UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

SUBJECT: EDSP Weight of Evidence Conclusions on the Tier 1 Screening Assays for the List 1 Chemicals

PC Code: See table, Attachment A Decision No.: NA Petition No.: NA
Risk Assess ment T y pe: NA
TXR No.: See table

, Attachment A
MRIDNo.:NA DP Barcode: NA Registration No.: NA Regulatory Action: NA
Case No.: NA
CAS No.: NA
40CFR: NA

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

# Fat**~~ fec** **~~ ~n** **~** Office of Pesticide Program

THR O UG H: Je ss Rowland

## ~ S

## ~~~ Deputy Divi s ion Director
Health Effects Division
Office of Pe s ticide Programs

TO: Jolene Trujillo Chemical Review Manager
Pe s ticide Re-Evaluation Division Office of Pesticide Programs Ver .A pr. 2010

EPA has completed its Weight of Evidence (WoE) assessment eval u ating results of the
Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. The WoE documents for the 52 chemicals are li sted in Attac hm ent A a l ong with the chemical
and report identifiers

.

Page I of2Page 2 of 2 Attachment A. EDSP List 1 Chemicals

Chemical Name PC Code TXR Number 2,4-D 030001 0057151
Abamectin 122804 0057152 Acephate 103301 0057153
Acetone 044101 0057154
Atrazine 080803 0057155
Benfluralin 084301 0057156
Bifenthrin 128825 0057157 Captan 081301 0057158 Carbaryl 056801 0057159
Carbofuran 090601 0057160
Chlorothalonil 081901 0057161 Chlorpyrifos 059101 0057162 Cyfluthrin 128831 0057163 Cypermethrin 109702 0057164
DCPA 078701 0057165
Diazinon 057801 0057166
Dichlobenil 027401 0057167
Dimethoate 035001 0057168
EPTC 041401 0057169
Esfenvalerate 109303 0057170 Ethoprop 041101 0057171
Fenbutatin-Oxide 104601 0057172
Flutolanil 128975 0057173 Folpet 081601 0057174 Glyphosate 417300 0057175 Imidacloprid 129099 0057176 Iprodione 109801 0057177 Isophorone 847401 0057178
Linuron 035506 0057179
Malathion 057701 0057180 Metalaxyl 113501 0057181 Methomyl 090301 0057182
Metolachlor 108801 0057183
Metribuzin 101101 0057184
MGK-264 057001 0057185 Myclobutanil 128857 0057186
Norflurazon 105801 0057150 o-Phenylphenol 064103 0057146 Oxamyl 103801 0057142
PCNB 056502 0057138
Permethrin 109701 0057149
Phosmet 059201 0057145 Piperonyl Butoxide 067501 0057141
Pronamide 101701 0057137 Propargite 097601 0057148 Propiconazole 122101 0057144 Pyriproxyfen 129032 0057140
Simazine 080807 0057136
Tebuconazole 128997 0057143 Tetrachlorvinphos (TCVP) 083701 0057147
Triadimefon 109901 0057139
Trifluralin 036101 0057135

### EDSP: WEIGHT OF EVIDENCE ANALYSI S OF POTENTIAL INTERACTION WITH ESTROGEN, ANDROGEN OR THYROID
PATHWAYS

CHEMICAL: CHLORTHA L DIMETHYL (DCPA)

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S E NVIRONMENTAL 

### P ROTECTION 

### A GENCYDCPA FINAL

ii TABLE OF CONTENTS

Abbreviations...................................................................................................iii
Executive Summary..........................................................................................1

I. Introduction.......................................................................................4

II. Sources of Scientific Data and Technical Information......................................5

A. EDSP Tier 1 Screening Assays............................................................5

B. Other Scientifically Rele vant Information (OSRI)......................................5

III. Weight of Evidence (WoE) Evaluation.......................................................5

A. EDSP Tier 1 Screening Assays.............................................................8

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9
1. Effects on Estrogen Pathway.....................................................9
2. Effects on Androgen Pathway..................................................13

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................16

IV. Committee’s Assessment of Weight of Evidence..........................................18

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................18

1. Tier 1 in vivo Assays.............................................................19

2. OSRI...............................................................................19

B. Estrogen Pathway...........................................................................20

C. Androgen Pathway.........................................................................21

D. Thyroid Pathway...........................................................................21

E. Conclusions.................................................................................21

V. EDSP Tier 2 Testing Recommendations....................................................21

VI. References.......................................................................................23

LIST OF TABLES: Table 1. Tier 1 Assay for DCPA........................................................................8
Table 2. Estrogenic/Anti-Est rogenic Pathway for DCPA.........................................10
Table 3. Androgenic/Anti-Andr ogenic Pathway for DCPA.......................................14
Table 4. Thyroid Pathway for DCPA................................................................17

APPENDIX 1. EDSP Tier 1 Screening Assays..........................................................25
APPENDIX 2. Other Scientifically Relevant Information (OSRI)................................35

APPENDIX 3. References Not Utiliz ed in the DCPA WoE Analysis.............................44DCPA FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

ANOVA Analysis of Variance
AOP Adverse Outcome Pathwa y A R Andro g en Rece p to r B max Bindin g at maximum BROD Benz y lox y resorufin-O-dealk y lase BUN Blood Urea Nitro g e n CA R Constitutive Androstane Rece p to r CFR Code of Federal Regulations

CG Cowper’s Gland

ChE Cholinesterase
ChEI Cholinesterase inhibition
CMC Carboxymethyl cellulose
CTA Comparative Thyroid Assay
CV Coefficient of Variation
CYP Cytochrome 450

DE R Data Evaluation Recor d DMSO Dimeth y l Sulfoxide DNA Deox y ribonucleic Aci d DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
E R Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase ) ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g enerationDCPA FINAL

iv Abbreviation Terminology

F2 Second filial g eneration
Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Intera g enc y Coordinatin g Committee on the Validation of Alternative K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

L Low dose LABC Levator Ani-Bulbocavernosus LAGDA Larval Am p hibian Growth and Develo p ment Assa y LC50 Lethal Concentration in 50% of test organisms

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

M Male
MDL Minimum Detection Level MEOGRT Medaka Extended One Generation Re p roduction Test
MH Medium hi g h
ML Medium low MoA Mode of Action MOE Mar g in of Ex p osure MRID Master Record Identifier

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated ConcentrationDCPA FINAL

v Abbreviation Terminology

MTD Maximum Tolerated Dose
N Ne g ative
NE Not examined/evaluate d
NF sta g e Nieuwkoo p and Faber’s Sta g in g Atlas NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level NS Not Statisticall y Si g nificant N R Not Re p orte d OCSPP Office of Chemical Safet y Pollution and Prevention OECD Organization for Economic Co-Operation and Development

OPP Office of Pesticide Programs

ORD Office of Research and Develo p ment OSCP Office of Science Coordination and Polic y OSRI Other Scientificall y Relevant Informatio n P Positive
P Parental g eneratio n PC Positive Control PC 10 Positive Control at 10% of response

PC 50 Positive Control at 50% of response

PND Post-Natal Da y POD Point of De p arture PPS Pre p utial Se p aration PROD Pentaox y resorufin-O-dealk y lase ( or de p ent y lase )
PX R Pre g nane X rece p to r QC Quality Control

RBA Relative Binding Affinity

RBC Red Blood Cells
RfD Reference Dose RPC max Relative to Positive Control at maximum

SAP Scientific Advisor y Panel SC Solvent Control

s.c Subcutaneous SDH Sorbitol deh y dro g enase SDWA Safe Drinking Water Act

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th SV Seminal Vesicles
T Th y roid ( hormonal p athwa y) T1WoERC EDSP Tier 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)DCPA FINAL

vi Abbreviation Terminology

TP Testosterone Pro p ionate
T R Th y roid Rece p to r TSH Thyroid Stimulating Hormone

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

VP Ventral Prostate VTG Vitello g eni n WoE Wei g ht-of-EvidenceDCPA FINAL

Page 1 of 44 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether a pesticide chemical inte racts with E, A, or T hormone pathways, the number and type of effects i nduced, the magnitude and pattern of responses observed across studies, taxa, and sexes we re considered. Additi onally, the conditions un der which effects occur were considered, in part icular, whether or not endocrine-rela ted responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On February 15, 2014 the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and th e Office of Science Coordination and Policy (OSCP) conducted a weight-of evidence (WoE) anal ysis of the potential interaction of DCPA with the E, A or thyroid T hormone pathwa ys. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

For DCPA, there was no convincing evidence to support potential interact ion with the estrogen pathway. Although DCPA increased estradiol produc tion in the Tier 1 steroidogenesis assay and the Tier 1 ER binding assay results were equivocal, the Tier 1 ER TA and aromatase assays were negative and there were no estrogen-related eff ects observed in the Ti er 1 uterotrophic and female pubertal assays or in the Part 158 ma mmalian toxicity studi es. There was a nontreatment-responsive decrease in fe cundity at the mid dose and a slig ht decrease in fertility at the high dose in the FSTRA; no other complement ary responses were observed across other endpoints within those treatment groups. In the wildlife Part 158 studies, in the bobwhite quail avian reproduction study, all observed reproductive effects were seen only at an overtly toxic dose, while in the mallard duck study decreases in viable embyros/hatching were observed (no other reproductive effects observe d). Therefore, there is a lack of redundancy across the battery,
with no or limited effects observed without complementary responses.

There was no convincing evidence of an intera ction with the androgen pathway. DCPA was found to be untestable (due to insolubility) in the Tier 1 AR binding assay and testosterone production was decreased In the Tier 1 steroidogen esis assay. DCPA was negative in theDCPA FINAL

Page 2 of 44 Hershberger assay and there were no treatment-related androgen effects in the male pubertal assay. There were no androgen-related effects observed in the mammalian Part 158 studies.
There was a decrease in fertili ty at the high dose in the FSTRA; however there was a lack of complementary responses across other endpoints. In the wildlife Part 158 studies, for the bobwhite quail reproduction study, al l effects were observed in th e presence of overt toxicity, while in the mallard decreases in viable embyr os/hatching were observe d (no other reproductive effects observed).

DCPA demonstrated a potential for interaction w ith the thyroid hormone pathway in the absence of overt or systemic toxicity. In the pubertal assays, serum T4 levels were dose-dependently
decreased in males at 500 mg/kg/day (78%) and 1000 mg/kg/ day (81%) and in females at 500 mg/kg/day (47%) and 1000 mg/kg/day (55%). In the amphibian metamorphosis assay (AMA), increased incidence of thyroid gland hypertroph y and follicular cell height increases were reported at the high concentration compared to the control. In addi tion, development changes
related to DPCA treatment seen in the AMA incl ude an increase in the normalized HLL on day 7
and 21.

In the mammalian Part 158 studi es, DCPA-induced thyroid effect s included thyroid follicular hypertrophy in a subchronic toxicity study in rats, and increased thyroid we ights, follicular cell hyperplasia/hypertrophy, decreased T4 levels and an increased incide nce of thyroid follicular cell neoplasms in both sexes of rats in a combin ed chronic toxicity/ carcinogenicity study.

In the EDSP Tier 1 male and female pubertal assa ys as well as in the OSRI, there was evidence for potential interaction with the thyroid pathway in studies conducted with adult animals, but no such data exists for potential thyr oid toxicity in the young animals.

For DCPA, the current point of departure (POD) of 10 mg/kg/day for human health risk
assessment is based on decreased level of T4 and increased incidence of histopathological lesions in the thyroid and liver at 10.0 mg/kg/day (LOAEL) in a chronic toxicity/carcinogenicity study in rats. This POD is used to derive the chronic Reference Dose (RfD) for dietary exposure risk assessments and the Margins of Expos ure (MOEs) for non-dietary exposure risk

assessments.

