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 71
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 71
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: 2, 4-DICHLOROPHENOXY ACETIC ACID (2,4-D) 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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2, 4-D FINAL 

ii 

TABLE OF CONTENTS 

Abbreviations...................................................................................................iii 

Executive Summary..........................................................................................1 

I. Introduction.......................................................................................3 

II. Sources of Scientific Data and Technical Information......................................4 

A. EDSP Tier 1 Screening Assays............................................................4 

B. Other Scientifically Relevant Information (OSRI)......................................4 

III. Weight of Evidence (WoE) Evaluation.......................................................4 

A. EDSP Tier 1 Screening Assays.............................................................7 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................8 

1. Effects on Estrogen Pathway.....................................................8 

2. Effects on Androgen Pathway..................................................14 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................19 

IV. Committee’s Assessment of Weight of Evidence..........................................23 

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................23 

1. Tier 1 in vivo Assays.............................................................24 

2. OSRI...............................................................................24 

B. Estrogen Pathway...........................................................................27 

C. Androgen Pathway.........................................................................27 

D. Thyroid Pathway...........................................................................27 

E. Conclusions.................................................................................28 

V. EDSP Tier 2 Testing Recommendations....................................................28 

VI. References.......................................................................................29 

LIST OF TABLES: 

 Table 1. Tier 1 Assays for 2,4-D......................................................................7 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for 2,4-D.........................................10 

 Table 3. Androgenic/Anti-Androgenic Pathway for 2,4-D.......................................15 

 Table 4. Thyroid Pathway for 2,4-D.................................................................21 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................32 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................39 

APPENDIX 3. References Not Utilized in the 2,4-D WoE Analysis....................................61 

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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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iv 

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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v 

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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vi 

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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Page 1 of 63 

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) perturbations. 

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 2, 4 dichlorophenoxy acetic acid (2,4-D) 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 October 22, 2014, the EDSP 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 2, 4-Dichlorophenoxy acetic acid (2,4-D) with the E, A or T signaling 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).

There was no convincing evidence of potential interaction of 2, 4-D with the estrogen pathway. 

The Tier 1 or OSRI in vitro assays were negative with the exception of a 20% increase in 

estradiol production in the in the steroidogenesis assay at 100 μM. The requirements for the 

EDSP Tier 1 in vivo mammalian uterotrophic and female pubertal assays were satisfied by the 

extended one-generation reproduction toxicity test (EOGRT) in rats (equivalent to the EDSP Tier 

2 study) considered as OSRI. No estrogen-related effects were observed in this study or in any 

of the mammalian Part 158 toxicity studies in the absence of overt toxicity. No effects were 

observed in the Part 158 bobwhite quail reproduction study. In the fish short-term reproduction 

assay (FSTRA), fecundity was decreased (34%) at a concentration of 96.5 mg a.i./L, and while 

an increase in the number of female ovaries at stage 2 (compared to stage 3 or 4) was observed 

for the 2,4-D treatment groups compared to the negative control, the incident rates were not 

dose-responsive, and there were no other effects observed in this study. 

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There was no convincing evidence of interaction of 2, 4-D with the androgen pathway. The Tier 

1 and OSRI in vitro assays were negative. The requirements for the Tier 1 in vivo mammalian 

Hershberger and male pubertal assays were satisfied by the extended one-generation 

reproduction test (EOGRT) in rats considered as OSRI. There were no androgen-related effects 

observed in this study. Additionally, there were no treatment-related effects in the FSTRA or 

avian reproduction studies, and there were no effects on the male reproductive parameters in the 

Part 158 mammalian studies in the absence of overt toxicity. 

In the amphibian metamorphosis assay (AMA), while there was a 15% decrease in HLL (at the 

high test concentration of 113 mg/L), there were no histological effects on the thyroid or effects 

on developmental stage. Some thyroid-related effects were seen in the mammalian Part 158 

studies. The effects were only seen in the presence of overt toxicity or at doses that exceeded the 

threshold for renal clearance of 2, 4-D in the rat, resulting in systemic toxicity. In the EOGRT, 

there were no treatment-related thyroid effects in males or females. Therefore, collectively the 

data across the Tier 1 and Part 158 data do not demonstrated convincing evidence for potential 

interaction with the thyroid pathway. 

Based on weight of evidence considerations, additional EDSP Tier 2 ecological testing is not 

recommended. It is noted that the agency already has a mammalian toxicity study (extended onegeneration reproduction test, EOGRT) that is equivalent to the EDSP Tier 2 study. 

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1. 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) perturbations. 

In addition to the available Tier 1 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 2,4 D 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 October 22, 2014, the EDSP 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 2, 4-D with the E, A or T signaling 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).

2, 4-D has a water solubility of 569 mg/L at 20°C and a octanol/water partition coefficient (log 

Kow) of 0.177. It has a vapor pressure of 1.4 x 10−7 mm Hg at 25°C and, therefore, 

volatilization is not expected to significantly contribute to the dissipation of 2, 4-D in the 

environment. The log Kow suggests that the potential for bioconcentration in aquatic organisms 

is low. The degradation of 2,4-D acid appears to be dependent on oxidative microbially-related 

mineralization in the terrestrial environment and photodegradation in water. Results from 

laboratory studies indicate rapid to moderately rapid degradation under aerobic soil conditions 

with half-lives ranging from 1.4 days to 12.4 days and a median half-life of 2.9 days. 2,4-D acid 

was stable to photodegradation in soil. 2,4-D acid was not stable in aerobic aquatic environments 

(t1/2=15.0 days) but was moderately persistent to persistent (t1/2=28.5 to 333 days) in anaerobic 

aquatic laboratory studies. 

The available information considered in determining the potential interaction of 2,4-D with the 

E, A, 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, 

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using the WoE approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The 

EDSP Tier 2 study recommendations are presented in Section V. 

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 as OSRI by the Test Order recipients and/or the public. This included 

studies published in the open literature and/or data submitted to support pesticide registration 

(e.g., Part 158 guideline studies). The Agency’s review of the initial OSRI is provided in the 

Report of the Endocrine Disruptor Review Team on 2, 4-D (USEPA, 2010). Since then, 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 hormonal 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 evaluate 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 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. 

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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 

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, an evaluation of 

conditions where 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 

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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 

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 with regard 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 2, 4-D are shown below in 

Table 1. 

Table 1. Tier 1 Assays for 2, 4-D. 

Tier 1 Assays Test 

Guideline Test Order Status 

ER Binding Assay (Rat uterine cytosol) OCSPP 

890.1250 Requirement Satisfied (MRID 48614303) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 

890.1300; 

OECD 455 

Requirement Satisfied (MRID 48614304) 

AR Binding Assay (Rat prostate 

cytosol) 

OCSPP 

890.1150 Requirement Satisfied (MRID 48614301) 

Steroidogenesis Assay (Human cell line 

H295R) 

OCSPP 

890.1550; 

OECD 456 

Requirement Satisfied (MRID 48614305) 

Aromatase Assay (human recombinant 

microsomes) 

OCSPP 

890.1200 Requirement Satisfied (MRID 48614302) 

Uterotrophic Assay (Rat) 

OCSPP 

890.1600; 

OECD 440 

Requirement satisfied by the Extended OneGeneration Reproduction Toxicity (EOGRT) 

Study (MRID 47972101) 

Hershberger Assay (Rat) 

OCSPP 

890.1400; 

OECD 441 

Requirement satisfied by EOGRT 

 (MRID 47972101) 

Pubertal Female Assay (Rat) OCSPP 

890.1450 

Requirement satisfied by EOGRT 

 (MRID 47972101) 

Pubertal Male Assay (Rat) OCSPP 

890.1500 

Requirement satisfied by EOGRT 

 (MRID 47972101) 

Fish Short-term Reproduction Assay 

OCSPP 

890.1350; 

OECD 229 

Requirement Satisfied (MRID 48317007) 

Amphibian Metamorphosis Assay 

(Frog) 

OCSPP 

890.1100; 

OECD 231 

Requirement Satisfied MRID 48317002) 

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B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis 

1. Effects on Estrogen Pathway 

The potential for 2, 4-D to interact with the estrogen 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-estrogenicor 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. 

2, 4-D was negative in the Tier 1 in vitro ER binding and ERTA assays. Literature studies 

considered as OSRI also showed no evidence for potential ER-related binding (Blair et al, 2002) 

or transactivation (Orton et al., 2009; Hurst and Sheahan, 2003; Kojima et al., 2004; Lemaire et 

al., 2006; and Sun et al., 2012). Additionally, Soto et al. (1995) reported that 2,4-D did not 

induce cell proliferation in ER-responsive MCF7 cells. 

2, 4-D was negative for aromatase inhibition in the Tier 1 aromatase assay. In the in vitro Tier 1 

steroidogenesis assay, 2,4-D treatment resulted in a 20% increase (p≤0.05) in estradiol 

production in all three runs at the highest concentration tested (10−4 M). However, in an OSRI in 

vitro study, Orton et al., (2009) reported no 2,4-D-related effects (up to 62.5 μM) in a frog 

oocyte assay evaluating ovarian steroidogenesis. 

The in vivo uterotrophic and female pubertal assays were not conducted because these 

requirements were satisfied by an extended one-generation rat reproductive test (EOGRT) 

considered as OSRI which is equivalent to the EDSP Tier 2 mammalian study. 

In the EORGT, although changes were noted in some estrogen-related endpoints, the effects 

were non-significant (NS) and /or non-dose-related, thereby there were no treatment-related 

estrogen effects. For example, increased absolute and relative uterine weights were observed at 

the adult P1 females (17%) and F1 offspring Set 1a (31 and 32%, respectively) at 600 ppm; 

however, these responses did not attain statistical significance. Non-statistically significant 

increases in absolute and relative uterine weights were also seen in the Set 3 F1 offspring at 300 

ppm (10% absolute and relative) and 600 ppm (10% absolute, 11 % relative). Increased ovarian 

weight (↑9%) was observed in the 600 ppm F1 Set 1a females, although statistical significance 

was not attained. In addition, non-significant decreases (9-10%) in pituitary weight were 

observed in the F1 Set 1a and 3 female offspring. There were no treatment-related effects on VO, 

anogenital distance, estrous cyclicity, gonadal histopathology or reproductive indices in the 

study. 

Page 16 of 71
2, 4-D FINAL 

Page 9 of 63 

In the other part 158 mammalian studies, the only potential -related effects observed occurred in 

the presence of overt toxicity. These effects included decreased ovarian weights in the 

subchronic (42%) and chronic/carcinogenicity (41%; NS) in the rat, decreased pituitary weight in 

the subchronic rat, and decreased F1b litter size/viability in the rat 2-generation reproduction 

study.

In the FSTRA, there was a 34% decrease in fecundity at the high treatment level (96.5 mg/L). 

Plasma VTG concentrations and GSI in females were not affected. While not concentrationdependent, an increase in the number of female ovaries at stage 2 (compared to stage 3 or 4) for 

the 2,4-D treatment groups compared to the negative control was evident. 

There were no effects on growth or reproduction up to 962 mg ae/kg-diet in a Part 158 

reproduction study with bobwhite quail and 2,4-D acid. In a Part 158 fathead minnow early life 

stage study with 2,4-D dimethylamine salt larval length was affected with a NOAEC of 14.2 mg 

ae/L (17.1 mg a.i./L); survival was also reduced at higher concentrations. In another early lifecycle study with fathead minnow and 2,-4-D ethylhexyl ester, larval survival was affected at 0.22 

mg a.i./L (NOAEC=0.0792 mg a.i./L), however, high variability in test concentrations was 

observed in this study.

Page 17 of 71
2, 4-D FINAL 

Page 10 of 63 

Table 2. Estrogenic/Anti-Estrogenic Pathway for 2,4-D

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for 2,4-D1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

EDSP Tier 1 

ER Binding (MRID 48614303) N ERTA (MRID 48614304) N Aromatase (MRID 48614302) N Steroidogenesis (MRID 48614305) P4Uterotrophic Requirement satisfied by OSRI (MRID 47972101); see below.Female Pubertal Rat Requirement satisfied by OSRI (MRID 47972101); see below.FSTRA (MRID 48317007) NE N N N 

Fcd: 

↓34% 

(H) 
2, 4-D FINAL 

Page 11 of 63 

Table 2. Estrogenic/Anti-Estrogenic Pathway for 2,4-D

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for 2,4-D1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

OSRI 

ER Binding (Blair et al., 2000) NRecombinant (hER) yeast-based assay/Frog ovulation assay (Orton et al., 

2009) 

 N N5

Recombinant (hER) yeast-based assay (Hurst and Sheahan, 2003) 

 N 

ERTA (Kojima, et al., 2004) N ERTA (Lemaire et al., 2006) N6ERTA (Sun et al., 

2012) N

Cell proliferation assay (Soto et al., 

1995) 

 N 

Subchronic toxicity (Rat; MRIDs 00101599 and 00102451) 

NE NE N NE 

Subchronic toxicity (Rat; MRID 41991501) 

NE N N N 
2, 4-D FINAL 

Page 12 of 63 

Table 2. Estrogenic/Anti-Estrogenic Pathway for 2,4-D

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for 2,4-D1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Subchronic toxicity (Mouse; MRID 41991502) 

NE N N N 

Subchronic inhalation toxicity (Rat; MRID 47398701) 

N N N NE 

Developmental toxicity (Rat; MRID 00130407 ) 

 N N 

Developmental toxicity (Rabbit; MRID 41747601) 

 N N 

Extended onegeneration reproduction (Rat, MRID 47972101)7

 N N N N N N N 

Two-generation reproduction (Rat; MRIDs 00150557 and 00163996) 

NE NE NE NE NE N 

Chronic/ Carcinogenicity (Rat; MRID 43612001) 

NE N N NE 

Carcinogenicity (Mouse; MRIDs 43879801 and 43597201) 

NE NE N NE 
2, 4-D FINAL 

Page 13 of 63 

Table 2. Estrogenic/Anti-Estrogenic Pathway for 2,4-D

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for 2,4-D1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Avian reproduction (Quail; MRID 45336401)

 N 

1 Key to responses: L=Low treatment, Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows (↓ or ↑) indica

response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight otherwise indicated. LW= liver 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 OS

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 

details see Section IV. A 

4. 1.2-fold increase in estradiol production at 100 μM. 5. Negative in an in vitro frog oocyte assay evaluating ovulatory response and ovarian steroidogenesis (conc. range: 0.00625- 62.5 μM6. Negative for ERα and ERβ transactivation at the single concentration evaluated (10 μM). 7. The extended one generation reproductive study included P1 and F1 generations, and the F1 generation was sub-divided into 5 Sub

to examine general systemic and thyroid toxicity (Set 1a), developmental neurotoxicity (Set 1b), developmental immunotoxicity (Sreproductive and endocrine toxicity (Set 3). 

P Positive finding N Negative finding (in vitro)/No effect (in vivo) NE Not examined 
2, 4-D FINAL 

Page 14 of 63 

2. Effects on Androgen Pathway 

The potential for 2,4-D to interact with the androgen 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. 

2, 4-D was negative (non-binder) in the Tier 1 in vitro AR binding assay and negative in the 

steroidogenesis assay. In in vitro literature studies considered as OSRI, 2,4-D also showed no 

evidence of competitive binding to the AR (Kim et al., 2005) or AR activation (Kojima, et al., 

2004; Orton et al., 2009; Sun et al., 2012). 

The Tier 1 in vivo Hershberger and male pubertal assays were not conducted because the 

requirements for these two assays were satisfied by an OSRI extended one-generation rat 

reproductive test (EOGRT) conducted with 2,4-D, which is equivalent to the EDSP Tier 2 

mammalian test. 

In the EOGRT, although changes were noted in some androgen-related endpoints, the effects 

were non-significant (NS) and /or non-dose-related, thereby there were no treatment-related 

androgen effects in the study. High-dose animals had non-statistically significant (NS) decreases 

in several AST weights [(prostate and epididymides (P1 and F1 Set 1a) and seminal vesicles 

(P1)]. Serum testosterone concentrations were not determined in the EOGRT, or any other Part 

158 guideline study with 2, 4-D. The age at PPS was increased (p<0.05) 1.6 days in the highdose F1 male offspring, but this delay was not considered biologically relevant since it was 

within the normal range of variability. Testes weights were decreased (NS) in P1 high-dose 

males, but were decreased (p<0.05) in all treated F1 PND 22 males by 14 to 15%, but did not 

show a dose response. No histopathological lesions were observed in the testes or epididymides. 

