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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

## ~ S

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

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

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

.

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

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

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

CHEMICAL: 2, 4-DICHLORO PHENOXY ACETIC ACID (2,4-D)

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCY2, 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 Rele vant 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-Est rogenic Pathway for 2,4-D.........................................10
Table 3. Androgenic/Anti-A ndrogenic 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 Utiliz ed in the 2,4-D WoE Analysis....................................612, 4-D FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

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

CG Cowper’s Gland

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

DE R Data Evaluation Recor d DMSO Dimeth y l Sulfoxide DNA Deox y ribonucleic Aci d DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
E R Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase ) ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g eneration2, 4-D FINAL

iv Abbreviation Terminology

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

K OW Octanol/Water Partition Coefficient

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

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

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

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated Concentration2, 4-D FINAL

v Abbreviation Terminology

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

OPP Office of Pesticide Programs

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

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

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

T4 Thyroxine (tetraiodothyronine)2, 4-D FINAL

vi Abbreviation Terminology

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

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

VP Ventral Prostate VTG Vitello g eni n WoE Wei g ht-of-Evidence2, 4-D FINAL

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 poten tial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interaction includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothala mic-pituitary thyroid (HPT) perturbations. In addition to the available Tier 1 assay data, ot her scientifically relevant information (OSRI), including general toxicity data a nd open literature studies of suffi cient quality were considered in this weight of evidence (WoE) assessment.

In determining whether 2, 4 di chlorophenoxy acetic acid (2,4-D) interacts with E, A or T hormone pathways, the number and type of e ffects induced, the magnitude and pattern of responses observed across studies, taxa and sexe s 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 system ic 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 we ight-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 Wo E 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 inte raction 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 steroidoge nesis assay at 100 μ M. The requirements for the
EDSP Tier 1 in vivo mammalian uterotrophic and female pube rtal assays were satisfied by the extended one-generation re production toxicity test (EOGRT) in ra ts (equivalent to the EDSP Tier 2 study) considered as OSRI. No estrogen-related effects were obs erved in this study or in any of the mammalian Part 158 toxicity studies in th e absence of overt toxicity. No effects were
observed in the Part 158 bobwhite quail reproduction study. In th e 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 fema le ovaries at stage 2 (compared to stage 3 or 4) was observed for the 2,4-D treatment groups compared to the ne gative control, the inci dent rates were not dose-responsive, and there were no othe r effects observed in this study.2, 4-D FINAL

Page 2 of 63 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. Additiona lly, there were no treatment-rela ted effects in the FSTRA or avian reproduction studies, and there were no eff ects on the male reproductive parameters in the
Part 158 mammalian studies in the ab sence of overt toxicity.

In the amphibian metamorphosis assay (AMA), wh ile 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-relate d effects were seen in the mammalian Part 158 studies. The effects were only seen in the presence of ove rt toxicity or at doses that exceeded the threshold for renal clearance of 2, 4- D in the rat, resulting in syst emic 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, a dditional EDSP Tier 2 ecolo gical testing is not recommended. It is noted that the agency alr eady has a mammalian toxicity study (extended onegeneration reproduction test, E OGRT) that is equivalent to the EDSP Tier 2 study.2, 4-D FINAL

Page 3 of 63 1. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide the necessary empirical data to evaluate the poten tial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interaction includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothala mic-pituitary thyroid (HPT) perturbations. In addition to the available Tier 1 data, othe r scientifically relevant information (OSRI), including general toxicity data a nd open literature studies of suffi cient 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 conditi ons under which effects occur were considered, in particular, whether or not endoc rine-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 we ight-of-evidence (WOE) analysis of the potential interaction of 2, 4-D with the E, A or T signaling path ways. 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 th e dissipation of 2, 4-D in the environment. The log Kow suggests that the pot ential 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 moderate ly 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 (t 1/2=28.5 to 333 days) in anaerobic aquatic laborato ry studies.

