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 Assessment Type: 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 & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

Environmental Fat~~fec~~~n ~ Office of Pesticide Program 

THROUGH: Jess Rowland ~ S ~~~ Deputy Division Director 

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

EPA has completed its Weight of Evidence (WoE) assessment evaluating results of the 

Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. 

The WoE documents for the 52 chemicals are listed in Attachment A along with the chemical 

and report identifiers. 

Page I of2 

Page 1 of 52
Page 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 

Page 2 of 52
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: CHLORTHAL DIMETHYL (DCPA) 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S ENVIRONMENTAL PROTECTION AGENCY

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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 Relevant 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-Estrogenic Pathway for DCPA.........................................10 

 Table 3. Androgenic/Anti-Androgenic 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 Utilized in the DCPA WoE Analysis.............................44 

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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 Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

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 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

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 Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

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Abbreviation Terminology 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

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Abbreviation Terminology 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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Executive Summary 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide 

the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen 

(E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism and 

antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary 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 interacts with E, A, or T hormone pathways, the 

number and type of effects induced, the magnitude and pattern of responses observed across 

studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur 

were considered, in particular, whether or not endocrine-related 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 the Office of Science Coordination and Policy 

(OSCP) conducted a weight-of evidence (WoE) analysis of the potential interaction of DCPA 

with the E, A or thyroid T hormone pathways. 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 interaction with the estrogen 

pathway. Although DCPA increased estradiol production in the Tier 1 steroidogenesis 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 observed in the Tier 1 uterotrophic and 

female pubertal assays or in the Part 158 mammalian toxicity studies. There was a nontreatment-responsive decrease in fecundity at the mid dose and a slight decrease in fertility at the 

high dose in the FSTRA; no other complementary 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 observed). 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 interaction 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 steroidogenesis assay. DCPA was negative in the 

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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 fertility 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, all effects were observed in the presence of overt toxicity, 

while in the mallard decreases in viable embyros/hatching were observed (no other reproductive 

effects observed). 

DCPA demonstrated a potential for 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 amphibian metamorphosis assay (AMA), 

increased incidence of thyroid gland hypertrophy and 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. 

In the mammalian Part 158 studies, DCPA-induced thyroid effects included thyroid follicular 

hypertrophy in a subchronic toxicity study in rats, and increased thyroid weights, follicular cell 

hyperplasia/hypertrophy, decreased T4 levels and an increased incidence of thyroid follicular cell 

neoplasms in both sexes of rats in a combined chronic toxicity/ carcinogenicity study. 

In the EDSP Tier 1 male and female pubertal assays 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 thyroid 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 Exposure (MOEs) for non-dietary exposure risk 

assessments. 

In general, since the POD and the toxicity endpoints of concern are based 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. Therefore, 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 nervous system from thyroid hormone disrupting chemicals. The 

specific purpose of this study is to establish a POD (i.e., NOAELs and LOAELs or benchmark 

doses) that may be used for human health risk assessment. The POD from this special study 

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would address the concern for the potential ability of DCPA to disrupt thyroid function in 

pregnant females and in the fetus or newborn. However, it is noted that the agency has issued a 

Data Call-In notice for this special study under the Registration Review program. 

For the wildlife, to add to the understanding of potential thyroid effects, 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 with both northern bobwhite quail and mallard 

duck are available for DCPA. In both species, effects on reproductive parameters were 

observed. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) 

are considered sufficient for evaluating potential reproductive effects to birds from DCPA 

exposure. Relative to EDSP, additional testing is not recommended. 

 

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I. Introduction 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide 

the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen 

(E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism and 

antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary 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 interacts with E, A, or T hormone pathways, the 

number and type of effects induced, the magnitude and pattern of responses observed across 

studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur 

were considered, in particular, whether or not endocrine-related 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 the Office of Science Coordination and Policy 

(OSCP) conducted a weight-of evidence analysis of the potential interaction of DCPA with the 

E, A or T hormonal pathways. 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).

DCPA has a water solubility of 0.5 mg/L, a partition coefficient (log octanol/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. Bioconcentration factors in bluegill sunfish are 1894, 777, and 

2574 in whole fish, edible tissue, and viscera, respectively, and depuration 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 toxicity studies and other published articles. These 

data are summarized in Sections III.A through III.C. An analysis of the data submitted to the 

Agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in 

Section IV. The EDSP Tier 2 studies recommendations are presented in Section V. 

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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 believed to be relevant to one or more of the Tier 1 

assays were submitted by the Test Order recipient. The Agency has also conducted a more recent 

search of available scientific literature 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 presented at the 2013 FIFRA 

Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how 

the individual lines of evidence are assembled and integrated along two concepts (i.e., 

complementarity and redundancy) within the conceptual framework of an adverse outcome 

pathway). Broadly, there are four main steps outlined in the guidance which provide the 

foundation for WoE evaluations. The first step is to the individual studies for their scientific 

quality and relevance in evaluating potential endocrine interaction(s). The second step is to 

integrate the data along different levels of biological organization while examining the extent of 

concordance of complementarity (i.e., the concordance of endpoints within an assay that 

measures multiple endpoints) and redundancy (i.e., the concordance of endpoints/responses 

across assays) in the observed responses across these 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 whether additional testing is needed 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 include published literature studies as well as 

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studies conducted under USEPA (often referred to as Part 158 data) or OECD guidelines 

submitted in support of pesticide registrations. Each 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 specific endpoint for discerning whether or not the 

chemical has interacted with the endocrine system, are considered. The collective response of the 

individual endpoints, as well as the conditions under which they were expressed, are considered 

when evaluating an overall indication of potential interaction as measured by the study. 

