chemical A (150mg/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 were 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 with 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 controls at p<0.05 
          ix. Pubertal Female Assay (Rat)
In a female pubertal assay, 16 Sprague-Dawley (Crl:CD[(R)][SD] IGS) 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 cycle.  Following sacrifice on PND 42/43, serum thyroxine (T4), and thyroid stimulating hormone (TSH) levels were analyzed by radioimmunoassay (RIA).  Adrenal, liver, thyroid, pituitary, and urogenital organ weights were recorded, and microscopic examinations of the uterus, ovaries, thyroid, and kidney were performed.
No treatment-related effects were observed on mortality, clinical signs, age at VO, mean age at first estrus, estrous cycle status, clinical chemistry, or gross or microscopic pathology.  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) by 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) by 7-11%.  Additionally, 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 by 13%.  These increased organ weights correlated with slight decreases in final body weights and slight increases in unadjusted organ weights.
At 50 mg/kg bw/day and above, serum T4 levels were slightly decreased (p<0.05) by 17-23%.  Serum TSH levels were similar to controls, and there were no effects observed on thyroid weights, or thyroid follicular cell colloid area or cell height.  Hormone values are shown in Table 13.
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 (%)
                                   Hormones
Serum T4, Total(ug/dL)
16
3.04
0.73
23.9
16
2.35* (↓23%)
0.58
24.5
16
2.51* (↓17%)
0.68
27.1
Serum TSH (ng/mL)
16
1.72
0.51
29.6
16
1.55
0.71
45.5
16
1.67
0.62
37.2
a Percent differences from controls are included in parentheses.  
*= p<= 0.05

At 100 mg/kg/day, mean estrous cycle length was increased (p<0.05) from 3.7 days to 4.6 days, and the percent regularly cycling was decreased (not significant [NS]) from 92% to 69%.  The mean age at first estrus and estrous cycle status were unaffected.  Because the effect on cycle 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 thyroid pathway in the female pubertal assay.

          x. Pubertal Male Assay (Rat)
In a male pubertal assay, 16 Sprague-Dawley (Crl:CD[(R)][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.  Animals were examined for preputial separation (PPS) daily beginning on PND 30 and weight at day of attainment was recorded.  Following sacrifice on PND 53/54, total serum testosterone, T4, and TSH levels were analyzed by RIA.  Adrenal, liver, thyroid, pituitary, and urogenital organ weights were recorded, and microscopic examinations of the testis, epididymis, thyroid, and kidney were performed.
No treatment-related effects were observed on mortality, clinical signs, body weights, body weight gains, age or weight at PPS, organ weights, hormone levels, clinical chemistry parameters, gross pathology, or microscopic examination of the thyroid, testes, epididymides, or kidney.  
A 15% (p<0.05) decrease in T4 levels was observed at the 50 mg/kg bw/day.   The minimal 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 and thyroid pathways in the pubertal male assay.

          xi. Fish Short-term Reproduction Assay
The in vivo 21-day short-term reproduction assay of Chemical A 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 months) were exposed to Chemical A at nominal concentrations of 0 (negative control) 0.01, 0.1 and 1.0 mg ai/L; mean-measured concentrations were <LOQ (<0.00083), 0.011, 0.120, and 0.820 mg ai/L. The test system was maintained at 24 to 25oC and a pH of 6.0 to 7.9.

In the control replicates, spawning frequency occurred at least every 4 days in two of the four replicates and fecundity in the control was 11 eggs/female/day/replicate (fecundity ranged from 6.2-16 with one of the four replicates having >=15 eggs/female/day).  Fertilization success in the control group averaged 91% (fertilization in the replicates ranged from 83-95%).   Fecundity was significantly reduced (p<=0.05) by 94% at the 0.820 mg a.i./L level compared to the control. Fertilization success was decreased 80% in the 0.820 mg a.i./L group compared to the control. 

Plasma VTG was significantly reduced (p<=0.05) relative to controls in female fish at the 0.011 and 0.820 mg a.i./L treatment levels, by 63 and 71%, respectively, but was not dose-responsive as the 37% reduction in the 0.120 mg a.i./L group, compared to the controls, was not statistically significant.  Male VTG levels were not significantly different from the control and did not exhibit a monotonic trend; while there was a 2665% increase in the mean VTG in the 0.120 mg a.i./L group compared to the control mean, this increase is due to 2 out of the 8 measured fish being above the mean control VTG by 2328 and 19,010%.  Due to the lack of consistency across males, the spikes in two of the fish were considered artifacts.

As shown in Table 14, significant reductions (p<=0.05) in gonado-somatic index (GSI) of 29 and 31%  was observed in male fish at the 0.120 and 0.820 mg ai/L levels, as well as a significant 42% reduction for female fish at the 0.820 mg ai/L level, relative to control.  Nuptial tubercle scores in males were also significantly reduced (p<=0.05) by 74% compared to the control at the 0.820 mg ai/L level with a decreasing trend, albeit weak, with increasing concentration; no tubercles were observed for females. Effects on gonadal histopathology were observed in female fish in the 0.820 mg a.i./L level and included an increased incidence of stage 0/1 ovaries with atrophy of perinucleolar and early cortical follicles. Male fish displayed an increased incidence and severity (as defined as ranging from minimal to moderate severity by the pathologist) of spermatogonia at all Chemical A-treated levels. The presence of a testis-ova was observed in one male at 0.120 mg a.i./L; however, given the low incidence, it is considered an artifact.
Chemical A exposure was additionally associated with a statistically significant reduction in body weight of female fish at the 0.120 and 0.820 mg a.i./L levels, with a slight decreasing trend observed. Overt morbidity including abnormal behavior and swimming was also observed in fish exposed to the 0.820 mg a.i./L level.  Overall mean survival values were 100, 96, 92, and 92%; however, the decreased survival with increasing exposure concentrations was not statistically significant (p>0.05).  Measurement technique of the male dorsal nape pad and sex steroids were described in the protocol, but results of these observations were not reported in the study. 
As shown in Table 15, several validity and performance criteria were not met for this study, where fecundity was less than the 15 eggs/female/day/replicate and spawning frequency was greater than every four days for two of the four control replicates, and fertilization success was less than 95% in control. Dissolved oxygen also dropped below the required minima percent saturation of 60%.  Although some control performance measures were less than recommended, in general, the performance was reasonable.
In general, there was a significant (p<0.05) decrease in fecundity 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.  Additionally, 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) was reported at 0.820 mg a.i./L along with a significant (p<0.05) decrease female body 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 cholinesterase 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 reflective of an endocrine-mediated response or are reflective of a compromised organism with limited ability to maintain reproductive function and homeostasis. However, given that there were responses in two endpoints (decreases in male GSI along with gonadal histopathology) that occurred in the absence of reported overt toxicity, they lack endocrine specificity, 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.
                                   Treatment
                                  (mg a.i./L)
                                  [measured]
                                Tubercle Score
                                      GSI
                      Gonadal Staging and Histopathology.
                                  Plasma VTG
                                       
                                    Median
                                 % Difference
                               Effect? (Yes/No)
% Difference
                    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
                                     -37.0
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 15:  Chemical A - Reproductive and HPG Endpoints[1] for Female Fathead Minnows.
Treatment
(mg a.i./L)
[measured]
                                   Fecundity
                               Fertility Success
                                Tubercle Score
                                      GSI
                      Gonadal Staging and Histopathology
                                  Plasma VTG

                                 % Difference
                                 % Difference.
Median
                                 % Difference.
Effect? (Yes/No)
                                 % Difference.
Control (dilution water only), <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[2]
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

                                 % Difference
                                 % Difference
                                 % Difference
                                 % Difference
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
          xii. Amphibian Metamorphosis Assay
The 21-day in vivo assay of Chemical A on amphibian metamorphosis of African clawed frogs (Xenopus laevis) was studied under flow-through conditions. Amphibian larvae (80 tadpoles per treatment, NF Stage 51) larvae were exposed to control and test chemical measured concentrations of 0.0076, 0.082 and 0.820 mg a.i./L.  The selection of test item concentrations was determined to be adequate according to guideline recommendations.  The test system was maintained at 21 to 23 °C and a pH of 7.9 to 8.2.
Chemical A did not significantly affect 7-day normalized hind-limb length (HLL) at 0.0076, 0.082 and 0.820 mg a.i./L.  Reductions in normalized HLL at day 21 in the 0.082 and 0.820 mg a.i./L treatment levels were similar, based on comparison to the negative control, but the effect was only statistically significant at the intermediate treatment level (p=0.045).   No significant change of median Nieuwkoop-Faber (NF) developmental stage was observed at 7 days or at 21 days at any treatment level. Asynchronous development was not observed in any of the treatments.   Body weight was significantly reduced on day 21 at the highest treatment level only (p=0.01); no other  effects on snout-vent length (SVL) or body weight were observed.  Thyroid gland hypertrophy was observed in the 0.820 mg/L treatment.  With respect to thyroid histopathology, mild follicular cell hypertrophy and hyperplasia were reported as common in both control and treatment specimens. Incidence of increased or decreased follicular lumen area was sporadic but again did not appear to be treatment-related. Clinical signs including discoloration and curvature of the spine (incidence values not reported) were observed in several tadpoles at each treatment concentration and in controls. Unless otherwise indicated, all effects are reported based on comparison to the negative control, and late stage tadpoles (NF Stage > 60) are excluded from analyses of continuous endpoints (SVL, body weight, and normalized HLL) due to the effects of normal tissue resorption during metamorphosis.
The absence of significant effects on thyroid-related endpoints in the AMA suggests that Chemical A did not interact with the amphibian HPT axis or its downstream thyroid hormonal pathway under these study conditions.

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 only 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 assays.   
                 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[(R)] Assay (Xenobiotic Detection Systems, 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 directly above the promoter confer estrogen responsiveness to the viral promoter and reporter gene.  The transfected cells are made stable by an antibiotic resistance gene resulting in the cell line used in the assay (BG1Luc4E2).  Chemicals that interact with estrogen-responsive DNA are identified by the luciferase gene response and quantified in terms of Relative Light Units (RLU).
The assays were conducted by growing the BG1Luc4E2 cells in medium for four days, transferring 2 x 10[5] cells/mL into 96-well plates and incubating at 37˚C with 5% CO2 for 24 h.  The medium was replaced, each test compound or an estrogen standard (E2) was added, and the plates were incubated at 37˚C with 5% CO2 for 24 h.  The medium was replaced and the cells were lysed with Promega lyse buffer.  Luciferase activity was measured via chemiluminescence with a Berthold[(R)] Microplate Luminometer in order to compare treatment RLU to an E2 standard.  For comparative purposes, the EC10 was used (Effective Concentration at which 10% of the maximum response was observed).  The data were not sufficient to allow calculation of an median effect concentration (EC50) at which 50% of the maximum response was observed.  The EC10 of E2 was 3.2 x10-12 M, which is within the expected 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 BG1Luc4E2 assay with a reported EC10 of 4.6 x10[-4] M, which is 8 orders of magnitude lower than the E2 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, epididymides, prostate, uterus, ovaries, thyroid, adrenal or the pituitary glands. No treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, uterus, ovaries, adrenal, thyroid or the pituitary glands. Clinical signs (hypersensitivity to sound and touch; hyperactivity and aggressiveness) were only observed at the highest dose.  
In 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, epididymides, prostate, uterus, ovaries, or the adrenal and thyroid glands.  No treatment-related histopathological lesions were seen in the testes, epididymides, prostate, uterus, ovaries, vagina, mammary, adrenal, thyroid or the pituitary glands 
In a combined chronic dietary toxicity/carcinogenicity study in rats, male and female Sprague Dawley rats 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, epididymides, prostate, uterus, ovaries, or the adrenal glands. No treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, uterus, ovaries, vagina, mammary, adrenal, thyroid or the pituitary glands. In a pre-natal 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% carboxy-methylcellulose by gavage on days 6 through 15 of gestation. Treatment-related findings seen only at the highest dose 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 cholinergic toxicity were noted.  
In a developmental neurotoxicity study, Chemical A was administered to 27 female Crl:CD[(R)] (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 (lactation) day (PND) 21.  The average daily 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 delayed at the high dose (45.0 1.88 days) compared to controls (43.1 1.41 days).  Vaginal opening was also delayed by at the high dose (33.82.28 days) compared to controls (32.51.10 days).  The mean body weight at attainment of puberty was similar between the treated and control groups for males and females. For maternal systemic 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 brain cholinesterase activities. For offspring systemic toxicity, the NOAEL was 2.36 mg/kg/day. The LOAEL is 24.2 mg/kg/day based reduced body weight, reduced body 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. There were no treatment-related changes in testicular or ovarian weights. No treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, coagulating glands, uterus, ovaries, vagina, cervix, or the pituitary glands. Slight decreases in male and female mating (86.7% vs. 96.7% in the control) as well as male and female 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 slightly 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 cycle length and periodicity, and sperm parameters were not evaluated in this study.   For parental/systemic 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 NOAEL is 0.67 mg/kg bw/day and the LOAEL 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 or T pathways, with the exception of the slight delay in PPS seen in the rat developmental neurotoxicity study.

                 3.  Ecotoxicity OSRI Data
In an early life-cycle toxicity study with sheepshead minnow, Cyprinodon variegatus, based on the most sensitive endpoints (length, dry weight, wet weight), the NOEC and LOEC were 0.0043 and 0.008 mg/L a.i., respectively. 

An early life-cycle 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 by any test concentration of Chemical A.

The chronic effects of Chemical A on fathead minnows and brook trout (Salvelinus fontinalis) were determined in flow-through systems with constant toxicant concentrations. Fathead minnows exposed to the lowest concentration tested (0.0032 mg/L) from 5 days after hatch through spawning had a significantly higher incidence of scoliosis (i.e., sideways curvature of the spine) than the control (p= 0.05). Hatch of their progeny was reduced by 30 percent at this test concentration. Yearling 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 ug/L and above. Neurological symptoms were evident in brook trout at 0.0024 mg/L and above early in the tests, but were rarely 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 tested (0.00055 mg/L) resulted in equally reduced growth rates for their progeny. Transfer of progeny between concentrations indicated that effects noted for progeny of both species at lower concentrations were the result of parental exposure alone and not the exposure of progeny following fertilization. 

In a partial life-cycle 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 test initiation) were exposed for 116 days and reproduction was monitored with a 32-33 day post-hatch exposure of the F1 generation. There was no effect on F1 hatching success or 4-week growth (LOAEC >0.0078 mg/L), but survival of 4-week old larvae was affected at 0.0078 mg/L with a NOAEC of 0.0035mg/L. 

In vivo acute toxicity testing with freshwater fish resulted in LC50 estimates as low as 0.090 mg/L; acute mortality data specific to fathead minnows indicate that this species is less sensitive (96-hr LC50=7.8 mg/L) than other freshwater species of fish. Acute toxicity testing with aquatic-phase amphibians (Rana boylii; LC50=7.5 mg/L) indicates that this taxon has a sensitivity similar to that of fathead minnow.  These data indicate that concentrations tested 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 highest 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 both males and females. Additionally, brain cholinesterase levels for females were depressed compared to the controls at all treatment levels. 

In a chronic life-cycle 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 estuarine/marine invertebrate, i.e., mysid 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 survival up to 0.0016 mg a.i./L. 

No additional data with frog or other amphibian species have been submitted.
Overall, the only effect observed in the OSRI ecotoxicity data that could be attributed to an interaction with the E, A or T pathways 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. 