The available information considered in determ ining the potential intera ction 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 stud ies and other published articles. These data are summarized in Sections III.A through III.C. An analysis of the data submitted to the agency,2, 4-D FINAL

Page 4 of 63 using the WoE approach outlined by the Agency (U SEPA, 2011), is presented in Section IV. The EDSP Tier 2 study recommendations ar e 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 believe d 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 T eam 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 c onsidered but not util ized 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 presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the indivi dual 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 endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to
evaluate whether additional te sting is needed based on the evidence and conclusions described
above.2, 4-D FINAL

Page 5 of 63 As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

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

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biol ogical organization? Fi nally, 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 systemic2, 4-D FINAL

Page 6 of 63 toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.

This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

For these WoE analyses, overt toxicity was gene rally 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 include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g ., organ weights/histopathology), biochemical ( e.g ., blood chemistry), and other clinical signs ( e.g ., lethargy) were also considered when evaluating overt toxicity, es pecially if the effects were extreme. In some instances, one parameter ( i.e ., death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

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

Page 7 of 63 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 bi ologically 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)

ER α 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)2, 4-D FINAL

Page 8 of 63 B. Effects on Hypothalamic-P ituitary-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 a ssays 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 reporte d 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 eviden ce for potential ER-related binding (Blair et al , 2002)
or transactiv ation (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 treatm ent 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 e ffects (up to 62.5 μM) in a frog oocyte assay evaluating ova rian 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 /o r non-dose-related, thereby ther e 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 Se t 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 p ituitary weight were observed in the F1 Set 1a and 3 female offsprin g. There were no treatmen t-related effects on VO, anogenital distance, estrous cyclicity, gonadal histopathology or reproductive indices in the study.2, 4-D FINAL

Page 9 of 63 In the other part 158 mammalian studies, the only pot ential -related effect s observed occurred in the presence of overt toxicity. These eff ects included decreased ovarian weights in the subchronic (42%) and chronic/carcin ogenicity (41%; NS) in the rat, decreased pituitary weight in the subchronic rat, and decrea sed F1b litter size/via bility in the rat 2-generation reproduction study.

In the FSTRA, there was a 34% decrease in fecund ity at the high treatment level (96.5 mg/L).
Plasma VTG concentrations and GSI in female s were not affected. While not concentrationdependent, an increase in the number of female ov aries at stage 2 (compared to stage 3 or 4) for the 2,4-D treatment groups compared to th e negative control was evident.

There were no effects on grow th or reproduction up to 962 mg ae/kg-diet in a Part 158 reproduction study with bobwhite qu ail 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 surviv al was affected at 0.22 mg a.i./L (NOAEC=0.0792 mg a.i./L), however, hi gh variability in test concentrations was observed in this study.2, 4-DFINALPage 10 of 63Table 2. Estrogenic/Anti-Estrogenic Pathway for 2,4-DLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for 2,4-D1Study Type/ LiteratureCitation 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) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1ER Binding(MRID 48614303)NERTA(MRID 48614304)NAromatase(MRID 48614302)NSteroidogenesis(MRID 48614305)P4UterotrophicRequirement satisfied by OSRI (MRID 47972101); see below.Female Pubertal RatRequirement satisfied by OSRI (MRID 47972101); see below.FSTRA(MRID 48317007)NENNNFcd:↓34%(H)NN2, 4-DFINALPage 11 of 63Table 2. Estrogenic/Anti-Estrogenic Pathway for 2,4-DLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for 2,4-D1Study Type/ LiteratureCitation 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) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRIER Binding (Blairetal., 2000)NRecombinant (hER)yeast-basedassay/Frog ovulationassay (Ortonet al.,2009)NN5Recombinant (hER)yeast-based assay(Hurst and Sheahan,2003)NERTA (Kojima,et al.,2004)NERTA (Lemaireetal., 2006)N6ERTA (Sunet al.,2012)NCell proliferationassay (Sotoet al.,1995)NSubchronic toxicity(Rat; MRIDs00101599 and00102451)NENENNENNSubchronic toxicity(Rat; MRID41991501)NENNNX(H)X(H)2, 4-DFINALPage 12 of 63Table 2. Estrogenic/Anti-Estrogenic Pathway for 2,4-DLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for 2,4-D1Study Type/ LiteratureCitation 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) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Subchronic toxicity(Mouse; MRID41991502)NENNNNNSubchronic inhalationtoxicity (Rat; MRID47398701)NNNNENNDevelopmentaltoxicity (Rat; MRID00130407 )NNNNDevelopmentaltoxicity (Rabbit;MRID 41747601)NNNX(H)Extended onegenerationreproduction (Rat,MRID 47972101)7NNNNN NNNNTwo-generationreproduction (Rat;MRIDs 00150557 and00163996)NENENENE NENX(X)X(H)Chronic/Carcinogenicity (Rat;MRID 43612001)NENNNEX(M,H)X(M, H)Carcinogenicity(Mouse; MRIDs43879801 and43597201)NENENNEN N2, 4-DFINALPage 13 of 63Table 2. Estrogenic/Anti-Estrogenic Pathway for 2,4-DLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for 2,4-D1Study Type/ LiteratureCitation 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) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Avian reproduction(Quail; MRID45336401)NN N1 Key to responses: L=Low treatment, Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑) indicate the direction of theresponse. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unlessotherwise indicated. LW= liver weight2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. 1.2-fold increase in estradiol production at 100 μM.5. Negative in anin vitrofrog oocyte assay evaluating ovulatory response and ovarian steroidogenesis (conc. range: 0.00625- 62.5 μM)6. Negative for ERαand ERβtransactivation 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 5Subsets at weaning (PND 21)to examine general systemic and thyroid toxicity (Set 1a), developmental neurotoxicity (Set 1b), developmental immunotoxicity (Sets 2a and 2b) andreproductive and endocrine toxicity (Set 3).P Positive findingN Negative finding (in vitro)/No effect (in vivo)NE Not examined2, 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-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