The second step in this WoE process is to formulate hypotheses and integrate 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 support a hypothesis that a chemical 

has the potential to interact E, A, or T pathways, are the concepts of complementarity and 

redundancy. These two concepts provide a basis for considering the plausibility, 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 interact with E, A, and T consisting of different 

levels of biological organization from a molecular initiating event, such as receptor binding, 

through potential adverse effects in apical endpoints such as sexual development 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 relevant, robust and consistent evidence in terms of 

complementarity and redundancy as well as biological 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 consideration of whether the responses were 

observed in the presence of overt or systemic toxicity?. The presence of overt and/or systemic 

toxicity introduces uncertainty in the ability to distinguish effects specifically related to an 

endocrine-related effect from a non-endocrine toxic response. 

This uncertainty in distinguishing whether the responses were endocrine-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, weight should be placed on signs of endocrine disruption in the 

presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in terms 

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of primary interactions with the endocrine system vs. secondary effects related 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 studies for both human and ecological risk 

assessments. Specifically, in these analyses, the effects that EPA considered to be potential 

evidence of overt toxicity included, but were not limited to: mortality; 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, especially 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/concentration 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 decreases 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 effects were generally considered as less severe 

forms of toxicity (e.g., changes in organ weights or blood chemistry). The circumstances for 

which a dose/concentration was considered overtly toxic for a particular 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/chemical properties and its 

known modes of action in its overall 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 determining 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 evidence supporting a conclusion; and, 5) what, if any, 

additional data are needed and why. Assessing the need for additional data is based on a caseby-case analysis which took all available toxicity data into account. 

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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 biologically 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 the presence of overt toxicity are discussed in the text for each 

respective pathway (E, A or T) but are not reported 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) 

ERα 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) 

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B. Effects on Hypothalamic-Pituitary-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-estrogenic, 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 reported 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 radiolabeled estradiol in the other two runs. 

Based on the results from the three 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 (17α-methyltestosterone) was lower than the 

expected values, indicating 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 seen 

for the very weak positive control 17α-methyltestosterone, therefore, DCPA was considered 

negative for ER transcriptional activation in this test system. In the Tier 1 steriodogenesis assay, 

DCPA treatment induced a 1.24- to 2.31-fold increase in estradiol production compared to 

controls. DCPA did not inhibit aromatase activity in the Tier 1 aromatase assay. No estrogenrelated effects were observed in the Tier 1 in vivo uterotrophic or female pubertal assays. 

In the FSTRA, fecundity was significantly decreased by 44% at the mid-dose (as compared to a 

non-significant 10% decrease in the 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 estrogen endpoints in the mammalian Part 158 studies. Effects 

observed in the bobwhite quail reproduction study 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 generation). In the mallard duck reproduction study, 

effects on egg viability (hatching) were observed in the absence of overt toxicity.

Page 17 of 52
DCPA FINAL 

Page 10 of 44 

Table 2: Estrogenic/Anti-Estrogenic Pathway for DCPA 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for DCPA1Study Type / Literature Citation 

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) 

EDSP Tier 1 Assay 

ER Binding (MRID 48615904) 

E

ERTA (MRID 448675901 &48443201) 

N4 

Aromatase (MRID 458615903) N Steroidogenesis (MRID No. 48615906) P Uterotrophic (MRID 48443203) N Female Pubertal Rat (MRID 48615905a) N N N N N N FSTRA (MRID No. 48670303) NE N N N 

Fcd: ↓44

(M); Frt

↓1.3% (H

OSRI 

Subchronic toxicity (Rats; MRID 41767901) N Subchronic toxicity (Mouse; MRID 41064801) 

N 

Chronic toxicity (Dog; MRID 00083584) 

N N 
DCPA FINAL 

Page 11 of 44 

Table 2: Estrogenic/Anti-Estrogenic Pathway for DCPA 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for DCPA1Study Type / Literature Citation 

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) 

Combined chronic toxicity / carcinogenicity (Rats; MRID 42731001) 

N N 

Carcinogenicity (Mouse; MRID 40958701) 

N N 

Two-generation reproduction toxicity (MRID 41750103) 

 N N 

Developmental toxicity (Rats; MRID 00160685) 

N N 

Developmental toxicity (Rats; MRID 00158010) 

N N 

Developmental toxicity (Rabbits; MRID 41054820) 

N N 

Developmental toxicity (Rabbits; MRID 45820101) 

N N 

Avian reproduction (Bobwhite quail; MRID 47550001) 

N N 
DCPA FINAL 

Page 12 of 44 

Table 2: Estrogenic/Anti-Estrogenic Pathway for DCPA 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for DCPA1Study Type / Literature Citation 

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) 

Avian reproduction (Mallard duck; MRID 47550002) 

N 

↓18% 

F1 

hatchin

(H) 