Finally, pituitary weights were decreased in several F1 offspring subsets with no 

histopathological findings; 14 and 10% decreases (NS) in pituitary weights were observed in the 

F1 Set 1a and PND 22 males, and a 9% decrease (p<0.05) was seen in the F1 Set 3 males at the 

high dose. However, the values were within the historical control range for the testing facility 

and were not associated with histopathology in the pituitary gland. 

The only other potentially androgen-related effects observed in the mammalian Part 158 toxicity 

studies occurred in the presence of overt toxicity. These include decreased testes weight and 

increased relative pituitary weights in the subchronic rat and decreased testes weight and 

testicular atrophy in the rat chronic toxicity/carcinogenicity study. In the FSTRA or Part 158 

avian reproduction study, there were no treatment-related effects observed in males. 

Page 22 of 71
2, 4-D FINAL 

Page 15 of 63 

 Table 3. Androgenic/Anti-Androgenic Pathway for 2,4-D

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for 2,4-D1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

EDSP Tier 1 

AR Binding (MRID 48614301) N Steroidogenesis (MRID 48614305) N Hershberger Requirement satisfied by OSRI (MRID 47972101); see below. Male Pubertal Rat Requirement satisfied by OSRI (MRID 47972101); see below. FSTRA (MRID 48317007) NE N N N N 
2, 4-D FINAL 

Page 16 of 63 

 Table 3. Androgenic/Anti-Androgenic Pathway for 2,4-D

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for 2,4-D1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

OSRI 

AR binding (Kim et al., 2005) N AR transactivation (Kojima et al., 2004) N AR transactivation (Orton et al., 2009) N AR transactivation (Sun et al., 2012) N Subchronic toxicity (Rat; MRIDs 00101599 and 00102451) 

 N N NE N NE NE 

Subchronic toxicity (Rat; MRID 41991501) 

 N N NE N N NE 

Subchronic toxicity (Mouse; MRID 41991502) 

 NE N NE N N NE 

Subchronic inhalation toxicity (Rat; MRID 47398701) 

 N N N N NE NE 

Developmental toxicity (Rat; MRID 

00130407 ) 

N 
2, 4-D FINAL 

Page 17 of 63 

 Table 3. Androgenic/Anti-Androgenic Pathway for 2,4-D

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for 2,4-D1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

Developmental toxicity (Rabbit; MRID 41747601) 

N 

Extended onegeneration reproduction (RatMRID 47972101)4 

 N N N N N N N N 

Two-generation reproduction (Rat; MRIDs 00150557 and 00163996) 

NE NE NE NE NE NE N 

Chronic/ Carcinogenicity (Rat; MRID 43612001) 

N N NE N NE NE 

Carcinogenicity (Mouse; MRIDs 43879801 and 43597201) 

NE N NE N N NE 

Avian reproduction (Quail; MRID 45336401) 

N 

1. Key to responses: L=Low treatment, Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows (↓ or ↑) indicat

response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (bulbocavernosus (LABC), Epididymides (E), Kidney weight (KW), Kidney histopathology (KH) 

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

this column. For details see Section IV. A 
2, 4-D FINAL 

Page 18 of 63 

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 

details see Section IV. A 

4. The extended one generation reproductive study included P1 and F1 generations, and the F1 generation was sub-divided into 5 Sub

to examine general systemic and thyroid toxicity (Set 1a), developmental neurotoxicity (Set 1b), developmental immunotoxicity (Sreproductive and endocrine toxicity (Set 3). 

P Positive finding N Negative finding (in vitro)/No effect (in vivo) 

NE Not examined 
2, 4-D FINAL 

Page 19 of 63 

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 redundancy and have the potential to detect 

changes in the HPT axis, i.e., the pubertal female and male (rat) assays, and the AMA (frog). 

The potential for 2, 4-D to interact with thyroid regulation are summarized in Table 4. The 

various targets of the thyroid pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for thyroid or HPT axis effects. This table also includes 

HPT-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. 

In the AMA with 2, 4-D, there were no histological effects on the thyroid, and the only 

developmental change was a decrease (p<0.05) in the Day 21 hind-limb length (HLL) value 

(15%) at the high test concentration (113 mg/L). 

The requirements for the Tier 1 in vivo male and female pubertal assays were satisfied by an 

OSRI rat extended one-generation rat reproductive test (EOGRT). In this study, thyroid 

parameters were evaluated at multiple life stages. Although changes were noted in some thyroidrelated endpoints, the effects were non-significant (NS) and /or non-dose-related, or seen at a 

dose that resulted in overt toxicity (10% decrease in body weight in male offspring), thereby 

there were no treatment-related thyroid effects in the EORGT. For example, non-significant 

decreases were observed in thyroid weights in several F1 offspring sub-groups (male and 

female), as well as a non-dose-responsive increase (NS) in thyroid weight in P1 gestational day 

(GD) 17 females. The decreased thyroid weights were present with no concomitant 

histopathological effects. Decreased follicle size was observed in 3/12 high-dose P1 GD 17 

females. Non-significant decreases in serum T4 concentrations generally were observed in 

several F1 offspring sub-groups (male and female) and in the P1 GD 17 females, with a few 

exceptions. Serum T4 concentrations were decreased (p<0.05) in the high-dose male F1 PND 22 

offspring at an overtly toxic dose. T4 levels were increased in the mid-dose male, and mid- and 

high-dose female F1 Set 1a offspring, but the changes were not statistically significant. Serum 

TSH concentrations were unchanged or increased (NS) in several mid- and high-dose F1 

offspring sub-groups (male and female) and in the P1 GD 17 females. Finally, non-significant 

decreases (9-10%) in pituitary weight were observed in the F1 Set 1a and 3 female offspring. 

Pituitary weights were decreased in several F1 offspring subsets with no histopathological 

findings; 14 and 10% decreases (NS) in pituitary weights were observed in the F1 Set 1a and 

PND 22 males. A 9% decrease (p<0.05) in pituitary weight was seen in the F1 Set 3 males at the 

high dose; however, the values were within the historical control range for the testing facility and 

were not associated with histopathology in the pituitary gland. 

Page 27 of 71
2, 4-D FINAL 

Page 20 of 63 

Thyroid effects were noted in several other Part 158 studies conducted with 2,4-D. In a 

subchronic oral toxicity study in rats, thyroid weights at termination were decreased (p<0.05) 

40% in males and increased (p<0.05; relative weight) 68% in females, with an increased 

incidence of thyroid hypertrophy observed in the high-dose females. Serum T4 concentrations 

were decreased for males and females in the high-mid- and high-dose groups, with a dose 

response observed in the males, but not the females. However, the observed thyroid-related 

effects in this study occurred at a dose that resulted in overt toxicity (clinical signs and/or ≥10% 

decrease in body weight). In a subchronic oral toxicity study with 2,4-D in mice, no effect on 

thyroid weights or histopathology was observed, but a decrease in serum T4 concentrations was 

observed in males and females in the high-mid- and high-dose groups (100 and 300 mg/kg/day, 

respectively). Finally, thyroid effects were noted in a chronic oral toxicity study with 2,4-D in 

rats. Thyroid weights were increased (p<0.05) in the high-dose (150 mg/kg/day) males, and mid 

(75 mg/kg/day)- and high-dose females, at 12 and 24 months. Histopathological changes in the 

thyroid were not noted, except for decreased secretory material in the follicles of high-dose 

females at the 12-month sacrifice only. Serum T4 concentrations were decreased (generally 

p<0.05) in all mid- and high-dose males and females. Overt toxicity (>10% body weight loss) 

was observed in females at the mid and high doses. 

In an open literature in vitro study, 2,4-D showed no thyroid receptor agonist or antagonist 

activity at concentrations from 0.003 to 3.0 mg/L in a receptor-related luciferase reporter gene 

assay in Vero (African green monkey kidney) cells (Sun et al., 2012). In Relyea, 2009, although 

there were no effects on survival to metamorphosis, mass at metamorphosis, or time to 

metamorphosis for either leopard frogs or gray tree frogs, it is noted that these endpoints are 

limited in their ability to inform potential thyroid-related perturbations. 

Page 28 of 71
2, 4-D FINAL 

Page 21 of 63 

 Table 4. Thyroid Pathway for 2,4-D.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for 2,4-D1

Study Type/ 

Literature 

Citation 

Thyroid Receptor Activation Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH Levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth 

(BW, SVL) 

Systemic Toxicity Observed2

Overt Toxicity Observed3

EDSP Tier 1 Assays 

Male 

Pubertal Rat Requirement satisfied by OSRI (MRID 47972101); see below. 

Female 

Pubertal Rat Requirement satisfied by OSRI (MRID 47972101); see below. 

AMA (Frog; 

MRID 

48317002) 

N HLL: ↓15% 

(Day 21, H) N N 

OSRI

Thyroid 

receptor 

transactivation 

(Sun et al., 

2012) 

N 

Subchronic 

toxicity (Rat; 

MRID 

41991501) 

N N N NE N 

X 

(MH, 

H) 

X 

(MH, H)

Subchronic 

toxicity 

(Mouse; MRID 

41991502) 

N N 

↓T4 

(MH, 

H) 

NE N 

X 

(MH, 

H) 

N 

Subchronic 

inhalation 

toxicity (Rat; 

MRID 

47398701) 

NE N NE NE NE N N 

Extended onegeneration 

reproduction 

(Rat MRID 

47972101)4

N N N N N X5 

 (H) 

X 

(males: H)

Twogeneration 

reproduction 

(Rat; MRIDs 

00150557 and 

00163996) 

NE NE NE NE NE N X (H) 

Page 29 of 71
2, 4-D FINAL 

Page 22 of 63 

 Table 4. Thyroid Pathway for 2,4-D.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for 2,4-D1

Study Type/ 

Literature 

Citation 

Thyroid Receptor Activation Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH Levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth 

(BW, SVL) 

Systemic Toxicity Observed2

Overt Toxicity Observed3

Chronic/ 

Carcinogenicit

y (Rat; MRID 

43612001)

↑18% 

(males

: H)

N 

↓32

% 

(mal

es: 

M) 

↓64

% 

(mal

es: 

H)5

NE NE 

X 

(femal

e: M, 

H; 

male: 

H)

X 

(female; 

MH, H) 

Carcinogenicit

y (Mouse; 

MRIDs 

43879801 and 

43597201)

NE N NE NE NE N N 

1. Key to responses: L=Low treatment, Medium treatment, MH=Medium-high 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. BW= Body weight; HLL=Hind limb length; SVL=Snout 

to vent length 

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. The extended one generation reproductive study included P1 and F1 generations, and the F1 generation was 

sub-divided into 5 Subsets at weaning (PND 21) to examine general systemic and thyroid toxicity (Set 1a), 

developmental neurotoxicity (Set 1b), developmental immunotoxicity (Sets 2a and 2b) and reproductive and 

endocrine toxicity (Set 3).4 Decreased follicle size in 3/12 Parental GD17 females. 2,4-D has been shown to 

exhibit non-linear toxicokinetics in mammals. Toxicokinetic information was used to select doses for the rat 

extended one-generation reproduction study. The high doses for both sexes in this study were selected to be 

at or slightly above the inflection point for non-linear toxicokinetics. Data from the extended one-generation 

study show that the high dose for males (800 ppm) and females (600 ppm) exceeded the threshold for 

saturation of renal clearance. 

5. T4 was decreased in both sexes at 6, 12, 18 and 24 months; p<0.05. The decrease in females occurred in the 

presence of overt toxicity. At 24 months, T4 level in males were decreased 32% and 64% at 75 and 150 

mg/kg/day, respectively. 

N Negative findings 

NE Not examined 

Page 30 of 71
2, 4-D FINAL 

Page 23 of 63 

IV. Committee’s Assessment of Weight of Evidence 

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

potential of 2, 4-D 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 a 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 Studies 

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and 

OSRI studies are described 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 31 of 71
2, 4-D FINAL 

Page 24 of 63 

1. Tier 1 in vivo Assays 

The requirements for the Hershberger, male and female pubertal assays, and uterotrophic assays 

with 2, 4-D were satisfied by OSRI (extended one-generation reproduction test, EOGRT). Overt 

toxicity was not observed in either the AMA or FSTRA. 

2. OSRI 

In an extended one-generation reproduction toxicity test in rats, systemic toxicity was noted, 

presenting primarily as effects on the kidney. P males administered 800 ppm 2, 4-D in the diet had 

increased absolute and relative (to body) kidney weights, with microscopic findings of degenerative 

lesions in the proximal convoluted tubules in the outer zone of the medulla. In the F1 generation, all 

PND 22 unselected treated (100, 300, and 800 ppm) males had decreased body weights (↓9%-10%), 

and the 800 ppm males had decreased absolute (↓37%) and relative (↓29%) adrenal weights, and 

absolute kidney (↓15%) and liver (↓18%) weights. In the F1 offspring observed to PND 70, the 800 

ppm males had decreased body weight (↓11%-17%) and body weight gains (↓11%-25%) throughout 

the study period, with the magnitude of the reduction diminishing with time of exposure. Both sexes 

displayed a slight increase in ALT and an increase in triglyceride levels at the high dose. The 800 

ppm males had a 10% decrease in terminal body weights, and decreases in liver (↓16%), pituitary 

(↓14%), and adrenal glands (↓12%). Increased absolute (↑9%) and relative (↑11%) kidney weights 

were observed in the 300 and 600 ppm females. An increased incidence of degeneration of the 

proximal convoluted tubule in the kidney was observed in the 300 and 800 ppm males and in the 600 

ppm females. Similar decreases in body weights were noted in pups used for immunotoxicity 

investigations (PND 67-73). 

In a guideline subchronic oral toxicity study in Fisher 344 rats, one 100 mg/kg/day male lost 

weight on Days 55 and 62, with subsequent weight recovery until Day 91. Decreased mean body 

weights were observed in the 150 mg/kg/day males and 100 and 150 mg/kg/day females 

beginning on Day 41, and in the 60 and 100 mg/kg/day males beginning on Day 59, all 

continuing through study termination. Terminal body weights were decreased in the 150 

mg/kg/day animals, with lesser decreases (NS) at 100 mg/kg/day. The primary target organ was 

the kidney with increases in kidney weights in all males, and 150 mg/kg/day females. A 

degenerative change was seen in the kidneys of male rats treated with ≥60 mg/kg/day. The 

degree of degeneration was more pronounced in the two highest dose groups. 

In a guideline subchronic oral toxicity study in Fisher 344 rats, clinical signs of toxicity (hunched 

posture, depressed activity, and few/no feces) occurred mainly in the 300 mg/kg/day females. 

Decreased (p<0.05) body weights were observed in the 100 and 300 mg/kg/day males and 

females, reaching 7-23% in males and 7-28% in females at Week 13. Decreased overall (Weeks 

0-13) body-weight gains were also noted in the 100 and 300 ppm males (↓9-37%) and females 

(↓11-57%). A corresponding decrease in food consumption was observed in both sexes at these 

doses. Changes (p<0.05) in absolute and/or relative organ weights [including adrenals (↑ males; 

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↓ females), testes with epididymides (↑), ovaries (↓), pituitary (↑ males; ↓ females), liver (↑

males and females), and thyroids/parathyroids (↑ males and females)] were observed primarily at 

300 mg/kg/day in both sexes; many of these changes may be attributable to decreased body 

weight. Gross findings, mainly in the high-dose group, included small testes and epididymis and 

opaque eyes (females). Treatment-related histopathological changes were observed primarily at 

300 mg/kg/day and included centrilobular hepatocellular hypertrophy, atrophy of the testes, 

hypertrophy of the zona glomerulosa (adrenal cortex, both sexes) and follicular cells (thyroid, 

females). 

In a guideline subchronic oral toxicity study in B6C3F1 mice, treatment-related effects at 100 

mg/kg/day included increased mean absolute and relative kidney weights in females. Treatmentrelated effects at 300 mg/kg/day included transient decreased (p<0.05) food consumption (up to 

Week 7) and histopathological changes in the liver characterized by nuclear hyperchromatism 

and decreased glycogen in periportal hepatocytes. 

In a guideline subchronic inhalation toxicity study in Sprague-Dawley rats, clinical signs 

associated with exposure at 1.0 mg/L included excessive salivation, labored breathing and 

chromodacryorrhea. Decreased body weights were observed in the 1.0 mg/L females from Day 

14 to the end of dosing (↓10%) and recovery (↓12%). Body weight gains were also reduced in 

these females throughout the study and recovery, and was accompanied by a reduction in food 

intake. 