Discussion 
     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 assays relevant to this analysis include the AR, binding, ER binding, ERTA, aromatase, and steroidogenesis assay.  Relevant in vivo assays include the male and female pubertal rat, uterotrophic, Hershberger, and the FSTRA assays.
Effects on Estrogen Pathway: Table 17 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 assays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects.


Table 17:  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
ER Binding
ER Activation
Steroidogenesis
Uterine Weight
Ovarian Weight
Ovarian/Gonad Staging and Histopathology 
Pituitary Weight
Estrous Cyclicity[b]
Age & Weight at VO
Fertility/Fecundity
Vitellogenin
Overt Toxicity Observed[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%
X
                                     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 days (p<0.05)


X
a Key to responses: [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  An "X" in this column indicates that the potential endocrine effect(s) observed in the assay occurred in the presence of overt toxicity

The EDSP Tier 1 battery of assays measure endpoints that are considered to be complementary within an assay and/or redundant across assays. The results for ER binding and aromatase inhibition were considered equivocal and were potentially confounded by the solubility limit of Chemical A. In a published literature study, submitted as OSRI (Kojima, 2005), Chemical A was weakly estrogenic (an EC10 of 4.6 x 10[-4] M) when the ER transactivation potential was evaluated in the E-CALUX test system; however, the chemical did not transactivate the ER in the EDSP ERTA assay when tested up to a cytotoxic concentration. In the steroidogenesis assay, Chemical A did not alter estradiol production. The equivocal in vitro responses in the ER binding and aromatase assays were not corroborated by altered organ or tissue level responses in the mammalian in vivo assays 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, effects on vitellogenin, gonadal histopathology, fertility, fecundity, and bodyweight 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 toxicity. Given the directionality (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 reproductive function and homeostasis.  Although control performance in this study was not entirely consistent with performance criteria, for fecundity and fertility, the study does not provide consistent evidence of estrogen pathway interaction across the various 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.    In a multi-generation study in rats, the only 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 days) was observed in the offspring.  In the OSRI ecotoxicity data, a reduction in the number of eggs was observed in freshwater fish 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 interacts with the E pathway. Chemical A was not an ER agonist up to cytotoxic concentrations in the ERTA assay.  The equivocal responses in the ER binding and aromatase assays 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 only 1.3 days 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 support potential interactions with estrogen signaling pathway. 

Effects on Androgen Pathway: Table 18 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 the androgen pathway.  The various targets of the androgen pathway across the relevant Tier 1 assays 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 effects 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 included abnormal behavior and significant body weight reductions consistent with cholinergic intoxication.   Given the directionality 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 intermediate 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, epididymis 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 days) 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 Hershberger 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 steroidogenesis), there is some uncertainty around the observed 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 only two endpoints affected in the absence of overt toxicity, and these are nonspecific responses 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.  
The in vitro responses in the AR binding and steroidogenesis assays (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 male pubertal assay, and in FSTRA, several endpoints were altered that could be consistent with androgen antagonism; however, many were in the presence of overt toxicity.
For the A signaling pathway, the in vitro assay results suggest that Chemical A has the 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), presumably with compensatory mechanisms, no androgen related effects were observed.  In the FSTRA, several endpoints were altered that could be consistent with androgen antagonism; 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.  

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
AR Binding
Steroidogenesis
Testosterone
Testes Weight
Gonad Staging and Histopathology
Epididymides Weight
Epididymides Histopathology 
Pituitary Weight
Accessory Sex Organ Weights/2° Sex Characteristics
Age and Weight at PPS
Vitellogenin
Overt Toxicity Observed[b]
                                  EDSP Tier 1
AR Binding
Binder











Steroidogenesis

 Test.










Hershberger








L:   SV 29% 
VP 26%












M: SV 37% 
VP  43%
 LABC  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
X

                                     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 Key to responses: [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.   SV= seminal vesicle; VP= ventral prostate; 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 overt toxicity. 

         ii. Effects 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 thyroid hormones (e.g., T4 and T3).  Thyroid hormones feedback to the hypothalamus or pituitary to regulate thyrotropin-releasing hormone and TSH, respectively, which, in turn, regulate hormone production by the thyroid gland.  The current Tier 1 screening battery does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid 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 pubertals 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 thyroid regulation. The various targets of the thyroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid 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 potential interaction with the HPT axis was a slight decrease (↓23% and 17%) in serum T4 levels observed in female pubertal rats treated with Chemical A at 50 and 100 mg/kg bw/day, respectively. A small decrease (↓15%) in T4 was also seen in the male pubertal assay 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 thyroid weights, pituitary weights, or thyroid histopathology in the male or female pubertal assays that might be anticipated if the compound were interacting with the HPT axis. Furthermore, the exposure period in the male pubertal assay is 10 days longer than the female pubertal assay, yet no compelling thyroid effects were observed in the male.   Thyroid gland hypertrophy 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 potential thyroid interaction in the AMA.  There were also no changes in thyroid weights, pituitary weights or thyroid histopathology in the mammalian Part 158 studies evaluated.  These studies included a subchronic study in rats treated with Chemical A for 13 weeks at doses up to 168 mg/kg bw/day and 2-year rat study and 1-year dog study treated with doses up to 10 and 9 mg/kg bw/day, respectively.  

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
Hormones 
(T4 and TSH)
Pituitary Weight
Thyroid Weight
Thyroid  Gross and Histopathology
Frog Development Stage
Hind Limb Length
Snout to Vent Length
Overt Toxicity Observed[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)



Equivocal
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:  Key to responses: [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.
Evidence of slight T4 depression in the female pubertal rats was not corroborated by complementary changes in TSH, thyroid weight or thyroid histopathology.  The decrease in T4 was not consistent with the magnitude of effects typically seen with thyroid toxicants.  There were also no biologically relevant changes in T4 levels in the pubertal males which are treated for 10 days longer than female rats in the pubertal assay.  The lack of thyroid pathway-related effects in the AMA and mammalian Part 158 studies reinforce the absence of thyroid interaction.  Based upon WoE evaluation of EDSP Tier 1 data and OSRI, Chemical A does not demonstrate a potential to interact with the thyroid pathway.  

Conclusions for Chemical A
All eleven assays comprising the EDSP Tier 1 battery were available for Chemical A. Chemical A was evaluated for several hypothesized endocrine modes of action for each specific E, A, and 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 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 pathways,  the number and type of effects induced, the magnitude of responses, and the  pattern of responses observed across studies, taxa, and sexes were 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 results in cholinergic toxicity.  

Integrating the in vitro and in vivo Tier 1 results, as well as consideration of relevant data beyond Tier 1 information (e.g., Part 158 standard toxicology studies) did not reveal convincing evidence of an interaction with the E or T pathways.  There was reasonable complementarity within assays and redundancy among responses across assays 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.  
With respect to the A signaling pathway, the in vitro responses in the AR binding and steroidogenesis assays (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 male pubertal assay, and in FSTRA, several endpoints were altered that could be consistent with androgen antagonism; 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.  


Case Study  --  Chemical S

A. Introduction
Chemical S is an organometal compound and has a mode of action involving inhibition of oxidative phosphorylation 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 vs. overt toxicity).
The available information considered to determine the potential interaction of Chemical S with the E, A and/or T pathways include all 11 EDSP Tier 1 studies and other scientifically relevant information (OSRI) such as 40CFR Part 158 guideline toxicity studies.  The data considered are summarized in Sections B, C and D of this chapter.  An analysis of the data submitted to the agency, using the WoE approach outlined by the Agency (USEPA, 2011b), is presented in Section E.  

B. Data Available for Chemical S
Chemical S is a relatively insoluble compound with a high log octanol-water partition coefficient (log Kow>5) and organic carbon partition coefficient (Koc~321,000 L/kg).  The compound is relatively stable to abiotic routes of degradation (i.e., hydrolysis and photolysis) 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 Koc is likely to partition to sediments.  Although the compound has a relatively high Kow, fish do not demonstrate appreciable bioconcentration factors suggesting that the compound may be metabolized by fish and/or its uptake may be limited.  The low solubility in water combined with the high Kow and Koc values render this chemical difficult to test in aquatic systems as the chemical would readily sorb to test containers.
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 20:  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 cytosol
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
                                  Negative[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
                                 Inadequate[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 exposure
              i.         ER Binding Assay (Rat uterine cytosol)
In the ER binding assay (98.75% a.i.), uterine cytosol 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 dimethyl 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, octyltriethoxysilane as a negative control, and 17-β-estradiol as the natural ligand reference material.
A saturation binding experiment demonstrated that the ER was present in the rat uterine cytosol at a reasonable concentration and functioning with appropriate affinity for the 17β-estradiol ligand.  The dissociation constant (Kd) for [[3]H]-17β-estradiol was 0.1153 nM, and the estimated average maximum binding (Bmax) was 45.75 fmol/100 μg protein for the single run with the batch of uterine cytosol that was prepared.  The Kd and the Bmax 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 Log IC50) was −8.94 Log 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 material showed that increasing concentrations of unlabeled 17β-estradiol generally displaced [[3]H]- 17β-estradiol in a manner consistent with one-site binding, as indicated by a mean Hill slope of approximately −0.9 to −0.8. 

Based on the results of three runs, Chemical S is classified as Not Interactive in the ER binding assay. 


              ii.  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 concentrations 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.  

A preliminary cytotoxicity test showed that Chemical S was cytotoxic in this assays system 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 (luciferase) gene products.  Luciferase 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,   however, the very weak agonist, 17α-methyltestosterone, did not achieve 50% transcriptional activation, therefore a PC50 could not be calculated.  Additionally, the response of the cells to 17α-methyltestosterone was weaker than the expected response, and the Log PC10 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 cytotoxic 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 (RPCmax) 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 transcriptional activation in this test system. 

              iii. iii. AR Binding Assay (Rat prostate cytosol)
In the AR binding assay, ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a saturation 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 x 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 positive control, and R1881 as the ligand reference standard.
The saturation binding  experiment demonstrated that the AR was present in the rat ventral prostate cytosol at a reasonable concentration and functioning with appropriate affinity for the R1881 ligand.  The dissociation constant (Kd) for [[3]H]-R1881 was 1.255 nM, and the estimated Bmax was 15.75 fmol/100 μg protein for the single run with the batch of prostate cytosol that was prepared.  The Kd and the Bmax were within the range for the performance criteria.  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 estimated average Log IC50 was −8.79 for R1881 and −4.34 for the weak positive control (dexamethasone).  Compared to R1881, the mean relative binding affinity (RBA) for the weak positive control was 0.0036%.  No precipitation of the test compound was visually observed at <=3.33 x10[−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 unlabeled R1881 generally displaced [[3]H]-R1881 in a manner consistent with one-site binding, as indicated by a mean Hill slope of approximately −1.0.  The lowest portion of the Chemical S across all runs was >75% at 3.3 x 10[−5] M (highest concentration tested).

Based on the results of three runs, Chemical S is classified as a "Non-binder" in the AR binding assay.


              iv. 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 uM 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 and estradiol levels were measured using HPLC-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 properly 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 reportedly conducted; however, only the results from 5 of the test runs were reported.  The remaining seven tests reportedly failed because either the QC plates did not meet the guideline criteria or because there was excessive variability in the test plates.  Increased cytotoxicity occurred in the Chemical S-treated cells at 1 uM (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 cytotoxicity 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 analytical method (10 pg/mL).  Inhibition of estradiol by 1 uM prochloraz slightly exceeded the 0.5-fold performance criteria in four runs (0.51- to 0.58-fold), and induction of testosterone by 10 uM 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 uM forskolin induced testosterone an average of 2.56-fold.  In addition, the between-plate coefficient of variation (CV) for testosterone was 38.2%, which is above the recommended limit of 30%.
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 uM 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 only 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 uM.  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 normalized for cell viability, there were no significant effects on testosterone levels at any Chemical S concentration in any 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 uM, and a slight decrease (p<0.05) of 0.87-fold in Run 3 at 0.1 uM.  
Based on the hormone responses in three runs, Chemical S was negative in the steroidogenesis assay.

              v.      Aromatase Assay (Human Recombinant Microsomes)
In an in vitro aromatase (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 activity.  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 for 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 activity 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-protein[−1]∙min[−1] at the highest tested concentration 10[−5] M.  The estimated Log IC50 for 4-OH ASDN averaged −7.23 Log M and the Hill Slope was −0.996.
For Chemical S, aromatase activity averaged 0.407 +- 0.061 nmol∙mg-protein[−1]∙min[−1] at the lowest tested concentration 10[−10] M and 0.270 +- 0.022 nmol∙mg-protein[−1]∙min[−1] at the highest tested concentration 10[−5] M.  The Log IC50 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 (88.5% activity) , the limited inhibition observed for Chemical S was generally 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 12 below). 
Figure 12.  Inhibition Response Curves for Chemical S From Each Test Run.
                                       
Based on the results of three runs, Chemical S is classified as Equivocal 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% purity) in corn oil was administered daily via oral gavage to groups of six adult ovariectomized, female Wistar (RccGab:WIST) rats at dose levels of 0 (vehicle), 10, or 75 mg/kg bw/day for three days from post-natal days (PND) 61/62 to 63/64.  A positive control group was treated with a daily dose of 17α-ethynyl estradiol (EE) at 0.3 ug/kg bw/day by subcutaneous injection.  Body weights were determined daily.  All animals were euthanized and necropsied on 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, respectively.  Terminal body weight was decreased (not significant, NS) by 5% in the 75 mg/kg bw/day group compared to vehicle controls.  This group displayed an overall (Days 1-4) body 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 body weight gain was decreased (NS) by 42% compared to controls.  Uterine weights (wet and blotted) in the Chemical S treatment groups were comparable to the vehicle controls. Body weights and overall body weight gain in the positive control (EE) group were comparable to the controls.  As expected, the absolute wet and blotted uterus weights for the EE group were increased (p<0.01) by 246 and 200%, respectively, over the 2 days of necropsy.  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.

              vi. vii.  Hershberger Assay (Rat)

The dose levels for the in vivo Hershberger assay were selected based on the results from a range-finding study 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 days.  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 displayed dyspnea and gasping.  Body weights, body weight gains, 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 leukocyte counts, total bilirubin, ALT 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 75 mg/kg bw/day was selected for the Hershberger Assay. 
In a Hershberger Assay screening for androgenic activity, Chemical S (97.85% purity) in corn oil was administered daily via oral gavage to groups of six 51-day old, castrated male Wistar (RccHan:WIST) rats at dose levels of 0 (vehicle), 10, or 75 mg/kg/day for 10 days.  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 subcutaneous (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 daily via oral gavage to groups of six 51-day old, castrated male Wistar (RccHan: WIST) 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 assays, body weights were determined daily and overall (Days 1-11) food consumption (g/rat/day) was determined.  Animals were dosed for 10 consecutive days and euthanized approximately 24 hours after the final dose administration to determine weights of the five androgen-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.  
Clinical 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, respectively, 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 study.  There were no treatment-related effects on weights of liver, kidneys, adrenals or any the accessory sex tissues of Chemical S-treated animals.  In the androgen agonist assay, there were no statistically significant (NS) differences in body 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) by 21% at 10 mg/kg bw/day, this solitary finding occurred only 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 (↑551%); and glans penis (↑76%), indicating that the test system 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.
In the anti-androgen assay, there were decreases (p<0.05) of 5-7% in body weights on Days 711% at 40 mg/kg bw/day+TP and 7-10% on Days 4-11 at 75 mg/kg bw/day+TP.  Overall body weight gains were also decreased (p<0.01) by 29 and 45% at 40 and 75 mg/kg bw/day, respectively, and food consumption was decreased (p<0.01) by 11-33% in all Chemical S-treated groups.  There were no treatment-related effects on weights of liver, kidneys 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) by 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 of 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 the 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 potential 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.  
Statistically 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 and 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 21 through 25.
 Chemical S was negative for androgenic activity, and positive for potential anti-androgenic activity at doses not associated with overt toxicity in the Hershberger assay.
Table 21:  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
                         Body Weight Gain (Days 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 were 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<0.01