In general, since the POD and the toxicity endpo ints of concern are base d on data obtained from adult animals, there usually would be a concern that the POD may not be protective of potential thyroid toxicity in the young. Ther efore, a special thyroid assay in pregnant animals, fetuses, postnatal animals, and adult animals is recommended. This special study should use a mechanistic approach to generate specific data on the thyroid ( i.e. , the primary target of DCPA) to protect the developing ner vous system from thyroid hormone disrupting chemicals. The specific purpose of this st udy is to establish a POD ( i.e. , NOAELs and LOAELs or benchmark doses) that may be used for hu man health risk assessment. The POD from this special studyDCPA FINAL

Page 3 of 44 would address the concern for th e potential ability of DCPA to disrupt thyroid function in pregnant females and in the fetus or newborn. Howe ver, it is noted that the agency has issued a
Data Call-In notice for this special study under the Regist ration Review program.

For the wildlife, to add to the understanding of potential thyroid e ffects, the EDSP Tier 2 Larval Amphibian growth and Development Assay (LAGDA) is recommended since there is no chronic aquatic amphibian toxicity data available. Chronic toxicity data with fish (surrogates for aquaticphase amphibians) are not currently available for DCPA.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for DCPA. In both speci es, effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potenti al reproductive effects to birds from DCPA exposure. Relative to EDSP, additional testing is not recommended.DCPA FINAL

Page 4 of 44 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether a pesticide chemical inte racts with E, A, or T hormone pathways, the number and type of effects i nduced, the magnitude and pattern of responses observed across studies, taxa, and sexes we re considered. Additi onally, the conditions un der which effects occur were considered, in part icular, whether or not endocrine-rela ted responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On February 15, 2014 the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and th e Office of Science Coordination and Policy (OSCP) conducted a weight-of evidence analysis of the potential interac tion of DCPA with the E, A or T hormonal pathways. The T1WoERC c onclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in

vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited
OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

DCPA has a water solubility of 0.5 mg/L, a pa rtition coefficient (log oc tanol/water) of 4.28, and a vapor pressure of 2.5  10

− 6 mm Hg at 20°C. DCPA is stable in water, and is stable to photolysis in water and on soil. Bioconcentrat ion factors in bluegill sunfish are 1894, 777, and 2574 in whole fish, edible tissue, and viscera, respectively, and depur ation appears to be complete after 14 days.

The available information considered in determining the potential interaction of DCPA with the E, A and/or T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxi city studies and other pu blished articles. These data are summarized in Sections III.A through I II.C. An analysis of the data submitted to the Agency, using the WoE approach outlined by th e Agency (USEPA, 2011), is presented in
Section IV. The EDSP Tier 2 studies recommendations are presented in Section V.DCPA FINAL

Page 5 of 44 II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix

1. B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 assays were submitted by the Test Order recipien t. The Agency has also conducted a more recent
search of available scientific l iterature for any additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies considered but not utilized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and approach used in the WoE determination on the potential of a
substance to interact with endocrine-related processes ( i.e., E, A, or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of eviden ce process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to the individual studie s for their scientific quality and relevance in evaluating potential en docrine interaction(s). The second step is to integrate the data along different levels of biological organizati on while examining the extent of concordance of complementarity ( i.e ., the concordance of endpoi nts within an assay that measures multiple endpoints) and redundancy ( i.e ., the concordance of endpoints/responses across assays) in the observed responses across th ese different levels of biological organization. The third step is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to evaluate wh ether additional testing is n eeded based on the evidence and
conclusions described above.

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well asDCPA FINAL

Page 6 of 44 studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a speci fic endpoint for discerning whether or not the chemical has interacted with the endocrine syst em, are considered. The co llective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indi cation of potential interaction as measured by the study.

The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data as well as characterizing the extent to which the available data suppor t a hypothesis that a chemical has the potential to interact E, A, or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, coherence, strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are
meant to evaluate whether or not a chemical can in teract with E, A, and T consisting of different levels of biological organization from a molecular initiating ev ent, such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment and fecundity at the whole organism level. The extent of expression of responses at higher levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher-levels of biological organization? Finally, under what conditions did the responses occur including co nsideration of whether the responses were observed in the presence of overt or systemic t oxicity?. The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.

This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in termsDCPA FINAL

Page 7 of 44 of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013). For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s current approach as used by OPP in reviewing 40 CFR Part 158 st udies for both human and ecological risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g ., organ weights/histopathology), biochemical ( e.g., blood chemistry), and other clinical signs ( e.g. , lethargy) were also considered when evaluating overt toxicity, es pecially if the effects were extreme. In some instances, one parameter ( i.e., death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g. , changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addressing the three main components described above is fundamental to the WoE process and in determ ining whether additional data are needed. In addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting data; 4) overall strength of eviden ce supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing th e need for additional data is based on a caseby-case analysis which took all availa ble toxicity data into account.DCPA FINAL

Page 8 of 44 The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and biol ogically relevant in regards to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, A, or T pathway. Effects that occurred in th e presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not re ported in the table for E, A or T.

A. EDSP Tier 1 Screening Assays

The Tier 1 assays submitted in response to the agency’s test order for DCPA are shown below in

Table 1

.

Table 1: Tier 1 Screening Assays for DCPA.

Tier 1 Assays Test Guideline Test Order Status

Estrogen Receptor (ER) Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48615904)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300;

OECD 455 Requirement Satisfied (MRID No. 48675901 & 48443201) Androgen Receptor (AR) Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No.48670302) Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550;
OECD 456 Requirement Satisfied (MRID No. 48615906) Aromatase Assay (Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48615903)

Uterotrophic Assay (Rat) OCSPP 890.1600;

OECD 440 Requirement Satisfied (MRID No. 48443203)

Hershberger Assay (Rat) OCSPP 890.1400;

OECD 441 Requirement Satisfied (MRID No. 48615902)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID No. 48615905)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID No. 48615905)

Fish Short-term Reproduction Assay OCSPP 890.1350;

OECD 229 Requirement Satisfied (MRID No. 48670303)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100;

OECD 231 Requirement Satisfied (MRID No. 48670304)DCPA FINAL

Page 9 of 44 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for DCPA to interact with the E pathway is summarized in Table 2. 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-estr ogenic, or HPG axis effects. This table also includes HPG-relevant findings from the data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reporte d in the table and not considered further in the WOE assessment.

In the Tier 1 ER binding assay, DCPA displaced >50% of the radiolabeled estradiol in one competitive binding run but less than 25% of the ra diolabeled estradiol in the other two runs.
Based on the results from the thr ee runs (one interactive run and two not interactive runs), DCPA was classified as equivocal in this assay. In the Tier 1 ERTA assay, the responsiveness of the cells to the very weak positive control chemical (17 α -methyltestosterone) was lower than the expected values, indicating a decreased sensitivit y of the assay to very weak agonists. Although the conditions of this assay were not optimal to detect very weak activity, DCPA responses were similar to those of the negative control corticoste rone and not comparable to the responses seen for the very weak positive control 17 α -methyltestosterone, therefore, DCPA was considered negative for ER transcrip tional activation in this test system. In the Tier 1 steriodogenesis assay,
DCPA treatment induced a 1.24- to 2.31-fold in crease in estradiol pr oduction compared to
controls. DCPA did not inhibit aromatase activ ity in the Tier 1 aromatase assay. No estrogenrelated effects were obs erved in the Tier 1 in vivo uterotrophic or female pubertal assays.

In the FSTRA, fecundity was significantly decrea sed by 44% at the mid-dose (as compared to a non-significant 10% decrease in th e high dose, and 7% increase in the low dose), and fertility was significantly decreased by 1.3% at the high- dose in the absence of overt toxicity.

There were no effects on estr ogen endpoints in the mammalian Part 158 studies. Effects observed in the bobwhite quail reproduction st udy included decreases in embryo viability, hatching and survival, but these effects co-occurred with overt toxicity (mortality, clinical signs, and reduced weight gain in the parental ge neration). In the mallard duck reproduction study, effects on egg viability (hatching) were observed in the absence of overt toxicity.DCPAFINALPage 10 of 44Table 2: Estrogenic/Anti-Estrogenic Pathway for DCPALines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for DCPA1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight / GSI Gonadal Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding (MRID48615904)EERTA (MRID448675901&48443201)N4Aromatase (MRID458615903)NSteroidogenesis (MRIDNo. 48615906)PUterotrophic (MRID48443203)NNNFemale Pubertal Rat(MRID 48615905a)NNNNNNAW5(H)NFSTRA (MRID No.48670303)NENNNFcd:↓44%(M); Frt:↓1.3% (H)NN6OSRISubchronic toxicity(Rats; MRID 41767901)NX(M,MH,H)NSubchronic toxicity(Mouse; MRID41064801)NX(H)NChronic toxicity(Dog; MRID00083584)NNNNDCPAFINALPage 11 of 44Table 2: Estrogenic/Anti-Estrogenic Pathway for DCPALines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for DCPA1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight / GSI Gonadal Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Combined chronictoxicity / carcinogenicity(Rats; MRID 42731001)NNX(ML,M,MH,H)X(H)Carcinogenicity(Mouse; MRID40958701)NNX(H)NTwo-generationreproduction toxicity(MRID 41750103)NNX(M,H)X(M, H)Developmental toxicity(Rats; MRID00160685)NNN NDevelopmental toxicity(Rats; MRID00158010)NNN NDevelopmental toxicity(Rabbits; MRID41054820)NNN NDevelopmental toxicity(Rabbits; MRID45820101)NNN NAvian reproduction(Bobwhite quail;MRID 47550001)NNNX(M, H)DCPAFINALPage 12 of 44Table 2: Estrogenic/Anti-Estrogenic Pathway for DCPALines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for DCPA1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight / GSI Gonadal Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Avian reproduction(Mallard duck;MRID 47550002)N↓18%F1hatching(H)N N1 Key to responses: L=Low treatment, M=Medium treatment, H=High treatment. Arrows (↓or↑) indicate the direction of the response. A shaded cellindicates that is parameter is not routinely evaluated oris not applicable in this assay. AW=adrenal weight2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an Xin this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. In the main assays, the responsiveness of the cells to the very weak positive control 17α-methyltestosterone was lower thanthe expected values, indicatinga decreased sensitivity of the assay to very weak agonists. Although the conditions of this assay were not optimal to detect very weak activity, DCPAresponses were similar to those of the negative control corticosterone and not comparable to the responses of 17α-methyltestosterone. DCPA is considerednegative in this assay.5. Relative adrenal weights↑14% (p≤0.05) and absolute adrenal weights↑17% at 1000 mg/kg/day.6. Decreases in male body weight observed; no other clinical signs of toxicity.P Positive findingsN Negative findings (in vivo)/ No effect (in vivo)E EquivocalDCPA FINAL

Page 13 of 44 2. Effects on Androgen Pathway

The potential for DCPA to interact with the A pathway is summarized in Table 3. The various targets of the androgen pathway across the relevant Ti er 1 assays are delineated so as to facilitate determination of potential for andr ogenic, anti-androgenic, or HPG axis effects. This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reporte d in the table and not considered further in the WOE assessment.

DCPA was found to be untestable in the Tier 1 AR binding assay according to the guideline criteria as precipitation was obser ved at concentrations above 10

− 6 M. In the Tier 1 steroidogenesis assay, testosterone production was decreased to 0.91- to 0.71-fold relative to the control. No effects on androgen-se nsitive tissue weights were obser ved in the Hershberger assay. In the male pubertal assay, non- dose dependent weight decreases were observed in the LABC ( ↓ 15% at 500 mg/kg/day, ↓ 14% at 1000 mg/kg/day) and semina l vesicles without coagulating fluid ( ↓ 17% at 500 mg/kg/day, ↓ 14% at 1000 mg/kg/day). The ch anges in AST weights were
considered to be not treatment related since the effects seen at 1⁄2 the limit dose were comparable to those seen at the limit dose (1000 mg/kg/day) and there were no effects on serum testosterone levels or gonadal histopathology. Fertility was decreased 1.3% in the FSTRA at the high test concentration; male body weights were significantly decreased at all treatment groups, but no other clinical signs of toxicity were noted.