2, 4-D was negative (non-b inder) 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 bi nding to the AR (Kim et al ., 2005) or AR activ ation (Kojima, et al

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

The Tier 1 in vivo Hershberger and male pubertal assa ys were not conducted because the requirements for these two assays were satisfi ed 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 /o r non-dose-related, thereby ther e were no treatment-related androgen effects in the study. High-dose animals ha d non-statistically signif icant (NS) decreases in several AST weights [(prostate and epididym ides (P1 and F1 Set 1a) and seminal vesicles (P1)]. Serum testosterone concentrations were not determined in the EOGRT, or any other Part158 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 tr eated 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 we re 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 hi stopathology 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 subc hronic rat and decrease d testes weight and testicular atrophy in the rat ch ronic toxicity/carcinogenicity study. In the FSTRA or Part 158 avian reproduction study, there were no treatment-related eff ects observed in males.2, 4-DFINALPage 15 of 63Table 3. Androgenic/Anti-Androgenic Pathway for 2,4-DLines 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 Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1AR Binding(MRID 48614301)NSteroidogenesis(MRID 48614305)NHershbergerRequirement satisfied by OSRI (MRID 47972101); see below.Male Pubertal RatRequirement satisfied by OSRI (MRID 47972101); see below.FSTRA(MRID 48317007)NENNNNN NE N2, 4-DFINALPage 16 of 63Table 3. Androgenic/Anti-Androgenic Pathway for 2,4-DLines 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 Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRIAR binding(Kimet al., 2005)NAR transactivation(Kojimaet al., 2004)NAR transactivation(Ortonet al., 2009)NAR transactivation(Sunet al., 2012)NSubchronic toxicity(Rat; MRIDs00101599 and00102451)N N NE N NE NEN XSubchronic toxicity(Rat; MRID41991501)N N NE N N NEX(MH, H)X(MH,H)Subchronic toxicity(Mouse; MRID41991502)NE N NE N N NEN NSubchronic inhalationtoxicity (Rat; MRID47398701)N N N N NE NEN NDevelopmentaltoxicity (Rat;MRID00130407 )NN N2, 4-DFINALPage 17 of 63Table 3. Androgenic/Anti-Androgenic Pathway for 2,4-DLines 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 Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Developmentaltoxicity (Rabbit;MRID 41747601)NNX(H)Extended onegenerationreproduction (RatMRID 47972101)4N N N NNNNNX(H;males)X(H;males)Two-generationreproduction (Rat;MRIDs 00150557and 00163996)NENENENENENENX(H)X(H)Chronic/Carcinogenicity (Rat;MRID 43612001)NNNENNENEX(H)X(H)Carcinogenicity(Mouse; MRIDs43879801 and43597201)NENNENNNEN NAvian reproduction(Quail; MRID45336401)NN N1. Key to responses: L=Low treatment, Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑) indicate the direction of theresponse. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unlessotherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Levator anibulbocavernosus (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 OSRI is indicated by an X inthis column. For details see Section IV. A2, 4-DFINALPage 18 of 633. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. The extended one generation reproductive study included P1 and F1 generations, and the F1 generation was sub-divided into 5Subsets at weaning (PND 21)to examine general systemic and thyroid toxicity (Set 1a), developmental neurotoxicity (Set 1b), developmental immunotoxicity (Sets 2a and 2b) andreproductive and endocrine toxicity (Set 3).P Positive findingN Negative finding (in vitro)/No effect (in vivo)NE Not examined2, 4-D FINAL