Table 22:  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 (Terminal)
                                       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
                         Body Weight Gain (Days 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 were 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<0.01

		Table 23:  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 were 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<0.01

Table 24:  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
Kidneys (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 were obtained from the study report.  Percent differences from vehicle controls (calculated by the reviewers) are included in parentheses.
	N=Number of animals in the group
	SD=Standard Deviation
	CV=Coefficient of Variation
 * Significantly different from controls at p<0.05
  
  
  
  
  
  
Table 25:  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
Kidneys (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 were obtained from the study report.  Percent differences from vehicle controls are included in parentheses.
N=Number of animals in the group
SD=Standard Deviation
CV=Coefficient of Variation
*Significantly different from controls at p<0.05
**Significantly different from controls at p<0.01

              vii. Pubertal Female Assay (Rat)
In a Female 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) 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 body weight at day of attainment were also recorded.  Following sacrifice of the rats on PND 42, blood was collected for clinical chemistry analyses, including selected hormones.  Total serum thyroxine (T4) and thyroid stimulating hormone (TSH) levels were determined using the enzyme-linked immunosorbent assay (ELISA).  Liver, adrenals, pituitary, thyroid, and urogenital organs were collected and weighed.  Histology examinations were performed on the uterus, thyroid, one ovary, one kidney, liver, pituitary, adrenals, and gross lesions.  Summary data are presented in Tables 26 through 31. 
No treatment-related effects were observed on mortality.  Thyroid 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 cycle 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 days.  The adjusted body weight at VO was increased (p<0.05) by 7%.  Mean cycle length was reduced to 3.5 days compared to 5.6 days in the vehicle control.  The number of females cycling was increased to 80% compared to the vehicle control (67%), but the number of females regularly cycling 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 cyclicity.  One animal in this group had a distended cervix; however, this finding was considered incidental.  Primordial follicular cell 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) by 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) by 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.  Minor decreases (p<0.05; within historical control range) were observed in serum total protein and serum albumin (↓8-10%).  
At 75 mg/kg bw/day, the following decreases (p<0.01; unless indicated otherwise) in unadjusted and adjusted organ weights were observed 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) thyroid (14% each).  Additionally, decreases (p<0.01) were noted in unadjusted, adjusted, 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) by 6.5-7.3 days.   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 mg/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). 
Chemical S significantly altered pubertal development at 75 mg/kg, a dose that had a number of overtly toxic indications.  However, there were signs of delayed puberty in the lower dose as well in the absence of overt toxicity. 
In the positive control group, slight to moderate salivation was observed (9/15).  Body weights were decreased (p<0.05) by 6-9% during PND 24-33; however, body weights were similar to the vehicle control thereafter.  Food consumption was decreased (p<0.01) by 21% during the measured interval of PND 22-29.  Unadjusted and adjusted ages at incomplete VO were increased (p<0.05) by 3.7 days, and unadjusted and adjusted ages at VO were increased (p<0.01) by 3.0-3.8 days.  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 regularly cycling 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) of 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 decreased (p<0.05) by 35%.
Chemical S was positive for E pathway in the absence of overt toxicity and negative for T pathway in the pubertal female assay.   




Table 26:  Selected Body Weight 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
                                      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
                                     10.5
                                      15
                                    120.68
                                     9.21
                                      7.6
a	Data were 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	Coefficient of Variation
*	Significantly different from controls at p<0.05.
**	Significantly different from controls at p<0.01.




Table 27:  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 were 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=Coefficient of Variation
**Significantly different from controls at p<0.01.






Table 28: 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
Age 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 were obtained from the study report, and were sometimes rounded by the reviewers.  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
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
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
                                      15
                                     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
                                     36.29
                                     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
                                      15
                                 198.1 (↓24)
                                     62.06
                                     31.3
                                      15
                                     226.3
                                     62.1
                                     27.4
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 were 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 weight ratio (relative to body weight)
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.

Table 30:  Estrous Cyclicity Data of Female Rats Treated with Chemical S in the Female Pubertal 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 were 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 different from controls at p<0.05.
Table 31:  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 (ug/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
ALT (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
ALP (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 were obtained from the study report.  Percent differences from controls are included in parentheses.

              viii. 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 rats 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 recorded.  Following sacrifice on PND 53, blood was collected for clinical chemistry analyses, including selected hormones.  Total serum testosterone, thyroxine (T4), and thyroid stimulating hormone (TSH) levels were determined using the enzyme-linked immunosorbent assay (ELISA).  Liver, adrenals, pituitary, thyroid, and urogenital organs were collected and weighed.  Microscopic examinations were performed on the right testes, right epididymis, thyroid, one kidney, liver, pituitary, adrenals, spleen, and gross lesions.
Summary data are presented in Tables 32 through 36. No treatment-related effects were observed on mortality or gross or microscopic pathology.  No effects were noted on the thyroid of the treated groups compared to vehicle controls:  thyroid 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.  Increased incidences (number affected / number 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 dose).  Unadjusted and adjusted age at partial PPS was increased (p<0.01) by only 2.2 and 2.4 days, respectively; this  effect was not corroborated with other evidence of delayed sexual development such as changes in androgen-dependent tissue weights or serum 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.  Body weights were decreased (p<0.05) by 10-22% from PND 27 to 53 compared to vehicle controls.  Adjusted and unadjusted final body weights were decreased (p<0.01) by 20% each, and adjusted and unadjusted overall body weight gains (PND 23-53) were decreased (p<0.01) by 26% each.  The adjusted body weight at PPS was decreased (p<0.05) by 11%.  Corresponding to the body weight decreases, food consumption was decreased (p<0.05) by 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 delayed (p<0.01) by 3.5 days compared to the control, and unadjusted and adjusted ages at PPS were delayed (p<0.01) by 5.9 days.  In 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 vesicle 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 epididymides (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 by 13% each and relative (to body) liver weight increased by 8%; (ii) unadjusted and adjusted kidney weight decreased 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) by 21%, and alkaline phosphatase (ALP) was decreased (p<0.01) by 34%.  These slight effects on ALT and ALP 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 body 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) by 2.8-3.0 days, and unadjusted and adjusted ages at PPS were increased (p<0.01) by 3.0-3.1 days.  The following differences (p<0.05) were observed 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 epididymides weights were decreased by 10% each; and (v) adjusted left and right testes weights were decreased by 6-7%.  Total T4 was decreased (p<0.01) by 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 single 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 pathways in the pubertal male assay. 
Table 32:  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)
                                     13.66
                                     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 were 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.

Table 33:  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 bw/day
                              Positive Control b
                                       
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      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** (↓27)
                                     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 were obtained from the study report. Percent difference from control is included in parentheses.
      b100 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
Table 34:  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
                              Positive 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 PPS (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
                                      169
                                     12.8
                                      7.6
                                       3
                                 145* (↓11)
                                     13.2
                                      9.1
                                      14
                                  174* (↑7)
                                     12.8
                                      7.3
                         Final Body Weight (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 unseparated (#/N)
                                     0/15
                                     0/15
                                     12/15
                                     1/15
      a Data were 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 weight 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.
Table 35:  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
                                      9.9
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
                                      296
                                     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
                                      282
                                     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, right (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
                                      219
                                     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 (mg)
                                       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
                                      8.4

a Data were 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 weight ratio (relative to body weight)
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


Table 36:  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 (ug/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
ALT (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
ALP (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
                                     2.17
                                     0.49
                                     22.4
                                      15
                                     1.86
                                     0.40
                                     21.7
a Data were obtained from the study report.  Percent difference from control is included in parentheses.
SD=Standard Deviation;  CV=Coefficient of Variation
*Significantly different from controls at p<0.05
**Significantly different from controls at p<0.01

              ix. 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 concentrations of 0 (negative control; NC), 0 (solvent control; SC; dimethylformamide (DMF) at 0.02mL/L), 0.16, 8.0 and 4.0 ug ai/L while mean-measured concentrations were <LOQ (<0.010), <LOQ, 0.0834, 0.527, and 1.66 ug ai/L, respectively.  The test system 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 days and fecundity in negative and solvent control was 26 and 34 eggs/female/day/replicate, respectively (range: NC=21-36, SC=24-40); fertilization success 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 ug a.i./L treatment group (Table 37).  Fecundity was not significantly 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 concentration compared to the negative control (Table 38). 
Male plasma vitellogenin (VTG) was significantly increased (p=0.045) by 90% relative to the negative control in the 1.66 ug 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 analyzed using a trend test (Jonckeere-Terpstra) and all reported values (analyzed using replicate means).  The individual fish values for male VTG are depicted in Figure 13. This figure illustrates that across the negative control and all Chemical S treatment groups, there is one individual 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 observed variability and potential outliers associated with male VTG measurements was presented in the FSTRA section of the May 2013 SAP white paper (USEPA 2013), and the results for Chemical S reflect that discussion. Furthermore, if induction of vitellogenesis 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 analysis 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 relatively 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)  compared to the negative control.  The median of the individual  is represented by the solid line, with the box representing 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<0.05) relative to control in male gonado-somatic index (GSI) was observed at the 1.66 ug ai/L group. Female GSI was not significantly (p>0.05) different.  Nuptial tubercle scores were also significantly reduced by 33% (p<0.05) in male fish at the 1.66 ug 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 sex steroids were not reported neither was information provided on dorsal nape pads.  Unless otherwise indicated, all effects are reported based on comparison to the negative (clean water) control. 
There was a significant (p<0.05) reduction in survival (50% reduction compared to negative control) at the 1.66 ug ai/L treatment level.  There appeared to be inadequate reporting of secondary sex characteristics and clinical signs. The study author reported pectoral fin hemorrhage in one male from the control group.  There were no incidences reported in the 1.66 ug 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 termination was not reported (Table 39).

The percent fertility in the negative control (93%) and solvent control (90%) was slightly less than the guideline criterion minimum of 95%.  Additionally, the dissolved oxygen dropped below 60% in several 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 days dissolved oxygen dropped below the minimum criteria. However, once a decrease was observed, aeration was added to each test chamber on Day 10.  Finally, the analytical 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 results 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 mortality. Additionally, for male VTG, while a significant increase was initially reported, upon further evaluation, it was not determined to be statistically different from the controls based on the range of reported values and the 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 phosphorylation resulting in the depletion of ATP, which may have affected the overall energy reserves of the fish and may have in turn impacted energy inputs into reproduction.  Given the mode of action for Chemical S along with the observed decreases in fertility and tubercles and increases in GSI which lack endocrine-specificity and were coincident with effects on survival, there is uncertainty whether these effects were due to a possible endocrine interaction with the A (antagonism) or E pathway (agonism) or due to the fish's response 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 FSTRA for impacts to E or T signaling modalities.
Table 37:  Chemical S - Reproductive and HPG Endpoints[1] for Male Fathead Minnow in the FSTRA
                                   Treatment
                                 (ug a.i./L)
                                [mean measured]
                                Tubercle Score
                                      GSI
                     Gonadal Staging and Histopathology[2]
                                  Plasma VTG
                                       
                                    Median
                                 % Difference
                               Effect? (Yes/No)
% Difference
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]
	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
                                       
                                   Treatment
                                 (ug a.i./L)
                                [mean measured]
                                   Fecundity
                               Fertility Success
                                Tubercle Score
                                      GSI
                     Gonadal Staging and Histopathology[2]
                                  Plasma VTG
                                       
                                 % Difference
                                 % Difference
                                    Median
                                 % Difference
                               Effect? (Yes/No)
                                 % Difference
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
                                     35.0
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 pathologist.
[3]  Statistically significant at p<0.05.
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 ug a.i./L.  Time-weighted average (TWA) concentrations were <0.010 (<LOQ; controls), 0.0156, 0.0711, and 0.307 ug a.i./L.  
The test system was maintained at 21.5 to 22.6°C and a pH of 7.9 to 8.4. Chemical S did not significantly 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 analysis 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 analysis for this reason.  Unless otherwise indicated, all effects are reported based on comparison to the negative (clean water) control.
No asynchronous development was observed in any treatment group.  Effects on thyroid gland histopathology were observed in both controls and the TWA 0.0156, 0.0711, and 0.307 ug a.i./L.  Histopathological effects included mild follicular cell hyperplasia and hypertrophy, though there was no apparent treatment-related effect because the incidence of these diagnoses was similar in controls and treatments.  The study author did not report clinical observations. 
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 from 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 Koc) of Chemical S in the test system make the results of this amphibian metamorphosis 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 from 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 ug/L) was higher than the guideline recommended level (<=10 ug/L); however, the analysis was last performed one year prior to the conduct of the study and it is unclear if the water analyzed 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 reverse 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.