1 Key to responses: L=Low treatment, M=Medium treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the resp

indicates that is parameter is not routinely evaluated or is not applicable in this assay. AW=adrenal weight 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the O

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 

details see Section IV. A 

4. In the main assays, the responsiveness of the cells to the very weak positive control 17

α-methyltestosterone was lower than the e

a decreased sensitivity of the assay to very weak agonists. Although the conditions of this assay were not optimal to detect very wresponses were similar to those of the negative control corticosterone and not comparable to the responses of 17

α-methyltestoste

negative 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 findings N Negative findings (in vivo) / No effect (in vivo)E Equivocal 
DCPA 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 Tier 1 assays are delineated so as to facilitate 

determination of potential for androgenic, 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 reported 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 observed 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-sensitive tissue weights were observed 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 seminal vesicles without coagulating 

fluid (↓17% at 500 mg/kg/day, ↓14% at 1000 mg/kg/day). The changes 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 mammalian Part 158 data. As noted 

previously, effects observed in the bobwhite quail reproduction study 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 generation). In the mallard 

duck reproduction study, effects on egg viability (hatching) were observed in the absence of 

overt toxicity.

Page 21 of 52
DCPA FINAL 

Page 14 of 44 

Table 3: Androgenic/Anti-Androgenic Pathway for DCPA 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for DCPA

Study Type / Literature Citation 

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) 

EDSP Tier 1 Assay 

AR Binding (MRID 48670302) 

U4

Steroidogenesis (MRID 48615906) P Hershberger (MRID 486159092; 48443202) N N N N Male Pubertal Rat (MRID 48615905b) N N N N N N N N FSTRA (MRID No. 48670303) NE N N Frt: 

↓1.3% 

OSRI 

Subchronic toxicity (Rats; MRID 41767901) 

N 

Subchronic toxicity (Mouse; MRID 41064801) 

N 

Chronic Toxicity (Dog; MRID 00083584) 

N N N N 

Combined chronic toxicity / carcinogenicity (Rats; MRID 42731001) 

N N 
DCPA FINAL 

Page 15 of 44 

Table 3: Androgenic/Anti-Androgenic Pathway for DCPA 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for DCPA

Study Type / Literature Citation 

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) 

Carcinogenicity (Mouse; MRID 40958701) 

N N 

Two-generation reproduction (MRID 41750103) 

N N 

Avian reproduction (Bobwhite quail; MRID 47550001) 

N N 

Avian reproduction (Mallard duck; MRID 47550002) 

↓18% 

F1 

hatchin

g (H) 

1. Key to responses: L=Low treatment, M=Medium treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the res

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 O

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 

details see Section IV. A 

4. DCPA is classified as untestable under the conditions of this assay because precipitation was observed at concentrations >10−6 M

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); no 

toxicity were observed. 

P Positive findings N Negative findings (in vivo) / No effect (in vivo) 

NE Not Examined U Untestable (i.e. compound is not soluble above 10-6 M)
DCPA FINAL 

Page 16 of 44 

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

The current EDSP Tier 1 battery does not have a specific in vitro assay to detect chemicals with 

the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it 

does include three in vivo assays that provide data to detect changes in the HPT axis, i.e., the 

female and male (rat) pubertal assays, and the AMA (frog). 

The potential for DCPA to interact with 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 thyroid 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 T4 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 and 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 the negative control. 

Significant differences between the negative control and solvent control were 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 noted at the mid-dose: 

compared to solvent control, there were effects approaching statistically significance (p = 0.06 or 

0.07) including increases in day-21 NF stage and 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 thyroid histopathology/developmental effects observed in 

the AMA co-occurred with overt toxicity (12.5 % increase in mortality) only at the high dose. 

In the Part 158 studies, thyroid follicular hypertrophy was observed 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 increases in thyroid weights (both sexes); increased 

follicular cell hyperplasia/hypertrophy (10 mg/kg/day males; ≥50 mg/kg/day both sexes); 

decreased T4 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 generation 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. 

Page 24 of 52
DCPA FINAL 

Page 17 of 44 

 Table 4: Thyroid Pathway for DCPA 

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for DCPA1

Study Type/ Literature 

Citation 

Thyroid Weight Thyroid Histo

pathology

T4 Levels 

TSH Levels Pituitary Weight Body Weight (BW) or SnoutVent-Length (SVL) Developmental Stage or Asynchronous Development or Hind Limb Len

gth 

Systemic Toxicity Observed2

Overt Toxicity Observed3

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) 

P4

(M) 

↓10% BW 

(L) and ↑4% 

SVL (M)

P5

(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) 

P6 P6 P6 

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 control. Thyroid gland hypertrophy and follicular cell height 

increases at the mid dose compared 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 findings 

Page 25 of 52
DCPA FINAL 

Page 18 of 44 

IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight of evidence (WoE) determination on the 

potential of DCPA to interact with endocrine related processes (i.e., E, A or T hormonal 

pathways) as well as recommendations 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 peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (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 analysis of those lines of evidence. 