There were no androgen-related effects observed in the mamma lian Part 158 data. As noted previously, effects observed in the bobwhite quail reproduction study included decreases in embryo viability, hatching and survival, but th ese effects co-occurred with overt toxicity (mortality, clinical signs, and reduced weight gain in the parental generation). In the mallard duck reproduction study, effects on egg viability (hatching) were observed in the absence of overt toxicity.DCPAFINALPage 14 of 44Table 3: Androgenic/Anti-Androgenic Pathway for DCPALines of Evidence Indicating Potential Interactionwith the Androgenic/Anti-Androgenic Pathway for DCPA1Study Type / LiteratureCitation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/2° Sex Characteristics Age and Weight at PPS Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding (MRID48670302)U4Steroidogenesis (MRID48615906)PHershberger (MRID486159092; 48443202)NNNNLW(H)5NMale Pubertal Rat(MRID 48615905b)NNNNNNNNN NFSTRA (MRID No.48670303)NENNFrt:↓1.3%NN6OSRISubchronic toxicity(Rats; MRID41767901)NX(M,MH,H)NSubchronic toxicity(Mouse; MRID41064801)NX(H)NChronic Toxicity(Dog; MRID00083584)NNNNN NCombined chronictoxicity /carcinogenicity(Rats; MRID42731001)NNX(ML,M,MH,H)XDCPAFINALPage 15 of 44Table 3: Androgenic/Anti-Androgenic Pathway for DCPALines of Evidence Indicating Potential Interactionwith the Androgenic/Anti-Androgenic Pathway for DCPA1Study Type / LiteratureCitation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/2° Sex Characteristics Age and Weight at PPS Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Carcinogenicity(Mouse; MRID40958701)NNX(H)NTwo-generationreproduction(MRID 41750103)NNX(M,H)NAvian reproduction(Bobwhite quail;MRID 47550001)NNNX(M, H)Avian reproduction(Mallard duck;MRID 47550002)↓18%F1hatching (H)N N1. Key to responses: L=Low treatment, M=Medium treatment, H=High treatment. Arrows (↓or↑) indicate the direction of the response. A shaded cellindicates that is parameter is not routinely evaluated or is not applicable in this assay.2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an Xin this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. DCPA is classified as untestable under the conditions of this assay because precipitation was observed at concentrations >10−6M.5. 20% increase in liver weight in the anti-androgenic phase of the Hershberger assay at 1000 mg/kg/day.6. Male body weights were decreased at all treatment levels, 0.0083, 0.025, and 0.075 mg/L, in the FSTRA (17-22%, p < 0.05); noother clinical signs oftoxicity were observed.P Positive findingsN Negative findings (in vivo)/ No effect (in vivo)NE Not ExaminedU Untestable (i.e. compound is not soluble above 10-6M)DCPA FINAL

Page 16 of 44 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry 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 provide data to de tect changes in the HPT axis, i.e ., the female and male (rat) pubertal assays, and the AMA (frog).

The potential for DCPA to interact w ith thyroid regulation is summarized in Table 4. The various targets of the T pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyr oid or HPT axis effects. This table also includes HPT-relevant findings from the available OSRI. Effects that occurred in the presence of overt toxicity are
discussed in the text but are not reported in the table and not considered further in the WOE

assessment.

Serum T 4 levels were dose-dependently decreased by 78-81% in the Tier 1 male pubertal assay, and by 47-55% in the Tier 1 female pubertal assay (at dose levels of 500 and 1000 mg/kg/day). No other thyroid-related effects were observed in the pubertal assays . In the AMA, thyroid gland histopathology observations included hypertrophy a nd follicular cell height increases at the mid and high doses compared to the negative and solvent controls. At day-7, in the mid-dose, growth effects included SVL and body weight increases of 10%, and 32%, respectively, as well as a 12% increase in the developmental endpoint (normalized HLL) compared to th e negative control. Significant differences between the negative contro l and solvent control we re observed for the day 21 growth and developmental endpoints and so the treatment groups were compared to the solvent control. The following development changes related to treatment were also not ed at the mid-dose: compared to solvent control, th ere were effects approaching sta tistically significance (p = 0.06 or 0.07) including increases in day-21 NF stage a nd normalized HLL (NF Stage median = 58 vs. 57 in solvent control, normalized HLL increase = 15 %). Additional growth effects compared to solvent control included 21-day SVL increase of 4% at the mid dose and 21-day wet weight decrease by 10% at the low dose. The thyroi d histopathology/developmen tal effects observed in the AMA co-occurred with overt toxicity (12.5 % incr ease in mortality) only at the high dose.

In the Part 158 studies, thyroi d follicular hypertrophy was observe d in the subchronic toxicity study in rats. In a combined chronic toxicity/ carcinogenicity study in rats , the following effects on the thyroid were also noted: treatment-related in creases in thyroid weight s (both sexes); increased follicular cell hyperplasia/hypertrophy (10 mg/kg/day males; ≥ 50 mg/kg/day both sexes);
decreased T 4 levels (10 mg/kg/day males; ≥ 50 mg/kg/day both sexes); and a treatment-related increase in thyroid follicular cell neoplasms ( both sexes). In a two ge neration reproduction study in rats, thyroid gland histological changes (basophilic clumped colloid and follicular cell hypertrophy) were observed at and above 250 mg/kg/ day in F0 and F1 males and/or females; thyroid hormones were not tested in this study. No thyroid effects were observed in the mouse or dog studies.DCPA FINAL

Page 17 of 44 Table 4: Thyroid Pathway for DCPA
Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for DCPA 1

Study Type/ Literature CitationThyroid WeightThyroidHistopathologyT4LevelsTSH LevelsPituitary WeightBody Weight(BW) or SnoutVent-Length(SVL)DevelopmentalStage orAsynchronousDevelopment orHind Limb LengthSystemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Male Pubertal (Rat) (MRID 48615905b) N N ↓ 78% L; ↓ 81% H N N N N N
Female Pubertal (Rat) (MRID 48615905a) N N ↓ 47% L; ↓ 55% H N N N N N

AMA (Frog) (MRID No. 48670304) P 4 (M) ↓ 10% BW (L) and ↑ 4%

SVL (M) P 5 (M) X (H)

OSRI

Subchronic toxicity (Rat; MRID 41767901) N P (H) X (M,MH,H) N

Subchronic toxicity (Mouse; MRID 41064801) N X (H) N

Chronic toxicity (Dog; MRID 00083584) N N N N

Combined chronic toxicity/ carcinogenicity (Rats; MRID 42731001) P 6 P 6 P 6 X (ML, M, MH, H) X

Carcinogenicity (Mouse; MRID 40958701) N N X (H) N

Two-generation reproduction (MRID 41750103) P (M, H) X (M, H) N

1. 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. 2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 3. The overt toxicity in the Tier 1 assays are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 4. Significant differences between the negative control and solvent control were reported, so all effects in the AMA in this table are in comparison to the solvent cont rol. Thyroid gland hypertrophy and follicular cell height increases at the mid dose compar ed to the solvent control. 5. The increases in the mid-treatment group were notable but not statistically significant (NF Stage median=58 vs. 57 in solvent control, Jonckheere p=0.07; normalized HLL increase=15%, Dunnett p=0.06). 6. Effect seen at doses ≥ 10 mg/kg/day (low dose) in males and ≥ 50 mg/kg/day (mid-low dose) in females. P Positive findings N Negative findingsDCPA FINAL

Page 18 of 44 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of DCPA to interact w ith endocrine related processes ( i.e., E, A or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing.. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 Assays and OSRI), then performing an integrated an alysis of those line s of evidence.

The WoE evaluation includes consid erations of biological plausibility of the findings from the different lines of evidence by examining the co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substance may contribute to the WoE evaluati on in helping el ucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively sequelae of systemic effects. E ndocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence eval uation of the results of the Tier 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues shou ld be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l disruptions in homeostasis” (USEPA, 2013).

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. In addition to the endocrine -related effects described above
for the in vivo Tier 1 assays and OSRI, other eff ects observed which may be considered overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. However, in other instances, mo re than one parameter was needed to determine overt toxicity. Effects which were considered to be signs of system ic toxicity were generally less severe forms of toxicity (e.g., changes in organ weights or blood chemistry).DCPA FINAL

Page 19 of 44 1. Tier 1 in vivo Assays

No adverse systemic toxicity or overt toxicity were seen when tested up to the limit dose (1000 mg/k/day) in the in the mammalian in vivo Tier 1 assays except for a 20% increase in liver weight in the antiandrogenic pha se of the Hershberger assay at the limit dose. In fish, male body weights were decreased at all treatment levels, 0.0083, 0.025, and 0.075 mg/L, in the FSTRA (17-22%, p<0.05). In frogs, survival was decreased at the highest treatmen t level, 0.58 mg/L in the AMA (12.5%, p<0.05).

2. OSRI

In the longer term toxicity studies with DCPA, thyroid toxicity was ch aracterized by decreased levels of thyroid hormone, microscopic thyroi d changes and increased thyroid weight. Liver toxicity included increased liver weight, elevated liver enzyme activity, increased cholesterol and liver hypertrophy. Data showed that the liver effect s are precursor events to the thyroid effects, with increased metabolism of thyroid hormone by the liver resulti ng in a compensatory stimulation of the thyroid.

In a subchronic mice study, absolute and relative liver weights were increased in males and females at the limit dose (1000 mg/kg/day); sign ificantly increased in cidence of minimal centrilobular hepatocyte enlargement was seen at the same dose in males and females. Thyroid hormones were not measured in this study.

In the subchronic rat study, absolute liver we ights increased in male s at the high dose (1000 mg/kg/day) and male relative liver weights increased at 150 and 1000 mg/kg/day; in females absolute and relative liver weights increased at doses ≥ 50 mg/kg/day but not in a dose-dependent manner. There were no changes in liver enzyme s. Changes in absolute and relative kidney weights were also seen at >150 mg/kg/day. Thyroid follicular cell hypertrophy was increased in males and females at 1000 mg/kg/day; clumped collo idal and cystic follicles were increased in thyroid in males at 1000 mg/kg/day. Thyroid hormones were not measured.

In the combined chronic toxicity /carcinogenicity study with rats, th ere was increased mortality in both sexes at the high dose (1000 mg/kg/day) and decreases in body weight in females at the mid dose (500 mg/kg/day) and in both sexes at the hi gh dose. Thyroid effects included decreases in T4 at various time intervals at doses >10 mg/ kg/day and T3 levels at doses >50 mg/kg/day. There were compensatory increases in thyroi d stimulating hormones in both sexes throughout the study, but increases did not show st atistical significance. Absolute and relative thyroid weights were increased in both sexes at the high dose. Histopathological change s included increases in thyroid follicular cell hyperpla sia, hypertrophy, and/or basophili c clumped colloid in males at >10 mg/kg/day and in females at >50 mg/kg/day. Thyroid tumors were seen in males (follicular cell adenomas only) and females (follicular cell adenomas and carcinomas) and liver tumors in females (adenomas, carcinomas, and cholangiocarcinomas). Possible evidence of liver injuryDCPA FINAL

Page 20 of 44 was shown by elevated serum GGT activity in bo th sexes at the mid ( 500 mg/kg/day) and high dose (1000 mg/kg/day) groups. Absolu te and relative liver weights we re variably increased in the two higher dose groups in both sexes. Histopa thological liver lesions included increased incidence of eosinophilic foci and increases in centrilobular hepatocyte swelling at doses of 10 mg/kg/day and greater.

In the carcinogenicity study in mice, liver toxicity manifested as alteratio ns in the liver enzymes (SDH, ALT) and increased liver weight and the pr esence of hepatic adenomas (not carcinomas) only in females. Thyroid hormones were not ev aluated and there were no treatment-related change in the thyroid glands.

In the rat 2-generation reproductive study, thyroid changes in 250 mg/kg/day and 1000 mg/kg/day F0 and F1 males and/or females incl uded basophilic clumped colloid and follicular cell hypertrophy. Centrilobular hepatocyte swelling occurred in 5000 ppm and 20000 ppm males and females. Thymic involution was increase d in 20000 ppm males. Thyroid hormones were not tested. Liver (centrilobular hepatocyte swelling) and lung (interstitial pneumonitis and thickened alveolar) microscopic pulmonary cha nges were observed in F0 and F1 males and
females at 250 and above.