In a guideline developmental toxicity study in pregnant Fischer 344 rats, dams treated at 75 

mg/kg/day displayed a decrease (NS) in body-weight gain during GD 6-15 (↓79%). The 

corrected body-weight gain was comparable among the groups. 

In a guideline developmental toxicity study in artificially-inseminated female New Zealand 

White rabbits, two 90 mg/kg/day does aborted (GDs 21 and 24) and were observed with ataxia, 

decreased motor activity, loss of righting reflex, extremities that were cold to the touch, and/or 

dried feces. Body weight gains were decreased at 90 mg/kg/day (↓27%) during the dosing 

period (GDs 6-19; NS). On GDs 7-8, the 10 and 30 mg/kg/day groups showed no body-weight 

gain, and the 90 mg/kg/day group displayed a body weight loss. On GDs 15-19, the 90 

mg/kg/day group displayed no body weight gains, and a decrease in the corrected body weight 

gains (↓23%; NS). 

In a guideline two-generation reproduction study in Fischer 344 rats, decreased (p<0.05) body 

weights were consistently observed at 80 mg/kg/day in the P males (↓3%) after Week 6 and in 

the P females (↓4%) by Week 12. The decreased body weights could not be attributed to reduced 

food consumption since food consumption and food consumption per gram body weight gain 

were slightly increased. Body weights were also decreased (p<0.05) in the P females during 

gestation of the F1a (↓5-6%) litters on GD 7, 13 and 20, and the F1b litters (↓10%) on GD 20. In 

the 80 mg/kg/day F1a pups, body weights were decreased (p<0.05) by 11-29% during PND 1-28 

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in the males, and by 15-25% during PND 4-28 in the females. In the 80 mg/kg/day F1b pups, 

body weights were decreased (p<0.05) by 22-42% in the males and 17-38% in the females 

during PND 1-28. In the 20 mg/kg/day F1b pups, body weights were decreased (p<0.05) in the 

males by 7% and 16% on PND 4 and 28, respectively, and in the females by 13% on PND 28. 

Additionally at 80 mg/kg/day the following differences (p,0.05) in gestation and survival were 

observed: (i) gestation duration was increased (p<0.01) for the F1b litters (22.5 days vs 21.9 

days); (ii) gestation survival index was decreased (p<0.01) for the F1b litters (31.7% vs 97.8%); 

(iii) the number of F1b pups born dead/dying by PND 1 was increased (110 treated vs. 5 

controls); (iv) F1b litter size was decreased (p<0.01; 5.1 treated vs. 9.5 controls); v) F1b pup 

viability was lower (p<0.01) throughout the weaning period; (vi) there were decreases (p<0.01) 

in F1b pup survival to LD 4 (86.3% treated vs. 100% controls); and (vii) survival to LD 28 was 

decreased (71.4% treated vs. 100% controls). 

In a guideline combined chronic toxicity/carcinogenicity study in Fischer 344 rats, body weights 

were lower than respective controls in females at 75 mg/kg/day (↓14%) and in males (↓8%) and 

females (↓26%) at 150 mg/kg/day at termination. Body weight gains were lower than respective 

controls in females at 75 mg/kg/day (↓24%) and in males (↓17%) and females (↓48%) at 150 

mg/kg/day. A corresponding depression in mean food consumption occurred in females at 75 

mg/kg/day (↓4%) and in males (↓5%) and females (↓12%) at 150 mg/kg/day. Statistically 

significant (p < 0.05) increases in plasma levels of alanine aminotransferase (ALT), aspartate 

aminotransferase (AST), alkaline phosphatase (AP) and/or cholesterol were seen in females at 75 

mg/kg/day and in males and females at 150 mg/kg/day at various time periods. After 12 months 

of treatment, treatment-related effects included: decreased hematopoiesis of the bone marrow of 

females at 150 mg/kg/day; altered tinctorial properties in the liver of females at 75 mg/kg/day 

and both sexes at 150 mg/kg/day; bilateral retinal degeneration of the eyes of females at 150 

mg/kg/day; multifocal alveolar histiocytosis in the lungs of females at 75 mg/kg/day and both 

sexes at 150 mg/kg/day; degeneration of the descending portion of the proximal convoluted 

tubules of the kidneys in both sexes at 75 mg/kg/day and 150 mg/kg/day; atrophy of the adipose 

tissue of females at 75 and 150 mg/kg/day; atrophy of the testes in males at 150 mg/kg/day; and 

decreased secretory material in the thyroid follicles of females at 150 mg/kg/day. After 24 

months of treatment, treatment-related non-neoplastic lesions were limited to the eyes, liver, 

lung, and the mesenteric fat. Eye lesions were characterized as slight to severe bilateral retinal 

degeneration and lenticular cataracts in both sexes at 150 mg/kg/day. Liver lesions manifested as 

increases in the size of hepatocytes, often accompanied by altered tinctorial properties that 

involved all hepatocytes within the hepatic lobule of both sexes at 150 mg/kg/day. Lesions of the 

respiratory system included subacute to chronic inflammation of the lungs in females at 75 

mg/kg/day and both sexes at 150 mg/kg/day. Atrophy of the adipose tissue was increased in both 

sexes at 150 mg/kg/day. It is interesting to note that lesions seen in the spleen, kidneys, testes 

and thyroid glands in rats sacrificed at 12 months were not seen in those sacrificed at 24 months. 

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In a guideline carcinogenicity study in B6C3F1 mice, the 300 mg/kg/day females had slightly 

lower (p<0.05) body weights at the 3- and 6-month intervals (↓4%). Body-weight gains were 

decreased (p<0.05) in the 5 (↓7%), 150 (↓6%), and 300 (↓14%) mg/kg/day females at the 3-

month interval, and the 300 mg/kg/day females had decreased (p<0.05) body-weight gain at the 

6- and 12-month (↓9%) intervals. Microscopically, there was an increased incidence of lesions in 

the kidneys of 62 and 120 mg/kg/day males and the 120 and 300 mg/kg/day females. 

In a guideline avian reproduction toxicity study, northern bobwhite quail (Colinus virginianus) 

fed diets containing concentrations of 0, 160, 400, and 1000 ppm [(mean-measured 

concentrations of 0 (basal diet), 147, 382, or 962 ppm)] for approximately 21 weeks did not 

display any signs of systemic or overt toxicity. 

B. Estrogen Pathway 

There was no convincing evidence of potential interaction of 2, 4-D with the estrogen pathway. 

The Tier 1 or OSRI in vitro assays were negative with the exception of a 20% increase in 

estradiol production in the in the steroidogenesis assay at 100 μM. The requirements for the Tier 

1 in vivo mammalian uterotrophic and female pubertal assays were satisfied by the extended onegeneration reproduction test in rats (equivalent to EDSP Tier 2 study) considered as OSRI. No 

estrogen-related effects were observed in this study or in any of the mammalian Part 158 toxicity 

studies in the absence of overt toxicity. No effects were observed in the Part 158 bobwhite quail 

reproduction study. In the FSTRA, fecundity was decreased (34%) at a concentration of 96.5 mg 

a.i./L, and while an increase in the number of female ovaries at stage 2 (compared to stage 3 or 

4) was observed for the 2,4-D treatment groups compared to the negative control, the incident 

rates were not dose-responsive, and there were no other effects observed in this study. 

C. Androgen Pathway 

There was no convincing evidence of interaction of 2, 4-D with the androgen pathway. The Tier 

1 and OSRI in vitro assays were negative. The requirements for the Tier 1 in vivo mammalian 

Hershberger and male pubertal assays were satisfied by the extended one-generation 

reproduction test in rats (equivalent to EDSP Tier 2 study) considered as OSRI. There were no 

androgen-related effects observed in this study. Additionally, there were no treatment-related 

effects in the FSTRA or avian reproduction studies, and there were no effects on the male 

reproductive parameters in the Part 158 mammalian studies in the absence of overt toxicity. 

D. Thyroid Pathway 

In the AMA, while there was a 15% decrease in HLL (at the high test concentration of 113 

mg/L), there were no histological effects on the thyroid or effects on developmental stage. Some 

thyroid-related effects were seen in the mammalian Part 158 studies. The effects were only seen 

in the presence of overt toxicity or at doses that exceeded the threshold for renal clearance of 2, 

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4-D in the rat, resulting in systemic toxicity. The requirements for the Tier 1 in vivo mammalian 

male and femal pubertal assays were satisfied by the extended one-generation reproduction test 

in rats (equivalent to EDSP Tier 2 study) considered as OSRI. In that study, there were no 

treatment-related thyroid effects in males or females. Therefore, collectively the data across the 

Tier 1 assays and Part 158 data do not demonstrated convincing evidence of potential interaction 

with the thyroid pathway. 

E. Conclusions 

The conclusion of the WoE evaluation is that 2, 4-D demonstrates no convincing evidence of 

potential interaction with the estrogen, androgen or thyroid pathways. 

V. EDSP Tier 2 Testing Recommendations 

Based on weight of evidence considerations, additional EDSP Tier 2 ecological testing is not 

recommended. It is noted that the agency already has a mammalian toxicity study (extended onegeneration reproduction) that is equivalent to the EDSP Tier 2 study. 

 

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VI. References 

Blair, R.M., Fang, H., Branham, W.S., Haas, B.S., Dial, S.L., Moland, C.L., Tong, W., Shi, L., 

Perkins, R. and Sheehan, D.M. (2000) The estrogen receptor relative binding affinities of 188 

natural and xenochemicals: structural diversity of ligands. Toxicological Sciences. 54:138-153. 

Gorzinski, S.J., Wade, C.E., Morden, D.C., Keyes, D.G., Wolfe, E.L., Dittenber, D.A., Kalnins, 

R.V., Schuetz, D.J. and Kociba, R.J. (1981a) Technical grade 2,4-Dichlorophenoxyacetic acid 

(2,4-D): results of a 13-week subchronic dietary toxicity study in the CDF Fischer 344 Rat. 

Unpublished study, Dow Chemical Company, Midland, NI. (MRID 00102451). 

Gorzinski, S.J., Wade, C.E., Morden, D.C., Keyes, D.G., Dittenber, D.A., Kalnins, R.V., 

Schuetz, D.J. and Kociba, R.J. (1981b) Purified 2,4-Dichlorophenoxyacetic acid (2,4-D): results 

of a 13-week subchronic dietary toxicity study in the CDF Fischer 344 Rat. Unpublished study, 

Dow Chemical Company, Midland, MI. (MRID 00101599). 

Hoberman, A.M. (1990) Developmental toxicity (embryo-fetal toxicity and teratogenic potential) 

study of 2,4-Dichlorophenoxyacetic acid (2,4-D) administered orally via stomach tube to New 

Zealand white rabbits. Project No. 320-003. Unpublished study, Argus Research Laboratories, 

Inc., Horsham, P A. (MRID 41747601). 

Hoffman, G.M. (2008) A 28-day subchronic inhalation toxicity study of 2,4-Dichloro 

phenoxyacetic acid in the rat via nose-only exposures. Project No. 07-6156. Unpublished study, 

Huntingdon Life Sciences, East Millstone, NJ. (MRID 47398701). 

Hurst, M.R., Sheahan, D.A (2003) The potential for oestrogenic effects of pesticides in 

headwater streams in the UK. The Science of the Total Environment 301:87-96. 

Jeffries, T.K., Yano, B.L., Ormand, J.R. and Battjes, J.E. (1995) 2, 4- Dichlorophenoxyacetic 

acid: chronic toxicity/oncogenicity study in Fischer 344 rats- Final Report. Project No. K002372-064F. Unpublished study, Dow Chemical Company, Midland, MI. (MRID 43612001). 

Kim, H.J., Park, Y.I. and Dong, M.S. (2005) Effects of 2,4-D and DCP on the DHT-induced 

androgenic action in human prostate cancer cells. Toxicological Sciences. 88:52-59. 

Kojima, H., Katsura, E., Takeuchi, S., Niiyama, K. and Kobayashi, K. (2004) Screening for 

estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using 

Chinese hamster ovary cells. Environmental Health Perspectives. 112:524-531. 

Lemaire, G., Mnif, W., Mauvais, P., Balaguer, P. and Rahmani, R. (2006) Activation of a- and 

P-estrogen receptors by persistent pesticides in reporter cell lines. Life Sciences. 79:1160-1169. 

Page 37 of 71
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Page 30 of 63 

Marty, M.S., Zablotny, C.L., Andrus, A.K., Boverhof, D.R., Bus, J.S., Perala, A.W., Saghir, S. 

and Yano, B.L. (2010) 2,4-D: an extended one-generation dietary toxicity study in Crl:CD(SD) 

rats. Unpublished Study Report of the Dow Chemical Company, Midland, Michigan. (MRID 

47972101). 

Mitchell, L.R., Beavers, J.B., Martin, K.H. and Jaber, M. (1999) 2,4-D acid: a reproduction study 

with the northern bobwhite. Final Report. Unpublished study by Wildlife International, Ltd. 181 

p. Lab Project Number: 467-106. (MRID 45336401). 

Orton, F., Lutz, I., Kloas, W. and Routledge, E.J. (2009) Endocrine disrupting effects of 

herbicides and pentachlorophenol: in vitro and in vivo evidence. Environ. Sci. Technol. 43:2144-

2150. 

Rodwell, D.E. (1983) A teratology study in Fischer 344 rats with 2, 4- dichlorophenoxyacetic 

acid- Final Report. Project No. WIL-81135. Unpublished study, WIL Research Laboratories, 

Inc., Ashland, OH. (MRID 00130407). 

Rodwell, D.E. and Brown, W.R. (1985) A Dietary Two-Generation Reproduction Study in 

Fischer 344 Rats with 2,4-Dichlorophenoxyacetic Acid: Final Report: Project No. WIL-81137. 

WIL Research Lab. Inc. (MRID 00150557). 

Rodwell, D.E. (1986) A dietary two-generation reproduction study in Fischer 344 rats with 2,4-

Dichlorophenoxyacetic acid- addendum to the Final Report. Project No. WIL-81137. 

Unpublished study, WIL Laboratories, Ashland, OH. (MRID 00163996). 

Schulze, G.E. (1991a) Subchronic toxicity study in rats with 2,4- Dichlorophenoxy-acetic acid. 

Project No. 2184-116. Unpublished study, Hazelton Laboratories America, Inc. Rockville, MD. 

(MRID 41991501). 

Schulze, G.E. (1991b) Subchronic toxicity study in mice with 2, 4- Dichlorophenoxy-acetic acid. 

Project No. 2184-117. Unpublished study, Hazelton Laboratories America, Inc. Vienna, VA. 

(MRID 41991502). 

Schwetz, BA et al. (1971) The effect of 2,4-Dichlorophenoxyacetic acid (2,4-D) and Esters of 

2,4-D on Rat Embryonal, Foetal and Neonatal Growth and Development. Fd. Cosmet. Toxicol. 

9:801-817. 

Soto, A.M., Sonnenschein, C., Chung, K.L., Fernandez, M.F., Olea, N. and Serrano, F.O. (1995) 

The ESCREEN assay as a tool to identify estrogens: an update on estrogenic environmental 

pollutants. Environmental Health Perspectives. 103 (Suppl 7):113-122. 

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Page 31 of 63 

Stott, W. (1995a) 2,4-Dichlorophenoxyacetic Acid: Dietary Oncogenicity Study in B6C3F1 

Mice Two-Year Final Report (Female). Lab Project Number K-002372-063F. The Dow 

Chemical Company. (MRID 43597201). 

Stott, W. (1995b) 2,4-Dichlorophenoxyacetic Acid: Dietary Oncogenicity Study in Male B6C3Fl 

Mice Two-Year Final Report. Lab Project Number K-002372-063MF. The Dow Chemical 

Company. (MRID 43879801). 

Sun, H., Si, C., Bian, Q., Chen, X., Chen, L., and Wang, X. (2012) Developing in vitro reporter 

gene assays to assess the hormone receptor activities of chemicals frequently detected in 

drinking water. J Appl Toxicol. 32(8):635-641. 

USEPA. (2005) Reregistration Eligibility Decision (RED) for 2,4-D. LIST A. CASE 0073. June, 

2005. EPA 738-R-05-002, Office of Pollution Prevention and Toxic Substances, U.S. 

Environmental Protection Agency, Washington, DC. 

USEPA. (2010) 2,4-Dichlorophenoxyacetic Acid (2,4-D): Report of the Endocrine Disruptor 

Review Team - Test Order #: EDSP-031001-120. December 10, 2010. Office of Chemical Safety 

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

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. (2012) 2,4-D: Revised Evaluation of Data Identified in NRDC Petition and Associated 

Documents. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection 

Agency, Washington, DC. 