A. 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 agency.  However, Chemical S has Part 158 mammalian and ecotoxicity 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 bw/day) for 3 months.  Testes weights and histopathology were evaluated. Body 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% from 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 and the LOAEL is 15 mg/kg bw/day based on elevated alkaline phosphatase in male rats. 
In a 2-year rat toxicity study, Chemical S (97% a.i.) was administered in the diet to CFE  rats (72/sex/group) for up to two years 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 thyroid glands. The diet was unpalatable to both males and females and resulted in decreases in body weights, body weight gains, and food consumption, especially during the early stages of the study.  There was a significant body 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 to decreased food efficiency for weeks 0-52.  It should be noted that the subchronic and 2-generation studies on reproduction generally 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 absolute 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 thyroid glands. The only effects on the intestines was a suggestion of an increased trend for soft stools, especially 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).  Mild 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 toxicologically 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/kg bw/day) for 18-months. Testes weights were obtained. Histopathological examinations were conducted for the gonads, prostate, uterus, pituitary and thyroid 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 body weights at 300 and 600 ppm were reduced by 5% and 16%-18%, respectively, and female body 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 LOAEL 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. ) by gavage in an aqueous solution at doses of 0, 15, 30, and 60 mg/kg bw/day on gestation days (GDs) 6-15, inclusive.  On GD 20, all surviving dams were euthanized and then necropsied, and all fetuses were examined externally, weighed and sacrificed.  Several high-dose animals exhibited diarrhea at varying times between GDs 6 and 20 (7/27 vs. 2/27 for controls). For maternal toxicity, the NOAEL was 15 mg/kg 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 incidences 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) by gelatin capsule at doses of 0, 1, 5, or 10 mg/kg bw/day on gestation days (GD) 6-18, inclusive.  On GD 29, all surviving does were sacrificed and the uteri were weighed and examined.  Treatment-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-, and high-dose groups, respectively.  Anorexia (subjective observations of not eating and passing few feces) was observed in 2/18, 2/18, 11/18, and 23/23 animals, respectively, and was often associated with abortion and/or whole litter loss.  Lesions of the gastric mucosa were found in the mid- and high-dose animals that died intercurrently, 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, the 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-related 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 loss/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 F0 and F1 rats at dietary concentrations of 0, 40, 75, 250, or 500 ppm.  The dietary concentrations corresponded to doses of 2.39, 4.47, 14.8, and 30.0 mg/kg bw/day for F0 males; 3.18, 5.92, 20.0, and 45.7 mg/kg bw/day for F1 males; 2.76, 5.13, 16.6, and 33.6 mg/kg bw/day for F0 females; and 3.68, 6.83, 23.9, and 54.1 mg/kg bw/day for F1 females averaged over the premating period only.  The F0 male and female rats received treated or control diets for 72 days prior to mating and during mating, gestation, and lactation of one litter.  The F1 pups were weaned onto the same diets as their parents and were fed their respective diets for 105 days prior to mating and during mating, gestation, and lactation of one litter.  The F0 males were sacrificed after 124-126 days of feeding, F0 females after 116-137 days, F1 males after 159-177 days, and F1 females after 160-178 days. Testes were weighed. Histopathological examinations were conducted for epididymides, testes, seminal vesicles, prostate, coagulating glands, ovaries, uterus, vagina, pituitary and thyroid glands. Sexual maturation, estrous cycle length and periodicity, 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 F0 or F1 male or female rats at any dose, or on body weights, body weight gain, food consumption, or food efficiency at 40, 75, or 250 ppm.  Body weights and overall body weight gain, food consumption, and food efficiency were significantly (p<0.05) decreased in both sexes and generations administered 500 ppm of the test material compared with the controls.  The F0 males weighed 8-19% less than controls throughout the premating/postmating period and gained 33% less weight; F1 males weighed 23-25% less and gained 24% less weight.  The F0 females weighed 7-12% less (except for week 1) and gained 41% less weight during the premating period, and F1 females weighed 18-30% less and gained 20% less weight.  The F0 and F1 males consumed 12% less food averaged over the entire premating period, F0 females consumed 13% less, and F1 females consumed 7% less.  
No treatment-related effects were observed on the following parameters for F1 or F2 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 weight 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, F1 pups weighed 6% (N.S.), 16%, 20%, and 24% less than controls on lactation days 4 (precull), 7, 14, and 21, respectively, and gained 28% less weight between day 0 and 21.  The F2 pups at 500 ppm weighed 13%, 19%, 22%, and 29% less, respectively, than controls and gained 33% less weight between day 0 and 21.  At 250 ppm, F1 pups had body weights similar to those of the controls throughout the lactation period; whereas, F2 pups weighed 9%, 12%, 9% (p<0.05), and 10% (p<0.05) less than controls on lactation days 4 (precull), 7, 14, and 21, respectively, and gained 11% less weight.  The F2 pup weights were statistically significantly decreased from control values on lactational day 14 and 21 at 250 ppm. Between lactational day 0- 4, F2 pups showed a weight gain decrement of 19% at 250 ppm.  
For parental/systemic toxicity, the NOAEL 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 body weights, body weight gain, food consumption, and food efficiency in F0 and F1 males and females.  For reproductive toxicity, the NOAEL was 30 mg/kg bw/day in males and 33.6 mg/kg bw/day in females; a LOAEL 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 F2 litters. 
Chemical S was administered in capsules to 8 control and 4 Beagle dogs/sex/group at 0, 2.5, 5, 15, 30 or 60 mg/kg/day for 2 years.  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.  In the 30 and 60 mg/kg bw/day groups vomiting and diarrhea continued after one year, 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 weights were significantly 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 body weight at 60 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 year of the study.  The NOAEL is 5 mg/kg bw/day and LOAEL is 15 mg/kg bw/day based on excess vomiting and diarrhea with a body 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 early life stage 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 ug/L and above.  The NOAEL values were 0.31 ug/L for larval survival and 0.61 ug/L for embryo 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 LC50 value for Chemical S using rainbow trout was 1.7 ug a.i./L (based on nominal concentrations) tested under static conditions.
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 reproductive 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 NOAEC 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 increase 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 ug/L (measured) or 25 ug/L (nominal).  There was a significant difference between the solvent control and the test chemical at the two highest treatment concentrations (50 and 100 ug/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 ug/L.  
No additional data with frog or other amphibian species have been submitted.

A. 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 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 assays relevant to this analysis include the AR, binding, ER binding, ERTA, aromatase, and steroidogenesis assay.  Relevant in vivo assays include the male and female pubertal rat, uterotrophic, Hershberger, and the FSTRA assays.
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 assays 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 assays.  Based on these results, Chemical S does not appear 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 Chemical 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) delays ( ~ 2 days) in vaginal opening (VO) and decreased body weight at VO were seen in the absence of overt toxicity.   At the high dose (75 mg/kg bw/day), estrogen-related effects were manifested as delayed VO (~ 7 days; p<= 0.01); decreased body weight at VO; decreased weights of the ovaries and uterus but in the presence of overt toxicity (salivation, and/or decreased body weights and food consumption).  In 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% decrease 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 consistent 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 markedly affected (50% overall mortality observed).  
The pesticidal mode of action of Chemical S involves the uncoupling of mitochondrial oxidative phosphorylation 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 irritation that compromises the integrity 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 the 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 uncertainty in whether the responses observed in the FSTRA are potentially 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 do not suggest an agonistic interaction 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 reported 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 perturbation of the HPG axis in the Female Pubertal assay. However, data from the remaining assays are not sufficiently compelling to support such a perturbation.     


Table 40:  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
                                       
 ER Binding
 ER Activation
 Steroidogenesis
 Uterine Weight
 Ovarian Weight
 Ovarian/Gonad Staging and Histopathology
 Pituitary Weight
 Estrous Cyclicity[b]
 Age & Weight at VO
 Fertility/Fecundity
 Vitellogenin
 Fish Gonadal Weight (GSI)
 Overt Toxicity Observed[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 days (V0); ↓ 7-12% BW
                                        
                                        
                                        
                                      X[d]
 
 FSTRA
 
 
 
 
 
 
 
 
                                        
                                        
                                        
                                        
                                        
                                        
                                        
                                       
                                        
                               ↓ (13%) ♂ H[e]
                               ↑(90%) ♂ H[e]
                               ↑(39%) ♂ H[e]
                                       X
                                     OSRI
 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 response; A shaded cell indicates that parameter is not routinely evaluated or is not applicable in the assay.
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. 
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) were in the absence of overt toxicity.
e A 50% decrease in survival was observed at this treatment level.

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 relevant Tier 1 assays 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 cytosol), 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 assays 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 assays.  
In the anti-androgenic phase of the Hershberger assay, significant 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 seminal 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, responses relevant to interaction with the androgen pathway consistent with antagonism included delayed preputial separation, decreases in the weights of seminal vesicles, ventral prostate, dorsolateral prostate, LABC, epididymides, 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 pathway 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 fertility, males GSI, and male tubercles) were observed only at levels with overt toxicity (increased mortality). However, as discussed above, there is uncertainty in whether these responses are due to a potential endocrine-interaction or from a response in decreased energy output.  
Increased testes weights (absolute and relative) were noted in the Part 158 chronic toxicity study in the rat; however, there were no accompanying 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. 
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 assays.






Table 41.  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
                                       
 AR Binding
 Steroidogenesis
 Testosterone
 Testes Weight
 Gonad Staging and Histopathology
 Epididymides Weight
 Epididymides Histopathology
 Pituitary Weight
 Accessory Sex Organ Weights/2° Sex Characteristics
 Age and Weight at PPS
 Vitellogenin
 Fish Gonadal Weight (GSI)
 Overt Toxicity Observed[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]
                                       
                                       X
                                     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 response; A shaded cell indicates that parameter is not routinely evaluated or is not applicable 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 weights) 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 were no effects at the low dose (10 mg/kg/day).   
e A 50% decrease in survival was observed at this treatment level.

     i. Effects 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 thyroid hormones (e.g., T4 and T3).  Thyroid hormones feedback to the hypothalamus or pituitary to regulate thyrotropin-releasing hormone and TSH, respectively, which, in turn, regulate hormone production by the thyroid gland.  The current Tier 1 screening battery does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid 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 pubertals 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 thyroid regulation. The various targets of the thyroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects.  

Table 42.  Thyroid Hormone Pathway for Chemical S
Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Chemical S[a]




Study Type/Literature Citation
Hormones         (T4 and TSH)
Pituitary Weight
Thyroid Weight
Thyroid Histopathology
Frog Development Stage
Hind Limb Length
Snout to Vent Length
Overt Toxicity Observed[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.  

There was no evidence that Chemical S has a potential to interact with the thyroid hormonal pathway.   The decreased thyroid and pituitary weights observed in the Female Pubertal assay at 75 mg/kg bw/day occurred in the presence of overt toxicity (salivation and decreases in body weights and food consumption).  There were no effects on the thyroid in the standard combined chronic toxicity/carcinogenicity studies in the rat and mouse.  
Adequate exposure was not achieved in the Amphibian Metamorphosis assay (AMA), and therefore was not successfully tested in this assay.  Testing in the AMA assay is limited by the physico-chemical characteristics of the test substance.  With respect to Chemical S, it belongs to a class of chemicals characterized by relatively low water solubility, high octanol-water partition coefficients (Kow) and high organic carbon partition coefficients (Koc).  While these compounds are relatively stable to both abiotic (hydrolysis and photolysis) and biotic (aerobic and anaerobic) degradation/metabolism, their low solubility and propensity to partition to organic matter render them difficult to test in aquatic systems.
Based upon WoE evaluation of EDSP Tier 1 screening assay and OSRI results, Chemical S did not demonstrate a potential to interact with the thyroid hormone pathway.  

Conclusions for Chemical S
All eleven assays comprising the EDSP Tier 1 battery were available for Chemical S. Chemical S was evaluated for several hypothesized endocrine modes of action for each specific E, A, and 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 assessment. 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 pathways,  the  number and type of effects induced, the magnitude 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 toxicity 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 Assays).  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 impacted the responses observed in the studies.  
Integrating the in vitro and in vivo Tier 1 results, as well as consideration of relevant data beyond Tier 1 information (e.g., Part 158 standard toxicology studies) revealed evidence of a potential interaction with the E or A pathways.  In the absence of overt toxicity, there was evidence of potential interaction with the androgen signaling pathway.  The complementary responses in the Hershberger assay were limited to significant decreases in only two androgen-dependent tissues.  Although androgen-related responses 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, there were no redundant responses across the in vivo assays to provide confirmatory evidence.  Likewise, Chemical S demonstrated a potential to interact with the estrogen signaling pathway in that only one response (delayed vaginal opening in the female pubertal assay) was significant.  Although there were responses in FSTRA that could be indicative 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 responses in the rat pubertal assay and AMA indicates that Chemical S does not interact with the thyroid hormonal pathway. 

        Case Study  --  Chemical J

B. Introduction
Chemical J is a triazole fungicide inhibiting sterol biosynthesis which in turn disrupts membrane function.  Chemical J also acts as a hepatic microsomal enzyme 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 modality (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 pathways include all 11 EDSP Tier 1 studies and other scientifically relevant information (OSRI) such as 40CFR Part 158 guideline toxicity studies.  The data considered are summarized in Sections B, C and D of this chapter.  An analysis of the data submitted to the agency, using the WoE approach outlined by the agency (USEPA, 2011b), is presented in Section E.  

Data Available for Chemical J
Chemical J is soluble in water and does not volatize appreciably. It is characterized as being moderately persistent as it is resistant (stable) to abiotic routes of degradation (i.e., hydrolysis and photolysis) and to anaerobic biotic metabolism.  The compound can slowly undergo aerobic metabolism with half-lives >70 days.  Based on a log octanol-water partition coefficient (log Kow) of approximately 3 and a relatively low propensity to sorb to organic matter, based on its organic carbon partition coefficient (Koc~520 mL/goc), Chemical J residues are not expected to bioaccumulate.

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. 
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 cytosol)

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 
Amphibian 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
3Classified as "Inhibitor" according to the Test Guideline OSCPP 890.1200

              i.               ER Binding Assay (Rat uterine cytosol)
In the ER binding assay, uterine cytosol from Sprague Dawley rats was used as the source of ER to conduct the binding assays. A saturation binding experiment was conducted prior to the competitive binding experiment to demonstrate that the ER in the isolated uterine cytosol was present in an adequate concentration and functioning with the appropriate affinity for the radiolabeled (tritium; [3]H) ligand.  The competitive binding experiment 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 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, octyltriethoxysilane as a negative control, and 17-β-estradiol as the natural ligand reference material.
In the saturation binding experiment, the maximum binding capacity (Bmax) was 59.28 fmol/100 ug protein and the dissociation constant (Kd) was 0.1032 nM.  The performance criteria were met for the saturation 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 (IC50) 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 generally performed as expected.  The negative control, octyltriethoxysilane, 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 dimethylsulfoxide (DMSO; 0.1%) for 24 hours in three independent runs.  The experiments were performed using 96-well plates and each Chemical J concentration was 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 by 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 (RPCmax) was 6.4% for the first run, 2.8% for the second run and 7.3% for the third run, with the associated maximum level of response (PCmax) of 10[−5] M for all three runs.
In runs 1 and 3, all performance criteria were met for the reference chemicals 17 β-estradiol, 17α-estradiol, and 17 α-methyltestosterone.  The Hill Slope values for 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 PC50 and Log PC10 values for 17 α-methyltestosterone were lower than the expected values for all three runs.  The Log PC50 values for each run were -7.6, -6.6 and -6.1 (expected range -6.0 to -5.1) and the LogPC10 values for the runs were -8.8, -8.9 and -8.8 (expected range -8.0 to -6.2).  These minor deviations did not negatively affect the interpretation of the study.
Chemical J was negative for ER transcriptional activation in this test system.  

      iii.  AR Binding Assay (Rat prostate cytosol)
In the AR binding assay, ventral prostate cytosol homogenate from Sprague Dawley rats was used as the source of AR to conduct the binding assays.  A saturation binding experiment was conducted prior to the competitive binding experiment to demonstrate that the AR in the cytosol 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 weak 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 (Bmax) was 3.245 fmol/100 ug protein and the dissociation constant (Kd) was 0.4641nM.  Although the Bmax fell slightly below the expected range of 10 to 150 fmol/100 ug protein and the Kd 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) and 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 saturation binding assay.  All other performance criteria and the competitive binding assays indicated acceptable performance of the assay. 
In the competitive binding experiment, Chemical J reduced the mean specific binding of the radiolabeled ligand at the 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 initial analysis suggested that the data for runs 2 and 3 did not fit the four-parameter, nonlinear regression model for analysis 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-analyzed 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.  
The mean estimated log IC50 was -3.5 M for the analysis 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 IC50 values for the strong positive control R1881 and the weak positive control (dexamethasone) were -9.0 and -4.4 M, respectively, 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.  
The results from the 3 competitive binding experiments, with the undefined bottom plateau, are summarized in 
Table 44 and shown graphically in Figure 14.  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 "Binder", albeit with a weak affinity, 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
Log IC50 (M)           R1881
                                    -8.986
                                    -8.958
                                    -8.994
                                    -8.978
                                                               Positive control
                                    -4.440
                                    -4.382
                                    -4.455
                                    -4.426
                                                                     Chemical J
                                    -3.419
                                   ~-3.830c
                                   ~-3.855c
                                    ~-3.848
IC50 (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]
Log 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 reviewer.
b Approximate estimates as 95% CI estimates were very wide
NA = Not applicable; 
r[2] = Goodness of fit; 
RBA (%) = relative binding affinity


Figure 14.  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
                                       

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
Log IC50 (M)	           Chemical J
                                    -3.552
                                    -3.486
                                    -3.493
                                    -3.510
IC50 (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 reviewer.
NA = Not applicable; r[2] = Goodness of fit; RBA (%) = relative binding affinity

Figure 15.  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.
                                       

              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 photoionization 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 properly to positive control agents at two concentration levels.  The positive controls included a known inhibitor (prochloraz) and a known inducer (forskolin) of estradiol and testosterone production.
Chemical J was not cytotoxic 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-fold of the solvent control values in all 3 runs at 100 uM 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 uM; however, the induction was not reproducible in the other two runs.  
Decreased (0.8-fold the solvent control; p<=0.05) estradiol levels were observed at 0.1 uM for Chemical J in runs 2 and 3, and significant decreases were observed in all 3 runs at concentrations >=1 uM (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 uM) in Run 2.  The effects of Chemical J on testosterone and estradiol production are summarized in Table and shown in Figure 16 and Figure 17.  
Guideline acceptability recommendations and requirements were generally met, including lack of cytotoxicity, 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 estradiol 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 uM prochloraz was 0.6-fold (> the 0.5-fold guideline limit). However, these exceptions were slight and did not impact the interpretation of the results.  
Based on the hormone responses in three runs, Chemical J was positive in the steroidogenesis assay.