Page 19 of 63 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it
does include three in vivo assays that provide redundancy an d 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 wi th thyroid regulation are summarized in Table 4. The various targets of the th yroid 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 ar e not reported in the ta ble and not considered further in the WOE assessment.

In the AMA with 2, 4-D, there were no histol ogical 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 con centration (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 stag es. Although changes were noted in some thyroidrelated endpoints, the effects were non-significant (NS) and /o r non-dose-related, or seen at a dose that resulted in overt toxicity (10% decr ease in body weight in male offspring), thereby there were no treatment-related thyroid effect s in the EORGT. For example, non-significant
decreases were observed in t hyroid weights in several F1 offspring sub-groups (male and female), as well as a non-dose-re sponsive increase (NS) in thyroi d 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 T 4 concentrations generally were observed in several F1 offspring sub-groups (male and fema le) and in the P1 GD 17 females, with a few exceptions. Serum T 4 concentrations were decreased (p< 0.05) in the high-dose male F1 PND 22 offspring at an overtly toxic dose. T4 levels we re increased in the mid-dose male, and mid- and high-dose female F1 Set 1a offspring, but the cha nges were not statistically significant. Serum TSH concentrations were unchanged or increas ed (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 obser ved 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 obs erved 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 hist orical control range for the testing facility and were not associated with histopa thology in the pituitary gland.2, 4-D FINAL

Page 20 of 63 Thyroid effects were noted in several other Part 158 studies conducte d with 2,4-D. In a subchronic oral toxicity study in rats, thyroid weights at term ination were decreased (p<0.05) 40% in males and increased (p<0.05; relative we ight) 68% in females, with an increased incidence of thyroid hypertrophy observed in the high-dose females. Serum T 4 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 fe males. However, the observed thyroid-related effects in this study occurred at a dose that resu lted 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 hist opathology was observed, but a decrease in serum T 4 concentrations was observed in males and females in the high-mi d- and high-dose groups (100 and 300 mg/kg/day, respectively). Finally, thyroid e ffects were noted in a chronic or al 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 an d 24 months. Histopathol ogical changes in the thyroid were not noted, except for decreased secr etory material in the follicles of high-dose females at the 12-month sacrifice only. Serum T 4 concentrations were decreased (generally p<0.05) in all mid- and high-dose males and fema les. 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 r eceptor agonist or antagonist activity at concentrations from 0.003 to 3.0 mg/L in a receptor-related lu ciferase 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 metamo rphosis, mass at metamorphosis, or time to metamorphosis for either leopard frogs or gray tree frogs, it is noted th at these endpoints are limited in their ability to inform potential thyroid-related perturbations.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-D 1

Study Type/ Literature CitationThyroid ReceptorActivationThyroid WeightThyroid: Grossand HistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmentalstage (±orasynchronous,HLL)Growth(BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

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 ↓ T 4 (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 X 5 (H) X (males: H)

Twogeneration reproduction (Rat; MRIDs 00150557 and 00163996) NE NE NE NE NE N X (H)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-D 1

Study Type/ Literature CitationThyroid ReceptorActivationThyroid WeightThyroid: Grossand HistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmentalstage (±orasynchronous,HLL)Growth(BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

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 sh aded cell indicates that is parameter is not routinely evaluated or is not applicable in this a ssay. 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. T 4 was decreased in both sexes at 6, 12, 18 and 24 m onths; 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 examined2, 4-D FINAL

Page 23 of 63 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of 2, 4-D to interact w ith endocrine related processes ( i.e., E, A, or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated analysis of those lines of evidence.