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. 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay; (OCSPP 890.1100) 

The 21-day assay (MRID 48317002) of 2,4-D (purity 98.6%) on amphibian metamorphosis of 

South African Clawed Frog (Xenopus laevis) was conducted under flow-through conditions. 

Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80/control and treatment group; 

20/replicate) were exposed to a negative control and test chemical nominal concentrations of 0.4, 

4.0, 40.0, and 100.0 mg a.i./L. Mean measured concentrations were <0.120 (<LOQ, negative 

control), 0.273, 3.24, 38.0, and 113 mg a.i./L. The test system was maintained at 21.9 to 22.7 °C 

and a pH of 7.0 to 7.8. 

Only one incidence of tadpole mortality occurred in the mid-low treatment group; the cause of 

death was unknown. No clinical signs of toxicity were noted. 

2,4-D did not affect Day 7 normalized (for snout-vent length) hind-limb length (HLL). However, 

there was a statistically significant (p<0.05) decrease of 15% in Day 21 normalized HLL at the 

highest treatment level compared with the negative control. There was no significant effect on 

median NF developmental stage, snout-vent length (SVL), or body weight at Day 7 or Day 21. 

Asynchronous development was not observed. There were no effects on thyroid gland 

histopathology. Late stage (>NF stage 60) tadpoles were observed in the negative control and in 

all treatment levels; consistent with the guideline recommendations, these tadpoles were excluded 

from analyses of growth and normalized HLL. 

Androgen Receptor (AR) Binding Assay; (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48614301), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment 

to measure the binding of a single concentration of [3

H]-R1881 (1 nM) in the presence of 

increasing concentrations (10−11 to 10−4

 M) of 2,4-D (98.5% purity). The test guideline 

recommends testing up to 10-3 M; however, the sponsor selected 10-4 M as the highest 

concentration based on in vivo toxicokinetic analyses in the rat; concentrations higher than 10−4

M were not relevant for testing in this assay as they are substantially above the inflection point 

for linear toxicokinetics (see Appendix B of study report). Ethanol was used as a solvent at a 

final concentration of <3%. A total of three independent runs were performed, and the assay 

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

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Saturation binding experiments were conducted to demonstrate that the AR in the rat prostate 

cytosol was present at adequate levels and functioning with appropriate affinity for the 

radiolabeled ligand. The saturation binding experiment resulted in a maximum binding capacity 

(Bmax) of 3.245 fmol/100 μg protein and the dissociation constant (Kd) was 0.4641 nM. 

Although these values were slightly below the range of values from the validation studies, the 

results were highly reproducible and all other performance criteria and the competitive binding 

assays indicated acceptable performance of the assay. The Scatchard plot indicated a linear 

response across the concentrations of ligand added. Nonspecific binding as a percent of total 

binding was less than 20% across the entire concentration range in the saturation binding assays 

(range 6.2-19.8%, with the exception of the high concentration (10 nM) in one assay, which was 

24.6%). 

There were no appreciable alterations in R1881 AR binding activity at 2,4-D concentrations 

ranging from 10-11 to 10-4 M in the competitive binding experiments, therefore, the log IC50 and 

relative binding affinity (RBA) for 2,4-D could not be calculated. The log IC50 values for R1881 alone and the positive control, dexamethasone, were −9.0 and −4.4 M, respectively. 

Compared to R1881, the RBA for dexamethasone was 0.0027 %. In all instances, R1881 and the 

positive control met the QC performance criteria established in the test guideline. 

Based on the results from the three runs, 2,4-D is classified as a Non-binder in the Androgen 

Receptor Binding Assay. 

Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48614302), 2,4-D (98.5% a.i., lot#: 2006 2433 

8006-USA) in ethanol (1%) was incubated with human recombinant aromatase and tritiated 

androstenedione (1-β [3

H(N)]-androst-4-ene-3,17-dione; [3H]ASDN) for 15 minutes at 37 °C to 

assess the potential of 2,4-D to inhibit aromatase activity. 2,4-D was tested at logarithmic 

concentrations from 10–10 M to 10−4 M in three independent runs. The test guideline 

recommends testing up to 10-3 M; however, the sponsor selected 10-4 M as the highest 

concentration based on in vivo toxicokinetic analyses in the rat. The sponsor considered 

concentrations higher than 10-4 M to be not relevant for testing in this assay as they are 

substantially above the inflection point for linear toxicokinetics (See Appendix B of the AR 

binding assay study report, MRID 48614301). 

Aromatase activity was determined by measuring the amount of tritiated water produced at the 

end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified 

using liquid scintillation counting (LSC). Three independent runs were conducted and each run 

included a full activity control, a background activity control, a positive control series (10−10 to 

10−5 M) using a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), and the test 

chemical series (10−10 to 10−4 M) with 3 repetitions per concentration. 

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Aromatase activity in the full activity controls ranged from 0.131 to 0.244 nmol·mgprotein−1

·min−1 for the three test runs, with a mean and standard deviation of 0.186±0.036 

nmol·mg-protein−1

·min−1. Activity in the background controls ranged from 7.31 to 11.51% of 

the full activity controls. The responses of full activity controls were outside of the 90 to 110% 

range in the 2nd and 3rd replicates in the Run 1 (86.6 and 113.8%, respectively). 

Results for the positive control were generally within the recommended ranges for the top of the 

curve, bottom curve, Hill slope, log IC50, and coefficient of variation for replicates of each 

concentration within runs, with the exception that the bottom of the curve in Runs 2 and 3 (-8.2 

and -7.2, respectively) exceeded the acceptable range (-5 to +6). Also, the coefficients of 

variations (%CVs) for replicates of each concentration of 2,4-D within a run were generally 

within the 15% guideline, with the exception that the overall %CV for the highest two 

concentrations (-45.2 and 415.9%) exceeded the acceptable limit. For 4-OH ASDN, the 

estimated log IC50 averaged −7.23 M and the slope was −0.92. 

For 2,4-D, aromatase activity averaged 0.195±0.045 nmol·mg-protein−1

·min−1 at the lowest 

tested concentration (10−10 M) and 0.168±0.025 nmol·mg-protein−1

·min−1 at the highest tested 

concentration (10−4 M). The data for 2,4-D were modeled for Run 1, and the goodness of fit (R2) 

value was 0.83; however, the 2,4-D data from Runs 2 and 3 could not be modeled. The average 

dose-response curve indicated that the aromatase activity of the test material at concentrations 

ranging from 10−10 M to 10−4

 M was essentially equivalent to the activity observed in the full 

activity controls. At 10–4 M, aromatase activity was approximately 91%. Since the average 

lowest portion of the activity response curve was greater than 75% activity, 2,4-D is classified as 

a non-inhibitor of aromatase activity up to the highest concentration tested (10-4 M). High CVs 

were observed for 4-OH ASDN at the two highest concentrations and at a single concentration 

for 2,4-D. Individual values were occasionally outside of the performance criteria ranges (with 

the mean value within range). 

Based on the data from the average response curve, 2,4-D is classified as a Non-inhibitor of 

aromatase activity in this assay. 

Estrogen Receptor (ER) Binding Assay; (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48614303) for 2,4-D (98.5%, Lot# 2006 2433 

8006-USA) , uterine cytosol from Sprague Dawley rats was used as the source of ER to conduct 

saturation binding and competitive binding experiments in this assay. 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 logarithmic increasing concentrations of 2,4-D from 10−11 to 10−4 M, 

rather than 10−10 to 10−3 recommended in the test guideline. The justification for lowering the 

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top test concentration was based on toxicokinetic data in the rat; concentrations higher than 10−4

M were not relevant for testing in this assay as they are substantially above the inflection point 

for linear toxicokinetics. Ethanol was used as a solvent at a final concentration of <3%. The 

assay included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative 

control, and 17β-estradiol as the natural ligand reference material, and three independent runs 

were performed on separate days. 

Summary data pertaining to the saturation binding experiment were reported separately in the 

study profile submitted by the test order recipient. The Kd for [3

H]-17β-estradiol was 0.1032 nM 

and the Bmax (nM) was 0.07097 for the prepared rat uterine cytosol used in these experiments. 

The Kd for the run was within the expected range of 0.03 to 1.5 nM, and the Bmax was within the 

expected range of 10-150 fmol/100 μg protein. The data produced a linear Scatchard plot. 

In the competitive binding experiment, no precipitation was observed at any concentration tested. 

The mean specific binding in the presence of 2,4-D was >95% at 2,4-D concentrations of ≤10−4 

M in all three runs. The estimated mean log IC50 and RBA was not calculated for 2,4-D as the 

percent binding inhibition did not reach 50% for any run. 

The estimated mean log IC50 for the natural ligand, 17β-estradiol, and the weak positive control 

(19-norethindrone) was −9.0 and −5.5 M, respectively. The mean RBA was 0.034% for 19-

norethindrone. All performance criteria were met for 17β-estradiol, 19-norethindrone and 

octyltriethoxysilane. 

2,4-D was tested over a concentration range that fully defined the top of the curve. The mean 

specific radioligand binding in the presence of 2,4-D was >95% at 2,4-D concentrations of 

≤10−4

 M. Based on the results from the three runs, 2,4-D is classified as Not Interactive in the 

Estrogen Receptor Binding Assay. 

ERα Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48614304), hERα-HeLa-9903 cells 

cultured in vitro were exposed to 2,4-D (98.5% a.i., Lot #2006 2433 8006 USA) at logarithmically 

increasing concentrations from 10−10 to 10−4

 M in DMSO (0.1%) for 24 hours. A total of four 

separate runs were performed. Each run was performed using 96-well plates and each 2,4-D 

concentration was tested in triplicate (3 wells/plate). Cells were exposed to the test agent for 

approximately 24 hours to induce reporter (luciferase) gene products. Luciferase expression in 

response to activation of the estrogen receptor was measured upon addition of a luciferase substrate 

and detection with a luminometer with acceptable sensitivity. 

2,4-D was tested up 10−4 M based on solubility, cytotoxicity and in vivo toxicokinetic analysis. 

There were deviations from expected performance criteria for all of the four reference chemicals, 

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but these deviations do not affect the interpretation of this study. The RPCMax was <0% for the 

first run, 8.8% for the second run, 5.3% for the third run, and 7.0% for the fourth run; the associated 

PCMax was 10−4

 M for runs 1-3 and 10-5 M for run 4. Because the RPCMax < PC10 in all assay runs, 

2,4-D 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 48317007) of 2,4-D with fathead minnow 

(Pimephales promelas) was conducted under flow-through conditions. Adult fish (20 spawning 

groups; 2 males and 4 females in each group; 4 groups/treatment; ca. 6 months old) were exposed 

to 2,4-D (98.6% purity) at nominal concentrations of 0 (negative control), 0.400, 4.00, 40.0, and 

100 mg a.i./L concentrations with corresponding mean-measured concentrations of <0.10 (<LOQ, 

negative control), 0.245, 3.14, 34.0, and 96.5 mg a.i./L, respectively. The test system was 

maintained at 24.5 to 25.2oC and a pH of 7.02 to 7.76. 

The single mortality observed during the assay occurred in a female in the high treatment group. 

There were no significant differences for male or female body weight or length relative the 

negative control. At test termination, observations of secondary sex characteristics were observed 

in the negative control and treated groups; no treatment-related effects were reported. Clinical 

signs included the loss of an eye, ascites, and scoliosis (bent tail) which were observed in single 

fish in the negative control or treatment groups. 

Spawning occurred in the negative control at least every 4 days in 3 of the 4 replicates, and mean 

fecundity was 29.8 eggs/female/day/replicate; fertility in the negative control was 96.9%. 

Fecundity was significantly decreased (Jonckheere-Terpstra; p<0.05) by 34% in the high treatment 

group with a non-significant (p>0.05) concentration-dependent trend of decreased fecundity in the 

lower treatment groups compared to the negative control. There were no significant differences 

for fertility between the 2,4-D treatments and the negative control. 

There were no significant differences (p>0.05) between the 2,4-D treatment groups and the 

negative control for male or female gonado-somatic index (GSI) or plasma vitellogenin (VTG). 

There was also no significant difference (p>0.05) for male nuptial tubercle scores; no tubercles 

were observed for females. No apparent treatment-related histopathological effects were observed 

in males and females. Although not concentration-dependent, an increase in the number of female 

ovaries that were observed as Stage 2 (compared to Stage 3 or 4) was reported for the 2,4-D 

treatments compared to the negative control. Plasma sex steroid concentrations were not reported. 

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Hershberger Assay; (OCSPP 890.1400) 

The requirement for a Tier 1 Hershberger assay is satisfied by OSRI: an extended dietary onegeneration reproductive toxicity study (MRID 47972101, 2010). The results from this study are 

presented with the OSRI in Appendix 2. 

Female Pubertal Assay; (OCSPP 890.1450) 

The requirement for a Tier 1 female pubertal assay is satisfied by OSRI: an extended dietary onegeneration reproductive toxicity study (MRID 47972101, 2010). The results from this study are 

presented with the OSRI in Appendix 2. 

Male Pubertal Assay; (OCSPP 890.1500) 

The requirement for a Tier 1 male pubertal assay is satisfied by OSRI: an extended dietary onegeneration reproductive toxicity study (MRID 47972101, 2010). The results from this study are 

presented with the OSRI in Appendix 2. 

Steroidogenesis Assay; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48614305), H295R cells cultured in vitro in 24-well plates 

were incubated with 2,4-D (98.5% purity, Lot # 2006 2433 8006-USA) at log concentrations of 

10−10 to 10−4

 M for 48 hours in triplicate in three independent experiments. Dimethyl sulfoxide 

(DMSO) was used as the vehicle, at a final concentration in the assay of 0.1%. 

Testosterone and estradiol levels were measured using LC-APPI-MS/MS. A Quality Control 

(QC) plate was run concurrently with each independent run of a test chemical plate to 

demonstrate that the assay responded properly to positive control agents at two concentration 

levels. The positive controls included a known inhibitor (prochloraz) and a known inducer 

(forskolin) of estradiol and testosterone production. 

Guideline acceptability recommendations and requirements were generally met, including lack 

of cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low 

%CV), and appropriate induction and inhibition with positive controls, with two exceptions. The 

required concentration for estradiol production (40 pg/mL) was not met in any run for blank or 

solvent control, with values ranging from 26.7 to 38.3 pg/mL, but the basal concentrations were 

greater than 2.5-fold of the minimum detection level (10 pg/mL) as required. Secondly, all three 

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runs of 1 μM prochloraz only reduced estradiol to 0.6-fold that of the solvent control (instead of 

0.5-fold). 

2,4-D had no effect on testosterone production at concentrations up to 10−4

 M, and no effect on 

estradiol production at concentrations up to 10−5

 M. At the highest concentration tested (10−4

M), 2,4-D increased (p≤0.05) estradiol levels by 20% (1.2-fold) in all three runs relative to the 

DMSO-treated cells. 

Based on hormone responses in each of the independent runs, 2,4-D treatment resulted in 

statistically significant and reproducible increases in estradiol production. 2,4-D treatment did 

not result in statistically significant and reproducible alterations in testosterone production. 

Uterotrophic Assay; (OCSPP 890.1600) 

The requirement for an uterotrophic assay is satisfied by OSRI: an extended dietary onegeneration reproductive toxicity study (MRID 47972101, 2010). The results from this study are 

presented with the OSRI in Appendix 2. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Published literature and Part 158 studies considered relevant for determining the potential 

interaction of 2,4-D with the endocrine system are summarized below. Given the breath of 

toxicity data available, the only Part 158 studies discussed in this appendix are those that fulfill 

current guideline requirements and are directly relevant to assessing potential endocrine effects 

(i.e. reproductive and developmental studies, subchronic and chronic toxicity studies, special 

mechanistic studies, etc.). In addition, recent re-evaluations of 2,4-D by the Agency (USEPA, 

2012) were considered in summarizing the available information. 

ER Binding 

In a non-guideline published study, Blair et al., 2000 screened a large, structurally diverse group 

of chemicals for estrogen receptor (ER) affinity using a standardized ER competitive-binding 

assay. The assay was tested using multiple chemicals including the EDSP List 1 chemicals 2,4-

D, atrazine, carbaryl, metolachlor and simazine; only data for the List 1 chemicals are presented 

herein. 