In bobwhite quail, mortality (9 -25%), weight loss (14-35%, p < 0.05), and clinical signs including emaciation, reduced reaction to external stimuli, lethargy, ruffled appearance, wing droop, and lower limb weakness were observed at 3170 and 8020 mg/kg-diet in a reproduction study. No effects were seen at 1280 mg/kg-diet.

B. Estrogen Pathway

There was no convincing evidence to support potent ial interaction with th e estrogen pathway. Although DCPA increased estradiol production in the Tier 1 steroi dogenesis assay and the Tier 1 ER binding assay results were equivocal, the Tier 1 ERTA and aromatase assays were negative and there were no estrogen-related effects obser ved in the Tier 1 uterotrophic and female pubertal assays or in the Part 158 mammalian to xicity studies. Th ere was a non-treatmentresponsive decrease in fecundity at the mid dose and a slight decrea se in fertility at the high dose in the FSTRA; no other complementary responses were observed across other endpoints within those treatment groups. In the w ildlife Part 158 studies, in the bobwhite quail avian reproduction study, all observed reproductive effects were seen only at an overtly toxic dose, while in the mallard duck study decreases in viable embyros/h atching were observed (no other reproductive effects observed). Therefore, there is a lack of redundancy across the ba ttery, with no or limited
effects observed without co mplementary responses.DCPA FINAL

Page 21 of 44 C. Androgen Pathway

There was no convincing evidence of an intera ction with the androgen pathway. DCPA was found to be untestable (due to insolubility) in the Tier 1 AR binding assay and testosterone production was decreased In the Tier 1 steroidogen esis assay. DCPA was negative in the Hershberger assay and there were no treatment-related androgen effects in the male pubertal assay. There were no androgen-related effects observed in the mammalian Part 158 studies.
There was a decrease in fertili ty at the high dose in the FSTRA; however there was a lack of complementary responses across other endpoints. In the wildlife Part 158 studies, for the bobwhite quail reproduction study, al l effects were observed in th e presence of overt toxicity, while in the mallard decreases in viable embyr os/hatching were observe d (no other reproductive effects observed).

D. Thyroid Pathway

DCPA demonstrated a potential interaction with the thyroid hormone pathway in the absence of overt or systemic toxicity. In the pubertal assays, serum T4 levels were dose-dependently
decreased in males at 500 mg/kg/day (78%) and 1000 mg/kg/ day (81%) and in females at 500 mg/kg/day (47%) and 1000 mg/kg/day (55%). In the AMA, increased incident of thyroid gland hypertrophy, follicular cell height increases were reported at the high concentration compared to the control. In addition, development changes related to DPCA treatment seen in the AMA include an increase in the normalized HLL on day 7 and 21.

DCPA also induced thyroid effects in several Part 158 studies. Thyr oid follicular hypertrophy was observed in a subchronic toxicity st udy in rats. In a combined chronic toxicity/carcinogenicity in rats, increa sed thyroid weights, follicular cell hyperplasia/hypertrophy, decreased T4 levels, and an increased incidence of thyroid follicular cell neoplasms were seen in both sexes.

E. Conclusions

Overall, there is no convincing evidence for inte raction with the estrog en or androgen pathway. However, there is evidence for a potential intera ction with the thyroid pathway in mammals and wildlife based on thyroid-related re sponses seen in the Tier 1 pube rtal assays and AMA, as well
as in the mammalian Part 158 studies.

V. EDSP Tier 2 Testing Recommendations

In the EDSP Tier 1 male and female pubertal assa ys as well as in the OSRI, there was evidence for potential interaction with the thyroid pathway in studies conducted with adult animals, but no such data exists for potential thyr oid toxicity in the young animals.DCPA FINAL

Page 22 of 44 For DCPA, the current point of departure (POD) of 1.0 mg/kg/day for human health risk
assessment is based on decreased level of T4 and increased incidence of histopathological lesions in the thyroid and liver at 10.0 mg/kg/day (LOAEL) in a chronic toxicity/carcinogenicity study in rats. This POD is used to derive the chronic Reference Dose (RfD) for dietary exposure risk assessments and the Margins of Expos ure (MOEs) for non-dietary exposure risk

assessments.

In general, since the POD and the toxicity endpo ints of concern are base d on data obtained from adult animals, there usually would be a concern that the POD may not be protective of potential thyroid toxicity in the young. Ther efore, a special thyroid assay in pregnant animals, fetuses, postnatal animals, and adult animals is recommended. This special study should use a mechanistic approach to generate specific data on the thyroid ( i.e. , the primary target of DCPA) to protect the developing ner vous system from thyroid hormone disrupting chemicals. The specific purpose of this st udy is to establish a POD ( i.e. , NOAELs and LOAELs or benchmark doses) that may be used for hu man health risk assessment. The POD from this special study
would address the concern for th e potential ability of DCPA to disrupt thyroid function in pregnant females and in the fetus or newborn. Howe ver, it is noted that the agency has issued a
Data Call-In notice for this special study under the Regist ration Review program.

For the wildlife, to add to the understanding of potential thyroid e ffects, the EDSP Tier 2 Larval Amphibian growth and Development Assay (LAGDA) is recommended since there is no chronic aquatic amphibian toxicity data available. Chronic toxicity data with fish (surrogates for aquaticphase amphibians) are not currently available for DCPA.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for DCPA. In both speci es, effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potenti al reproductive effects to birds from DCPA exposure. Relative to EDSP, additional testing is not recommended.DCPA FINAL

Page 23 of 44 VI. References

Akhurst, L.C. (2011). Chlorthal dimethyl (DCP A) estrogen receptor transcriptional activation (Human Cell Line (HeLa-9903)). Huntingdon Life Sciences, Cambridgeshire, England. Laboratory report number BDG0009. Unpublished. (MRID 48443201).

Akhurst, L.C. (2011). Chlorthal dimethyl (DCPA) H295R steroidogenesis assay. Huntingdon Life Sciences Ltd., Alconbury, Huntingdon, Cambri dgeshire, England. La boratory Study No.: BDG0013. Unpublished. (MRID 48615906)

Benz, G., Lucas, F., and Lovell, R. (1990). A two generation reproductio n study in rats with technical DCPA. Ricerca, Inc.: Lab Project Number: 173088-0176-TX-003: 88-0176. Unpublished. (MRID 41750103).

Chun, J., Lucas, F., and Killeen, J. (1989). A tera tology study with technical DCPA in rabbits. Ricerca, Inc.: Proj. ID 87-3204, 1116- 87-0067-TX-002. Unpublished. (MRID 41054820).

Donington, S. (2011). Amended Final Report, Chlorthal Dimethyl (DCPA): Hershberger bioassay in the castrated rat (o ral administration). Huntingdon Li fe Sciences, Cambridgeshire, England. Laboratory Project Study ID : BDG0016. Unpublished. (MRID 48615902).

Donington, S. (2011). Chlorthal Dimethyl (DCPA) : Hershberger bioassay in the castrated rat (oral administration). Huntingdon Life Sciences , Cambridgeshire, England. Laboratory Project Study ID: BDG0016. Unpublished. (MRID 48443202).

Frizell, E.R. and Parker, R.M. (2011). Chlort hal dimethyl (DCPA): a ssessment of pubertal development and thyroid function in juvenile male and female rats. Huntingdon Life Sciences, East Millstone, NJ. Laboratory Report # 10-4359. Unpublished. (MRID 48615905 a & b).

Ford, W. (1986). A teratology study in rats with technical DCPA. SDS Biotech Corp.: Document No. 712-5TX-85-0039-003: Repor t SDS-893. Unpublished. (MRID 00160685).

Ford, W. (1986). A 90-day dietary study in mice with technical DCPA. SDC Biotech Corp.:
Document No.: 696-5TX-84- 0082-002. Unpublished. (MRID 41064801).

Frey, L.T., et al. (2003). Chlo rthal-Dimethyl (DCPA): A Reproduction Study with the Northern Bobwhite. Unpublished study performed by Wildlife Internationa l Ltd., Easton, MD. Laboratory Project No. 246-115. Study sponsored by Amvac Chemical Corporation, City of Commerce, CA. Study initiated June 17, 2002 and submitted May 28, 2003. (MRID 47550001).

Frey, L.T., et al. (2003). Chlo rthal-Dimethyl (DCPA): A Reproduction Study with the Mallard. Unpublished study performed by Wildlife Interna tional Ltd., Easton, MD. Laboratory Project No. 246-116. Study sponsored by Amvac Chemical Corporation, City of Commerce, CA. Study initiated June 17, 2002 and submitted June 20, 2003. (MRID 47550002).

Hazleton, L.W., and Dieterich, W.H. (1963). Final report: two- year dietary feeding — dogs. Diamond Shamrock Agricultural Chemicals: Lab Study No.: CDL:090443-T. Unpublished. (MRID 00083584).DCPA FINAL

Page 24 of 44 Lucas, F., and Killeen, J. (1988). A combined chr onic toxicity and tumorigenicity study in mice with technical DCPA. Ri cerca, Inc.: Document N o. 1098-85-0057-TX-002. Unpublished. (MRID 40958701).

Lucas, F., and Benz, G. (1991). A 90-day feeding study in rats with technical DCPA. Ricerca, Inc.: Lab Project Number: 89-0208. Unpublished. (MRID 41767901).

Lucas, F., Mizens, M., and Laveglia, J. (1993). A combined chronic toxi city/oncogenicity study in rats with DCPA. Ricerca, Inc.: Lab Project Number : 3339-90-0005-TX-006. Unpublished. (MRID 42731001).

Matthews, A. (2011). Chlorthal dimethyl (DCP A): estrogen receptor binding assay using rat uterine cytosol (ER-RUC). Huntingdon Life Sciences, Huntingdon, Cambridgeshire, UK. Laboratory Study No.: BDG0008. Unpublished. (MRID 48615904).

Matthews, A. (2011). Chlorthal Dimethyl (D CPA): Aromatase (Human Recombinant) Assay. Huntingdon Life Sciences Ltd (Huntingdon Re search Centre [Woolley Rd, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS, UK ]). Laboratory Study Number: BDG0007. Unpublished. (MRID 48615903).

Sahrif, I. (2011). Chlorthal dimethyl (DCPA): ut erotrophic bioassay in ov ariectomised rats (oral administration). Huntingdon Life Sciences, Camb ridgeshire, England. Laboratory Project Study ID: BDG0014. Unpublished. (MRID 48443203).

Schneider, S.Z., K.H. Martin, T.Z. Kendall a nd H.O. Krueger. (2012). Chlorthal dimethyl (DCPA): Fish short-term reproductio n Assay with the fathead minnow ( Pimephales promelas ). Wildlife International, Ltd., Easton, Mary land. Lab Study No.: 246A-101A. Unpublished. (MRID 48670303).

Schneider, S.Z., T.Z. Kendall, and Krueger, H.O. (2012). Chlorthal dimethyl (DCPA): amphibian metamorphosis assay. Wildlife Inte rnational, Ltd., Easton, MD. Laboratory report number 246A-102. Unpublished. (MRID 48670304).

USEPA (2011). Endocrine Disruptor Screeni ng Program — Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.

USEPA (2013) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting held July 30 - August 1, 2013 on Weight-of-Ev idence: Evaluating Resu lts of EDSP Tier 1 Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

Willoughby, J.A. (2012). Chlorthal dimethyl: andr ogen receptor binding (rat prostate cytosol). CeeTox Inc., Kalamazoo, MI. Laboratory Study No.: 9150V-100601ARB. Unpublished. (MRID 48670302).DCPA FINAL

Page 25 of 44 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (Frog); OCSPP 890.1100

The 21-day assay (MRID 48670304) of DCPA on am phibian metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 larvae per co ntrol and treatment group) were exposed to negative and solvent controls (0.020 mL/L di methylformamide; DMF) and DCPA (98.3% purity) at nominal concentrations of 0.0083, 0.025, and 0.075 mg a.i./L. Mean-measured concentrations were <0.00400 (<LOQ; negativ e and solvent contro ls), 0.0071, 0.021, and 0.58 mg a.i./L, respectively. The test system was maintained at 2 1.0 to 22.4°C and a pH of 7.9 to 8.4.