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

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

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. Generally, one parameter (i .e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concen tration to be considered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were ge nerally less severe forms of toxicity (e.g., changes in orga n weights or blood chemistry).2, 4-D FINAL

Page 24 of 63 1. Tier 1 in vivo Assays

The requirements for the Hershberger, male and fe male pubertal assays, a nd uterotrophic assays with 2, 4-D were satisfied by OSRI (extended one-generation reproductio n 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 ma les 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 t oxicity study in Fisher 344 rats, one 100 mg/kg/day male lost weight on Days 55 and 62, with subsequent we ight 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 prim ary 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 pronoun ced in the two highest dose groups.

In a guideline subchronic oral toxi city study in Fisher 344 rats, clin ical signs of toxicity (hunched posture, depressed activity, and few/no feces) oc curred mainly in the 300 mg/kg/day females. Decreased (p<0.05) body weights were obser ved in the 100 and 300 mg/kg/day males and females, reaching 7-23% in males and 7-28% in females at Week 13. Decr eased overall (Weeks 0-13) body-weight gains were also no ted 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 re lative organ weights [including adrenals ( ↑ males;2, 4-D FINAL

Page 25 of 63 ↓ 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 ch anges may be attribut able to decreased body weight. Gross findings, mainly in the high-dose group, included small testes and epididymis and opaque eyes (females). Treatment-related histop athological changes were observed primarily at 300 mg/kg/day and included cen trilobular hepatoce llular hypertrophy, atr ophy of the testes, hypertrophy of the zona glomerulosa (adrenal co rtex, both sexes) and fo llicular cells (thyroid, females).

In a guideline subchronic oral t oxicity study in B6C3F1 mice, tr eatment-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 transi ent 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 inhala tion toxicity study in Sprague -Dawley rats, clinical signs associated with exposure at 1.0 mg/L include d 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 recover y, and was accompanied by a reduction in food
intake.

In a guideline developmental toxi city study in pregnant Fische r 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 toxi city study in artificially-ins eminated 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 we re 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 we ight gains, and a decrease in the corrected body weight gains ( ↓ 23%; NS).

In a guideline two-genera tion reproduction study in Fischer 344 rats, decreas ed (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 we ights were also decreased (p <0.05) in the P fe males during gestation of the F 1a ( ↓ 5-6%) litters on GD 7, 13 and 20, and the F 1b litters ( ↓ 10%) on GD 20. In the 80 mg/kg/day F 1a pups, body weights were decreased (p<0.05) by 11-29% during PND 1-282, 4-D FINAL

Page 26 of 63 in the males, and by 15-25% during PND 4-28 in the females. In the 80 mg/kg/day F 1b 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 F 1b pups, body weights were d ecreased (p<0.05) in the males by 7% and 16% on PND 4 and 28, respectiv ely, 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 F 1b litters (22.5 days vs 21.9 days); (ii) gestation survival i ndex was decreased (p<0.01) for the F 1b litters (31.7% vs 97.8%); (iii) the number of F 1b pups born dead/dying by PND 1 wa s increased (110 treated vs. 5 controls); (iv) F 1b litter size was decreased (p<0.01; 5.1 treated vs. 9.5 controls); v) F 1b pup viability was lower (p<0.01) throughout the weani ng period; (vi) there were decreases (p<0.01)
in F 1b pup survival to LD 4 (86.3% treated vs. 100% controls); and (vii) survival to LD 28 was decreased (71.4% treate d vs. 100% controls).

In a guideline combined chronic toxicity/carcinogenici ty study in Fischer 344 rats, body weights were lower than respective contro ls in females at 75 mg/kg/day ( ↓ 14%) and in males ( ↓ 8%) and females ( ↓ 26%) at 150 mg/kg/day at termination. Body we ight 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 aminotrans ferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP) and/or choleste rol 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: decr eased hematopoiesis of the bone marrow of females at 150 mg/kg/day; altere d tinctorial properties in the liv er of females at 75 mg/kg/day and both sexes at 150 mg/kg/day; bilateral retinal degeneration of the ey es 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; dege neration of the descending porti on 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-neopla stic lesions were limited to the eyes, liver, lung, and the mesenteric fat. Eye lesions were char acterized 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 hepato cytes, often accompanied by alte red 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 chroni c 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 inte resting to note that lesions seen in the spleen, kidneys, testes and thyroid glands in rats sacrifi ced at 12 months were not seen in those sacrificed at 24 months.2, 4-D FINAL

Page 27 of 63 In a guideline carcinogenicity study in B6C3F1 mice, the 300 mg/kg/day females had slightly lower (p<0.05) body weights at th e 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 ha d 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 a nd 300 mg/kg/day females.