Uterine cytosol from ovariectomized Sprague-Dawley rats was used as the ER source for the 

competitive-binding assay. Test compounds were first screened at high concentrations to 

determine whether the compounds competed with [3

H]-estradiol (E2) for the ER. Compounds 

that demonstrated affinity for the ER were further assayed with a wide range of concentrations to 

determine the IC50 and relative binding affinity (RBA) values of the compounds. 

Adult (~245 days old; retired breeders), non-pregnant female Sprague-Dawley rats (NCTR:SDN, 

an outbred albino rat stock originating from CRL:CD [SD]BR rats) were ovariectomized (mean 

n = 14 rats/cytosol batch) 10 days prior to receptor preparation. After sacrifice by CO2

asphyxiation, the uteri were excised, trimmed of excess fat and mesentery, weighed and placed 

in ice-cold TEDG buffer that included 10 mM Tris, 1.5 mM EDTA, 10 mM dithiothreitol, and 

10% glycerol (pH 7.4). The pooled uteri were homogenized at 4°C in fresh, ice-cold TEDG 

buffer (1 g tissue/10 mL buffer). The resulting homogenate was centrifuged at 105,000 × g for 

60 min at 4°C, separated into 3-mL portions, and stored at –70°C until used in the competition 

assays. 

The test compounds 2,4-D (99% purity), atrazine (98%), carbaryl (99%), metolachlor (98.7%), 

and simazine (99%) were obtained from Supelco. The preparation of the test compounds 

including the solvent used was not reported in the article. 

In the competitive binding assay, 1 × 10–9 M [3

H]-E2 (10 μL) was incubated with 10 μL of 

increasing concentrations of the test compound, 50 μL of uterine cytosol preparation, and 230 μL 

of 50 mM Tris buffer in duplicate tubes, and incubated at 4°C for 20 hours. After incubation, 

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750 μL of a cold 60% hydroxylapatite (HAP) slurry in 50 mM Tris buffer was added to separate 

the bound ligand from the free ligand, and the tubes were incubated in an ice water bath for 20 

minutes, and then centrifuged at 4 °C. The resulting HAP pellet was resuspended in 2.0 mL of 

cold 50 mM Tris buffer and centrifuged again. After three washes, the supernatant was 

discarded and 2.0 mL of cold ethanol was added to extract the radiolabeled E2 from the HAP. 

Tubes were incubated on ice for 15 min, centrifuged at 4°C, and the resulting supernatant was 

decanted into vials containing 10 mL of scintillation cocktail. Radioactivity was measured with 

a liquid scintillation analyzer. 

Each assay also included a “zero” tube (no competitor added; representing the total binding of 

[

3

H]-E2; average ~15,000 dpm) and E2 standard curve (1 × 10–7, 1 × 10–8, 1 × 10–9, 3.33 × 10–10, 

1 × 10–10, and 3.33 × 10–11 M concentrations) for quality control. The 1 × 10–7 M E2 tube 

contained a 100-fold molar excess of radioinert E2 (compared to [3

H]-E2), and represented nonspecific binding (NSB; average ~800 dpm). Radioactivity counts (dpm) of the NSB tubes were 

subtracted from all tubes prior to calculation of percent bound [3

H]-E2. Data for each test 

compound and the E2 standard curve were plotted as percent bound [3

H]-E2 versus molar 

concentration, and the IC50 (50% inhibition of [3H]-E2 binding) for each test compound was 

determined. The RBA for the test compound was calculated by dividing the IC50 of E2 by the 

IC50 of the competitor and was expressed as a percentage (E2=100). 

Compounds with a log RBA of >0 were considered strong binders by the study authors, 

moderate binders had a log RBA between 0 and –2, and weak binders had a log RBA of <–2. As 

expected, all of the tested steroidal estrogens, synthetic estrogens, and anti-estrogens 

demonstrated an affinity for the ER under the conditions of the assay. 17β-Estradiol had a mean 

IC50 of 8.99 × 10−10 M. 

RBAs for the EDSP List 1 chemicals 2,4-D, atrazine, carbaryl, metolachlor, and simazine could 

not be calculated as the EC50 estimates were >1.00 × 10−4

 (2,4-D, atrazine, carbaryl, and 

metolachlor) or >3.33 × 10−5

 (simazine). Therefore, these compounds were not considered 

binders of the ER. 

ER and AR Activation 

In a published, non-guideline study, Kojima et al., (2004) screened and evaluated 200 pesticides 

(including 2,4-D), covering nine chemical classes, for their potential to interact with two human 

estrogen receptor subtypes (hERα and hERβ) and one human androgen receptor (hAR) in 

transactivation assays with Chinese hamster ovary cells (CHO-K1 cells). 

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The human estrogen receptor (hERα) and androgen receptor (hAR) expression vectors 

(pcDNAERα and pZeoSV2AR, respectively) were constructed as described previously (Kojima 

et al, 2003). The hERβ expression vector was constructed by cloning the hERβ cDNA with 

reverse transcriptase-polymerase chain reaction from human placental RNA. The sequence was 

verified and inserted into the mammalian expression vector pcDNA3.1Zeo(-) creating 

pcDNAERβ. The construction of the estrogen or androgen responsive elements containing 

reporter plasmid (pGL3-tkERE and pIND-ARE, respectively) was described previously (Kojima 

et al, 2003). pRL-SV40 containing the Renilla luciferase gene was used as an internal control for 

transfection efficiency. 

For detection of hERα or hERβ activity, CHO cells were transfected with pcDNAERα or 

pcDNAERβ, 50 ng pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were 

transfected with pZeoSV2AR, pIND-ARE and pRL-SV40. After a 3 h transfection period cells 

were dosed with various concentrations of test compounds (95-100% purity) or with 0.1% 

DMSO (vehicle control) in complete medium. For measurement of antagonistic activity to 

hERα, hERβ and hAR, either 10-11 or 10-10 M 17β-estradiol (E2, >97% purity) or 10-10 M 5αdihydrotestosterone (DHT, 95% purity) was added to the cell cultures along with the test 

compound, respectively. Cells were incubated for 24 hours and luciferase activity was measured 

with a luminometer and was normalized based on the activity of cotransfected pRL-SV40. At 

least three independent experiments were conducted for each compound. 

Agonistic activities were evaluated by relative activity expressed as 20% relative effective 

concentration (REC20), the concentration of the compound showing 20% of the activity of 10-10

M E2, 10-9 M E2, or 10-9 M DHT for ERα, ERβ, and AR, respectively. Antagonistic activities 

were evaluated by relative activity expressed as 20% relative inhibitory concentration (RIC20), 

the concentration of the compound showing 20% inhibition of the activity of 10-10 M E2, 10-9 M 

E2, or 10-9 M DHT for ERα, ERβ, and AR, respectively. The range of concentrations tested was 

≈ 10-8 to 10-5 M; all pesticides were tested at concentrations ≤ 10-5 M to avoid cytotoxicity. 

When the activity of the test compound was higher than the REC20 the pesticide was judged to be 

positive for activity. When the activity of the test compound was higher than the RIC20 the 

pesticide was judged to be positive for inhibitory activity. The statistical significance of 

differences was evaluated with a two-tailed Student’s t-test with a nominal level of significance 

of p < 0.05. 

E2 demonstrated dose-dependent transactivation of ERα and ERβ which indicated that both 

receptors can be activated at very low hormone concentrations. The maximal ERα activity was 

achieved at 10–10 M E2 or more, exhibiting approximately 10-fold that of the control solvent. 

The maximal ERβ activity induced was 8.5-fold that of the solvent control at 10–9 M E2 or more. 

Thus, E2 was more potent for ERα than for ERβ. From the dose–response curves, REC20 values 

of E2 for ERα and ERβ were determined to be 2.5 × 10–12 M and 5.3 × 10–12 M, respectively. 5αDHT caused dose-dependent transactivation of AR and was detectable from 10–11 M DHT and 

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plateaued at 10–9 M DHT. The maximum induction was 21-fold that of the control solvent. The 

REC20 value of DHT for AR was 3.1 × 10–11 M. 2,4-D was negative for both ER (α and β) and 

AR agonist and antagonist activity in this test system. 

ER Activation 

In a published, non-guideline study, Lemaire et al. (2006) evaluated 49 pesticides (including 

2,4-D; purity 95-100%) covering nine chemical classes, for ERα and ERβ activation or inhibition 

in stable reporter cell lines, HELN ERα and ERβ (HeLa cells stably transfected with an EREdriven luciferase plasmid). 

For the ERα and ERβ competitive-binding assay, ERα and ERβ cells were seeded at a density of 

1.5×105 cells per well in 24-well tissue culture plates. Cells were labeled with 0.1 nM [3H]-E2

([3

H]-17β-estradiol, 41.3 Ci/mmol specific activity) in the presence or absence of increasing 

concentrations of cold E2 (as control) or each of the tested compounds in 400 μl of DMEM 

supplemented with 6% DCC-FBS. 

The effect of pesticides on cell viability was assessed with a 3-[4,5-dimethylthiazol-2-yl]-2,5-

diphenyl-tetrazolium bromide (MTT) toxicity assay. Treatment with 2,4-D, as well as all other 

pesticides, did not result in cytotoxicity in the study. 

To evaluate the estrogenic potential of pesticides, HELN ERα and HELN ERβ were used to 

identify the active compounds and discriminate between ERα and ERβ activities. To determine 

ERα and ERβ agonistic activity of pesticides, the relative transactivation activity on HELN ER 

cells was computed as the ratio of luciferase reporter gene induction values of each compound at 

a concentration of 10 μM to the luciferase reporter gene induction value of E2 at 10 nM. 

Antagonistic effects of pesticides in the ERα and ERβ transactivation assay were determined 

over a range of five concentrations from 10−7

 to 10−5. 

To confirm the ER antagonist effects in the transactivation assays reflected an inhibition of E2 

binding to ERα and/or ERβ, whole cell competitive binding assays were conducted with the 

HELN ERα and ERβ cells. The binding affinity for ER of anti-estrogenic pesticides was 

assessed with 0.1 nM [3H]-E2 as a tracer. As expected, non-radiolabelled E2 displaced [3H]-E2 by 

50% at a concentration of 0.12 nM for ERα and 0.18 nM for ERβ, and ICI 164,384 inhibited 

[

3

H]-E2 binding by 50% at the concentration of 2.5±0.5 nM for ERα and 3.3±1.0 nM for ERβ. 

2,4-D was reported negative with a percent activity in ERα and ERβ of 8.4 ± 1.4% and 10.2 ± 

2.1%, respectively, which was not statistically different from the solvent DMSO (9.3 ± 1.3% and 

10.8 ± 1.6%, respectively). 

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ER Activation 

In a published, non-guideline study, Hurst and Sheahan, 2003) screened 26 pesticide 

compounds (including 2,4-D) and storm water from two agricultural catchments for estrogenic 

activity using an in vitro recombinant yeast-based reporter assay. The assay screens for the 

binding and activation of human estrogen receptor (hER-α) by measuring estrogenic activity 

relative to 17β-estradiol (E2). 

The pesticide standards used in the study were obtained from Dr. Ehrenstorfer (GmbH); purities 

ranged 95-99.5%. The yeast assay uses a genetically modified yeast strain in which hER-α has 

been integrated into the yeast genome, together with expression plasmids carrying estrogenresponsive elements (EREs) which control the expression of the reporter gene Lac-Z. In the 

presence of estrogens or chemicals with estrogenic activity, the yeast synthesizes β-galactosidase 

which is secreted into the assay medium. The β-galactosidase breaks down the chromogenic 

substrate chlorophenol red β-galactopyranoside (CPRG) into a product that is quantified by 

absorbance. 

For the initial screening, yeast cells were combined in microtitration plates with pesticide 

standards dissolved in ethanol at 12 different concentrations (not reported). A dilution series of 

E2, as the positive control and estrogenic standard, and an ethanol solvent blank were assayed 

along with the pesticide standards. An estrogenic response was defined as an increase by the 

pesticide standard of at least 0.1 optical density (OD) units color (at 540 nm) in comparison with 

the solvent control. The estrogenic potency was reported as the potency relative to E2, where E2 

has a potency value of 1; the lowest observed effect concentration (LOEC) for an estrogenic 

response was also determined. 

In this test system, 2,4-D did not demonstrate an estrogenic response up to cytotoxic 

concentrations. A toxic response (inhibition of yeast growth) was observed at 50 mg/L. 

ER Activation/Proliferation 

In a published non-guideline study, Soto et al, 1995, using the E-SCREEN assay assessed the 

potential estrogenicity of environmental chemicals using the proliferative effect of estrogen and 

estrogenic chemical on and estrogen-sensitive target cell line. This quantitative in vitro assay 

compares the cell number achieved by similar inocula of human breast cancer estrogen-sensitive 

(MCF-7) cells in the absence of estrogens (negative control), and in the presence of 17β-estradiol 

(E2, positive control) and a range of concentrations of chemicals suspected to be estrogenic. The 

assay was developed based on the premise that a human serum-borne molecule specifically 

inhibits the proliferation of human estrogen sensitive cells and estrogens or estrogen-like 

chemicals induce cell proliferation by blocking this inhibitory effect. The aim of the work was 

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to: 1) validate the E-SCREEN Assay, 2) screen a variety of chemicals, 3) examine whether the 

effects of different chemicals act cumulatively and 4) assess the reliability of the assay for use as 

a screening tool. 

Proliferation rates are measured by comparing the cell yield achieved by similar inocula 

harvested simultaneously during the late exponential phase of proliferation. The proliferative 

effect is measured as the ratio between the highest cell yield obtained with the test chemical and 

the hormone free control. In this experimental design, MCF-7 cell yields were measured after 6 

days of exposure and differences between treatment and positive (E2) control were apparent after 

4 days. Estrogenic activity was assessed by 1) relative proliferative potency (RPP) which is the 

ratio between the minimal concentration of estradiol needed for maximum cell yield and the 

minimum dose of the test compound needed to achieve a similar effect, and 2) measuring the 

relative proliferative effect (RPE) which is 100 times the ratio between the highest cell yield 

obtained with the chemical and with E2l. The RPE allows one to quantitatively compare the 

agonist effect relative to E2. Results were expressed as mean ± SE. Proliferation yield 

experiments were conducted in duplicate wells with a minimum of 5 replicated. Differences 

between groups were assessed by analysis of variance and the a posteriori Shaffe’s test. A p 

value ≤ 0.05 was regarded as significant. 2,4-D was tested over a concentration range of 10-9 to 

10-3 M and found to have no activity in the E-SCREEN Assay. 

ER, AR and TR Transactivation 

In a published, non-guideline study, Sun, et al. (2012) developed receptor-related luciferase 

reporter gene assays to evaluate and compare estrogen receptor (ER), androgen receptor (AR) 

and thyroid hormone receptor (TR) activities of target chemicals. The assay was tested using 

2,4-dichlorophenoxyacetic acid (2,4-D), chlorpyrifos, di-2-ethylhexyl-phthalate, and bisphenol 

A; only data for 2,4-D are discussed herein. 

All the assays utilized Vero (African green monkey kidney) cells which do not contain the 

endogenous receptors. For the ER Reporter Gene Assay, the cells were transfected with pERETATA-Luc, rERα/pCl, and phRL-tk (used as internal control to assess transfection efficiency 

and cytotoxicity of test chemicals). For the AR Reporter Gene Assay, the cells were transfected 

with pMMTV-Luc, AR/pcDNA3.1, and phRL-tk, and for the TR Reporter Gene Assay, the cells 

were transfected with pUAS-tk-luc, pGal4-L-TR, and phRL-tk. 

Agonistic activity was reported as the 20% relative effective concentration (REC20), the 

concentration at which the tested chemical showed 20% of the maximum activity of the positive 

control (E2, testosterone, or T3). Antagonistic activity was reported as the 20% relative 

inhibitory concentration (RIC20), the concentration at which the tested chemical induced a 20% 

reduction of activity in E2, testosterone, or T3. The REC20 and RIC20 were calculated using 

nonlinear regression analysis with a sigmoid dose-response (variable slope) curve. Datasets 

were tested for homogeneity of variance and normality; all datasets met these criteria. Data were 

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then analyzed by one-way ANOVA followed by Duncan’s multiple comparisons test when 

appropriate. Statistical significance was determined at p<0.05. Hormone agonistic treatments 

were compared to the vehicle (DMSO) control group and antagonistic treatments were compared 

to the positive control (E2, testosterone, or T3) group. 

The cytotoxicity of the test chemicals was determined prior to conducting the receptor assays. 