Larval survival on Day 7 ranged from 90 to 100% in the controls and treatment groups. By Day 21, survival was 97.5 and 96.3% in the negative and solvent controls , respectively, and 95.0, 97.5, and 87.5% in the low, mid and high treatment groups, respectively. At the high treatment group, the observed mortality of 12.5% was a statis tically significant increase from the negative control (Fisher’s Exact Test p< 0.05). These high treatment group mortalities occurred in all replicates and 8 of the 10 mortal ities occurred on Days 5 and 6. Cu rved/crooked (bent) tails were observed in 18-30 (36 to 53%) su rviving tadpoles among both contro ls and all treatment groups. It is unclear if the low food ration (which was half of the recommended ration during the definitive portion of the test) influenced the c ondition of the test organisms; however, due to
similar incidence rates in both the negative and solvent controls and treatment groups, it does not appear to be a treatment-related effect.

At the mid treatment level, DCPA significan tly increased (p<0.05) Day 7 hind-limb length (HLL; ↑ 23%), normalized (for snout-vent length) HLL ( ↑ 12%), wet weight ( ↑ 32%), and snoutvent length (SVL; ↑ 10%) compared to the negative cont rol. By Day 21, DCPA significantly increased (p<0.05) HLL ( ↑ 14, 43 and 24%) and wet weight ( ↑ 8, 28 and 20%) relative to the negative control at the low, mid and high treatment levels, respectively. Additionally, Day 21 SVL was significantly increased (p<0.05) by 3 to 9% and Day 21 normalized HLL was significantly increased (p<0.05) by 11 -32% relative to the negative control at all treated levels. No significant acceleration or de lay of NF development stage was observed on either Day 7 or 21, and no asynchronous development was observed. Three late-stage (NF stage >60) tadpoles (two from the mid treatment and one from the high treatment group) were excluded from analyses of continuous endpoint s (SVL, weight, and normalized HLL). Effects on thyroid gland histopathology, including thyroid gland hypertrophy and follicular cell height increases, were observed at the mid and high treatment groups.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. The reviewer found no significant differences be tween the negative control and the solvent control for any e ndpoints on Day 7. However, signif icant differences between theDCPA FINAL

Page 26 of 44 negative and solvent controls were detected (p <0.05; 2-sided t-test) on Day 21 for wet weight, SVL, and HLL. Therefore, it is unclear to what extent the results observed may be related to the presence of the solvent. Treatment groups were compared to the solven t control for the Day 21 endpoints. There were no statistically significan t differences between the solvent control and treatment groups for development stage or norma lized HLL, however the increases in the mid treatment group were notable (NF Stage median =58 vs. 57 in solvent control, Jonckheere p=0.07; normalized HLL increase=15%, Dunnett p=0.06). For HLL and SVL, there were significant increases (Dunnett p<0.0 5) in the mid treatment group. For wet weight, the lowest treatment group was significantly decreased (M ann-Whitney p=0.03) comp ared to the solvent
control. While effects were st ill observed in the mid-treatmen t group compared to the solvent control, the results were more pronounced when compared to the negative control.

AR Binding Assay (rat pros tate cytosol); OCSPP 890.1150

In an androgen receptor (AR) binding assay (MRID 48670302), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment, which measured the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (10

− 12 to 10

− 3 M) of DCPA (98.3% a.i ., Batch# 090614-2). Dimethyl sulfoxide (DMSO) was used as the solvent vehicle at a final a ssay concentration of approximately 3.2%. A total of three independe nt runs were performed, and each run included dexamethasone as a weak positive control, a nd R1881 as the ligand reference standard.

Saturation binding data were not provided in the original stud y report; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. The dissociation constant (K d ) for [ 3 H]-R1881 was
0.613±0.041 nM and the estimated B max was 0.817±0.049 fmol/100 μg protein for the single batch of prostate cytosol used for th is assay. The mean and individual K d and B max values were below the expected ranges repor ted in the EPA validation program (0.685 to 1.57 nM and 7 to 16 fmol/100 μg protein, respectively). Confiden ce in these numbers is high according to the goodness of fit (R 2 = 0.957-0.984) and the small variation among runs.

In the competitive binding experiment, precip itation of DCPA was visually observed at
concentrations of 10

− 5 to 10

− 3 M in Run 1 and 10

− 5 and 10

− 4 M in Run 2; therefore, the data at
these concentrations were not evaluated. Alth ough no precipitation was obs erved at the highest concentration tested in Run 3, the 10

− 5 M concentration was not evaluated due to the precipitation observed at this level in the first two runs. Therefore, 10

− 6 M was the highest
concentration evaluated in all three runs.DCPA FINAL

Page 27 of 44 In all three runs, specific binding of [ 3 H]-R1881 was 84.2-102.3% at DCPA concentrations of

10

− 12 to 10

− 6 M.

The estimated average log IC 50 s for R1881 (

− 9.0 M) and the weak positive control, dexamethasone (

− 4.6 M), were within the expected ranges, and the mean RBA for the
dexamethasone was 0.0040%. Confidence in the numb ers for the reference standards is high as
there was no variation in the IC 50 values between runs. The solv ent control responses indicated no drift in the study assay, and the performance criter ia were generally met.

DCPA is classified as untestab le under the conditions of this assay because precipitation was
observed at concentrations >10

− 6 M.

ER Binding Assay (Rat ut erine cytosol); OCSPP 890.1250

In an estrogen receptor (ER) binding assa y (MRID 48615904), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct saturation and competitive binding experiments. A saturation binding experiment wa s conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radio-labeled reference estr ogen prior to conducting ER competitive binding experiments. The competitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of increasing concentrations of DCPA (logarithmic increase from 10 -10 to 10 -4 M). DMSO was used as the vehicle at a final concentration of 2%. 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.

For the saturation binding assay, the appropriate protein concentration was determined to be 0.5 mg/mL as it bound 26% of the [ 3 H]-17 β -estradiol (within the Gu ideline range of 25-35% binding). The K d for [ 3 H]-17 β -estradiol was 0.380 nM, and the estimated B max was 0.1287 nM for the prepared rat uterine cytosol. The K d for each run was within the expected range of 0.03

to 1.5 nM.

For the competitive binding assa y, the appropriate protein concen tration was determined to be 0.35 mg/mL as it bound approximately 11% (by interpolation) of the [ 3 H]-17 β -estradiol (within the Guideline range of 10-15% binding). The mean IC 50 from the three runs was 9.7 × 10 -10 M
for 17 β -estradiol and 3.57 × 10 -6 M for the weak positive control, 19-norethindrone. The mean relative binding affinity (RBA) was 0.026% for 19-norethindrone. DCPA di splaced >50% of the
radiolabeled estradiol in one competitive binding run with an IC 50 of 5.16 × 10 -5 M, and a corresponding RBA of 0.0014%. However, in th e remaining two competitive runs, DCPA displaced <25% of the radiolabeled estradiol. Based on the resu lts from the three runs (oneDCPA FINAL

Page 28 of 44 interactive run and two not interactive runs), DC PA is classified as Equivocal in the Estrogen Receptor Binding Assay.

ER α Transcriptional Activation Assay (H uman cell line HeLa 9903); OCSPP 890.1300

In an estrogen receptor transcriptio nal activation assay (MRID 48675901, and 48443201) conducted by Huntingdon Life Sciences, hER α -HeLa-9903 cells cultured in vitro were exposed to DCPA (98.3% a.i., Lot # 090614-2) at concen trations increasing logarithmically from 7.5 ×

10

− 11 to 7.5 × 10

− 5 or 3.75 × 10

− 11 to 3.75 × 10

− 5 in DMSO (0.1% v/v) for approximately 24 hours. The experiments were performed using 96 -well plates and each DCPA concentration was tested in triplicate (3 wells/plate). Cells were exposed to DCPA for 23±1 hours to induce reporter (luciferase) gene produc ts. Luciferase expression in response to activation of the estrogen receptor by DCPA was measured upon add ition of a luciferase substrate and detection with a luminometer (sensitivity not reported). DCPA was tested up to the limit of solubility, 7 ×

10

− 5 M. No cytotoxicity was observed at any concentration.

The mean RPC max for DCPA was 6.8%, 5.7% and 2.0% fo r the first, second, and third runs, respectively, and the associated PC max values were 7.5 × 10

− 5 , 3.5 × 10

− 5 , and 3.5 × 10

− 5 M. Precipitation was observed in the first run at the PC Max (7.5 × 10

− 5 M).

In the main assays, the responsiveness of th e cells to the very weak positive control 17 α

methyltestosterone was lower than expected, indi cating a decreased sensitivity of the assay to very weak agonists. Although the conditions of this assay were not optimal to detect very weak activity, DCPA responses were similar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to reach a maximum 41.3-75.2% PC. DCPA was only able to reach a ma ximum of 2.0-6.8% PC when tested up to the highest concentration possible based on cytotoxicity. Because the RPC Max < PC 10 in all three assay runs, DCPA was considered negative for estr ogen receptor transcripti onal activation in this
test system.

Fish Short-term Reproduction Assay (FSTRA); OCSPP 890.1350

The 21-day short-term reproduction assay (MRID 48670303) of DCPA (chlorthal dimethyl) with fathead minnows ( Pimephales promelas) was conducted under flow-t hrough conditions. Adult fish, 20 spawning groups (2 males and 4 females in each group; 6 months old), were exposed to DCPA (98.3% purity) at nominal concentrations of 0 (nega tive control), 0 [solvent control; dimethylformamide (DMF; 20 μL/L)], 0.0083, 0.025, and 0.075 mg a.i./L; mean-measured concentrations were <LOQ (<0.005), <LOQ (<0.005), 0.0074, 0.022, and 0.058 mg a.i./L, respectively. The test system was maintained at 24.0 to 25.9 o C and a pH of 7.9 to 8.5.DCPA FINAL

Page 29 of 44 Unless otherwise noted, the DCPA treatment groups were compared to the negative control. There were no significant effect s (p>0.05) on survival. Male body weight exhibited significant (Jonckheere-Terpstra; p<0.05) decreases of 17, 22 and 20% at the low, mid, and high treatment levels, respectively, relative to the negative control; female body weights and male and female body lengths in treated fish were similar to cont rols. No apparent trea tment-related effects on clinical signs (i.e., beha vioral and other subletha l effects) were reported relative to the negative
control.

Spawning frequency in controls wa s at least every 4 days in 2 a nd 1 of the 4 replicates in the negative and solvent control, respec tively, and fecundity was 20.8 and 16.2 eggs/female/day/replicate in the negative and solvent control, resp ectively. Fertilization success was 98.2% in the negative controls and 98.9% in the solvent controls. There was a significant (Dunnett’s; p<0.05) reduction in fecundity of 46% at the mid treatment le vel relative to the negative control, and there was a slight but si gnificant (Jonckheere-Te rpstra; p<0.05) reduction in fertility of 1.3% at the hi gh treatment level. Fecundity e ffects were not concentrationresponsive. Compared to the ne gative control, fecundity was decreased by 22% in the solvent control and 10% in the high dose, and increased by 7% in the lo w dose (all of which were not statistically significant).

There were no significant effects (p>0.05) on male or female vitell ogenin (VTG), male or female gonadal-somatic index (GSI), or male tubercle sc ores (no tubercles detected in females) when treated groups were compared to the nega tive control. Plasma testosterone and 17 β -estradiol (sex steroids) were not measured in this study.

One male fish showed the pres ence of testis-ova in one gonad in the mid treatment group; additionally, another male was observed to be small and had increased spermatogonia with decreased secondary spermatocytes and spermatids. These observations were isolated to the mid
treatment level and no other males in this trea tment or the next highe r treatment levels had remarkable findings, therefore, these observations were considered not to be related to DCPA exposure. There were no apparent treatment-relate d effects on secondary sex characteristics or gonadal staging. Fat pad (dorsal na pe pad) scores among males in all treatment groups tended to be slightly lower than both controls, but it was not concentration-dependent nor considered
treatment related.