Table 46:  Mean (+-SD) Hormone Concentrations following Treatment with Chemical J for 48 Hours.[a]
                          Nominal Concentration (uM)
                                     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 The lower limit of quantification (LLQ) was 25 pg/mL for testosterone and 10 pg/mL for estradiol.

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


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

              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 activity.  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 amount 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 background 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 0.090 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 activity controls.  The responses of the full activity 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 IC50, 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 performance 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 Log IC50 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 IC50 of the test material 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 level 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 presented in Table 47, along with the overall standard deviation and % CV.  Inhibition response curves for Chemical J from each run are shown in Figure 18.
Based on the results of three, Chemical J is classified as inhibitor in the aromatase assay. 
Table 47:  Effect of Chemical J on Aromatase Activity (as Percent of Control) From Independent Runs [a]
Chemical
                                     Run 1
                                     Run 2
                                     Run 3
                                     Mean
                                      SEM
                                      %CV
                                 Log IC50 (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 18.  Aromatase Inhibition Response Curves for Chemical J from Each Test Run.

                                       
 

      vi. Uterotrophic Assay (Rat)
In the in vivo uterotrophic assay conducted to screen for potential estrogenic activity, Chemical J was administered daily 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 days (PND) 19-21.  A positive control group was treated with a daily dose of 17α-ethynyl estradiol (EE) at 10 μg/kg bw/day by oral gavage.  Body weights were determined daily.  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 significant increase in VO was noted in the Chemical J-treated females.  At 475 mg/kg bw/day, body weights were decreased (p<0.05) by 15% on Day 3, resulting in a 59% decrease (p<0.05) in overall (Days 1-4) body weight gains relative to the vehicle control.  Uterine 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) by 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.

              i. Hershberger Assay (Rat)
In the in vivo Hershberger assay testing for androgenic activity, Chemical J was administered daily 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), 25, 125, or 500 mg/kg bw/day for 10 consecutive days.  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, additional groups of seven 55-day old castrated male Sprague Dawley rats/group were dosed daily via oral gavage with Chemical J in corn oil at 25, 125, or 500 mg/kg bw/day for 10 consecutive days 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 for 10 days in conjugation with a daily s.c. dose of TP (0.4 mg/kg bw/day).
Body weights, body weight gains, and food consumption were measured.  Animals were euthanized approximately 24 hours after the final dose administration.  The five androgen-dependent tissues (i.e., 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 in either the agonist or antagonist assays on mortality, clinical signs of toxicity, body weights, body weight gains, food consumption, adrenals and kidneys 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 dose 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%), indicating that the test system 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 any dose. The decrease in LABC weight at 500 mg/kg bw/day is not considered 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%), LABC (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 system was sensitive to an anti-androgenic response.
Liver weights were increased by 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-androgenic 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 hypertrophy, 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 were seen in two or more of the androgen responsive tissues. 
Chemical J was negative for androgenicity and anti-androgenicity in the Hershberger assay.
              ii. Pubertal Female Assay (Rat)
In a Female 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) 22 to 42.  Animals were examined for vaginal opening (VO) daily beginning on PND 22, and weight at day of attainment was recorded.  Following sacrifice on PND 42, total thyroxine (T4) and thyroid stimulating hormone (TSH) levels were determined using chemiluminescent immunoassay and radioimmunoassay, respectively.  At termination, weights were measured for liver, kidneys, thyroid, adrenals, pituitary, and urogenital organs, and microscopic examinations were performed on the ovaries, uterus, thyroid, and kidneys from the control and high-dose groups and on livers from all dose groups. 
At 400 mg/kg bw/day, treatment-related increases (p<0.05) were observed in alanine aminotransferase (ALT; ↑24%) and gamma glutamyltransferase (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/day.  Animals in these dose groups also displayed treatment-related histopathological effects consisting of very slight to slight hepatocyte hypertrophy with altered tinctorial properties (increased cytoplasmic eosinophilia) of centrilobular and midzonal hepatocytes, very slight multifocal chronic inflammation, and very slight to slight increased number of mitotic figures of hepatocytes.  These findings showed a dose-dependent increase in incidence and severity, and were indicative of hepatotoxicity at 400 mg/kg bw/day.  Based on these findings in liver and on evidence of hepatic necrosis at 500 mg/kg bw/day in a concurrently submitted Hershberger study, the high dose in the current study appears to be an adequate high dose. 
There were no significant treatment-related changes or alterations on mortality, clinical signs of toxicity, body weights, body weight gains, age of attainment of VO, body weight at VO, mean age at first estrus, mean cycle length, percent cycling, percent regular cycling, organ weights, serum T4 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 thyroid-related endpoints 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, thyroxine (T4), and thyroid stimulating hormone (TSH) levels were analyzed using radioimmunoassays (TSH) or chemiluminescent assays (testosterone and T4).  Liver, kidney, pituitary, adrenals, thyroid, and urogenital organ weights were recorded, and microscopic examinations of the thyroid, testes, epididymides, kidneys, and liver were performed.
As shown in Table 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, body weight grain, weight 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 pituitary 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% decrease 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 thyroid, but increased thyroid follicular cell heights and decreased colloid area were noted at 400 mg/kg bw/day.   No changes were noted in serum T4 levels (Table 51).
Significant (p<0.05) increases were observed 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, respectively.  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 hypertrophy with altered tinctorial properties in centrilobular and midzonal hepatocytes (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 hepatocytes (2/15 control, 9/15 at 400 mg/kg bw/day).  A significant increase (53%) was also noted in gamma glutamyl transferase (GGT) at the high dose.

It appears the high 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 treatment related changes were seen at the low dose (50 mg/kg bw/day). 
At 200 mg/kg bw/day, delays in PPS, decreases in testosterone levels, changes in two androgen sensitive organs (seminal vesicle + coagulating gland and the ventral prostate) were reported. At 400 mg/kg bw/day, the observed changes were: delayed PPS, decreases in testosterone, decreases in four androgen sensitive tissues (seminal vesicle + coagulating gland, ventral prostate, dorsolateral prostate, and LABC complex).  Changes in the thyroid histopathology 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.


Table 48: Preputial 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.

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 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
                                      (%)
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
Epididymis, 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
Epididymis, 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
                                      NA
Testis, right (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 weight 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.
Table 50:  Additional 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 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
                                      (%)
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
Thyroid, 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 mean 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 weight 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.

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 T4, Total (ug/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 parentheses) the %CV is less.
SD = Standard Deviation; 
CV= Coefficient of Variation
*Significantly different from controls at p<0.05.



     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 system 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. 
Spawning frequency in the negative control occurred at least every 4 days and fecundity 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, respectively.  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, respectively, while male GSI was significantly (p<0.05) increased 79% at the 3.3 mg a.i./L level. 
Female plasma vitellogenin (VTG) was significantly reduced (p<0.05) by 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 testosterone 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 biologically significant. Histopathology on female gonads reported severe oocyte 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 proportions 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 reported in both the control and treatment groups. Additionally, 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 statistical 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.
In summary, at the highest treatment group, i.e., 3.3 mg a.i./L, there was a significant (p<0.05) decrease in fecundity and female plasma VTG and estradiol, along with a significant increase in male and female GSI. Additionally, alterations 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 body 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, fecundity, T and E), Chemical J is considered to be positive for potential interactions with the E and A pathways.
Table 52:  Chemical J - Reproductive and HPG Endpoints for Male Pimephales promelas in the FSTRA
                                       
                                   Treatment
                                  (mg a.i./L)
                                  [measured]
                                Tubercle Score
                                      GSI
                     Gonadal Staging and Histopathology[1]
                                  Plasma VTG
                                   Plasma T
                                   Plasma E2
                                       
                                    Median
                                 % Difference
                               Effect? (Yes/No)
                                 % Difference
                                 % Difference
                                 % Difference
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
                                    -18.82
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

Table 53.  Chemical J -Reproductive and HPG Endpoints for Female Pimephales promelas in the FSTRA

Treatment
(mg a.i../L)
[measured]
Fecundity
Fertility Success
Tubercle Score
GSI
Gonadal Staging and Histopathology[1]
Plasma VTG
                                   Plasma T
                                   Plasma E2

                                   % Diffe.
                                    % 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[2]
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
                                       
                                 % Difference
                                 % Difference
                                 % Difference
                                 % Difference
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

 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 system was maintained 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 delays (p<0.05) were observed at Chemical J concentrations associated with growth inhibition and clinical signs of toxicity.   On Day 7, developmental stage was significantly (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 asynchronous development reported on Days 7 or 21. There were no treatment-related effects on thyroid gland histopathology at any treatment level.
Some transient abnormal behaviors (floating on the surface, lying on the bottom of the tank, inverted or irregular swimming, lack of surfacing activity, and being nonresponsive 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 any 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 analysis.  Additionally, there 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 delays, in the absence of thyroid histopathological effects, makes it difficult to interpret whether Chemical J has the potential to interact with the hypothalamic-pituitary-thyroid (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 likely to interact with the thyroid pathway in the AMA.
Other Scientifically Relevant Information (OSRI) for Chemical J
The registrant elected to conduct the 11 Tier 1 screening assays, therefore, no OSRI were submitted to the agency. However, Chemical J has Part 158 mammalian and ecotoxicity 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). Evaluations included, but were not limited to, clinical observations and mortality, body weight, food consumption, hematology, clinical chemistry, organ weights, gross necropsy 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 thyroid for males.  Additionally, effects on the liver and kidneys were observed (i.e., changes in weights and histopathology) at the 150 mg/kg bw-day dose.

In a 1-year feeding study, 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 histological 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 thyroid or pituitary weights were reported.  There were no histological changes in the testes, seminal vesicles, prostate, ovary, uterus, thyroid or pituitary compared to the control group.  Liver effects including, hepatocellular hypertrophy and increased liver weight were observed 14.28 and 15.68 mg/kg bw- day for males and females, respectively.

In a 2-year 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-dose 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 weeks 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, 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 necropsy 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 in testicular atrophy was observed at 39.21 mg/kg bw/day.  Microscopically, 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 the prostate, ovaries, uterus, mammary gland, pituitary, or thyroid at any dose tested.   No changes in ovary or thyroid weights were reported.

In a 2-year supplemental carcinogenicity study, Chemical J was administered to 50 male and 50 female Sprague Dawley 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 necropsy and histopathology. At 125 mg/kg bw/day, Absolute and relative testes weights were significantly 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 testes, 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 hypospermia and cellular debris in the epididymis of treated males.  Liver effects including hypertrophy and vacuolization were also observed at 125 mg/kg bw/day. 

In a 2-year feeding/oncogenicity study, groups of 110 male and 110 female Crl:CD-1(ICR)BR mice (Mus musculus) were administered technical 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 separately in which 25 mice/sex were evaluated at 24 months. Evaluations included, but was not limited to, clinical observations and mortality, body weight, food and water consumption, clinical chemistry, gross necropsy and histopathology. No effects on the weights of the thyroid, ovaries or pituitary were observed.  There were no histological changes in the testes, seminal vesicles, prostate, ovary, uterus, mammary, thyroid or pituitary compared to the controls. Effects on the liver (increases in liver enzyme (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/oncogenicity study, groups of 60 female Crl:CD-1 (ICR)BR mice were administered technical Chemical J at dietary concentrations of 0 or 2000 ppm (393.5 mg/kg bw-day). Ten mice/group were 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 study 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 necropsy and histopathology. Clinical chemistry and urine analyses were not performed.
No effects on the weights of the thyroid, ovaries or pituitary were observed.  There were no histological changes in the testes, seminal vesicles, prostate, ovary, uterus, mammary, thyroid or pituitary compared to the controls.  Effects 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 of 0, 31.3, 93.8, 312.6 or 468.9 mg/kg bw/day by gavage on gestation days 6-15.  For maternal toxicity, the NOAEL 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 NOAEL was 93.8 mg/kg bw/day and the developmental toxicity LOAEL was 312.6 mg/kg bw/day, based on increased incidences of 14[th] rudimentary ribs and 7[th] cervical ribs.  In a range-finding study, 100% maternal 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 days 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 observations 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.   In a range-findings study, maternal mortality was observed at doses of 464 and 700 mg/kg bw/day, and the maternal and developmental toxicity NOAELs for that study 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 hypertrophy was observed in the P2 males.  This was supported by slight but statistically significant increases in liver weights in males.  At 1000 ppm, centrilobular hepatocellular hypertrophy was observed in both sexes in the P1 and P2 generations.  These were again supported by slight but statistically significant increases in liver weights.  For parental systemic 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 hypertrophy and increases in liver weights.  At 1000 ppm, an increase in the number of stillborn or percent born dead pups was observed in both generations.  In addition, multifocal or diffuse testicular atrophy was observed in males in the P2 generation.  Increased necrotic spermatocytes/ spermatids or decreased spermatozoa and atrophy of the prostate were also observed in these animals. Sexual maturation, estrous cycle length and periodicity, and sperm parameters were not evaluated in this study.   For reproductive toxicity, the NOAEL was 200 ppm (10 mg/kg 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 atrophy of the testes and prostate.  At 1000 ppm, there was a slight decrease in pup weight gain during lactation.  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 body 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 spermatocytes/spermatids or decreased spermatozoa and atrophy of the prostate).  Additionally, decreased testes weight, testicular atrophy and effects on spermatogenesis were observed in the rat chronic/carcinogenicity studies.

2.  Ecotoxicity OSRI Data
In an early life-cycle toxicity test with fathead minnow (Pimephales promelas) after 35 days 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 basis, Chemical J is classified as "moderately toxic" to freshwater and estuarine/marine fish with a 96-hour LC50 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 with mallard ducks  and Northern bobwhite quail with Chemical J, no effects on adult birds, hatchlings or reproductive parameters was observed at any concentration tested. The NOAEC was 260 mg/kg-diet, the highest concentration tested.
In a one-generation reproduction study with mallard ducks and Northern bobwhite quail with Chemical J, no effects on reproduction were observed at any concentration tested. The NOAEC was 60 mg/kg-diet, the highest concentration tested.
In a chronic 28-day toxicity test with the mysid shrimp exposure to Chemical J at concentrations between 2.58 and 85.6 μg ai/L did not cause any significant decreases on F0 survival, F0 male or female body length, number of young produced per female, survival of F1 mysids, or F1 male or female body length. 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 ug a.i./L, but not at the highest concentration of 85.6 ug 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.
In evaluating the available ecotoxicity Part 158 data for Chemical J for endpoints relevant to the E, A or T pathway, reproduction did not appear to be altered (birds and mysids), except for a significant decrease in the time to first brood compared to the control in the chronic mysid shrimp study.  