The WoE evaluation includes considerations of biological plausibility of the findings from the 

different lines of evidence by examining the consistency, 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 evaluation in helping elucidate if 

effects seen in the Tier 1 assay are related to perturbations of the endocrine system per se or 

alternatively sequelae of systemic effects. Endocrine modes of action may elicit a number of 

phenotypic consequences other than those 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 are 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 evaluation of the results of the Tier 1 assays stated that “In summary, 

the Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in 

the presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in 

terms of primary interactions with the endocrine system vs. secondary effects related to toxicity 

in non-endocrine organs or overall 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 below. In addition to the endocrine-related effects described above 

for the in vivo Tier 1 assays and OSRI, other effects 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, more than one parameter was needed to determine 

overt toxicity. Effects which were considered to be signs of systemic toxicity were generally less 

severe forms of toxicity (e.g., changes in organ weights or blood chemistry).

Page 26 of 52
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 phase 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 treatment 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 characterized by decreased 

levels of thyroid hormone, microscopic thyroid 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 effects are precursor events to the thyroid effects, 

with increased metabolism of thyroid hormone by the liver resulting 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); significantly increased incidence 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 weights increased in males 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 enzymes. 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 colloidal 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, there 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 high 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 thyroid stimulating hormones in both sexes throughout the 

study, but increases did not show statistical significance. Absolute and relative thyroid weights 

were increased in both sexes at the high dose. Histopathological changes included increases in 

thyroid follicular cell hyperplasia, hypertrophy, and/or basophilic 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 injury 

Page 27 of 52
DCPA FINAL 

Page 20 of 44 

was shown by elevated serum GGT activity in both sexes at the mid (500 mg/kg/day) and high 

dose (1000 mg/kg/day) groups. Absolute and relative liver weights were variably increased in the 

two higher dose groups in both sexes. Histopathological 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 alterations in the liver enzymes 

(SDH, ALT) and increased liver weight and the presence of hepatic adenomas (not carcinomas) 

only in females. Thyroid hormones were not evaluated 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 included basophilic clumped colloid and follicular 

cell hypertrophy. Centrilobular hepatocyte swelling occurred in 5000 ppm and 20000 ppm males 

and females. Thymic involution was increased in 20000 ppm males. Thyroid hormones were 

not tested. Liver (centrilobular hepatocyte swelling) and lung (interstitial pneumonitis and 

thickened alveolar) microscopic pulmonary changes 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 potential interaction with the estrogen pathway. 

Although DCPA increased estradiol production in the Tier 1 steroidogenesis 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 observed in the Tier 1 uterotrophic and female 

pubertal assays or in the Part 158 mammalian toxicity studies. There was a non-treatmentresponsive decrease in fecundity at the mid dose and a slight decrease in fertility at the high dose 

in the FSTRA; no other complementary 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 observed). Therefore, there is a lack of redundancy across the battery, with no or limited 

effects observed without complementary responses. 

Page 28 of 52
DCPA FINAL 

Page 21 of 44 

C. Androgen Pathway 

There was no convincing evidence of an interaction 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 steroidogenesis 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 fertility 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, all effects were observed in the presence of overt toxicity, 

while in the mallard decreases in viable embyros/hatching were observed (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. Thyroid follicular hypertrophy 

was observed in a subchronic toxicity study in rats. In a combined chronic 

toxicity/carcinogenicity in rats, increased 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 interaction with the estrogen or androgen pathway. 

However, there is evidence for a potential interaction with the thyroid pathway in mammals and 

wildlife based on thyroid-related responses seen in the Tier 1 pubertal 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 assays 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 thyroid toxicity in the young animals. 

Page 29 of 52
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 Exposure (MOEs) for non-dietary exposure risk 

assessments. 

In general, since the POD and the toxicity endpoints of concern are based 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. Therefore, 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 nervous system from thyroid hormone disrupting chemicals. The 

specific purpose of this study is to establish a POD (i.e., NOAELs and LOAELs or benchmark 

doses) that may be used for human health risk assessment. The POD from this special study 

would address the concern for the potential ability of DCPA to disrupt thyroid function in 

pregnant females and in the fetus or newborn. However, it is noted that the agency has issued a 

Data Call-In notice for this special study under the Registration Review program. 

For the wildlife, to add to the understanding of potential thyroid effects, 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 with both northern bobwhite quail and mallard 

duck are available for DCPA. In both species, effects on reproductive parameters were 

observed. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) 

are considered sufficient for evaluating potential reproductive effects to birds from DCPA 

exposure. Relative to EDSP, additional testing is not recommended. 

Page 30 of 52
DCPA FINAL 

Page 23 of 44 

VI. References 

Akhurst, L.C. (2011). Chlorthal dimethyl (DCPA) 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, Cambridgeshire, England. Laboratory Study No.: 

BDG0013. Unpublished. (MRID 48615906) 

Benz, G., Lucas, F., and Lovell, R. (1990). A two generation reproduction 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 teratology 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 (oral administration). Huntingdon Life 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). Chlorthal dimethyl (DCPA): assessment 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: Report 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). Chlorthal-Dimethyl (DCPA): A Reproduction Study with the Northern 

Bobwhite. Unpublished study performed by Wildlife International 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). Chlorthal-Dimethyl (DCPA): A Reproduction Study with the Mallard. 

Unpublished study performed by Wildlife International 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). 

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Lucas, F., and Killeen, J. (1988). A combined chronic toxicity and tumorigenicity study in mice 

with technical DCPA. Ricerca, Inc.: Document No. 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 toxicity/oncogenicity study 

in rats with DCPA. Ricerca, Inc.: Lab Project Number: 3339-90-0005-TX-006. Unpublished. 