Hershberger Assay (Rat); OCSPP 890.1400

The Hershberger Assay consists of androgenic a nd anti-androgenic components. To screen for potential androgenic activity, DCPA (98.3% a.i. , Lot # 090614-2) in 0.5% methylcellulose was administered daily via oral gava ge to ten 54- to 58-day old, castr ated male Sprague Dawley ratsDCPA FINAL

Page 30 of 44 at dose levels of 0 (vehicle), 300 or 1000 (limit dose) mg/kg/day. An androgenic positive control group consisted of ten castrated rats exposed to 0.4 mg/kg/day testoste rone propionate (TP) by subcutaneous injection.

To screen for potential anti-androgenic ac tivity, DCPA (98.3% a. i., lot # 090614-2) in 0.5% methylcellulose was administered daily via or al gavage to ten 54- to 58-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300, or 1000 mg/kg/day in conjunction with a daily dose of reference andr ogen TP at 0.4 mg/kg/day by subcutaneous injection. The anti-androgenic pos itive control group consisted of ten castrated rats exposed to 0.4 mg/kg/day TP and 3.0 mg/ kg/day flutamide (FT).

For both components of the assay, the animal s were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration to determine weights of the five androgen-dependent tissues.

All animals survived until schedul ed termination, and there were no clinical signs of toxicity at
the dose levels tested.

In the androgen agonist assay, there were no e ffects on body weights or b ody weight gains in the DCPA treated groups. Animals dosed with 0.4 mg/kg/day TP had increased body weights (p<0.05) on Day 11 by 8%, and cumulative body wei ght gains were increased (p<0.01) for Days 1-11 ( ↑ 44%). There were no statistically significant increases in any of the accessory sex organs
of animals dosed with DCPA. Animals in the positive control group (TP treated) had accessory sex organ weight increases (p< 0.05) as follows: 530% in semi nal vesicles; 522% in ventral prostate; 181% in the levator ani/bulbocavernosus (LABC) mu scle complex; 285% in Cowper’s gland; and 49% in glans penis. Liver weights in the 0.4 mg/kg/day TP group were increased 12% (p<0.01). The CV value for 1000 mg/kg/day gl ans penis (25%) was slightly higher than the maximum permissible value (22%), but CVs for the remaining tissues were below the maximum
recommended values.

In the anti-androgen assay, there were no treatment-related effects on body weights in any dose groups, and there were no effects on body wei ght gains in the 100 mg/kg/day group or the positive control group. However, cumulative body weight gains were increased in the 300 mg/kg/day ( ↑ 28%, p<0.05%) and 1000 mg/kg/day ( ↑ 38%, p<0.01) dose groups. At 1000 mg/kg/day, liver weight was incr eased by 20% (p<0.01). A statistic ally significant decrease in organ weight was observed in only one androgen se nsitive tissue in animal s treated with DCPA: at 1000 mg/kg/day, the weights of Cowp er’s glands were decreased 25% (p ≤ 0.05). Animals in the positive control group (TP + FT) had decreased (p ≤ 0.05) accessory sex organ weights as follows: 62% in seminal vesicles; 59% in vent ral prostate; 46% in LABC; 58% in Cowper’s gland; and 20% in glans penis. Live r weights were unchanged in this group.DCPA FINAL

Page 31 of 44 Statistically significant weight changes were not seen in two or more of the five androgen sensitive tissues. DCPA was negative for a ndrogenicity and anti-androgenicity in the Hershberger assay.

Pubertal Female A ssay (Rat); OCSPP 890.1450

In a Female Pubertal Assay (MRID 48615905), 15 Sprague-Dawley (Crl:CD ® [SD] IGS BR) rats/dose group were treated daily via oral gavage with chlorthal dimethyl (DCPA; 98.3% a.i., lot # 090614-2) in 0.5% aqueous methyl cellulose at doses of 0, 500, or 1000 (limit dose) mg/kg/day from post-natal day (PND) 22 to 42. Animals we re examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at da y of attainment was recorded. Following sacrifice on PND 42, total thyroxine (T4) and th yroid stimulating hormone (TSH) levels were analyzed using radioimmunoassays. Pituitary, adre nal glands, liver and urogenital organ weights were recorded, and microscopic examinations of the ovaries, uterus, t hyroid/parathyroids, and kidneys were performed.

There were no clinical signs of toxicity related to treatment, and no effects of treatment on mortality, body weights, body wei ght gains, age of attainment of VO, weight at VO, food consumption, mean age at first vaginal estrus , mean cycle length, percent cycling, percent regularly cycling, clinical chemistry parame ters, or gross and microscopic pathology.

Relative (to body) kidney we ights were increased (p ≤ 0.05) by 6% at 500 mg/kg/day, and absolute, adjusted, and relative (to body) kidney weights were increased (p ≤ 0.05) by 7-9% at 1000 mg/kg/day. Relative (to body) liver weights were increased (p ≤ 0.05) by 5-6% at 500 mg/kg/day and above. At 1000 mg/kg/day, absolu te, adjusted, and relative (to body) adrenal weights were increased (p ≤ 0.05) by 14-16%. Serum T 4 levels were decreased (p ≤ 0.001) by 47- 55% at 500 mg/kg/day and above. However, plasma TSH levels were not affected by treatment and there were no changes in thyroid histology.

The doses tested were adequate since the hi gh dose was the Limit Dose (1000 mg/kg/day).

Pubertal Male Assay (Rat); OCSPP 890.1500

In a Male Pubertal Assay (MRID 48615905), 15 Sprague-Dawley (Crl:CD ® [SD] IGS BR) rats/dose group were treated daily via oral gavage with chlorthal dimethyl (DCPA; 98.3% a.i., lot # 090614-2) in 0.5% aqueous methyl cellulose at doses of 0, 500, or 1000 (limit dose) mg/kg/day from post-natal day (PND) 23 to 53. Animals we re examined for preputial separation (PPS) daily beginning on PND 30, and age and weight at day of attain ment was recorded. Following sacrifice on PND 53, total seru m testosterone, thyroxine (T 4 ), and thyroid stimulating hormoneDCPA FINAL

Page 32 of 44 (TSH) levels were analyzed using radioimmunoassa ys. The weights of ad renals, liver, pituitary, thyroid, and urogenital organs we re recorded, and microscopic examinations of the testes, epididymides, thyroid, and kidneys were performed.

DCPA was tested up to the limit dose (1000 mg/ kg/day). At 1000 mg/kg/day, the adjusted body weight on the day of acquisition of PPS was increas ed (p<0.05) by 10%. Th e age of attainment of PPS at all doses was comparable to cont rols and within the acceptable range of the performance criteria. Treatment-related organ weight effects were limited to decreases (p<0.05) at 500 and 1000 mg/kg/day in the adjusted weig hts of the levator ani and bulbocavernosus muscle (LABC) complex (14-15%) and the semina l vesicles and coagul ating glands without fluid (14-17%). The unadjusted values for all or gan weights in the contro l group were within the acceptable range of the performance criteria. Tr eatment-related effects on hormone levels were limited to decreases (p<0.001) in T 4 levels (78-81%) at 500 and 1000 mg/kg/day. TSH and
testosterone levels were unaffect ed by treatment. The hormone values for the control group were within the acceptable range of the performance crit eria. All thyroids examined in all dose groups
were considered within normal limits of cont rol tissues. There were no treatment-related histopathological findings not ed in the testes, epididymid es, or kidneys at any dose.

Steroidogenesis Assay (Human cell line H295R); OCSPP 890.1550

In a steroidogenesis assay (M RID 48615906), H295R cells cultured in vitro in 24-well plates
were incubated with DCPA

, (98.3% purity, Lot # 090614-2) at l og concentrations from 0.0001 to 100 μM in triplicate for 48 hours. Dimethyl sulf oxide (DMSO) was used as the vehicle, at a
final concentration of 0.1%.

Testosterone and estradiol c oncentrations were measured using ELISA methods. Four independent experiments were pe rformed. 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 concen tration levels; positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production. The highest suitable concentration of DCPA in a ll three runs was 20 μM due to cytotoxicity and precipitation at higher concentrations.

Minimum basal hormone production was met in all blank and solvent contro l (SC) wells. Basal hormone production on the QC plates met th e fold-increase criteria in SC of ≥ 2.5-fold for estradiol above the minimum detection limit (MDL) of the assay, but two of the four runs were
below the ≥ 5-fold recommended increase for testosterone (4.46- to 4.65-fold). Exposure to 10 μM forskolin induced testosterone production 1. 51- to 2.02-fold (average 1.86-fold) over SC which is below the Guideline criteria ( ≥ 2-fold). Estradiol pr oduction was induced by ≥ 7.5-fold (actual 7.78- to 14.93-fold) over SC. Exposure to 1 μM prochloraz in hibited synthesis ofDCPA FINAL

Page 33 of 44 estradiol by ≥ 50% ( ≥ 65%) compared to the SC; inhibition of testosterone synthesis was reduced by ≥ 50% in only one run of four. The decreases ranged from 26 to 61%, with a mean decrease of only 43%. Hormone measurement sensitivity on the test plates met Guideline requirements with the exception of Run 2 for te stosterone (4.78-fold increase ove r the MDL instead of 5-fold). Although the estradiol data met the performance criteria reco mmended by the Guideline, the poor inhibition responses after incubation with 1 μM prochloraz may indicate an analytical problem. Interference by DCPA with the ELISA assays was ruled out, as hormone crossreactivity tests met performance criteria.

The % CVs for absolute testosterone and estradio l concentrations in the SC well replicates for within- and between-plate determinations on the QC and test chemical plates met the performance criteria of ≤ 30% as recommended by the Guideline.

The significant fold-induction of estradiol concen trations across the three runs ranged from 1.24- to 2.31-fold, with the maximum induction of 2.3-fold achieved at 20 μM. The apparent inhibition of testosterone in Run 2 was not concentration-dependent, and the results were dissimilar from Runs 1 and 3, therefore, a fourth run was conducted and analyzed for testosterone only. Testosterone production was significantly de creased 0.91- to 0.71-fold of
control.

Based on the hormone responses in at least three independent runs, DCPA treatment resulted in statistically significant and repr oducible decreases in testosterone production and increases in estradiol production.

Uterotrophic Assay (Rat); (OCSPP 890.1600)

In an uterotrophic assay (MRID 48443203) conducted to screen for potential estrogenic activity, DCPA (98.3% a.i., lot # 090614-2) in 0.5% methylcellulose was ad ministered daily via oral gavage to groups of 61- to 68-day old ovariectomi zed female Sprague-Dawley rats at dose levels of 0 (vehicle), 100, 300, and 1000 (limit dose) mg/kg/day. A positive control group was treated
with 17 α -ethynyl estradiol (EE) by daily subcutaneous injection at a dose level of 0.3 mg/kg/day. All animals were terminated and necropsied ap proximately 24 hours after the final dose on PND
65-72 to determine wet and blotted ut erine weights, and liver weights.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in animals from any dose groups. Body weights in the 100, 300, and 1000 mg/kg/day dose groups were comparable to the contro ls throughout the duration of the assay. Animals dosed with 0.3 mg/kg/day EE had decreased (p<0.01) body weight gains for Days 1-5 ( ↓ 71%). Uterine weights in the dose groups were comparable to the controls . Absolute wet and blotted uterus weights forDCPA FINAL

Page 34 of 44 the 0.3 mg/kg/day EE group were increased (p<0.001) by 944% and 588%, respectively. These increased uterine weights we re in the expected range.

No statistically significant changes were seen in uterine weights in this assay. DCPA was negative in the uterotrophic assay.DCPA FINAL

Page 35 of 44 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

90-Day (Subchronic) Oral Toxicity in Rodents (Rat )

In a subchronic toxicity study in rats ( MRID 41767901 ), DCPA (98.0%, lot # 10148, T-170-2) was fed in the diet for 90 days. Dietary concentr ations were adjusted to provide constant doses of 0, 10, 50, 100, 150, or 1000 mg/kg/day throughout the study. There were 15 CD VAF/Plus SD rats/sex/dose group. Two sate llite groups of 10 ra ts/sex/group received 0 or 1000 mg/kg/day for 60 days and were used for evaluations of lung toxicity.