In a guideline avian reproduction toxi city 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 syst emic or overt toxicity.

B. Estrogen Pathway

There was no convincing evidence of potential inte raction 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 steroidoge nesis assay at 100 μ M. 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 st udy) considered as OSRI. No estrogen-related effects were obser ved 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 d ecreased (34%) at a co ncentration of 96.5 mg a.i./L, and while an increase in the number of fe male ovaries at stage 2 (compared to stage 3 or 4) was observed for the 2,4-D treatment groups comp ared 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 (equivale nt to EDSP Tier 2 study) consid ered 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 st udies, 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 t hyroid or effects on developmental stage. Some thyroid-related effects were seen in the mammalia n Part 158 studies. The effects were only seen in the presence of overt toxicity or at doses th at exceeded the threshold for renal clearance of 2,2, 4-D FINAL

Page 28 of 63 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-generat ion 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 fema les. Therefore, collec tively the data across the Tier 1 assays and Part 158 data do not demonstr ated 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 estroge n, androgen or thyr oid pathways.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations, a dditional EDSP Tier 2 ecolo gical testing is not recommended. It is noted that the agency alr eady has a mammalian toxicity study (extended onegeneration reproduction) that is equi valent to the EDSP Tier 2 study.2, 4-D FINAL

Page 29 of 63 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 estrog en receptor relative binding affinities of 188
natural and xenochemicals: stru ctural 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-Di chlorophenoxyacetic acid (2,4-D): results of a 13-week subchronic dietary toxicity study in the CDF Fischer 344 Rat. Unpublished study, Dow Chemical Co mpany, Midland, NI. (MRID 00102451).

Gorzinski, S.J., Wade, C.E., Morden, D.C., Ke yes, 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 st udy in the CDF Fischer 344 Rat. Unpublished study, Dow Chemical Company, Midland, MI. (MRID 00101599).

Hoberman, A.M. (1990) Developmental toxicity (e mbryo-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 i nhalation toxicity study of 2,4-Dichloro phenoxyacetic acid in the rat via nose-only e xposures. 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 Battj es, J.E. (1995) 2, 4- Dichlorophenoxyacetic acid: chronic toxicity/oncogeni city 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 huma n 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 activi ties in 200 pesticides by in vitr o 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 . 79:1160-1169.2, 4-D FINAL

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-gen eration dietary toxicity study in Crl:CD(SD) rats. Unpublished Study Report of the Dow Ch emical 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. U npublished 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 Fisc her 344 rats with 2, 4- dichlorophenoxyacetic acid- Final Report. Project No. WIL-81135. Unpub lished study, WIL Research Laboratories, Inc., Ashland, OH. (MRID 00130407).

Rodwell, D.E. and Brown, W.R. (1985) A Di etary Two-Generation Reproduction Study in Fischer 344 Rats with 2,4-Dichlorophenoxyace tic Acid: Final Report: Project No. WIL-81137. WIL Research Lab. Inc. (MRID 00150557).

Rodwell, D.E. (1986) A dietary two-generati on reproduction study in Fi scher 344 rats with 2,4- Dichlorophenoxyacetic acid- addendum to the Final Report. Project No. WIL-81137. Unpublished study, WIL Laboratorie s, 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 Labor atories 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-Dichlorophenox yacetic 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 iden tify estrogens: an update on estrogenic environmental pollutants. Environmental Health Perspectives . 103 (Suppl 7):113-122.2, 4-D FINAL

Page 31 of 63 Stott, W. (1995a) 2,4-Dichlorophenoxyacetic Ac id: Dietary Oncogenicity Study in B6C3F1 Mice Two-Year Final Report (Female). La b 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., Ch en, L., and Wang, X. (2012) Developing in vitro reporter gene assays to assess the hormone receptor act ivities of chemicals fr equently detected in drinking water. J Appl Toxicol . 32(8):635-641.