(MRID 42731001). 

Matthews, A. (2011). Chlorthal dimethyl (DCPA): 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 (DCPA): Aromatase (Human Recombinant) Assay. 

Huntingdon Life Sciences Ltd (Huntingdon Research Centre [Woolley Rd, Alconbury, 

Huntingdon, Cambridgeshire, PE28 4HS, UK]). Laboratory Study Number: BDG0007. 

Unpublished. (MRID 48615903). 

Sahrif, I. (2011). Chlorthal dimethyl (DCPA): uterotrophic bioassay in ovariectomised rats (oral 

administration). Huntingdon Life Sciences, Cambridgeshire, England. Laboratory Project Study 

ID: BDG0014. Unpublished. (MRID 48443203). 

Schneider, S.Z., K.H. Martin, T.Z. Kendall and H.O. Krueger. (2012). Chlorthal dimethyl 

(DCPA): Fish short-term reproduction Assay with the fathead minnow (Pimephales promelas). 

Wildlife International, Ltd., Easton, Maryland. 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 International, Ltd., Easton, MD. Laboratory report 

number 246A-102. Unpublished. (MRID 48670304). 

USEPA (2011). Endocrine Disruptor Screening Program — Weight of Evidence: Evaluating 

Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical 

Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC. 

USEPA (2013) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting 

held July 30 - August 1, 2013 on Weight-of-Evidence: Evaluating Results 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: androgen receptor binding (rat prostate cytosol). 

CeeTox Inc., Kalamazoo, MI. Laboratory Study No.: 9150V-100601ARB. Unpublished. (MRID 

48670302). 

 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (Frog); OCSPP 890.1100 

The 21-day assay (MRID 48670304) of DCPA on amphibian metamorphosis of African clawed 

frog (Xenopus laevis) was conducted under flow-through conditions. Amphibian larvae at 

Nieuwkoop-Faber (NF) stage 51 (80 larvae per control and treatment group) were exposed to 

negative and solvent controls (0.020 mL/L dimethylformamide; 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; negative and solvent controls), 0.0071, 0.021, and 0.58 

mg a.i./L, respectively. The test system was maintained at 21.0 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 statistically 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 mortalities occurred on Days 5 and 6. Curved/crooked (bent) tails were 

observed in 18-30 (36 to 53%) surviving tadpoles among both controls 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 condition 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 significantly 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 control. 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 delay 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 endpoints (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 effects are reported based on comparison to the negative (clean 

water) control. The reviewer found no significant differences between the negative control and 

the solvent control for any endpoints on Day 7. However, significant differences between the 

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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 solvent control for the Day 21 

endpoints. There were no statistically significant differences between the solvent control and 

treatment groups for development stage or normalized 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<0.05) in the mid treatment group. For wet weight, the lowest 

treatment group was significantly decreased (Mann-Whitney p=0.03) compared to the solvent 

control. While effects were still observed in the mid-treatment group compared to the solvent 

control, the results were more pronounced when compared to the negative control. 

AR Binding Assay (rat prostate 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 assay concentration of 

approximately 3.2%. A total of three independent runs were performed, and each run included 

dexamethasone as a weak positive control, and R1881 as the ligand reference standard. 

Saturation binding data were not provided in the original study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. The dissociation constant (Kd) for [3H]-R1881 was 

0.613±0.041 nM and the estimated Bmax was 0.817±0.049 fmol/100 μg protein for the single 

batch of prostate cytosol used for this assay. The mean and individual Kd and Bmax values were 

below the expected ranges reported in the EPA validation program (0.685 to 1.57 nM and 7 to 16 

fmol/100 μg protein, respectively). Confidence in these numbers is high according to the 

goodness of fit (R2

 = 0.957-0.984) and the small variation among runs. 

In the competitive binding experiment, precipitation 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. Although no precipitation was observed 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. 

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

10−12 to 10−6 M. 

The estimated average log IC50s 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 numbers for the reference standards is high as 

there was no variation in the IC50 values between runs. The solvent control responses indicated 

no drift in the study assay, and the performance criteria were generally met. 

DCPA is classified as untestable under the conditions of this assay because precipitation was 

observed at concentrations >10−6

 M. 

ER Binding Assay (Rat uterine cytosol); OCSPP 890.1250 

In an estrogen receptor (ER) binding assay (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 was 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 estrogen prior to conducting ER competitive binding 

experiments. The competitive binding experiment was conducted to measure the binding of a 

single concentration of [3

H]-17β-estradiol (1 nM) in the presence of increasing concentrations of 

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]-17β-estradiol (within the Guideline range of 25-35% 

binding). The Kd for [3H]-17β-estradiol was 0.380 nM, and the estimated Bmax was 0.1287 nM 

for the prepared rat uterine cytosol. The Kd for each run was within the expected range of 0.03 

to 1.5 nM. 