Mortality and clinical signs were unaffected by treatment. Body weights were comparable among treatment groups, but body weight gain was decreased in high-dose males (92% of controls) and females (86% of controls) and was accompanied by decreased food consumption in high-dose females. There were no treatment-rela ted effects on hematology, clinical chemistry, or urinalysis. Thyroid hormone levels were not determined.

At necropsy, white foci were s een in lungs of high-dose males (8 vs 0 in controls) and high-dose females (6 vs 0 in controls). There was a dos e-related increase in foamy macrophages in the lungs seen microscopically, prin cipally in high-dose males and females, which correlated with the white foci seen grossly in lungs.

Absolute and relative liver weights were increase d, principally in high-dose males and females. Liver weights were also incr eased in 50-150 mg/kg/day females, but not in a dose-related
manner. There were dose-related increases in centrilobular hypertrophy of the liver seen microscopically, in the three higher dose male and female dose groups.

Absolute and relative kidney weights were increase d in the two highest dose groups in males. Renal epithelial hyperplasia was increased in hi gh-dose males; two animals in this dose group had kidneys with a granular surface at necr opsy. Thyroid follicular cell hypertrophy was increased in high-dose males and females; clumped colloid and cystic follicles were increased in thyroids of high-dose males.

The NOAEL is 50 mg/kg/day and the LOAEL is 100 mg/kg/day based upon centrilobular hypertrophy of the liver. At 1000 mg/kg/day there were gross and microscopic lesions of lungs and kidneys and microscopic lesions in thyroids. Thyroid hormone s were not evaluated in this study, however, the purpose of a subchronic rat study is to sel ect doses for the rat chronic toxicity/carcinogenicity study.

90-Day (Subchronic) Oral T oxicity in Rodents (Mice)

In a 90-day toxicity study in mice ( MRID 41064801 ), DCPA (96.7%, batch # 893-0501) was
administered in the diet at concentrati ons of 0, 625, 1250, 2500, or 7500 ppm in males and 0, 1000, 2500, 5000, or 10000 ppm in females. Corresponding doses were 0, 100, 199, 406, orDCPA FINAL

Page 36 of 44 1235 mg/kg/day in males and 0, 223, 517, 1049, or 2198 mg/kg/day in females. There were 15 CD-1 mice/sex/dose group.

There were no treatment related effects on mo rtality, clinical signs , body weight, hematology parameters, clinical chemistry, or urinalyses. There was an incr ease of enlarged livers in highdose males (13/15 vs 3/15 in controls) and fema les (11/15 vs 1/15) noted on gross examination.
Absolute and relative liver weights were increased in high-do se males and females (115-130% of control values). There was an increased incidence of centril obular hepatocyte enlargement, classified “minimal,” in 10,000 ppm males (14/15 vs 6/15 in controls) and 10,000 ppm females (11/15 vs 0/15 in controls). The NOAEL is 406 mg/kg/day in males and 1049 mg/kg/day in females. The LOAEL is 1235 mg/kg/day in males and 2198 mg/kg/day in females based upon centrilobular hepatocyte enlargement.

Chronic Oral Toxicity in Non-Rodents (Dog)

In a chronic toxicity study ( MRID 00083584 ), beagle dogs were administered 0, 2.5, 25, or 250 mg/kg/day DCPA in the feed for two years. Adve rse effects were not observed. Therefore, the

NOEL was ≥ 250 mg/kg/day.

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat)

In a combined chronic/carcinogenicity study in rats ( MRID 42731001 ), DCPA (97.7%, batch # 10148) was administered in the diet for 104 weeks. There were 70 SD CD VAF/Plus ® rats/sex/dose group. Concentration of DCPA in the diet was adju sted to provide doses of 0, 1, 10, 50, 500, or 1000 mg/kg/day throughout the study. Test materi al contained 0.13% hexachlorobenzene (HCB) as a contaminant a nd doses of HCB were calculated to be 0, 0.0013, 0.013, 0.065, or 1.3 mg/kg/day.

There was increased mortality in males in the 1000 mg/kg/day group during the second year of the study (73% mortality vs 52% mortality in controls). Mortality rates in 1000 mg/kg/day females were decreased in comparison to controls (40% vs 52% in controls). Males in the two highest dose groups had signs of poor physical health such as anogenital staining, thin appearance, material around the mouth, increases in few feces, soft feces, dark urine, and red urine/penile discharge. Females in th e two highest dose groups appeared thin.

Body weight decrements occurred during the second year of the study for females in the 500 mg/kg/day group (81% of control body weight at the end of the study), and in males and females in the 1000 mg/kg/day group (86% and 72% of control body weight , respectively).

There were no changes in hematology parameters in the first year of the study. At termination, erythrocyte counts were decrease d in males and females (87% of controls for both sexes).DCPA FINAL

Page 37 of 44 Thyroid toxicity was shown by decreases in the t hyroid hormone, T4. At various time intervals, T4 levels were significantly de creased (p < 0.01) in 10 mg/kg/day males (62% of control levels at 83 weeks), in 50 mg/kg/day males and females (43% and 45% of controls, respectively, at 104 weeks), in 500 mg/kg/day males and females (16% and 19% of controls, respectively, at 104 weeks), and 1000 mg/kg/day males and females ( 10% and 15% of controls, respectively, at 104 weeks). The thyroid hormone, T3, was decreased in 1000 mg/kg/day males at 52 weeks (77% of control levels) and in 50 mg/ kg/day and greater female groups. There were compensatory increases in thyroid stimulat ing hormones in both sexes thro ughout the study, but increases did
not show statisti cal significance.

Absolute and relative thyroid we ights were increased in 1000 mg/kg/day males after both one year and two years of treatment (approximately 124-134% of controls). In the 1000 mg/kg/day female group, relative thyroid weights were incr eased at one year and two years (120% and 147% of controls, respectivel y. Histopathological changes included increases in thyroid follicular cell hyperplasia, hypertrophy, and/or basophilic clumped colloid in the 10 mg/kg/day and greater male dose groups at interim and termin al sacrifices. Thyroid histology was altered in
females at doses of 50 mg/kg/day and greater.

Possible evidence of liver inju ry was shown by elevated seru m GGT activity in 500 mg/kg/day females (368% of control activity at 104 week s) and in 1000 mg/kg/day males and females (435% and 616% of controls, respectively). C holesterol was elevated in the two higher dose groups, though the toxicological si gnificance of this finding is not clear. Absolute and relative liver weights were variably increased in the two higher dose groups in males and females. Histopathological liver lesions incl uded increased incidence of eo sinophilic foci and increases in centrilobular hepatocyte swelling at doses of 10 mg/kg/day and greater.

Evidence of kidney injury was shown by variable increases in BUN and creatinine in the two higher male dose groups. Absolute and relative ki dney weights were variably increased in the two higher male and female dose groups. Seve rity of chronic nephropathy in males was increased in males in the three higher dose groups . Both incidence and severity of chronic nephropathy were increased in females in the three higher dose groups. The study pathologist stated that exacerbation of this common aging lesi on was apparently the main cause of death or moribundity in the high-dose group.

Several dose-related microscopic changes seen in the lungs, principally in the two higher dose groups, included pneumonitis (granulomatous or interstitial), thicke ned alveolar walls, accumulation of foamy macrophages, and choleste rol clefts. The foam y macrophages correlated with white foci seen at necropsy.

No changes to the eyes were attributed to tr eatment after ophthalmological examinations. On histopathological examination, re tinal atrophy was increased in eyes of females at doses of 10 mg/kg/day and greater. A subseque nt chronic toxicity study in ra ts with DCPA found no ocularDCPA FINAL

Page 38 of 44 toxicity at 20,000 ppm DCPA in the diet (approximately 1000 mg/kg/ day) after treatment for two years (MRID 41750102, Tox Doc 0008373).

In males, thyroid follicular cell adenomas we re increased (1, 2, 2, 8, 10, 7, in respective dose groups), but thyroid follic ular cell carcinomas were not increased (1, 1, 1, 0, 1, 0). In females, incidences were as follows: thyroid follicul ar cell adenomas (1, 1, 2, 4, 1, 4) and thyroid follicular cell carcinomas (0, 0, 1, 0, 1, 4).

Hepatocellular adenomas were increased in fe males (0, 0, 1, 1, 5, 7 in respective dose groups) and hepatocellular carcinomas were also increa sed (0, 0, 1, 0, 3, 3 in respective dose groups). The incidence for hepatocholangiocarcinomas in females was 0, 0, 0, 0, 0, 2 in respective dose groups. There was no treatment-related increase in liver tumors in males. This study was used to determine the Q1* for DCPA of 1.49 x 10-3 based upon the three combined types of liver tumors in females (Bernice Fisher memo, 12/20/94).

The NOAEL is 1 mg/kg/day and the LOAEL is 10 mg/kg/day based upon d ecreased level of the thyroid hormone, T4, and increased in cidences of thyroid and liver hi stological changes in males.

Chronic Oral Toxicity/Carcin ogenicity in Rodents (Mice)

In a carcinogenicity study in mice ( MRID 40958701 ), DCPA was administered in the diet to CD-1 mice for 104 weeks. Purity of test compound was 96.7% and the batch # was 5TX-85- 0057 (130985, 190686/11, 170687/2), lot # JK8401. Di etary doses were 0, 100, 1000, 3500, or 7500 ppm, equivalent in males to 0, 12, 123, 435, or 930 mg/kg/day; and in females to 0, 15, 150, 510, or 1141 mg/kg/day.

There were no treatment-related effects upon survival, clinical si gns, body weight, food consumption, or hematology.

Liver enzymes were slightly elevated in females, more pronounced in week 76 than in week 102. GPT enzyme activities in females during week 76 were 24, 20, 55, 57, 41 and in week 102 were 33, 28, 51, 31, 54 mU/mL in respective dose groups. SDH activities in females in week 76 were 18.5, 15.1, 35.0, 47.0, 36.5 and in week 102 were 22.1, 22.6, 39.5, 26.5, 47.1 mU/mL. Liver enzyme activities in male treatment groups were comparable to controls. There were slight increases in cholesterol in high-dos e females, principally at week 76 (156% of controls), an event of uncertain toxicological significance.

Absolute liver weights were increased in high-dose males at termination (130% of control value) and in high-dose females at termination (126%). Relative liver weight s were increased at termination in high-dose males (138% of controls ) and high-dose females (119%). Incidence of centrilobular hepatocyte enlargement (minimal) in high-dose males was increased relative toDCPA FINAL

Page 39 of 44 controls at the interim sacrifices of weeks 27, 53, and 79, but not at terminal sacrifice. Incidence of centrilobular hepatocyte enlargem ent was not increased in females.

There were no changes in thyroi d histology in either sex. Thyr oid hormones were not evaluated. Incidence of corneal opacity was 0, 1, 2, 3, 5 in males. Based upon evalua tion of ocular effects in a chronic rat study (MRID 41750102), it was concl uded that this was not a treatment related effect and may have been due to ocular irritation from the powdered diet (memo, 9/12/90, HED document 0008095).

Hepatic adenomas were increased in females (2, 0, 2, 3, 8 in respective dose groups), but carcinomas were not increased (0, 1, 0, 1, 1). In males, the incidences were as follows: hepatic adenomas (14, 16, 13, 9, 22) and carcinomas (6, 6, 9, 8, 11). Toxicity in this study was minimal and the mice could clearly have tolerated a larg er dose. Dosing was considered adequate to evaluate the carcinogenicity of DCPA.

Increases in liver enzyme activit ies occurred only in females, were of slight magnitude, did not exhibit a good dose-response relationship, and were unaccompanied by histological signs of hepatocellular injury. Although li ver enzymes showed transient elevations in the 150 and 510 mg/kg/day female groups at week 76, the 1141 mg/kg/day female group had elevated SDH and GPT activity at both weeks 76 and 102. The NOAEL is 510 mg/kg/day and the LOAEL is 1141 mg/kg/day based upon elevated liver enzymes and in creased liver weight in high-dose females.