The test chemicals did not affect the viability or proliferation of the Vero cells with or without 

E2, testosterone, or T3. All three positive controls exhibited appropriate concentration-dependent 

responses in the respective assays. Based on the concentration-response curves, the EC50 values 

were 1.78×10–6 mg/L for E2, 3.32×10–4 mg/L for testosterone, and 2.09×10–3 mg/L for T3. 

2,4-D showed no ER, AR or TR agonist or antagonist activity at 0.003-3.0 mg/L in this test 

system. The authors, however, suggested that 2,4-D may have acted as a synergist at 3.0 mg/L 

in the antagonist assay, significantly (p<0.01) enhancing the effects of testosterone. 

AR Activation 

In a published, non-guideline study, Kim et al., 2005, characterized the androgenic activity of 

2,4-D (>98% pure) and its metabolite 2,4-dichlorophenol (DCP) with 5α-dihydrotestosterone 

(DHT) using an in vitro mammalian system with prostate cancer cell lines. Additionally, the 

molecular mechanisms of phenoxy compound action related AR activation were investigated. 

Two human prostate cancer cell lines were used in the study: AR expressed 22Rv1 (derived 

from a relapsed tumor) and androgen-independent PC3 (androgen-insensitive cell line used for 

validation). African green monkey kidney cells (COS-1) were used for the in vitro AR-binding 

assay. 

In the cell proliferation assay, the 22Rv1 cells were seeded in 96-well plates (3 × 103 cells/well) 

and DHT was added to the experimental medium (phenol red-free RPMI 1640 containing 10% 

dextran charcoal-stripped FBS) 24 hours later. The bioassay was terminated on the fourth day 

and the cells were fixed and stained with sulforhodamine-B (SRB). Briefly, the cells were 

treated with 10% trichloroacetic acid and incubated at 4°C for 1 hour, washed with phosphatebuffered saline (PBS), and the proteins stained with 0.4% SRB at room temperature for 1 hour. 

The wells were washed with acetic acid, air dried, and the bound dye solubilized with 10 mM 

Tris base (pH 10.5). The amount of SRB product in each well was determined with a plate 

reading spectrophotometer at 570 nm; the assays were performed in triplicate for each cell line. 

In the transactivation assays, the 22Rv1 and PC3 cells (1.2 × 105

) were seeded in 24-well plates 

and transfected with mouse mammary tumor virus luciferase vector (MMTV-Luc) and pCMVhAR expression vector or with MMTV-Luc alone using ExGene 500. After transfection (24 

hours), DHT and the test chemical were added at specified concentrations with the experimental 

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medium. After another 24-hour incubation, the cells were lysed and the extracts were assayed 

for luciferase activity with a dual-luciferase reporter system and liquid scintillation counter 

luminometer. Luciferase activity was normalized for transfection efficiency using pRL-TK as 

the internal control. Cell cytotoxicity was determined by using the colorimetric MTS cell 

cytotoxicity assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay). 

For the competitive steroid binding assays, whole-cell binding assays were performed. Briefly, 

COS-1 cells were transfected with pCMV-hAR in the presence of lipofectamine reagent. The 

cells were placed in serum-free, phenol red-free medium and incubated with [3

H]5α-DHT for 2 

hours at 37°C in the presence and absence of increasing concentrations of the test compounds. 

Nonspecific binding of [3

H]5α-DHT was determined by adding a 100-fold molar excess of 

unlabeled 5α-DHT. Cells were washed in PBS, harvested in buffer containing 2% SDS, 10% 

glycerol, and 10 mM Tris (pH 6.8), and the radioactivity was determined with a scintillation 

counter. 

For fluorescence imaging, the PC3 cells were cultured and the GFP-AR expression vector was 

transiently transfected with ExGene500 reagent. After 30 hours, the transfected cells were 

mixed with fresh medium containing 10% dextran charcoal-stripped FBS, and treated for 30 min 

with the vehicle (ethanol), 10 nM DHT, or 10 nM DHT in combination with each of the test 

chemicals also at 10 nM. Next, the cells were fixed with 10% formaldehyde, rinsed with PBS, 

dried, and fluorescence was analyzed with a confocal laser system connected to a fluorescence 

microscope. For the evaluation of GFP-AR localization, the relative nuclear and cytoplasmic 

fluorescence in >100 cells per condition from three experiments was determined and scored 

according to a 5-grade nuclear localization score: exclusive nuclear fluorescence (4); nuclear 

fluorescence exceeding cytoplasmic fluorescence (3); equivalent nuclear and cytoplasmic 

fluorescence (2); cytoplasmic fluorescence exceeding nuclear fluorescence (1); and exclusive 

cytoplasmic fluorescence (0). 

The study authors concluded that 2,4-D and DCP in combination with DHT have androgenic 

activity in cell proliferation and androgen-induced transactivation, possibly through promotion of 

AR translocation to the nucleus. In the in vitro assay systems, while 2,4-D or DCP alone did not 

show androgenic activity, 2,4-D or DCP with DHT exhibited synergistic androgenic activities. 

Co-treatment of 2,4-D or DCP with DHT stimulated cell proliferation by 1.6-fold, compared to 

DHT alone. In transient transfection assays, androgen-induced transactivation was also 

increased to a maximum of 32-fold or 1.28-fold by co-treatment with 2,4-D or DCP with DHT, 

respectively. However, 2,4-D and DCP had no effect on either mRNA or protein levels of the 

AR. In a competitive AR binding assay, 2,4-D and DCP inhibited androgen binding to the AR 

(up to 50% at concentrations of approximately 0.5 mM for both compounds). The nuclear 

translocation of green fluorescent protein-AR fusion protein in the presence of DHT was further 

promoted with the addition of 2,4-D and DCP. The authors therefore concluded that the results 

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indicate that 2,4-D and DCP enhancement of DHT-induced AR transcriptional activity may be 

attributable, at least in part, to the promotion of AR nuclear translocation. 

ER/AR Activation and Ovarian Steroidogenesis 

In a published study, Orton et al. (2009), investigated eleven pesticides (including 2,4-D (>97% 

pure)) for AR/ER related activity in a recombinant yeast screen. The same chemicals were also 

evaluated for effects on the ovulatory response and ovarian steroidogenesis in Xenopus oocytes. 

In the yeast (anti-) estrogen screen (YES) and yeast (anti-) androgen screen (YAS), activation of 

transfected human estrogen or androgen receptor results in a color change in the media as 

measured by absorbance at 540 nm. Turbidity was monitored to check for any cytotoxic effects. 

It was reported that 2,4-D did not elicit (anti-) estrogenic or (anti-) androgenic activity in this test 

system (no data presented). 

 

In the ovulation assay, ovaries from sexually mature Xenopus laevis were removed, cultured in 

Modified Barth’s Medium, and cut into 10 oocyte fragments. The oocytes were incubated with 

6.25, 12.5, 25, and 50 IU of human chorionic gonadotropin (hCG) or control media for 20 h. 

The media was sampled and hormone analysis was performed by RIA. After incubation, a 

submaximal concentration of hCG (~60% of maximum ovulatory response) was chosen for coincubation with pesticides at concentrations of 6.25 and 62.5 μM. 2,4-D had no significant effect 

on the ovulatory response or steroid hormone production in this ovulation assay (no data 

presented). 

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

A 13-week subchronic oral toxicity study (MRIDs 00101599 and 00102451, 1981) with 2,4-D 

(97.73% purity; Lot/Batch # V602 Tank) in Fischer 344 rats was conducted on 15 

animals/sex/group administered at target doses of 0, 15, 60, 100 or 150 mg/kg/day. Statistical 

significance is presumed to be α=0.05, but is illegible in the available copy of the DER. 

All rats survived until scheduled termination. Although body weights and food consumption 

were recorded throughout the study, achieved doses were not reported. No overt toxicity was 

observed, but one male in the high-mid-dose group (100 mg/kg/day) lost weight on two days 

mid-study (Days 55 and 62), with subsequent weight recovery until Day 91. Decreased mean 

body weights were observed beginning on Day 41 in the high-dose males, and high-mid- and 

high-dose females, and continuing through study termination. Decreased mean body weight also 

was observed beginning on Day 59 in the low mid- and high-mid-dose males, and continuing 

through study termination. Body weight gains were decreased (NS) in the high-dose animals, 

and food consumption was occasionally decreased in the high-dose animals throughout the 

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study. Terminal body weights were decreased in the high-dose animals, with lesser decreases 

(NS) in the high-mid-dose group. 

Concentrations of T4 were increased in the high-mid-dose males, and the low and low-mid-dose 

females, but decreased in the high-mid- and high-dose females. The primary target organ was 

the kidney (see below) with concomitant increases in kidney weights in all males, and females in 

the high-dose group. Liver weights were affected in the high-dose animals, but these changes 

were secondary to decreased terminal body weights. 

Histopathology was conducted on 10 animals/sex/group. A degenerative change was seen in the 

kidneys of all male rats treated with ≥60 mg/kg/day (low-mid dose) and in 1/10 male rats in the 

low-dose group. The degree of degeneration was more pronounced in the two highest dose 

groups. The original pathology report of this study stated that there was an increased epithelial 

cytoplasmic homogeneity of the convoluted tubules in the kidneys of male rats treated with 2,4-

D at ≥60 mg/kg. The interpretation of this finding was that there were decreased amounts of 

protein absorption droplets, which are primarily α2u-globulins which are sex-dependent urinary 

proteins of hepatic origin. Decreased amounts of protein droplets could represent changes in the 

nutritional status of the rats, a metabolic change in the liver cells where α2u-globulins are 

produced, or a direct effect on the kidneys. 

The dose of 60 mg/kg/day exceeded the maximum-tolerated dose as evidenced by cellular 

alterations in the kidneys of both sexes. The 15 mg/kg/day dose was considered the NOEL for 

female rats; in male rats this dose produced no effects except for a slight increase in relative 

kidney weight. 

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

In a subchronic oral toxicity study (MRID 41991501, 1991), 10 Fischer 344 rats/sex/group were 

administered 2,4-D (96.1% purity; Lot/Batch #909) in the diet for 13 weeks at target doses of 0, 

1, 15, 100, and 300 mg/kg/day. Body weights and food consumption were recorded throughout 

the study. Average daily compound intake during the study was 0.93, 13.98, 93.93, and 

278.39 mg/kg/day for males and 0.96, 14.39, 96.16, and 293.42 mg/kg/day for females. 

There were no treatment-related deaths. Clinical signs of toxicity occurred mainly in the highdose females, and demonstrated as hunched posture, depressed activity, and few/no feces 

(included four high-dose males at Week 1). There was a dose-related increase in the incidence 

of pale/opaque eyes in both sexes. Decreased (p<0.05) body weights were observed during the 

study in the high-dose males and females, with the magnitude of the deficit increasing with time 

(males ↓15%/23% and females ↓21%/28% at Weeks 6/13, respectively). Decreased body-weight 

gains (Weeks 0-6: males ↓28%/females ↓50%; Overall: males ↓37%/females ↓57%) were 

observed in the high-dose animals. At the high mid-dose, decreased body weight (↓7% at 

Week 13; both sexes) and body-weight gain (Overall: males ↓9%/females ↓11%) were observed. 

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A corresponding decrease in food consumption was observed in both sexes at the two highest 

doses. 

Decreased (p<0.05) T3 and T4 concentrations were observed at the high-mid- and high-doses at 

both intervals, except for T3 concentrations in the high-mid-dose males (Weeks 6 and 13) and in 

the high-dose females at Week 6 (NS). Changes (p<0.05) in absolute and/or relative organ 

weights [including adrenals (↑ males; ↓ females), testes with epididymides (↑), ovaries (↓), 

pituitary (↑ males; ↓ females), liver (↑ males and females), and thyroids/parathyroids (↑ males 

and females)] were observed primarily at the high-dose in both sexes, and many of these changes 

may be attributable to decreased body weight. Gross findings, mainly in the high-dose group, 

included small testes and epididymis and opaque eyes (females). Treatment-related 

histopathological changes were observed primarily in the high-dose animals and included 

centrilobular hepatocellular hypertrophy, atrophy of the testes, hypertrophy of the zona 

glomerulosa (adrenal cortex, both sexes)’ and follicular cells (thyroid, females). Many of the 

lesions correlated well with the alterations observed in hematology and clinical chemistry 

parameters and/or organ-weight data of the high-dose animals. 

The LOAEL is 100 mg/kg/day based on decreased body weight/body-weight gain, alterations in 

some hematology and clinical chemistry [decreased T3 (females) and T4 (both sexes)] 

parameters, and cataract formation in females. The NOAEL is 15 mg/kg/day.

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

In a 90-day oral toxicity study (MRID 41991502, 1991), male and female B6C3F1 mice were 

fed diets containing 2,4-D (96.1% purity; Lot/Batch #909) at target doses of 0, 1, 15, 100, or 

300 mg/kg/day for 13 weeks. Body weights and food consumption were recorded throughout the 

study. The average daily compound intake during the study was 0.98, 14.71, 98.2, and 

292.61 mg/kg/day for males and 0.99, 14.84, 98.85, and 295.88 mg/kg/day for females. 

There were no adverse treatment-related effects on survival, body weight, body weight gain, 

food consumption, and gross pathology up to the high intermediate dose (100 mg/kg/day). 

Treatment-related effects at the high-mid dose included decreased glucose (p<0.05; females) and 

T4 concentrations (statistical analysis confounded; males), and increases in mean absolute and 

relative kidney weights (females). Treatment-related effects at 300 mg/kg/day included transient 

decreased (p<0.05) food consumption (up to Week 7); decreased glucose (p<0.05; females) and 

T4 concentrations (statistical analysis confounded; males); and histopathological changes in the 

liver characterized by nuclear hyperchromatism and decreased glycogen in periportal 

hepatocytes. Under the conditions of this study, a NOEL of 15 mg/kg/day and a LOEL of 

100 mg/kg/day is established for the 90-day oral toxicity of 2,4-D to male and female mice. The 

LOEL is based on decreased glucose and T4 concentrations, and increased mean and relative 

kidney weights, observed at 100 mg/kg/day. 

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28-Day (Subchronic) Inhalation Toxicity in Rodents (Rat) 

In a subchronic inhalation toxicity study (MRID 47398701, 2008), 2,4-D (99% purity, Lot/Batch 

#2006 24833 8006-USA) was administered to 10 or 20 SD CD® rats/sex/concentration by 

dynamic (nose only) exposure at concentrations of 0, 0.05, 0.10, 0.30 or 1.00 mg/L for 6 hours 

per day, 5 days/week for 28 days (for a total of at least 20 exposures). Body weights and food 

consumption were recorded throughout the study. 

After nose-only inhalation exposure, 2,4-D was associated with portal-of-entry effects that 

consisted of squamous metaplasia and epithelial hyperplasia with increased mixed inflammatory 

cells within the larynx. The incidence and severity of the effects were increased in a dose-related 

manner, and the effects persisted after the 4-week recovery period, although the incidence and 

severity were reduced. Clinical signs associated with exposure at the high dose included 

excessive salivation (Day 13 and subsequently), labored breathing (Day 13 and subsequently), 

and chromodacryorrhea (Day 12 and intermittently thereafter). A slight decrease in body weight 

was observed in the high-dose females by Day 14 and continued throughout the remainder of the 

dosing (↓10%) and the recovery periods (↓12%). Body-weight gain was reduced in the highdose females throughout the study and recovery period, and was accompanied by a reduction in 

food intake. Alkaline phosphatase concentrations were increased (p<0.05 and 0.01, respectively) 

in the mid-high and high-dose females, and aspartate aminotransferase concentrations were 

increased (p<0.01) in the high-dose females, but no correlating microscopic pathology findings 

in the liver were observed. Lung weights were unaffected by treatment. High-dose females 

displayed slight reductions in spleen (p<0.05) and thymus (NS) weights; organ weights were 

comparable among the male groups. 

The systemic toxicity LOAEL is 1.0 mg/L/day, based on increased alkaline phosphatase and 

aspartate aminotransferase concentrations, and decreased spleen weights, in females. The 

NOAEL is 0.30 mg/L/day. The LOAEL for portal-of-entry effects (squamous metaplasia and 

epithelial hyperplasia with increased mixed inflammatory cells within the larynx; not totally 

resolved following a 4-week recovery period) is 0.05 mg/L, the lowest dose tested. A NOAEL 

for portal-of-entry effects was not determined. 

Prenatal Development in Rodents (Rat) 

In a developmental toxicity study (MRID 00130407, 1983), pregnant Fischer 344 rats (35/group) 

were administered 2,4-D (97.5% purity; Lot/Batch #3/82) by gavage at doses of 0 (corn oil), 8, 

25, and 75 mg/kg/day from gestation day (GD) 6 through 15. All surviving animals were 

euthanized on GD 20. 