Discussion

          ii. 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 pathways related to steroidogenesis or sex steroid signaling.  The Tier 1 in vitro assays relevant to this analysis include the AR, binding, ER binding, ERTA, aromatase, and steroidogenesis assay.  Relevant in vivo assays include the male and female pubertal rat, uterotrophic, Hershberger, and the FSTRA assays.

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 the estrogen pathway across the relevant Tier 1 assays 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 suggesting 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 enzyme 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 steroidogenesis 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 steroidogenesis which would result in decrease estrogen levels are corroborated by 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 fecundity (↓98%).  An increase in gonadal oocyte atresia in females was also observed at this high concentration.  Additionally, a significant increase in male GSI (↑80%) along with alterations in gonadal histopathology was reported.  
Significant increases in female testosterone (↑200%) and GSI (↑41%) were reported at the next lower concentration which may suggest an inhibition of aromatase activity.  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 synthesis.
Therefore, based upon WoE evaluation of EDSP Tier 1 data, Chemical J may alter steroidogenesis which then interferes with the estrogen signaling pathway and the HPG axis.

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
ER Binding
ER Activation
Steroidogenesis
Uterine Weight
Ovarian Weight  or  Fish GSI
Ovarian/Gonad Staging and Histopathology 
Pituitary Weight
Estrous Cyclicity[b]
Age & Weight at VO
Fertility/Fecundity
Vitellogenin
Estradiol
Overt Toxicity Observed[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♀


                                     OSRI
Subchronic toxicity (Rat) 




N
N







Chronic toxicity (Dog)



N
N
N







Chronic toxicity (Rat)



N
N
N







Chronic toxicity (Dog)



N
N
N







Carcinogenicity (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; arrows (↓ or ↑) indicate the direction of the response; A shaded cell  indicates that parameter was 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. 


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 assays are delineated so as to facilitate determination of potential for androgenic, anti-androgenic, 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 by in the in vitro steroidogenesis assay which reported a significant decrease in testosterone 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-dependent tissue weights (↓15-41%) and finally 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 males 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.  Thus, there was redundancy between the male pubertal and FSTRA in that alterations in androgen-dependent tissues were reported in both assays.  Furthermore, as discussed in the estrogen pathway section, alterations in several levels of biological organization for the females were reported which included alterations to enzyme activity up to the apical endpoint of fecundity.  
Chemical J was classified as a "binder" in the AR binding assay; based on the analysis 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 Hershberger 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-generation reproduction rat Part 158 study, multifocal or diffuse testicular atrophy, increased necrotic spermatocytes/spermatids or decreased spermatozoa and atrophy of the prostate were observed in these animals.  Decreased testes weight, testicular atrophy and 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 steroidogenesis which then interferes with the androgen signaling pathway and the HPG axis.

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
AR Binding
Steroidogenesis
Testosterone
Testes Weight or Male Fish GSI

Gonad Staging and Histopathology
Epididymides Weight
Epididymides Histopathology 
Pituitary Weight
Accessory Sex Organ Weights/2° Sex Characteristics
Age and Weight at PPS
Fecundity/fertility
Vitellogenin
Overt Toxicity Observed[b]
                                  EDSP Tier 1
AR Binding
P












Aromatase

P











Steroidogenesis

P











Hershberger








N




Male Pubertal Rat


↓(49%)M
↓(85%)H
No effect
No effect
No effect
No effect
↓(8%)L;
↓(10%)M
↓(10%)H
↓(15-19%)  M;
↓(16-41%) H
↑(1.7d) H



FSTRA


↑ (200%) M ♀
↑ (80%)
Present





↓
(98%)
H♀
↓
(88%)
H♀

                                     OSRI
Subchronic toxicity (Rat) 



No effect

No effect
No effect
No effect
No effect
No effect




Chronic toxicity (Dog)



No effect
No effect
No effect
No effect






Chronic toxicity (Rat)



 (23%) M;
(19-42%); H[b]
Testes atrophy

Present






Carcinogenicity (Mouse)



No effect
No effect

No effect






Two-generation reproduction  (Rat)




No effect
Testes atrophy

No effect

Present

Present


Avian Reproduction










No effect


Aquatic Invertebrate Reproduction










No effect


a  Key to responses:  Positive (P), negative (N) or equivocal (E) observation; arrows (↓ or ↑) indicate the direction of the response; A shaded cell indicates that parameter was not evaluated or is not applicable; L, M, and H refer to low, middle and high test concentration, respectively; ♂ = male; ♀ = female
b 125 mg/kg bw/day dose in supplemental chronic/carcinogenicity study.
          iii. Effects 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 thyroid hormones (e.g., T4 and T3).  Thyroid hormones feedback to the hypothalamus or pituitary to regulate thyrotropin-releasing hormone and TSH, respectively, which, in turn, regulate hormone production by the thyroid gland.  The current Tier 1 screening battery does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid 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 pubertals 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 thyroid regulation.  The various targets of the thyroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects.
There were no changes to the thyroid pathway endpoints in the female pubertal study.  In the male pubertal assay, alterations in thyroid histopathology were reported and included increased thyroid follicular cell heights and decreased colloid area.  There was also a significant decrease in pituitary weights. Additionally, 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 thyroid weight or serum T4 levels.  A significant decrease (8-10%) in pituitary weight in the 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 in 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 the thyroid pathway in the AMA. 
In a three-month feeding Part 158 study with male and female COBS-CD (SD) BR rats, increases in the number of small follicles of thyroid 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 the follicle which is a normal response to maintain homeostasis of the thyroid hormone system.
Overall, while there were changes in thyroid histology in the male rat pubertal assay as well as in one Part 158 rat feeding study, which are likely normal homeostatic responses of the thyroid hormone system  There was no evidence of thyroid 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 and OSRI, there is no evidence that Chemical J results in significant changes in thyroid hormones through interaction with the HPT axis.


Table 57:  Thyroid Pathway for Chemical J
Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Chemical J[a]
Study Type /
Literature Citation
Hormones (T4 and TSH)
Pituitary Weight
Thyroid Weight
Thyroid  Gross and Histopathology
Frog Development Stage
Hind Limb Length
Snout to Vent Length
Overt Toxicity Observed[b]
                                  EDSP Tier 1
Male Pubertal (Rat)
                                   No effect
                          ↓ (8%) L; ↓ (10%) M. H
                                   No effect
                                       P
                                       
                                       
                                       
                                       
Female Pubertal (Rat)
                                   No effect
                                  No 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




Carcinogenicity (Mouse)


No effect
No effect




   a  Key to responses:  Positive (P), negative (N); arrows (↓ 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 two of the three treatment groups, at the lowest treatment groups, at the lowest treatment groups, where there was an no overt toxicity observed, there were no indications that the effects observed (delayed development and growth) were due the chemical's interaction with the thyroid pathway.

Conclusions for Chemical J
All eleven assays comprising the EDSP Tier 1 battery were available for Chemical J. Chemical J was evaluated for several hypothesized endocrine modes of action for each specific E, A, and 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 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 pathways, the  number and type of effects induced, the magnitude 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 toxicity was present. 

Integrating the in vitro and in vivo Tier 1 results, as well as consideration of relevant data beyond 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 pathways 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 assays.  Additionally, the negative responses in the ERB, ERTA and uterotrophic assays 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 steroidogenesis (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 directly interact on the androgen receptor.  However, there were complementary responses within the male pubertal and FSTRA assays and redundancy across biological levels of organization between those two assays.  The results in the in vivo assays, along with the positive results in the steroidogenesis assay demonstrate that Chemical J may have the potential to interact with the androgen system via a perturbation to steroidogenesis.    

For the T pathway, the AMA and female pubertal assays were negative.  The only finding was a change in thyroid histology in male rats which is likely 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 thyroid-mediated endpoints were not affected.  Based on this evidence, Chemical J does not appear to interact with the HPT axis.

        Case Study  --  Chemical N

A. Introduction
Chemical N, a cyclic unsaturated ketone which has irritant properties, is one of four chemicals selected from the 21 chemicals used in the EDSP assay and battery performance analysis to serve as case studies for the current WoE analysis for Tier 1 screening. The available information considered to determine the potential interaction of Chemical N with the E, A and/or T pathways included all 11 EDSP Tier 1 studies and OSRI, such as the Part 158 mammalian and ecotoxicity data, when available.  
In the case of Chemical N, issues included potential endocrine effects observed in a mammalian Tier 1 assay (male pubertal) that occurred only near the limit dose (1000 mg/kg/bw/day), an effect on fish vitellogenin that was not corroborated by other effects at that test concentration, 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. An analysis of the data submitted to the agency, using the WoE approach outlined by the Agency (USEPA, 2011b), is presented in Section E. 

Data Available for Chemical N
Chemical N is relatively soluble in water with a low log octanol-water partition coefficient (log Kow~2).  The chemical can persist in the environment although it undergoes aerobic degradation.  Based on the chemical-physical characteristics of the compound, it is considered moderately mobile and can move to surface water via runoff/erosion.  With its relatively low Kow, the compound is not expected to bioconcentrate in aquatic organism.

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. 

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 cytosol)
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
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 pathways
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 effects
          ii. ER Binding Assay (Rat uterine cytosol)
In the ER binding assay for Chemical N, uterine cytosol 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 concentrations (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 concentration in the cytosol preparation and functioning with the appropriate affinity 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, octyltriethoxysilane as a negative control, and 17-β-estradiol as the natural ligand reference material.
In the Saturation Binding Experiment, the maximum binding capacity (Bmax) was 59.28 fmol/100 μg protein and the dissociation constant (Kd) was 0.1032 nM. In the Competitive Binding Experiment, the estimated mean Log IC50 was −8.917 Log M for 17β-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 criteria were met.  Chemical N had no effect on specific binding of the [[3]H]-ligand; therefore, a Log IC50 and RBA could not be calculated for Chemical N. 
Based on the results of three runs, Chemical N is classified as Not Interactive in the ER binding assay. 

ER Transcriptional Activation Assay
In the ER transcriptional activation assay, hERα-HeLa-9903 cells were exposed to Chemical N (98.7% a.i.) at logarithmically increasing concentrations from 10−9 M to 10[−3]M in dimethylsulfoxide (DMSO; 0.1%) in three independent 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 products. Luciferase 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 limit of 10[−3] M. In 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-methyltestosterone (very weak agonist), and corticosterone (negative).   The reference chemical results adequately met the test guideline performance criteria. . For test chemical N, the RPCmax was 5.9% for the first run, 7.8% for the second run and 9.9% for the third run. Because the RPCmax < PC10 in all three runs, 
Chemical N was negative for ER transcriptional activation in this test system.
             AR Binding Assay (Rat prostate cytosol)
In the in vitro AR binding assay, ventral prostate cytosol from Sprague 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 increase 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 saturation binding experiment was conducted in duplicate and summarized in a submitted study profile.  In the Saturation Binding Experiment, the maximum binding capacity (Bmax) was 3.245 fmol/100ug protein and the dissociation constant (Kd) was 0.4641 nM.  These values were below the range of values from the validation studies (0.685-1.57 nM).  The Scatchard plot indicated a linear response across the concentrations of ligand added. Nonspecific binding as a percent of total binding was less than 20% across the entire concentration range in the saturation binding assays (range 6.2-19.8%), with the exception of the high concentration (10 nM) in one assay, which was 24.6%.
In the competitive binding experiment, the estimated mean Log IC50s 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 affinity (RBA) for the weak positive control was 0.0027%.  All performance criteria were met.  
Chemical N had no effect on specific binding of the [[3]H]-ligand; therefore, a Log IC50 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 assays, Chemical N is classified as a "Non-Binder" in the AR binding assay. 

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 100 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) plate 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 inducer (forskolin) of estradiol and testosterone production.
The guideline 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 production in the presence of 10 uM forskolin met induction requirements.  Although testosterone inhibition was greater than the required 50% in the presence of 1 uM prochloraz, inhibition of estradiol only reached 40% in Runs #2 and #3.  Because all other guideline requirements were met, these results were deemed acceptable.
No solubility or cytotoxicity 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 vehicle controls in the test assays 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 reviewers consider these data acceptable.  
No effect on testosterone production was observed at any 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 slight 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 steroidogenesis assay.

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 (LSC).  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 curve, 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 IC50 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.
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-protein[−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 activity controls.  The 10[ - 3] M dose level reduced the aromatase activity by approximately 20%.
Based on the results of three runs, Chemical N is classified as a Non-Inhibitor in the aromatase assay.

Uterotrophic Assay (Rat)
In the uterotrophic assay conducted 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 days (PND) 56-58.  A positive control group was treated with 17α-ethynyl estradiol (EE) in corn oil by s.c. injection at a dose level of 0.27 ug/kg/day.  :  The high dose (50 mg/kg/day) was selected based on the results of a range-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 euthanized 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 any Chemical N treated groups.  No effects on body 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) by 32% compared to the vehicle control.  
In the positive control group (EE), overall body weight gains were decreased (p<0.05) by 28% on Days 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) group were increased (p<0.05) by 86% each.  
No statistically significant increase in uterine weight was seen in this study. Hence, Chemical N was  negative in the Uterotrophic assay.

             Hershberger Assay (Rat)
In the in vivo Hershberger assay testing for androgenic activity, Chemical N (>=98.7+-0.1%) was administered daily 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 days.  The study included an androgenic positive control group of seven castrated male rats dosed daily 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 daily via oral gavage with Chemical N in corn oil at 100, 400, or 800 mg/kg/day for 10 consecutive days in conjunction with a daily s.c. injection of TP in corn oil at 0.4 mg/kg/day.  The control group for anti-androgenic activity consisted of seven castrated male rats dosed daily with TP (0.4 mg/kg/day) by s.c. injection, and the positive anti-androgen control group consisted of seven castrated male rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg/day for 10 days in conjugation with a daily s.c. dose of TP (0.4 mg/kg/day).
Body weights, body 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 (LABC), Cowper's glands or glans penis), adrenals, kidneys, and liver were weighed and macroscopically examined. 
No adverse treatment-related effects were observed on mortality, clinical signs of toxicity, body weights, body 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 kidneys in the androgenic and anti-androgenic studies.  Liver weights were increased (p<=0.05) by Chemical N treatment in both the androgenic and anti-androgenic portions of the study by 27 - 28% at 400 mg/kg/day and by 32 - 34% at 800 mg/kg/day.  The high dose was selected based on the results of a range-finding study in which at a 1000 mg/kg/day dose, there was excessive toxicity as evidenced by significant body 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 system 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-treated group.  These data indicate that the test system was sensitive to an anti-androgenic response.  Flutamide administration did not affect food consumption or body weight gains.  
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 with 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 administered the vehicle by oral gavage only while the other group was administered the vehicle by oral gavage and s.c. injection.  Body weights in animals given corn oil by both the oral and s.c. routes were similar to the body weights in animals given corn oil by oral gavage only.  However, a minor decrease (p<=0.05) in overall (Days 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 by gavage only.  Minor decreases (p<=0.05) of 6 - 8% of food consumption during the intervals of Days 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 any organ weight between the two control groups. 
Chemical N was negative for androgenicity and anti-androgenicity in the Hershberger assay.