Developmental Toxicity in Rodents (Rat)

In a developmental toxicity study in rats ( MRID 00160685 ), DCPA (96.7%, batch JK-8401) was administered to pregnant SD Crl:COBS ® CD rats (25/sex/dose group) by gavage from gestation days 6-15. Dose groups we re 0, 500, 1000, and 2000 mg/kg/day.

There were no treatment-related effects on survival, clinical signs, body weight, food consumption, or cesarean parameters in maternal rats. The maternal NOAEL is ≥ 2000 mg/kg/day, the highest dose tested. The maternal LOAEL is >2000 mg/kg/day.

There were no treatment-related effects on de velopmental parameters. The developmental
NOAEL is ≥ 2000 mg/kg/day, the highest dose tested. The developmental LOAEL is >2000 mg/kg/day.

Developmental Toxicity in Rodents (Rat) - Metabolite

In a developmental toxicity study in rats ( MRID 00158010 ), TPA (>99%, batch #12826-87-12)
was administered to Crl:COBS CD (SD) BR rats by gavage from gestation days 6-15. Doses were 0, 625, 1250, or 2500 mg/kg/day. There were 25 pregnant female rats per dose group.DCPA FINAL

Page 40 of 44 There was no effect upon maternal mortality. Maternal clinical signs incl uded excess salivation and soft or liquid stools, accompanied by reddened anal region and red-colored mucus in feces. These signs may be due to irritation as the compound has two carboxylic acid functional groups.
There were no treatment-related effects upon body weight, gross necropsy, or caesarean sectioning of dams. The maternal NOAEL is 1250 mg/kg/day and the maternal LOAEL is 2500 mg/kg/day based upon soft stools and excess salivation.

There was no effect upon developmental paramete rs and no developmental toxicity was noted. The developmental NOAEL is ≥ 2500 mg/kg/day, the highest dose tested.

Developmental Toxicity in Non-Rodents (Rabbit)

In a developmental toxicity study in rabbits ( MRID 41054820 ), DCPA (95.5%, lot JK8401) was administered to HRA:(NZW)SPF rabbits by gavage from gestation days 7-19. Dose groups were 0, 125, 250, and 500 mg/kg/day with 20 rabbits per dose group.

There were no treatment-related effects on survival, clinical signs, body weight, food consumption, cesarean parameters, or developmenta l parameters in any of the dose groups. The
maternal NOAEL is ≥ 500 mg/kg/day, the highest dose test ed. The developmental NOAEL is

≥ 500 mg/kg/day, the highest dose tested. This study is classified acceptable/guideline. Due to the lack of toxicity, this st udy alone does not satisf y guideline requirements for a developmental study, but should be considered in conjunction with another developmental study in rabbits (MRID 45820101) which used higher doses.

In a developmental toxicity study in rabbits ( MRID 45820101), DCPA (96.5%, lot JK8401) was administered to HRA: (NZW) SPF rabbits by gavage from gestation days 6-19. Dose groups were 0, 500, 1000, and 1500 mg/kg/day with 20 rabbits per dose group.

Mortality was high in all treatment groups (1, 4, 13, 12 deaths in respective dose groups). Clinical signs (ataxia, decreased motor activity, loss of righting reflex) and gastric ulcers were
associated with animals that died or aborte d. Weight gains during the dosing period were decreased in treatment groups (0.16, 0.10, -0.03, 0.10 kg in respective groups). Food consum ption during the dosing period was slightly decreased in the mid-dose group (39.6, 39.2, 32.5, 41.4 g/kg/day in respective dose groups). Cesarean-section and developmental parameters we re not affected by treatment, although due to the high maternal mortality, definite conclu sions cannot be reached in this regard.

The maternal NOAEL is <500 mg/kg/day and the ma ternal LOAEL is 500 mg/kg/day, the lowest dose tested, and is based upon maternal mortality.DCPA FINAL

Page 41 of 44 Two-Generation Reproduction Toxicity in Rodents (Rat)

In a 2-generation reproduction study in rats ( MRID 41750103 ), DCPA (96-98%, batch # JK8401 and 10148) was administered in the feed to SD CD VAF/Plus rats. The F0 parental generation produced two litters, F1a and F1b. The F1b generation was mated to produce two litters, F2a and F2b. There were 35 rats/sex/dose group in the F0 and F1 generations with a ten week growth phase for the F0 generation before the fi rst mating and a ten week growth phase for the F1b generation before the first mating. There we re 20 rats/sex/dose grou p in the F2b generation
which were observed for a six week growth period.

Dietary concentrations were 0, 1000, 5000, or 20000 ppm (equivalent to 0, 50, 250, or 1000 mg/kg/day using a 0.05 mg/kg/day per ppm conversion factor). Doses were changed to 0, 200, 500, or 20000 ppm on day 0 of lactation for the F2b litters (equivalent to 10, 25, or 1000 mg/kg/day using a 0.05 conversion factor) in or der to ensure a NOAEL for F2 pup body weight
decrements.

Compound consumption was estimated by conver ting dietary ppm to mg/kg/day using a 0.05
conversion factor. This was done because f ood consumption during the reproduction phase of the study was not reported. Based on food and compound consumption, parental doses at the time of mating were approximately 45, 233, 952 mg/kg/day in males and 0, 63, 319, 1273 mg/kg/day in females for the 1000, 5000, and 20000 ppm dose groups, respectively. Doses during the growth phase of the F2b gene ration (calculated with food and compound consumption) were higher than parental doses at the time of mating: 17, 43, 1932 mg/kg/day in males and 18, 47, 1946 mg/kg/day in females in the 200, 500, and 20000 ppm groups.

Parental toxicity: Body weight wa s not affected by treatment in pa rental F0 and F1 males. Body weights in F0 dams in the 2000 ppm group were approximately 92-94% of controls during pregnancy. Body weights in F1 dams in the 5000 and 20000 ppm groups were 92-95% of controls during pregnancy.

Kidney toxicity occurred in 5000 ppm and 20000 ppm males and females of F0 and F1 generations. F1 5000 ppm and 20000 ppm males had red urine in the cages. Urinalyses of
affected animals found red blood cells in urine in the 20000 ppm males. Kidneys in 5000 ppm males and 20000 ppm males and females appear ed abnormal at gross necropsy and had microscopic changes indicating an ea rly onset of chronic nephropathy.

Pulmonary toxicity occurred in 5000 ppm a nd 20000 ppm F0 and F1 males and females. Interstitial pneumonitis and thickened alveolar walls were seen microscopically. Small, white foci on the lungs correlated w ith foamy macrophages seen microscopically in 5000 ppm and 20000 ppm females and in 1000 ppm and higher fema le dose groups. Phospholipidosis/ alveolar proteinosis was diagnosed by electron microscopy.DCPA FINAL

Page 42 of 44 Thyroid changes in 5000 ppm and 20000 ppm F0 and F1 males and/or females included basophilic clumped colloid and follicular cell hypertrophy. Centrilobular hepatocyte swelling occurred in 5000 ppm and 20000 ppm males and fema les. Thymic involution was increased in 20000 ppm males. Thyroid hor mones were not tested.

The parental NOAEL is 50 mg/kg/day and the parental LOAEL is 250 mg/kg/day based upon body weight decrements, gross and microscopic ch anges in kidneys and lungs, and microscopic changes in liver and thyroids.

Reproductive toxicity: There we re no treatment-related eff ects upon reproductive indices. Mating index, fertility index, pre gnancy rates, and litter size were not affected by treatment.

The stillborn index was increased in the 20000 pp m F2b group, but was comparable to historical control ranges and was not attributed to treatment. The reproductive NOAEL is ≥ 1000 mg/kg/day, the highest dose tested.

Offspring toxicity: On day 21 of lactation th ere were body weight decrements in the 5000 ppm F1a and F1b groups (92 and 89% of controls, respectively) and the 20000 ppm F1a and F1b groups (81 and 84%) of controls. On lactati on day 21, there were body weight decrements in 5000 ppm F2a pups (89% of controls) and in 20000 ppm F2a and F2b pups (76-77% of controls). Pup body weights in the 500 ppm F2 b litters were not affected by treatment. Body weight decrements in weaned pups were accompanied by decreased food consumption in F1 animals but
not in F2 animals.

There were no treatment-related effects seen at pup necropsy. The offspring NOAEL is 50 mg/kg/day and the offspring LOAEL is 250 mg/ kg/day based upon pup body weight decrements.

Avian Reproduction Toxicity

In two reproductive toxicity studies in avian species, mallard duck ( MRID 47550002 ) and Northern bobwhite quail ( MRID 47550001 ) were administered DCPA in diet over 21 weeks at levels of 0, 1280, 3170, and 8020 mg a.i./kg-diet.

Bobwhite quail was the more sensitive species te sted. Fourteen treatment-related mortalities were observed during the study, in cluding 8 at the highest treatm ent level. A NOAEC of 1280 mg a.i./kg-diet and a LOAEC of 3170 mg a.i./kg-diet were determined based on mortality, survivor weight, signs of toxicity, and effect s on reproduction and offspring. Treatment-related clinical signs of toxici ty were also observed at the 3170 and 8020 mg a.i./kg levels and included thinness or emaciation, reduced reaction to extern al stimuli, a ruffled appearance, lethargy, wing droop, and lower limb weakness. Treatment related reductions in multiple reproductive parameters were detected at the top two treatme nt levels, including the ratios of live 3-week embryos to viable embryos, number hatched to li ve 3-week embryos, and hatchling survivors toDCPA FINAL

Page 43 of 44 eggs set and to number hatched. Furthermore, at the highest treatment level, the reviewer’s analysis detected significant reductions in eggs laid, eggs set, and viable embryos. A slight reduction in food consumption was also observed, but this was not statis tically significant.

In the mallard duck study, No mortalities or overt signs of toxicity were observed in the control or in any of the treatment concentrations during the test. There were no treatment-related effects on any adult or offspring parameter at the 1280 a nd 3170 mg a.i./kg diet le vels. At the 8020 mg a.i./kg level, there was a treatment-related re duction (not statistically significant) in the percentage of viable embryos of eggs set (75 %) compared to the control (92%). A NOAEC of 3170 mg ai/kg-diet and a LOAEC of 8020 mg ai /kg-diet were determined based on embryo viability (i.e., # eggs hatched/eggs set).DCPA FINAL

Page 44 of 44 APPENDIX 3: References Not Utiliz ed in the DCPA WoE Analysis

In 2009, after public review and comment, a fina l list of 67 chemicals and schedule for issuing
Test Orders for the EDSP Tier 1 screening battery was made av ailable in a Federal Register Notice issued October 21, 2009 (74 FR 54422). The ag ency’s review of the initial data submitted as “other scientifically releva nt information (OSRI) was provided in the Report of the Endocrine Disruptor Review Team (USEPA, 2010).

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies previously submitted to the agency for registra tion/reregistration, published literature articles and/or Tier 1 assays. The agency also c onducted a more recent search (2009 to 2014) of
available scientific literature fo r any additional relevant informa tion for their weight of evidence (WoE) evaluations. These articles were evalua ted in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/s cience/efed/policy_guidance/team _authors/endangered_species _reregistration_workgroup/PDF_ro t/esa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature To xicity Studies to Suppor t Human Health Risk Assessment ( http://www.epa.gov/pesticides /science/lit-studies.pdf).

The following published and unpublished references were considered for use in the WoE analysis for DCPA but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the com pound of interest was not us ed in the study; 3) the test material was not adequately described; 4) a formulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only da ta on invertebrates.

Davidson, C., Stanley, K., and Simonich, S.M. (2012) Contaminant residues and declines of the Cascades frog (Rana cascadae) in the California Cascades, USA. Environ Toxicol Chem . 31(8):
1895-1902.

Navarro, A., Weißbach, S., Faria, M., Barata, C ., Piña, B., and Luckenbach, T. (2012). Abcb and Abcc transporter homologs are expressed and activ e in larvae and adults of zebra mussel and induced by chemical stress. Aquat Toxicol . 122-123:144-52.

Weldon, R.H., Barr, D.B., Trujillo, C., Bradma n, A., Holland, N., and Eskenazi, B. (2011). A pilot study of pesticides and PCBs in the breast milk of women residing in urban and agricultural communities of California. J Environ Monit . 13(11):3136-3144.