For the competitive binding assay, the appropriate protein concentration was determined to be 

0.35 mg/mL as it bound approximately 11% (by interpolation) of the [3H]-17β-estradiol (within 

the Guideline range of 10-15% binding). The mean IC50 from the three runs was 9.7 × 10-10 M 

for 17β-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 displaced >50% of the 

radiolabeled estradiol in one competitive binding run with an IC50 of 5.16 × 10-5 M, and a 

corresponding RBA of 0.0014%. However, in the remaining two competitive runs, DCPA 

displaced <25% of the radiolabeled estradiol. Based on the results from the three runs (one 

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interactive run and two not interactive runs), DCPA is classified as Equivocal in the Estrogen 

Receptor Binding Assay. 

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

In an estrogen receptor transcriptional activation assay (MRID 48675901, and 48443201) 

conducted by Huntingdon Life Sciences, hERα-HeLa-9903 cells cultured in vitro were exposed 

to DCPA (98.3% a.i., Lot # 090614-2) at concentrations 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 products. Luciferase expression in response to activation of the 

estrogen receptor by DCPA was measured upon addition 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 RPCmax for DCPA was 6.8%, 5.7% and 2.0% for the first, second, and third runs, 

respectively, and the associated PCmax 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 PCMax (7.5 × 10−5 M). 

In the main assays, the responsiveness of the cells to the very weak positive control 17αmethyltestosterone was lower than expected, indicating 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 17α-methyltestosterone, which was able to reach a maximum 

41.3-75.2% PC. DCPA was only able to reach a maximum of 2.0-6.8% PC when tested up to the 

highest concentration possible based on cytotoxicity. Because the RPCMax < PC10 in all three 

assay runs, DCPA was considered negative for estrogen receptor transcriptional 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-through 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 (negative 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 25.9o

C and a pH of 7.9 to 8.5. 

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Unless otherwise noted, the DCPA treatment groups were compared to the negative control. 

There were no significant effects (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 controls. No apparent treatment-related effects on 

clinical signs (i.e., behavioral and other sublethal effects) were reported relative to the negative 

control. 

Spawning frequency in controls was at least every 4 days in 2 and 1 of the 4 replicates in the 

negative and solvent control, respectively, and fecundity was 20.8 and 16.2 

eggs/female/day/replicate in the negative and solvent control, respectively. 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 level relative to the 

negative control, and there was a slight but significant (Jonckheere-Terpstra; p<0.05) reduction 

in fertility of 1.3% at the high treatment level. Fecundity effects were not concentrationresponsive. Compared to the negative control, fecundity was decreased by 22% in the solvent 

control and 10% in the high dose, and increased by 7% in the low dose (all of which were not 

statistically significant). 

There were no significant effects (p>0.05) on male or female vitellogenin (VTG), male or female 

gonadal-somatic index (GSI), or male tubercle scores (no tubercles detected in females) when 

treated groups were compared to the negative control. Plasma testosterone and 17β-estradiol (sex 

steroids) were not measured in this study. 

One male fish showed the presence 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 treatment or the next higher treatment levels had 

remarkable findings, therefore, these observations were considered not to be related to DCPA 

exposure. There were no apparent treatment-related effects on secondary sex characteristics or 

gonadal staging. Fat pad (dorsal nape 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 and 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 gavage to ten 54- to 58-day old, castrated male Sprague Dawley rats 

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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 testosterone propionate (TP) by 

subcutaneous injection. 

To screen for potential anti-androgenic activity, DCPA (98.3% a.i., lot # 090614-2) in 

0.5% methylcellulose was administered daily via oral 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 androgen TP at 0.4 mg/kg/day by subcutaneous 

injection. The anti-androgenic positive 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 animals 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 scheduled termination, and there were no clinical signs of toxicity at 

the dose levels tested. 

In the androgen agonist assay, there were no effects on body weights or body 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 weight 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 seminal vesicles; 522% in ventral 

prostate; 181% in the levator ani/bulbocavernosus (LABC) muscle 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 glans 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 weight 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 increased by 20% (p<0.01). A statistically significant decrease in 

organ weight was observed in only one androgen sensitive tissue in animals treated with DCPA: 

at 1000 mg/kg/day, the weights of Cowper’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 ventral prostate; 46% in LABC; 58% in Cowper’s 

gland; and 20% in glans penis. Liver weights were unchanged in this group. 

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Statistically significant weight changes were not seen in two or more of the five androgen 

sensitive tissues. DCPA was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

Pubertal Female Assay (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 were examined for vaginal opening (VO) daily 

beginning on PND 22, and age and weight at day of attainment was recorded. Following 

sacrifice on PND 42, total thyroxine (T4) and thyroid stimulating hormone (TSH) levels were 

analyzed using radioimmunoassays. Pituitary, adrenal glands, liver and urogenital organ weights 

were recorded, and microscopic examinations of the ovaries, uterus, thyroid/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 weight 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 parameters, or gross and microscopic pathology. 