Pubertal Female Assay (Rat)

In the Female Pubertal Assay, 16 Sprague Dawley rats/dose group were treated daily 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 (PND) 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 thyroxine (T4) levels were determined using a chemiluminescent immunoassay and thyroid stimulating hormone (TSH) levels were determined using a radioimmunoassay; serum was also analyzed for levels of selected clinical chemistry parameters.  Adrenal, liver, pituitary, thyroid, and urogenital organ weights were recorded and microscopic examinations of the ovaries, uterus, thyroid, and kidneys 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 injury.  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 attainment of VO, mean age at first vaginal estrus, mean cycle length, percent cycling, or percent regularly cycling.  There were no dose-related effects on serum T4 or TSH. 
At 800 mg/kg/day, adjusted, absolute, and relative liver weights were increased (p<0.05) by 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 thyroid.  
Serum urea nitrogen levels were decreased (p<0.05) in the 50, 200, and 800 mg/kg/day dose groups (↓17%, ↓25%, and ↓25%, respectively).  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) by 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 within 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 thyroid pathways in the female pubertal assay.

Pubertal Male Assay (Rat)
In the Male 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 for preputial separation (PPS) daily beginning on PND 30 and weight at day of attainment was recorded.  Following sacrifice on PND 53, total T4 and testosterone levels were determined using chemiluminescent immunoassays and TSH levels were determined using a radioimmunoassay.  Serum was also analyzed for levels of selected clinical chemistry parameters.  Adrenal, liver, pituitary, thyroid, and urogenital organ weights were recorded, and microscopic examinations of the testes, epididymides, thyroid, and kidneys 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 59, at 800 mg/kg/day, a statistically significant (p<0.05) delay in the age of attainment of PPS (1.8 days, adjusted for body weight on PND 23) was observed compared to the controls.  Although the unadjusted age at attainment of PPS was increased in the 800 mg/kg/day group (+1.7 days 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.  Body weights, body weight gains, and body weight at attainment of PPS were similar across all dose groups. 

Table 59:  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 body 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
Body 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 body 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 body 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
Body 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 were obtained 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=Not applicable; Not reported; *Significantlydifferent from controls at (p<0.05)
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 vesicles weights in the 50 and 200 mg/kg/day groups were similar to controls.  There were no dose-related effects on the weights of kidneys, pituitary glands, adrenal glands, ventral prostate, dorsolateral prostate, levator ani-bulbocavernosus muscle (LABC), epididymides, testes, or thyroid.  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 by 16-20%, and at 800 mg/kg/day by 32%-33% compared to controls.
As shown in Table 60, at 800 mg/kg/day, the level of serum testosterone was decreased (p<0.05) by 58% compared to the control.  Levels were also decreased at 50 and 200 mg/kg/day; however, the decreases were not statistically significant.  Serum urea nitrogen 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) by 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 T4, serum TSH, or the other evaluated clinical chemistry parameters. 
Chemical N was positive for the A pathway and negative for the T pathway in the pubertal male assay. 

Table 60:  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 T4, Total (ug/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
Creatinine (umol/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
Alkaline phosphatase
(u/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 Data were 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.
Fish Short-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) were 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, respectively.  The test system 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 significantly (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 any treatment group compared to the controls.  However, the study author reported reduced feeding and an observable decrease in body weight in high-dose males compared to the controls.
Spawning frequency occurred at least every 4 days and fecundity 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 significantly (p<0.05) reduced 44 and 77% of control at the 7.62 and 79.8 mg ai/L treatment levels, respectively, 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 significantly different (p>0.05) from the control; tubercles were not observed in any female. Although not analyzed statistically, there were no reported treatment-related histopathology observations in males, but there was a reported increase in incidence of mild to moderate oocyte atresia at the 79.8 mg ai/L treatment level compared to the female control fish.  Sex-steroids were not measured. Observation of dorsal fat (nape) pads was reported but further analysis 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; however, 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 reductions 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 body 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 VTG in 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.  Therefore, while the reported VTG decrease could be due to an interaction of Chemical N with endocrine function, the veracity or biological significance of this is uncertain.  Conceptually, a decrease in female  VTG could be reflective of direct estrogen 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:  Reproductive and HPG Endpoints for Female Fathead Minnow in the FSTRA
                                 Mean-Measured
                                   Treatment
                                  (mg a.i./L)
                                  [measured]
                                   Fecundity
                               Fertility Success
                                Tubercle Score
                                      GSI
                      Gonadal Staging and Histopathology
                                  Plasma VTG
                                       
                                 % Difference
                                 % Difference
                                    Median
                                 % Difference
                               Effect? (Yes/No)
                                 % Difference
                          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]
Abbreviations: Conc. Concentration.  [GSI] Gonado-Somatic 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.

Table 62: Chemical N - Reproductive and HPG Endpoints for Male Fathead Minnow in the FSTRA.
                                 Mean-Measured
                                   Treatment
                                  (mg a.i./L)
                                  [measured]
                                Tubercle Score
                                      GSI
                     Gonadal Staging and Histopathology[1]
                                  Plasma VTG
                                       
                                    Median
                                 % Difference
                               Effect? (Yes/No)
                                 % Difference
                                       
                          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
                                    -88.63
                                       
Abbreviations: Conc. Concentration.  Diff. Difference.   [GSI] Gonado-Somatic 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
                                       
                                 % Difference
                                 % Difference
                                 % Difference
                                 % Difference
                    Control (dilution water only), 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.
LOQ=<0.199 mg ai/L.
Amphibian 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 system 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 had no significant effect (p>0.05) on normalized hind-limb length (HLL) at any treatment level (up to including 59.0 mg a.i./L) on Day 7 or 21.  Non-normalized HLL was also analyzed and showed no effects at either time point.  Further, there was no significant acceleration or delay of median NF developmental stage on Day 7 or 21. No asynchronous development was observed in any treatment group. Mild follicular cell hypertrophy was observed in the control and all treatment groups and did not appear to be treatment-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 reported based on comparison to the negative (clean water) control.  Analysis 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 individuals from the control and Chemical N-treated groups which matured beyond 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 any 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, respectively). At test termination, tadpoles with bent tails were observed in the control (17%) and all treatment groups (20, 29, and 17%) and, like thyroid gland observations, did not appear to be related to Chemical N exposure.  No apparent cause of this common malformation was reported.  Potentially 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 any iodide supplementation was provided beyond 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 analyzed for pesticides, heavy 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 CaCO2) was slightly higher than recommended in the guideline (40-48 mg/L as CaCO2).  Ultimately, the presence of bent tails in these tadpoles was not considered to have impacted the study conclusions in this case. The absence of significant effects on thyroid-related endpoints in the AMA suggests that Chemical N did not interact with the amphibian HPT axis or its downstream thyroid hormonal pathway under these study conditions.
Table 64:  Developmental and Thyroid Gross Pathology/Histopathology Endpoints1,2 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
Abbreviations: Diff. Difference.  NA Not applicable.
[1] Unless otherwise indicated, effects are reported based on comparison to the clean water control.  Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.
2 Unless otherwise specified, effects are considered statistically significant at p < 0.05.
3 Hind-limblength is normalized to snout-vent length (SVL).
[H] Several tadpoles were excluded from the analysis of Day 21 endpoints for wet weight, SVL, HLL, and norm HLL. Excluded individuals comprised 13, 8, 10, and 18% of the control, 0.584, 5.63, and 59.0 mg ai/L treatment group total sample sizes, respectively. Late stage individuals ranged from NF 61 to 62 with no apparent pattern of occurrence or dose-dependency.

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
                                       
                                 % Difference
                                       p
                                 % Difference
                                       p
                                 % Difference
                                       P
                                 % Difference
                                       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  Unless otherwise indicated, effects are reported based on comparison to the negative (clean water) control.
2  Unless otherwise specified, effects are considered statistically significant at p < 0.05.
H Several tadpoles were excluded from the analysis of Day 21 endpoints for wet weight, SVL, HLL, and norm HLL. Excluded individuals comprised 13, 8, 10, and 18% of the control, 0.584, 5.63, and 59.0 mg ai/L treatment group total sample sizes, respectively. Late stage individuals ranged from NF 61 to 62 with no apparent pattern of occurrence or dose-dependency.


Other Scientifically Relevant Information (OSRI) for Chemical N
                 1. Mammalian OSRI Data
The registrant elected to conduct the entire Tier 1 assay. However, limited in vitro and mammalian in vivo data were submitted as OSRI.  These studies are discussed below

Chemical N was evaluated in a ERα yeast two-hybrid (Y2H) system.  The system 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 concentration (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 ERα 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 purity) 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 only males rats in high dose group.  No effect on testes or thyroid weight was observed.  No histopathological findings were seen in the testes, prostate, ovaries, uterus, pituitary or thyroid.  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.

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 thyroid weight was observed.  No histopathological findings were seen in the testes, prostate, seminal vesicle, ovaries, uterus, pituitary or thyroid.  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 50 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 carcinogenicity 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 thyroids.  No histopathological effects were seen in the testes, prostate, ovaries, uterus, mammary, or thyroid. 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 at dose levels of 0, 250 or 500 mg/kg bw/day, 5 days 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 mesenchymal tumors of the integumentary system (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 thyroids were observed.  No histopathological effects were seen in the testes, prostate, ovaries, uterus mammary, or thyroid. There was no evidence of carcinogenicity of Chemical N in female B6C3Fl mice. 
In a developmental toxicity study, Chemical N [96.8% purity] was administered by 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 days 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 ppm and the LOAEL was 115 ppm, based on the significantly decreased fetal crown-rump distance in female fetuses. 
                 2.                  Ecotoxicity OSRI Data
No ecotoxicity OSRI were available for review.

       Discussion
              i.               Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis

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 assays relevant to this analysis include the AR, binding, ER binding, ERTA, aromatase, and steroidogenesis assays.  Relevant in vivo assays include the male and female pubertal rat, uterotrophic, Hershberger, and the FSTRA assays.
Effects on Estrogen Pathway: Table 66 below summarizes the results of the available EDSP studies and endpoints relevant for determining the potential for Chemical N to interact with the estrogen signaling pathway.  Effects on body weight or size are noted; such effects are typically in response to generalized toxicity but could be endocrine mediated.  Any other signs of overt toxicity or cytotoxicity in relevant in vitro and in vivo assays are identified to help distinguish responses due to generalize toxicity versus endocrine activity and to provide a characterization of the observed responses and any uncertainties.
Chemical N did not produce responses in the in vitro assays, the in vivo Uterotrophic assay, or the female pubertal rat assays indicative of estrogen agonism or antagonism, or disruption of steroidogenesis. There were, however, some responses that may potentially be related to interaction with the estrogen or androgen pathways reported in FSTRA.   Plasma VTG 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).  No 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 significantly 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: fecundity (↓94%), fertilization success (↓70%), female plasma VTG (↓77%), and a slight increase in oocyte atresia which could be consistent with impaired reproduction and reduced VTG.    While reduced feeding and lower body 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 pathways 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 reduction in plasma VTG, increase in oocyte 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, the only 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.  

.  
Table 66:  Estrogenic/Anti-Estrogenic Pathway for Chemical N
Lines of Evidence Indicating Potential for Interaction with the Estrogen/Anti-Estrogenic Pathway for Chemical Na.
                        Study Type/Literature Citation
                                  ER Binding
                                 ER Activation
                                   Aromatase
                                Steroidogenesis
                                Uterine Weight
                 Ovarian Weight or Gonado-Somatic Index (GSI)
                              Gonadal Staging and
                                Histopathology
                               Pituitary Weight
                              Estrous Cyclicityb
                             Age & Wight at VO
                                   Fecundity
                             Fertilization success
                                 Vitellogenin
                         Overt  Toxicity observed  [b]
                                  EDSP Tier 1
ER Binding
                                Not Interactive
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
ERTA
                                       
                                   Negative
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
Aromatase
                                       
                                       
                                 Non-Inhibitor
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
Steroidogenesis
                                       
                                       
                                       
                                   Negative
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
Uterotrophic
                                       
                                       
                                       
                                       
                                   No effect
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
Female
Pubertal Rat
                                       
                                       
                                       
                                       
                                   No effect
                                   No effect
                                   No effect
                                   No effect
                                   No effect
                                   No effect
                                       
                                       
                                       
                                       
FSTRA
                                       
                                       
                                       
                                       
                                       
                                   No effect
                              ↑Oocyte atresia H
                                       
                                       
                                       
                                   ↓94% H
                                   ↓70% H
                                  ↓44% ♀M
                                  ↓77% ♀H
                                       
                                     OSRI
Yeast 2-hybrid assay ERα
                                   Negative
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
ERα transcriptional activation 
                                   Negative
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
Subchronic toxicity (Rat)
                                       
                                       
                                       
                                       
                                       
                                       
                                   No effect
                                       
                                       
                                       
                                       
                                       
                                       
                                       
Subchronic toxicity (Dog)
                                       
                                       
                                       
                                       
                                       
                                       
                                   No effect
                                       
                                       
                                       
                                       
                                       
                                       
                                       
Carcinogenicity (Rat)
                                       
                                       
                                       
                                       
                                       
                                   No effect
                                   No effect
                                       
                                       
                                       
                                       
                                       
                                       
                                       
Carcinogenicity (Mouse)
                                       
                                       
                                       
                                       
                                       
                                   No effect
                                   No effect
                                       
                                       
                                       
                                       
                                       
                                       
                                       
Developmental toxicity (Rat)
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
Developmental toxicity (Mouse)
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
a Key to responses: [L] Low treatment, [M] Medium treatment, [H] High treatment.  Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that parameter is not routinely evaluated or is not applicable 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.


Effects on Androgen Pathway: Table 67 summarizes the results of the available EDSP studies and endpoints relevant for determining the potential for Chemical N to interact with the androgen signaling pathway.  Effects on body weight or size are noted; such effects are typically in response to generalized toxicity but could be endocrine mediated as a decrease in T could is often associated with a decrease in body weight.  Any other signs of overt toxicity or cytotoxicity in relevant in vitro and in vivo assays 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 assays 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 only 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 perturbation or another unspecified mechanism.