There were no treatment-related deaths. Clinical signs were comparable among the groups. 

Body weights were comparable among the groups throughout the study, but dams at the highdose displayed a decrease (NS) in body-weight gain during the dosing period (79% of control for 

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GD 6-15; 57% of control for GD 6-10). The corrected body-weight gain was comparable among 

the groups. 

There was a slight decrease in pregnancy rate with increasing dose, but the numbers of corpora 

lutea, implantations, and live fetuses were comparable among the groups, and there were no dead 

fetuses. The number of resorptions, as well as pre- and post-implantation losses, were not 

adversely affected by treatment. The number of the dams with 100% resorptions was similar 

(0 to 2) among study groups. Fetal body weight and crown-rump length were comparable among 

the groups, as was the sex ratio. 

There were no treatment-related differences in the incidence of fetal external or visceral 

malformations. Although none of the increases attained statistical significance, they were 

attributed to treatment since some of the findings were also observed in the F1b pups of dams fed 

2,4-D at 80 mg/kg/day (actual dose 75 mg/kg/day) in a 2-generation reproduction study (MRIDs 

00150557 and 00163996) in the same strain of rat. Additionally, skeletal findings [2nd wavy 

ribs, lumbar ribs] and missing sternebrae were observed in another developmental toxicity study 

in the SD rat (Schwetz et al., 1971) at a comparable 2,4-D dose of 87.5 mg/kg/day. 

The maternal toxicity NOAEL is 25 mg/kg/day, and the maternal toxicity LOAEL is 

75 mg/kg/day, based on decreased body-weight gain. The NOAEL for developmental toxicity is 

25 mg/kg/day, and the developmental toxicity LOAEL is 75 mg/kg/day, based on an increased 

incidence of skeletal abnormalities. 

Prenatal Development in Non-Rodents (Rabbit) 

In a developmental toxicity study (MRID 41747601, 1990), artificially-inseminated female New 

Zealand White rabbits (20/group) were administered 2,4-D (96.1% purity; Lot/Batch #909) at 

doses of 0 (aqueous 0.5% methylcellulose), 10, 30, and 90 mg/kg/day from GDs 6 through 18. 

All surviving does were euthanized on GD 29. 

There were no treatment-related deaths. Two high-dose does aborted (GDs 21 and 24). 

Treatment-related clinical signs of toxicity were observed in the does that aborted; ataxia was 

observed in both does, and decreased motor activity, loss of righting reflex, extremities that were 

cold to the touch, and dried feces were observed in the doe that aborted on GD 21. Body weights 

were comparable among the groups throughout the study, but body-weight gain was decreased at 

the high-dose (↓27%) during the dosing period (GDs 6-19; NS). On GDs 7-8, the low- and middose groups showed no body-weight gain, and the high-dose group displayed a negative bodyweight gain. On GDs 15-19, the high-dose group displayed no body-weight gain, and corrected 

body-weight gain was decreased at the high-dose (↓23%; NS). 

Pregnancy rates were comparable among the groups; similar numbers of corpora lutea, 

implantations, and live fetuses were observed among the groups with no dead fetuses. One 

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control doe had 100% resorptions, and the number of resorption, and pre- and post-implantation 

losses, were comparable among the groups. Gravid uterine weights also were comparable 

among the groups. 

No differences were observed in mean fetal body weight due to treatment. At the high-dose, 

there was an increase (p<0.05) in the percent of live male fetuses (71.2%) compared to control 

(52.8%) and the other dose groups (low: 54.4%; mid: 59.4%). There were no apparent 

differences in the incidence of external, visceral, or skeletal variations, anomalies, retardations, 

or malformations among the groups. 

The maternal toxicity NOAEL is 30 mg/kg/day, and the maternal toxicity LOAEL is 

90 mg/kg/day based on abortions, decreased body-weight gain, and clinical signs of toxicity. 

The developmental toxicity NOAEL is 30 mg/kg/day, based on abortions at the developmental 

toxicity LOAEL of 90 mg/kg/day. 

Extended One-Generation Reproduction in Rodents (Rat) 

In an extended dietary one-generation reproductive toxicity study (MRID 47972101), 2,4-

dichloro phenoxyacetic acid (2,4-D; 97.85%-98.6% a.i.; lot # 2006 2433 8006-USA) was 

administered to 27 Crl:CD(SD) young adult rats/sex/dose via the diet at dose levels of 0, 100, 

300, or 600 (females)/800 (males) ppm [equivalent to 0, ≈5, 15, or 30 (females)/40 (males) 

mg/kg bw/day] for approximately four weeks prior to mating and continuing through mating (up 

to 2 weeks), gestation, and lactation. P1 males were exposed for a minimum of 11 weeks 

including 7 weeks from the initiation of the mating phase. P1 females were exposed until 

lactation day 22 (LD22). A satellite group of P1 females (12/dose) were subject to the same 

exposures as the P1 females on the main study (exposure for 4 weeks during the pre-mating, up 

to 2 weeks during the mating period and during gestation until termination on gestation day 17 

(GD 17). Satellite males were not exposed to dietary 2, 4-D except during co-housing with 

satellite females during the mating period. 

Doses were selected based on a combined analysis of toxicity and toxicokinetic (TK) data 

collected from the 2,4-D range-finding/TK study (MRID 47417901) and titration studies (MRID 

47417902). The high dose was set at or slightly above the threshold for nonlinear kinetics. The 

range-finding study confirmed a gender-based difference in the renal clearance of 2,4-D in adult 

rats, and different high-dose levels were selected for the sexes. The male high dose was 40 

mg/kg/day (dietary concentration of 800 ppm), slightly higher than the inflection point for 

nonlinear TK in male pups from PND 35 to adulthood. The female high dose was 30 mg/kg/day 

(600 ppm), which was clearly higher than the inflection point in female pups and adults 

throughout the entire (range-finding) study. 

P1 Generation: A comprehensive evaluation of P1 male and P1 female reproductive system was 

conducted, including an evaluation of gonadal function, the estrous cycle, sperm parameters, 

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mating performance, conception, gestation, parturition and lactation, as well as survival, growth 

and development of the offspring. Selected systemic toxicity parameters were also evaluated in 

the P1 males and P1 females. 

Satellite GD 17 Females: A satellite group of P1 females (12/dose) was included for assessments 

of selected systemic toxicity parameters, clinical chemistry/hematology, thyroid hormone levels, 

thyroid weights, plasma 2, 4-D levels, histopathology, and selected reproductive parameters 

during gestation (corpora lutea and implantation numbers). 

F1 Generation: F1 offspring were evaluated for potential effects on the nervous system, immune 

system, reproductive and endocrine systems, thyroid function, and other systemic toxicity 

parameters. 2, 4-D plasma levels were also assessed in the F1 offspring. In-life parameters in all 

F1 offspring included clinical observations, body weights, feed consumption, anogenital 

distance, nipple retention and puberty onset. Selected F1 offspring were divided into three 

different groups (Sets 1, 2, and 3) at weaning (postnatal day 21; PND 21). Each set of F1 

offspring was maintained on the test diet until PND 60 (Set 1b F1 offspring), ≈PND 70 (Sets 1a 

and 2a F1 offspring), or ≈PND 90-139 (Sets 2b and 3 F1 offspring). 

Set 1a (10/sex/dose): assessment of general systemic and thyroid toxicity, which included 

clinical chemistry/hematology parameters, thyroid hormone assessment, and urinalysis (males 

only). Post-mortem evaluations in Set 1a (PND70) included gross pathology, organ weights and 

histopathology on a wide range of tissues, including thyroids. 

Set 1b (10/sex/dose): developmental neurotoxicity (DNT) assessment, which included functional 

observational battery (FOB), motor activity and acoustic startle response (ASR). On PND 60, Set 

1b animals were perfused for central nervous system (CNS) and peripheral nerve neuropathology 

evaluation and brain morphometry. A special stain (Luxol Fast Blue) was used to evaluate brain 

myelination. 

Set 2a (10sex/dose): assessment of potential developmental immunotoxicity (DIT): examination 

of humoral immune function using the sheep red blood cell (SRBC) antibody-forming cell 

(AFC) assay on PND 70-74. 

Set 2b (10/sex/dose): assessment of potential developmental immunotoxicity (DIT): examination 

of innate cellular immunity using the natural killer cell (NK) assay on PND 87-93. 

Set 3 (23-27/sex/dose): assessment of reproductive/endocrine toxicity, which included estrous 

cycle evaluation and post-mortem evaluations that focused on reproductive organs, sperm 

assessment, and ovarian follicle counts on PND 139. TK analyses were conducted on Set 3 males 

and females on PND 63 and 84 to determine plasma 2, 4-D levels. 

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In addition, selected pups culled on PND 4 were used to assess thyroid hormone levels. 

Additional data were gathered from F1 offspring not assigned to Sets 1-3. On PND 22, 

unselected weanlings were either perfused for examination of neuropathology (12/sex/dose) or 

euthanized for assessment of systemic toxicity, which included thyroid hormone assessment, 

organ weights, and post-mortem examinations (gross pathology and histopathology) in 

10/sex/dose. 

Reproductive and selected data from the F1 generation were used to assess whether a second 

generation would be produced. None of the criteria were met (Table 1), and a second generation 

was not assessed in this study. 

P1 Adult Rats: There were no treatment-related deaths or clinical signs of toxicity in either sex 

of P1 adults. Body weights and body-weight gains were comparable among the groups during 

the pre-mating and mating phases (both sexes) and during gestation and the latter part of 

lactation (dams). Prior to dietary adjustment of 2, 4-D concentration during the second week of 

lactation, the 600 ppm dams displayed a decrease in body weight (LD 7; ↓5%) and body-weight 

gain (LD 1-4; ↓64%), which is consistent with reduced food intake during the first week of 

lactation. The reduction in food intake can be attributed to the increase in the actual dose (≈65 

mg/kg/day) above the targeted level (30 mg/kg/day) during this time. After dietary adjustment, 

food intake for the 600 ppm dams was above control levels. 

There were no apparent treatment-related effects on hematology, differential white blood cell 

counts, and prothrombin time, and clinical chemistry and urinalysis parameters were comparable 

among the groups (both sexes). P1 males displayed increased kidney weights (absolute and 

relative) at 800 ppm, which were accompanied by histopathological findings (degenerative lesion 

in the proximal convoluted tubules in the outer zone of the medulla) and are consistent with 

previous findings that the kidney is a target organ. There were no treatment-related findings in 

the P1 female kidney. Decreased reproductive and accessory sex gland weights were observed at 

300 ppm and/or 800 ppm. These changes, however, are related to the concurrent control being 

outside of the laboratory historical control range. P1 females at 600 ppm displayed increased 

uterine weights (↑17%, both absolute and relative), although statistical significance was not 

attained. There were no alterations in estrous cycle pattern in the 600 ppm P1 females compared 

to the control, and no significant difference in mean estrous cycle length in P1 females at any 

dose level compared to the control. There were no significant, treatment-related effects on sperm 

motility or progressive motility, no differences in testicular spermatid and epididymal sperm 

counts, and no differences in the proportion of abnormal sperm. Male and female mating, 

conception, fertility, and gestation indices were comparable among the groups, and postimplantation loss was comparable among the groups. Both the time to mating and gestation 

length were comparable among the groups. 

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GD 17 Satellite Females: All P1 satellite females survived to scheduled sacrifice, and body 

weights were comparable among the groups. Hematology and clinical chemistry parameters were 

comparable among the groups. Reproductive indices and the numbers of corpora lutea and 

implantations were comparable among the groups. There was a slight increase in resorptions at 

600 ppm (0.9 vs 1.5), although there was wide variability (standard deviations exceed the 

means). There was a slight increase in post-implantation loss at 600 ppm (9.2 vs 5.5). It should 

be noted that this observation was not corroborated since post-implantation losses in the P1 

adults of the definitive study were comparable amongst all dose groups. Both the 100 ppm and 

600 ppm females displayed an increase in thyroid weight (↑9%), but there was no dose-response. 

There were no statistically significant, treatment-related differences in serum T3, T4, or TSH in 

the GD 17 satellite females. Although the 600 ppm GD 17satellite females displayed the 

predicted pattern of thyroid hormone changes (↓ T3 and ↓ T4 with ↑TSH levels) that suggest 2, 

4-D exposure may adversely affect thyroid function at doses above the renal saturation clearance, 

the thyroid effects noted below renal saturation are not considered sufficiently robust to be 

adverse. 

F1 Offspring: There were no treatment-related effects on the numbers of live or dead F1 pups 

born/litter or on pup survival or sex ratio. Slightly lower body weights were observed in the 600 

ppm pups during early lactation, which coincided with the dams decreased food intake LD 1-4 

and LD 4-7). Pup body weight (600 ppm) remained lower in the 600 ppm pups (↓6%) during 

PND 14-21. There was no significant, treatment-related difference in absolute or relative 

anogenital distance in either sex and no differences in nipple/areolae retention between control 

and high-dose groups in either sex. F1 males at 800 ppm displayed a 1.6 days delay in preputial 

separation (well within normal variability), which was accompanied by a very slight reduction in 

body weight compared to the control (↓2.1 grams; 99% of control). The age at vaginal opening 

was comparable among the groups of F1 females. 

F1 Offspring Thyroid Assessments: PND 4 - There were no statistically-significant differences 

in serum T3, T4, or TSH in PND 4 culled pups. T4 was reduced to a similar extent in both sexes 

at the 300 ppm (↓14%-15%) and 600 ppm/800 ppm (↓12%-14%) dose levels, and female PND 4 

pups showed an increase in TSH (↑19%) at 600 ppm. F1 PND 22 Weanlings - F1 PND 22 males 

displayed a statistically-significant reduction (↓28%) in T4 at 800 ppm, and F1 PND 22 females 

displayed a non-statistically significant reduction (↓20%) in T4 at 600 ppm. T3 was reduced in 

the males at 300 ppm (↓19%) and 800 ppm (↓13%), but there was no dose response. F1 PND 62-

64 - Both sexes displayed increased TSH at 300 ppm (↑26%) and at 800 ppm (males ↑23%))/600 

ppm (females ↑24%)), although the increase in males was not dose-related and none of the 

differences in thyroid hormone levels were statistically significant. T4 was decreased at 800 ppm 

in males (↓13%). Though these findings suggest that 2, 4-D exposure may adversely affect 

thyroid function at doses above the renal saturation clearance, the thyroid effects noted below 

renal saturation are not considered sufficiently robust to be adverse. 

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F1 Unselected Offspring (PND 22 weanlings): There were no effects on survival of the 

unselected weanlings used for systemic toxicity (non-perfused). All treated males displayed a 

decrease in body weight (↓9%-10%) compared to the control males. Decreased adrenal weights 

were observed in males at 800 ppm (absolute ↓37% and relative ↓29%). The decreases in kidney 

(↓15%), liver (↓18%), testes (↓15%), and thyroid (↓14%) weights observed in males at 800 ppm 

were slightly greater than the body-weight deficit of 10%. Organ weights were comparable 

among the groups of females. There were no significant differences in perfused absolute brain 

weights, cerebral lengths and widths or cerebellar lengths and widths in perfused F1 PND 22 

weanlings of either sex. There were no neuropathological observations attributed to treatment in 

the perfused F1 PND 22 weanlings, and no treatment-related changes in myelin in either males at 

800 ppm or females at 600 ppm. 

F1 Offspring Set 1a (PND 70): All Set 1a pups survived to scheduled sacrifice. Males at 800 

ppm displayed decreased body weight (↓11%-17%) and body-weight gains (↓11%-25%) 

throughout the study period, with the magnitude of the reduction lessening with time of 

exposure. Females displayed comparable body weight/gain among the groups. Platelet counts 

were reduced in the 800 ppm males but not in the females at any dose level. Both sexes 

displayed a slight increase in ALT (↑18%/25%) and an increase in triglyceride (↑31%/43%) 

levels. Although some of the decreases in organ weights observed in the 800 ppm males may be 

attributed to the 10% decrease in body weight at termination, the decreases in liver (↓16%), 

pituitary (↓14%), and adrenal glands (↓12%) might be related to treatment. Increased uterine 

weights (↑31% absolute and ↑32% relative) were observed at 600 ppm. Although statistical 

significance was not attained, the finding is considered treatment-related since a similar increase 

was observed at 600 ppm in the P1 and Set 3 F1 females. Increased ovarian weight (↑9%) was 

observed in the 600 ppm F1 Set 1a females, although statistical significance was not attained. 