Relative (to body) kidney weights 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, absolute, adjusted, and relative (to body) adrenal 

weights were increased (p≤0.05) by 14-16%. Serum T4 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 high 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 were examined for preputial separation (PPS) 

daily beginning on PND 30, and age and weight at day of attainment was recorded. Following 

sacrifice on PND 53, total serum testosterone, thyroxine (T4), and thyroid stimulating hormone 

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(TSH) levels were analyzed using radioimmunoassays. The weights of adrenals, liver, pituitary, 

thyroid, and urogenital organs were 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 increased (p<0.05) by 10%. The age of attainment 

of PPS at all doses was comparable to controls 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 weights of the levator ani and bulbocavernosus 

muscle (LABC) complex (14-15%) and the seminal vesicles and coagulating glands without 

fluid (14-17%). The unadjusted values for all organ weights in the control group were within the 

acceptable range of the performance criteria. Treatment-related effects on hormone levels were 

limited to decreases (p<0.001) in T4 levels (78-81%) at 500 and 1000 mg/kg/day. TSH and 

testosterone levels were unaffected by treatment. The hormone values for the control group were 

within the acceptable range of the performance criteria. All thyroids examined in all dose groups 

were considered within normal limits of control tissues. There were no treatment-related 

histopathological findings noted in the testes, epididymides, or kidneys at any dose. 

Steroidogenesis Assay (Human cell line H295R); OCSPP 890.1550 

In a steroidogenesis assay (MRID 48615906), H295R cells cultured in vitro in 24-well plates 

were incubated with DCPA, (98.3% purity, Lot # 090614-2) at log concentrations from 0.0001 to 

100 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a 

final concentration of 0.1%. 

Testosterone and estradiol concentrations were measured using ELISA methods. Four 

independent experiments were performed. A Quality Control (QC) plate was run concurrently 

with each independent run of a test chemical plate to demonstrate that the assay responded 

properly to positive control agents at two concentration levels; positive controls included the 

known inhibitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production. 

The highest suitable concentration of DCPA in all three runs was 20 μM due to cytotoxicity and 

precipitation at higher concentrations. 

Minimum basal hormone production was met in all blank and solvent control (SC) wells. Basal 

hormone production on the QC plates met the 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 production was induced by ≥7.5-fold 

(actual 7.78- to 14.93-fold) over SC. Exposure to 1 μM prochloraz inhibited synthesis of 

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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 testosterone (4.78-fold increase over the MDL instead of 5-fold). 

Although the estradiol data met the performance criteria recommended 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 estradiol 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 concentrations 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 decreased 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 reproducible 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 administered daily via oral 

gavage to groups of 61- to 68-day old ovariectomized 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 17α-ethynyl estradiol (EE) by daily subcutaneous injection at a dose level of 0.3 mg/kg/day. 

All animals were terminated and necropsied approximately 24 hours after the final dose on PND 

65-72 to determine wet and blotted uterine 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 controls 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 for 

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the 0.3 mg/kg/day EE group were increased (p<0.001) by 944% and 588%, respectively. These 

increased uterine weights were in the expected range. 

No statistically significant changes were seen in uterine weights in this assay. DCPA was 

negative in the uterotrophic assay. 

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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 concentrations 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 satellite groups of 10 rats/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-related effects on hematology, clinical chemistry, 

or urinalysis. Thyroid hormone levels were not determined. 

At necropsy, white foci were seen in lungs of high-dose males (8 vs 0 in controls) and high-dose 

females (6 vs 0 in controls). There was a dose-related increase in foamy macrophages in the 

lungs seen microscopically, principally in high-dose males and females, which correlated with 

the white foci seen grossly in lungs. 

Absolute and relative liver weights were increased, principally in high-dose males and females. 

Liver weights were also increased 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 increased in the two highest dose groups in males. 

Renal epithelial hyperplasia was increased in high-dose males; two animals in this dose group 

had kidneys with a granular surface at necropsy. 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 hormones were not evaluated in this 

study, however, the purpose of a subchronic rat study is to select doses for the rat chronic 

toxicity/carcinogenicity study. 

90-Day (Subchronic) Oral Toxicity 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 concentrations 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, or 

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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 mortality, clinical signs, body weight, hematology 

parameters, clinical chemistry, or urinalyses. There was an increase of enlarged livers in highdose males (13/15 vs 3/15 in controls) and females (11/15 vs 1/15) noted on gross examination. 

Absolute and relative liver weights were increased in high-dose males and females (115-130% of 

control values). There was an increased incidence of centrilobular 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. Adverse 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 adjusted to provide doses of 0, 1, 

10, 50, 500, or 1000 mg/kg/day throughout the study. Test material contained 0.13% 

hexachlorobenzene (HCB) as a contaminant and 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 the 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 decreased in males and females (87% of controls for both sexes). 

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Thyroid toxicity was shown by decreases in the thyroid hormone, T4. At various time intervals, 

T4 levels were significantly decreased (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 stimulating hormones in both sexes throughout the study, but increases did 

not show statistical significance. 

Absolute and relative thyroid weights 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 increased at one year and two years (120% and 

147% of controls, respectively. 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 terminal sacrifices. Thyroid histology was altered in 

females at doses of 50 mg/kg/day and greater. 

Possible evidence of liver injury was shown by elevated serum GGT activity in 500 mg/kg/day 

females (368% of control activity at 104 weeks) and in 1000 mg/kg/day males and females 

(435% and 616% of controls, respectively). Cholesterol was elevated in the two higher dose 

groups, though the toxicological significance 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 included increased incidence of eosinophilic 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 kidney weights were variably increased in the 

two higher male and female dose groups. Severity 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 lesion 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), thickened alveolar walls, 

accumulation of foamy macrophages, and cholesterol clefts. The foamy macrophages correlated 

with white foci seen at necropsy. 