Table 67:  Androgenic Pathway for Chemical N
Lines of Evidence Indicating Potential for Interaction with the Androgenic/Anti-Androgenic Activity for Chemical Na.
                                       
                        Study Type/ Literature Citation
                                  AR Binding
                                       
                                Steroidogenesis
                                 Testosterone
                  Testes Weight or Gonado-Somatic Index (GSI)
                              Gonadal Staging and
                                Histopathology
                                 Epididymides
                                    Weight
                                 Epididymides
                                Histopathology
                               Pituitary Weight
                          Accessory Sex
Organ Weights
                             Age and Weight at PPS
                         Secondary Sex Characteristics
                                 Vitellogenin
                         Overt Toxicity Observed[b]  
AR Binding
                                Not Interactive
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
Steroidogenesis
                                       
                                   Negative
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
Hershberger
                                       
                                       
                                       
                                   No effect
                                       
                                   No effect
                                   No effect
                                       
                                   No effect
                                       
                                       
                                       
                                       
Male
Pubertal Rat
                                       
                                       
                                   ↓58% H
                                   No effect
                                   No effect
                                   No effect
                                   No effect
                                   No effect
                                   ↓24% H
                                      SV
                                   ↑1.8 H
                                       
                                       
                                       
FSTRA
                                       
                                       
                                 Not measured
                                   No effect
                              ↑Oocyte atresia H
                                       
                                       
                                       
                                       
                                       
                                   No effect
                                  ↓44% ♀M
                                  ↓77% ♀H
                                       
                                     OSRI
Subchronic toxicity (Rat)
                                       
                                       
                                       
                                       
                                   No effect
                                       
                                       
                                       
                                   No effect
                                       
                                       
                                       
                                       
Subchronic toxicity (Dog)
                                       
                                       
                                       
                                       
                                   No effect
                                       
                                       
                                       
                                   No effect
                                       
                                       
                                       
                                       
Carcinogenicity (Rat)
                                       
                                       
                                       
                                       
                                   No effect
                                       
                                       
                                       
                                   No effect
                                       
                                       
                                       
                                       
Carcinogenicity (Mouse)
                                       
                                       
                                       
                                       
                                   No effect
                                       
                                       
                                       
                                   No effect
                                       
                                       
                                       
                                       
a Key to responses: [L] Low treatment, [M] Medium treatment, [H] High treatment; d Days; [Sem ves]  Seminal vesicles + coagulating glands.  Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that parameter is not routinely evaluated or is not applicable 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 vesicle

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 of reproduction and associated endpoints may be reflective of a non-endocrine MoA, given the observations of reduced feeding in male fish and reduced body weight in both males and females.  For example, the Agency's Assessment Tool for Ecological Risk (ASTER) 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 complementarity 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 male 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 hypothesis 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 Thyroid Hormone Pathway

In general, regulation of the HPT axis is comparable to the HPG axis, except that the feedback relationship involves thyroid hormones (e.g., T4 and T3).  Thyroid hormones feedback to the hypothalamus or pituitary to regulate thyrotropin-releasing hormone and TSH, respectively, which, in turn, regulate hormone production by the thyroid gland.  The current Tier 1 screening battery does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid 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 pubertals and the AMA). 

Thyroid Hormones: Table 68 summarizes the results of the available EDSP studies and endpoints relevant for determining the potential for Chemical N to interact with the HPT axis or thyroid hormone signaling pathway.  Effects on body 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 assays in the Tier 1 battery relevant to this modality.
The Tier 1 assays showed no evidence of interaction with the HPT axis or thyroid hormonal pathway in pubertal rats or metamorphosing amphibians.  The slight reductions in amphibian body weight and size and effects 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 thyroid hormonal pathway.  
Overall, the Tier 1 screening data suggest that Chemical N does not interact with the HPT axis or thyroid hormonal pathway. 

Table 68: Thyroid Pathway for Chemical N
Lines of Evidence Indicating Potential for Interaction with the Thyroid Pathway for Chemical N[a]
                                       
                         Study Type/Literature Ciation
Developmental Stage (Amphibian)
Asynchronous Development (Amphibian)
Hind Limb Length b (Amphibian)
Thyroid Weight
Thyroid Histopathology
T4
TSH
Pituitary Weight
Body Weight (BW)
or Amphibian
Snout-Vent Length (SVL)
Overt Toxicity Observed[b]   
Male Pubertal (Rat)
                                       
                                       
                                       
                                   No effect
                                   No effect
                                   No effect
                                   No effect
                                   No effect
                                   No effect
                                       
Female Pubertal (Rat)
                                       
                                       
                                       
                                   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
                                       
                                       
                                       
                                       
                                       
Carcinogenicity (Rat)
                                       
                                       
                                       
                                   No effect
                                   No effect
                                       
                                       
                                       
                                       
                                       
Carcinogenicity (Mouse)



                                   No effect
                                   No effect





      a Key to responses: [L] Low treatment, [M] Medium treatment, [H] High treatement.  Arrows (↓ or ↑) indicate the direction of the response; A shaded cell indicates that parameter is not routinely evaluated or is not applicable in the assay.
      b Hind-limb length is normalized to snout-vent length prior to analysis.


Conclusions for Chemical N
The absence of effects in the mechanistic in vitro, uterotrophic, and Hershberger assays 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 pathways through perturbation of the HPG axis.  For the estrogen pathway there were no redundant responses across assays, 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 assays (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 responses occurred near limit doses of Chemical N.  Given the high doses or aquatic concentrations needed to elicit these responses and the reduced feeding and body weights observed at high test level in fish, there is some uncertainty as to whether the observed 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 thyroid hormonal pathway.

  1. 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 pathways using Tier 1 data OSRI.  Because the EDSP WoE guidance is based on concepts routinely employed in the agency's WoE evaluations, the purpose of this analysis 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 analysis was on the WoE process for determining (1) whether or not there is a chemical interaction with E, A, or T signaling pathways,(2)  at what level of biological organization is that interaction expressed  (e.g., receptor/target binding, cell responses, enzyme activity, organ response etc), and under what conditions (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 system.  Determining responses to questions 1 and 2 is the fundamental analysis 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 of 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 analysis presented to the May 2013 SAP represented a broad range of physical-chemical properties, chemical classes and pesticidal modes of action and associated toxicities.  The further subsample used as case studies for this WoE analysis also spans a broad range of physical-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 assays potentially impacting the analysis of complementarity within assays and redundancy across the battery of assays.
The four case studies presented in Sections 6-9 are illustrative of the varying degrees of complementarity and redundancy observed within and across the Tier 1 assays responses.  The case studies also demonstrate the varying 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 hypothesis-based approach.  This hypothesis-based analysis is supported by the AOP/MoA conceptual framework that promotes a systematic and transparent approach to integrate all relevant types of data in characterizing responses at different levels of biological organization (e.g., receptor/target binding, cell responses, enzyme activity, organ response, males and female system integrity, development (thyroid) and comparative system integrity) (see SAP 2010; SAP 2011b; SAP 2012a, b).  Inherent within the AOP/MoA concept is the importance of understanding the adaptive or homeostatic capacity of biological systems and their 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 system may compensate, or depending on the degree of perturbation may become unstable, and therefore, dysfunctional.   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 endpoints in vivo.  
The evaluation of the experimental data concerning E, A, or T hypotheses 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/MoA, 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 illustrate this hypothesis-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.  
The determination whether a chemical is interacting with a particular pathway is based on an analysis of endpoints that are identified in each of the EDSP Tier 1 assays (Table 3).  While the in vitro studies contain comparatively few endpoints, the in vivo Tier 1 studies have multiple endpoints and in some cases have decision logic trees 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 consistent and transparent process for evaluating studies across study 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 observation of effects; the EDSP Tier 1 assays provide guidance on selecting maximum tolerated dose (MTD) or the limit dose used. In achieving an adequate high dose, it is recognized that some systemic (overt) toxicity may be observed or conversely that relatively 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 majority 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 assays 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. Likewise, in the case of Chemical S, the majority of Tier 1 in vivo responses were decreases in the measured endpoints and were largely expressed in the presence of overt toxicity.  This could be consistent with its pesticidal mode of action as an uncoupler of mitochondrial oxidative phosphorylation resulting in depletion of ATP.

 Chemical A

In applying the WoE guidance framework, the case study 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 as measured the in vitro Tier 1 assays.  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 magnitude of organ weight decreases and number of tissues affected.  Chemical A, however, did not produce any organ weight or histological effects in androgen-dependent tissues when evaluated in the Tier 1 male rat pubertal assay and other mammalian toxicology 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 effects 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, several 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 specificity as to their diagnostic ability (i.e., GSI and histology). Using the EDSP WoE hypothesis-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 steroidogenesis). 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 fish 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 (particularly altered steroidogenesis).  

Chemical S

The case study with Chemical S is an example of a difficult to test substance given its physical-chemical properties. The low water solubility combined with the high log Kow (>5) and Koc (~321,000 L/kg) values makes this chemical difficult to test in aquatic systems. Chemical S is also a severe irritant to the gastrointestinal tract, which is a potential source of overt toxicity in mammalian assay systems. The low palatability of or a gut irritation induced by Chemical S can limit the absorption and systemic 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. 

Although the in vitro Tier 1 assays were negative for Chemical S and the MIE 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 anti-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 of responses in the male pubertal assay consistent with an anti-androgenic interaction, but these effects only occurred near or at higher overtly toxic doses.   The minor increase in PPS at the lower dose was not corroborated with other evidence of delayed 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 weight 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 mammalian Tier 1 data (Hershberger and male pubertal assays), Chemical S has the potential to interact with the androgen pathway.  The negative in vitro data may indicate that a different MIE may be occurring or that an activated metabolite is responsible for the in vivo effects.  There is a greater degree of complementarity and redundancy in responses but at doses that are likely compromising the integrity of the gastro-intestinal tract. At lower doses, fewer responses are observed. Thus, when the Tier 1 data 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 overt toxicity, i.e., effects on male GSI and secondary sex characteristics (tubercles) were only observed at concentration with effects on survival.  The situation is similar to the rat in the sense that fewer responses are found at lower doses that are not overtly toxic. With respect to the estrogen pathway, there was limited complementarity and redundancy found among the available data in that only one estrogen related response was observed in the female pubertal assay (delayed 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., effects on survival).   The absence of complementary and redundant responses in the rat pubertal assays and AMA provides some support for a lack of interaction with the thyroid 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 concentrations, there is uncertainty 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 data in that there were no effects found relevant the T hormonal pathway.  

Consistent with the low solubility of Chemical S, a solvent (dimethylformamide) was used in an attempt to achieve test concentrations sufficient to challenge the test species in both the FSTRA and AMA.  In 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, physical-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 variability deviates from performance standards.    

Chemical 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 types of data are integrated along the biological continuum (i.e., in vitro and in vivo Tier I assays), 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 steroidogenesis, 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 (decrease 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 hypothesis in that other mammalian studies reported gonado-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 (gavage 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 hypothalamic-pituitary-thyroid 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 thyroid histology (increased thyroid follicular cell height and decreased colloid area) in the male pubertal assay, as well as, in one Part 158 rat feeding study (increase in number of small follicles). Given that Chemical J is known to induce microsomal enzymes, these changes are likely 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 thyroid-mediated developmental effects in the amphibian Tier 1 assay.  The lack of thyroid-related responses across taxa demonstrates that Chemical J does not alter HPT axis. 

Chemical N

In the case of Chemical N, the mechanistic in vitro studies, as well as, the in vivo Uterotrophic and Hershberger assays were all negative.  Nonetheless, there were limited complementary and redundant responses in the rat pubertal and fish Tier 1 assays with respect to potential interaction of Chemical N with the HPG axis.  These responses, 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, fecundity, 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 assays as well as effects on androgen-sensitive tissues (seminal vesicles) and delayed 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 likely exceeded or overwhelmed given that effects only occurred near limit doses.  

        Concluding Remarks

Because of the complexity of some of the in vivo assays where measurement endpoints can span multiple levels of biological organization and degrees of specificity as direct measures of interactions with endocrine-mediated processes, decision frameworks have evolved to assist reviewers in making determinations.  These decision frameworks 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 Figure 2). A tiered decision logic tree (Figure 3) has been developed for the fish assay depicting what is contained in the FSTRA guidance and provides a means of consistently and transparently identifying potential interactions with the E or A pathways. The extent of complementarity among measurement endpoints with any assay though must be considered in the context of the direction and magnitude of change as well as whether there are confounding effects such as overt toxicity or whether effects are observed at levels that are environmentally relevant.  Also, measurement endpoints can represent varying degrees of specificity 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 analyses are drawn from the subset of chemicals that received the first set of Tier 1 test orders based on their potential for exposure and not endocrine disrupting potential. Similar to the 21 chemicals that were used in the assay and battery performance analysis, the WoE case studies represent a range of physical-chemical properties and pesticidal modes of action and associated toxicities.  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 ensuring that an adequate range of exposure levels 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 specifically related to an endocrine-mediated AOP.  
The approach articulated within the 2011 EDSP WoE guidance document relies on an hypothesis-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 pathways. This conceptual framework promotes the systematic use of a battery of assays and other relevant information that are designed to evaluate particular responses along specific endocrine pathways. The scientific determination of an endocrine interaction focuses on comparing responses to what is known about the underlying 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, and consistency of the body 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 with respect to the level of biological organization and the conditions in which these interactions occurred. The decision logic used as part of the analyses provides a means of ensuring clear and consistent interpretations of potential interactions based on individual endpoints and combinations of endpoints within and across assays. 

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USEPA (2005). Guidelines for Carcinogen Risk Assessment. Risk Assessment Forum, Washington, DC. EPA/630/P-03/001F. Accessed online at http://www.epa.gov/raf/publications/pdfs/CANCER_GUIDELINES_FINAL_3-25-05.PDF

USEPA (2008). Meeting Minutes of the FIFRA Scientific Advisory Panel to review and consider the Endocrine Disrupter Screening Program (EDSP) Proposed Tier 1 Screening Battery. SAP Minutes No. 2008-03. Accessed online at 
http://www.epa.gov/scipoly/sap/meetings/2008/march/minutes2008-03-25.pdf

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USEPA (2009). An Effects-based Expert System to Predict Estrogen Receptor Binding Affinity for Food Use Inert Ingredients and Antimicrobial Pesticides: Application in a Prioritization Scheme for Endocrine Disrupting Screening. Office of Pesticide Programs, U.S. EPA, Washington, DC. Available at: http://www.regulations.gov/#!document Detail;D=EPA-HQ-OPP-2009-0322-0002 
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USEPA (2010) Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting on Re-evaluation of the Human Health Effects of Atrazine:  Review of Experimental and In Vitro Studies and Drinking Water Monitoring Frequency.  SAP minutes No. 2010-04.  Accessed online at
http://www.epa.gov/scipoly/sap/meetings/2010/april/042610minutes.pdf
USEPA (2011a) Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting on Integrated Approaches to Testing and Assessment Strategies:  Use of new Computational and Molecular Tools. SAP minutes No. 2011-04.   Accessed online at http://www.epa.gov/scipoly/sap/meetings/2011/may/052411minutes.pdf
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USEPA (2011b).  Endocrine Disruptor Screening Program Weight-of-Evidence:  Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing.  Office of Chemical Safety and Pollution Prevention. U.S Environmental Protection Agency, Washington DC. September 14, 2011. http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2010-0877-0021  
USEPA (2012a) Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting on Re-evaluation of Human Heath effects of Atrazine:  Review of Non-Cancer Effects and Drinking Water Monitoring Frequency.  SAP Minutes No. 2010-07.  Accessed online at: http://www.epa.gov/scipoly/sap/meetings/2010/september/091410minutes.pdf
USEPA (2012b) Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting on Comparative Effects Methodology Developed by the Office of Pesticide Programs and the Office of Water.  SAP minutes No. 2012-02.  Accessed online at: http://www.epa.gov/scipoly/sap/meetings/2012/january/013112minutes.pdf
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USEPA (2013a). Prioritization of the Endocrine Disruptor Screening Program Universe of Chemicals for an Estrogen Receptor Adverse Outcome Pathway Using Computational Toxicology Tools, Office of Pesticide Programs, U.S. EPA, Washington, DC. Available at: http://www.epa.gov/scipoly/sap/meetings/2013/012913meeting.html#materials
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USEPA  (2013b).  Endocrine Disruptor Screening Program SAP Review of EDSP Tier 1 Screening:  Assay and Battery Performance. May 21  -  23, 2013. http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPP-2013-0075-0003
Zoeller RT, Fenner-Crisp P, Patton DE. (2005). Using Mode of Action and Life Stage Information to Evaluate the Human Relevance of Animal Toxicity Data. Critical Reviews in Toxicology  Vol 35, No. 8-9, Pages 663-771.

                                 

				



			
                                  Appendix-1
                                       
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