Increased kidney weights (↑9% absolute and ↑11% relative) were observed in the females at 300 

ppm and 600 ppm, although there was no dose-response and kidney weights were comparable 

among the male groups. Decreased thymus weights (↓12% absolute and ↓10% relative) were 

observed in females at 600 ppm and in Set 3 females at 600 ppm (↓14% absolute and ↓13% 

relative). An increased incidence of degeneration of the proximal convoluted tubule in the kidney 

was observed in males at 300 ppm and 800 ppm and in females at 600 ppm. Regarding the 

terminal stage of estrous, 2 of 10 females at 300 ppm and 3 of 10 females at 600 ppm displayed 

proestrus, whereas none of the 10 females in the control and 100 ppm groups displayed 

proestrus. 

F1 Offspring Set 1b (PND 54-56): There were no significant differences in body weight/gain in 

either sex. There was an increase in the level of urination in all treated male groups compared to 

the control group, but there was no dose response. Developmental neurotoxicity results were not 

applicable to EDSP evaluation. 

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F1 Offspring Set 3 (PND 90 or 139): Reproductive Toxicity: There were no treatment-related 

deaths or clinical signs of toxicity. Terminal body weights were comparable among the groups 

(both sexes). No significant differences were observed in mean estrous cycle length at any dose 

level compared to the control. There were no significant, treatment-related effects on the 

numbers of small follicles, growing follicles, or total follicles. There were no significant, 

treatment-related effects on sperm motility or progressive motility, no differences in testicular 

spermatid and epididymal sperm counts, and no differences in the proportion of abnormal sperm 

between the control and 800 ppm males. Absolute (↓9%) and relative (↓8%) pituitary gland 

weights were significantly lower in the 800 ppm males and absolute (↓9%) and relative (↓10%) 

pituitary gland weights were non-significantly lower in the 600 ppm females. There was no 

associated histopathology in the pituitary glands. Uterine weights were increased at 300 ppm 

(↑10% absolute and ↑10% relative) and 600 ppm (↑10% absolute and ↑11% relative) compared 

to the controls. Thymus weights were decreased (↓14% absolute and ↓13% relative) in females at 

600 ppm, although statistical significance was not attained. No histopathological changes were 

observed in the pituitary or thymus in either sex. A degenerative lesion was observed in the 

kidney (proximal convoluted tubule) in both sexes at 300 ppm and at 600 ppm/800 ppm. Ovarian 

follicle counts were comparable between the control and 600 ppm females (PND 139). 

The parental systemic LOAEL is 800 ppm (45.3 mg/kg bw/day in males), based on 

nephrotoxicity manifested as increased kidney weights, and degenerative lesions in the proximal 

convoluted tubules in the main study P1 rats. The parental systemic NOAEL is 300 ppm (16.6 

mg/kg bw/day in males). No toxicologically relevant effects were identified in P1 females or in 

the GD 17 satellite female groups at the highest dose tested (600 ppm; 40.2 mg/kg/day). 

The thyroid toxicity NOAEL is established at 800/600 ppm (45.3 mg/kg/day in males and /40.2 

mg/kg/day in females), the highest dose tested. The thyroid effects noted in the database were 

considered to be adaptive. 

The offspring (F1 adults) LOAEL is 800/600 ppm (55.6 mg/kg bw/day in males and 46.7 

mg/kg/day in females), based on kidney toxicity manifested as increased kidney weights and 

increased incidence of degeneration of the proximal convoluted tubules. The offspring NOAEL 

is 300 ppm (20.9/ mg/kg bw/day in males and 23.3 mg/kg/day in females). 

The F1 offspring (PND 22) LOAEL is 800/600 ppm, based on decreased body weight observed 

throughout lactation. The offspring NOAEL is 300 ppm. The dose on a mg/kg/day basis for the 

PND 22 F1 offspring was not calculated. 

The reproductive LOAEL is > 800/600 ppm (45.3 mg/kg bw/day in males, 40.2 mg/kg bw/day in 

females), based on the lack of effect on estrous cyclicity, (P1 females, satellite GD 17 dams, Set 

3 F1 offspring) or reproductive indices (mating, fertility, time to mating, gestation length, preand post-implantation loss, number of corpora lutea (satellite GD 17 dams), sperm parameters, 

ovarian follicle counts, and reproductive organ histopathology). The reproductive NOAEL is 

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800/600 ppm (45.3 mg/kg bw/day in males, 40.2 mg/kg bw/day in females), the highest dose 

tested. 

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat) 

In a combined chronic toxicity/carcinogenicity study (MRID 43612001, 1995), 50 Fischer 

344 rats/sex/group were administered 2,4-D (96.4%; Lot/Batch #909) in the diet for up to 

24 months at target concentrations of 0, 5, 75, and 150 mg/kg/day. The achieved doses were 

respectively 4.77, 73.15, and 144.98 mg/kg/day in males and 4.89, 73.11, and 143.52 mg/kg/day 

in females. Additionally, 10 rats/sex/group were sacrificed at 12 months (interim sacrifice data 

were reported in MRID 43293901). 

There were no treatment-related deaths or clinical signs of toxicity. Decreased (p<0.05) body 

weight was observed throughout the study in the high-dose group (males ↓4 to 8%; females ↓10 

to 26%) and in mid-dose females (↓10 to 14%). At study termination, body weight decreases 

(p<0.05) in the high-dose males and females were 8 and 26%, respectively. The mid-dose 

females also displayed a decrease (p<0.05) in body weight at study termination of 14%. Bodyweight gains were decreased (p<0.05) throughout the study in mid- and high-dose females, and 

high-dose males. Consistent with the decreased body-weight gains were decreases in food 

consumption in the same groups.

There were dose-related decreases (p<0.05) in T4 values throughout the study in both sexes at the 

mid- and high-doses; decreases at 6- and 18-months in mid-dose males were NS. No treatmentrelated effects were observed on T3 concentration at any dose. Thyroid weights were increased 

(p<0.05) in the mid-dose females and in both sexes at the high-dose at the 12-month interim 

sacrifice. At study termination, thyroid weights were increased (p<0.05) in both sexes at the 

mid- and high-doses, although the increase for mid-dose males was NS, and the increase at the 

mid-dose for both sexes was generally greater than at the high-dose. Decreased (p<0.05) testes 

weights were observed at the high-dose at both the interim and terminal sacrifices, and at the 

mid-dose at study termination (NS). Similarly, decreased (NS at 12 months; p<0.05 at 

24 months) ovarian weights were observed at the high-dose at both sacrifice times and in the 

mid-dose females at study termination. The decreases in testes and ovarian weights are 

consistent findings in other studies on 2,4-D and its salts/esters. 

Microscopically, there were increased incidences of lesions in the liver (altered tinctorial 

properties in mid-dose females, and both sexes at high dose), and thyroid (hyperplasia in highdose males; hypertrophy and epithelial cells in high-dose females) at the interim sacrifice. At 

study termination, there was an increased incidence of increased size of the hepatocytes with 

altered tinctorial properties in both sexes at the high-dose. Tumor incidence was not affected by 

treatment. 

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The LOAEL is 75 mg/kg/day based on multiple endpoints, including decreased body-weight 

gain and food consumption in females, alterations in hematology and clinical chemistry 

parameters (including decreased T4 in both sexes), increased thyroid weights (both sexes at study 

termination), and decreased testes and ovarian weights. There was no treatment-related increase 

in the incidence of any tumor. The NOAEL is 5 mg/kg/day. 

Carcinogenicity in Rodents (Mouse) 

In a carcinogenicity study (MRID 43879801 and 43597201, 1995), 50 B6C3F1 CRL BR 

mice/sex/group were administered 2,4-D (96.4% purity; Lot/Batch #909) in the diet for 

104 weeks at concentrations of 0, 5 (both sexes), 62 (males)/150 (females), and 120 (males)/ 

300 (females) mg/kg/day. Additionally, 10 mice/sex/group were sacrificed (interim) after 

52 weeks. Body weights and food consumption were recorded throughout the study. The 

average achieved doses were 5.0, 61.9 or 128.6 mg/kg/day for male mice, respectively, and 5.01, 

149.83 or 310.01 mg/kg/day for female mice, respectively. 

There were no treatment-related deaths or clinical signs of toxicity in either sex. Body weight, 

body-weight gain, and food consumption were comparable among the male groups throughout 

the study. Females at the high-dose had a slightly lower body weight at the 3- and 6-month 

intervals (p<0.05; ↓4%). Body-weight gains were decreased (p<0.05) at all doses in the females 

at the 3-month interval (↓7%, ↓6%, and ↓14% at the low-, mid-, and high-dose, respectively), 

and the high-dose females had decreased (p<0.05) body-weight gain at the 6- and 12-month 

(↓9%) intervals. There were no consistent changes in food consumption in the female groups. 

Thyroid parameters were not monitored, and reproductive organ weights were not evaluated. 

Gross pathology findings were comparable among the groups in both sexes. Microscopically, 

there was an increased incidence of lesions in the kidneys of both sexes in the mid- and highdose groups. There was no treatment-related increase in any tumor type in either sex. 

The LOAEL of 62 mg/kg/day (males)/150 mg/kg/day (females) is based on increased absolute 

and/or relative kidney weights, and an increased incidence of renal microscopic lesions. There 

was no treatment-related increase in the incidence of any tumor type. The NOAEL is 

5 mg/kg/day. 

Avian Reproduction Test (Quail) (OCSPP 850.2300) 

Northern Bobwhite quail (Colinus virginianus) were fed diets containing 2,4-D (96.9% purity; 

Lot/batch not reported) at nominal concentrations of 0, 160, 400, and 1000 ppm (mean-measured 

concentrations of 0 (basal diet), 147, 382, or 962 ppm) for approximately 21 weeks in an avian 

reproduction study (MRID 45336401, 2000). There were no treatment-related mortalities, signs 

of toxicity, effects on adult body weight or feed consumption, or gross abnormalities observed 

upon necropsy. In addition, there were no treatment-related effects on reproductive parameters. 

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(The reviewer detected a significant reduction in eggs set/eggs laid at the highest treatment dose, 

962 ppm. Despite being statistically significant, this endpoint was only reduced 4% and was not 

considered to be biologically significant by the reviewer.) The NOAEC was 962 ppm, the 

highest concentration tested, and the LOAEC was not determined (>962 ppm). 

Fish Early Life-Cycle Toxicity Test (850.1400) 

In an early life-cycle toxicity test with fathead minnow (Pimphales promelas), embyros (<24hrs 

old) were exposed to 2,4-D dimethylamine salt (2,4-D DMA; 99.3%) (MRID 41767701). 

Hatching success, larval survival, and growth were measured. The most sensitive parameter 

measured was length with effects observed at ≥28.4 mg a.i./L with no effect observed at 17.1 mg 

a.i./L (14.2 mg a.e./L). 

In another early life-cycle toxicity test with fathead minnow (MRID 41737305), embryos were 

exposed to 2,4-D ethylhexyl ester (94.7%) for 32-days, at mean measured concentrations of 0.08, 

0.13, 0.12, 0.22, 0.45 and 1.96 mg a.i./L. Hatching success, larval survival, and growth were 

measured. The most sensitive parameter measured was larval survival with a NOAEC of 0.12 mg 

a.i./L (0.0792 mg a.e./L) and a LOAEC of 0.22 mg a.i./L. However, large variability in measured 

test concentrations were observed. 

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APPENDIX 3: References Not Utilized in the 2, 4-D 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 2,4-D 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. 

Coady, K., Marino, T., Thomas, J., Sosinski, L., Neal, B., and Hammond, L. (2013). An 

evaluation of 2,4-dichlorophenoxyacetic acid in the Amphibian Metamorphosis Assay and the 

Fish Short-Term Reproduction Assay. Ecotoxicol Environ Saf. 90:143-150. 

Erickson J.D., Mulinare J., McClain P.W., Fitch T.G., James L.M., McCleam A.B., and Adams 

M.J. Jr. (1984) Vietnam veterans' risks for fathering babies with birth Defects. JAMA. 

252(7):903-12. 

Evangelista de Duffard, A.M., Orta, C. and Duffard, R. (1990) Behavioral changes in rats fed 

diet containing 2,4-dichlorophenoxyacetic butyl ester. NeuroToxicology. 11:563-572. 

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Evangelista de Duffard, A.M., Bortolozzi, A. and Duffard, R. (1995) Altered behavioral 

responses in 2,4-Dichlorophenoxyacetic acid treated and amphetamine challenged rats. 

NeuroToxicology. 16:470-488. 

Hwang, U-G. (2002) Effect of 2,4-dichlorophenoxyacetic acid on vitellogenin synthesis and E2-

ER binding affinity or hepatocytes in rainbow trout (Oncorhynchus mykiss). Han'guk Yangsik 

Hakhoechi 15( 1): 31-37. (English abstract) 

Jungbauer, A. and Beck, V. (2002) Yeast reporter system for rapid determination of estrogenic 

activity. Journal of Chromatography. B 777:167-178. 

Kim, H.C., Kim, W.D., Kwon, T.H., Kim, D.H., Park, Y.I., and Dong, M.S. (2002) Mechanism 

of phenoxy compounds as an endocrine disruptor. J. Toxicol. Pub. Health 18, 331-339. 

Lamb, J.C., Marks, T.A., Gladen, B.C., Allen, J.W., and Moore, J.A. (1981) Male fertility, sister 

chromatid exchange, and germ cell toxicity following exposure to mixtures of chlorinated 

phenoxy acids containing 2,3,7,8-tetrachlorodibenzo-p-dioxin. J Toxicol. Environ. Health. 8(5-

6):825-34. 

Lamb, J.C., Moore, J.A., Marks, T.A., and Haseman, J.K. (1981) Development and viability of 

offspring of male mice treated with chlorinated phenoxy acids and 2,3, 7 ,8-tetrachlorodibenzop-dioxin. J Toxicol. Environ. Health. 8(5-6):835-44. 

Mayes, M.A., et al. (1990) 2,4-Dichlorophenozyacetic acid: evaluation of the toxicity to early 

life stages of the fathead minnow, Pimephales promelas Rafinesque. Environmental Toxicology 

and Chemistry Research Laboratory, Health and Environmental Sciences, The Dow Chemical 

Company, Midland, MI, 48674. Study sponsor: Technical Committee Industry Task Force II on 

2,4-D Research Data. Indianapolis, IN. Project ID No.: ES-DR-0002-2297-10. (MRID 

41737304) 

Oakes, D.J., Webster, W.S., Brown-Woodman, P.D., and Ritchie, H.E. (2002) A study of the 

potential for a herbicide formulation containing 2,4-D and picloram to cause male-related 

developmental toxicity in rats. Toxicol Sci. 68(1 ):200-6. 

Pochettino, A.A., Bongiovanni, B., Duffard, R.O., and Evangelista de Duffard, A.M. (2013) 

Oxidative stress in ventral prostate, ovary, and breast by 2,4-dichlorophenoxyacetic acid in preand postnatal exposed rats. Environ Toxicol. 28(1):1-10. 

Saghir, S.A., Zablotny, C.L., Bus, J.S., Marty, M.S., Perala, A.W., and Yano, B.L. (2008) A 

dietary dose range-finding and pharmacokinetic study of2,4-Dichlorphenoxyacetic acid (2,4-D) 

in the pregnant Crl:CD(SD) rat and its offspring in preparation for a subsequent F1-extended 

one-generation toxicity study in rats. Unpublished Report of The Dow Chemical Company, 

Midland, MI. Report Number HET K-002372-130. (MRID 47417901) 

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Saghir, S.A., Perala, A.W., and Clark, A. J. (2008) A dietary titration study of 2,4-

dichlorophenoxyacetic acid (2,4-D) pharmacokinetics in female CRL:CD(SD) rats. Laboratory 

Project Study ID 071210. Unpublished Report of Toxicology & Environmental Research and 

Consulting, The Dow Chemical Company, Midland, MI. (MRID 47417902) 

Schreinemachers, D. M. (2010). Perturbation of lipids and glucose metabolism associated with 

previous 2,4-D exposure: a cross-sectional study of NHANES III data, 1988-1994. 

Environmental health : a global access science source. 9:11. 

Van den Berg, K.J., van Raaij, J.A., Bragt, P.C., and Notten, W.R. (1991) Interactions of 

halogenated industrial chemicals with transthyretin and effects on thyroid hormone levels in 

vivo. Arch Toxicol. 65(1):15-9.

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