No changes to the eyes were attributed to treatment after ophthalmological examinations. On 

histopathological examination, retinal atrophy was increased in eyes of females at doses of 10 

mg/kg/day and greater. A subsequent chronic toxicity study in rats with DCPA found no ocular 

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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 were increased (1, 2, 2, 8, 10, 7, in respective dose 

groups), but thyroid follicular cell carcinomas were not increased (1, 1, 1, 0, 1, 0). In females, 

incidences were as follows: thyroid follicular cell adenomas (1, 1, 2, 4, 1, 4) and thyroid 

follicular cell carcinomas (0, 0, 1, 0, 1, 4). 

Hepatocellular adenomas were increased in females (0, 0, 1, 1, 5, 7 in respective dose groups) 

and hepatocellular carcinomas were also increased (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 decreased level of the 

thyroid hormone, T4, and increased incidences of thyroid and liver histological changes in males. 

Chronic Oral Toxicity/Carcinogenicity 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. Dietary 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 signs, 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-dose 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 weights 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 to 

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controls at the interim sacrifices of weeks 27, 53, and 79, but not at terminal sacrifice. Incidence 

of centrilobular hepatocyte enlargement was not increased in females. 

There were no changes in thyroid histology in either sex. Thyroid hormones were not evaluated. 

Incidence of corneal opacity was 0, 1, 2, 3, 5 in males. Based upon evaluation of ocular effects 

in a chronic rat study (MRID 41750102), it was concluded 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 larger dose. Dosing was considered adequate to 

evaluate the carcinogenicity of DCPA. 

Increases in liver enzyme activities 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 liver 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 increased 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 were 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 developmental 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. 

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There was no effect upon maternal mortality. Maternal clinical signs included 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 parameters 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 developmental parameters in any of the dose groups. The 

maternal NOAEL is ≥500 mg/kg/day, the highest dose tested. 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 study alone does not satisfy 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 aborted. Weight gains during the dosing period were 

decreased in treatment groups (0.16, 0.10, 

-0.03, 0.10 kg in respective groups). Food consumption 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 were not affected by treatment, although due to 

the high maternal mortality, definite conclusions cannot be reached in this regard.

The maternal NOAEL is <500 mg/kg/day and the maternal LOAEL is 500 mg/kg/day, the lowest 

dose tested, and is based upon maternal mortality. 

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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 first mating and a ten week growth phase for the 

F1b generation before the first mating. There were 20 rats/sex/dose group 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 order to ensure a NOAEL for F2 pup body weight 

decrements. 

Compound consumption was estimated by converting dietary ppm to mg/kg/day using a 0.05 

conversion factor. This was done because food 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 generation (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 was not affected by treatment in parental 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 appeared abnormal at gross necropsy and had 

microscopic changes indicating an early onset of chronic nephropathy. 

Pulmonary toxicity occurred in 5000 ppm and 20000 ppm F0 and F1 males and females. 

Interstitial pneumonitis and thickened alveolar walls were seen microscopically. Small, white 

foci on the lungs correlated with foamy macrophages seen microscopically in 5000 ppm and 

20000 ppm females and in 1000 ppm and higher female dose groups. Phospholipidosis/ alveolar 

proteinosis was diagnosed by electron microscopy. 

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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 females. Thymic involution was increased in 

20000 ppm males. Thyroid hormones 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 changes in kidneys and lungs, and microscopic 

changes in liver and thyroids. 

Reproductive toxicity: There were no treatment-related effects upon reproductive indices. 

Mating index, fertility index, pregnancy rates, and litter size were not affected by treatment. 

The stillborn index was increased in the 20000 ppm 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 there 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 lactation 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 F2b 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 tested. Fourteen treatment-related mortalities 

were observed during the study, including 8 at the highest treatment 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 effects on reproduction and offspring. Treatment-related 

clinical signs of toxicity were also observed at the 3170 and 8020 mg a.i./kg levels and included 

thinness or emaciation, reduced reaction to external stimuli, a ruffled appearance, lethargy, wing 

droop, and lower limb weakness. Treatment related reductions in multiple reproductive 

parameters were detected at the top two treatment levels, including the ratios of live 3-week 

embryos to viable embryos, number hatched to live 3-week embryos, and hatchling survivors to 

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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 statistically 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 and 3170 mg a.i./kg diet levels. At the 8020 mg 

a.i./kg level, there was a treatment-related reduction (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). 

 

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APPENDIX 3: References Not Utilized in the DCPA WoE Analysis 

In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing 

Test Orders for the EDSP Tier 1 screening battery was made available in a Federal Register 

Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted 

as “other scientifically relevant 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 registration/reregistration, published literature articles 

and/or Tier 1 assays. The agency also conducted a more recent search (2009 to 2014) of 

available scientific literature for any additional relevant information for their weight of evidence 

(WoE) evaluations. These articles were evaluated in accordance with the agencies Evaluation 

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

(http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species

_reregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance 

for considering and Using Open Literature Toxicity Studies to Support 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 compound of interest was not used 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 adequately described; 7) only an abstract of the study 

was available; 8) the reference is a review article or book chapter and does not contain primary 

study data; 9) insufficient information was available to adequately assess the validity of the study 

results; 10) the 40 CFR Part 158 guideline study was 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) the study contained only data 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 active 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., Bradman, 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. 

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