USEPA. (2005) Reregistration E ligibility Decision (RED) for 2,4- D. LIST A. CASE 0073. June, 2005. EPA 738-R-05-002, Office of Pollution Pr evention 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 Screeni ng Program – Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.

USEPA. (2012) 2,4-D: Revised Evaluation of Data Identified in NRDC Petition and Associated Documents. Office of Chemical Safety and Pollu tion Prevention. U.S. Environmental Protection Agency, Washington, DC.

USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght 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. Envir onmental Protection Agency, Washington, DC.2, 4-D FINAL

Page 32 of 63 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosi s 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 laev is) 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 c oncentrations 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 treatmen t group; the cause of
death was unknown. No clinical si gns 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 signifi cant (p<0.05) decrease of 15% in Day 21 normalized HLL at the highest treatment level compared with the negati ve control. There was no significant effect on median NF developmental stage, snout-vent leng th (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) tadpole s were observed in the negative control and in all treatment levels; consistent with the guideli ne recommendations, these tadpoles were excluded from analyses of growth and normalized HLL.

Androgen Receptor (AR) Bindi ng 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). Et hanol 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 contro l, and R1881 as the ligand reference standard.2, 4-D FINAL

Page 33 of 63 Saturation binding experiments were conducted to demonstrate that the AR in the rat prostate cytosol was present at adequate levels and functioning with appropria te affinity for the radiolabeled ligand. The saturation binding expe riment resulted in a maximum binding capacity (B max ) of 3.245 fmol/100 μg protein a nd the dissociation constant (K d ) was 0.4641 nM. Although these values were slightly below the range of values fr om the validation studies, the results were highly reproducible and all othe r 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 th e 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 IC 50 and relative binding affinity (RBA) for 2,4- D could not be calculated. The log IC 50 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 criteri a 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 (MRI D 48614302), 2,4-D (98.5% a.i., lot#: 2006 2433 8006-USA) in ethanol (1%) was incubated with human recombin ant aromatase and tritiated androstenedione (1- β [ 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) for 15 minutes at 37 °C to assess the 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 tes ting in this assay as they are substantially above the inflec tion point for linear toxicokine tics (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 concentra tion of chemical. Tritiated water was quantified using liquid scintillation counting (LSC). Thr ee independent runs were conducted and each run included a full activity c ontrol, 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.2, 4-D FINAL

Page 34 of 63 Aromatase activity in the fu ll 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 re sponses of full activity controls were outside of the 90 to 110% range in the 2 nd and 3 rd replicates in the Run 1 ( 86.6 and 113.8%, respectively).

Results for the positive control were generally within the recommended ra nges for the top of the curve, bottom curve, Hill slope, log IC 50 , and coefficient of varia tion 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 replicat es 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 th e acceptable limit. For 4-OH ASDN, the estimated log IC 50 averaged

− 7.23 M and the slope was

− 0.92.

For 2,4-D, aromatase activity av eraged 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 modele d for Run 1, and the goodness of fit (R 2 ) 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 ac tivity 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 appr oximately 91%. Since the average lowest portion of the activity res ponse 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 ou tside of the performance criteria ranges (with the mean value within range).

Based on the data from the average response curv e, 2,4-D is classified as a Non-inhibitor of aromatase activity in this assay.

Estrogen Receptor (ER) Bindi ng 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 ra ts 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]-17 β -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 the2, 4-D FINAL

Page 35 of 63 top test concentration was based on toxicokinetic data in the ra t; 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 c ontrol, octyltriethoxys ilane as a negative control, and 17 β -estradiol as the natural ligand referen ce material, and three independent runs were performed on separate days.

Summary data pertaining to the saturation binding experiment we re reported separately in the study profile submitted by the test order recipient. The K d for [ 3 H]-17 β -estradiol was 0.1032 nM
and the B max (nM) was 0.07097 for the prepared rat uterin e cytosol used in th ese experiments.
The K d for the run was within the expect ed range of 0.03 to 1.5 nM, and the B max was within the expected range of 10-150 fmol/100 μ g protein. The data produced a linear Scatchard plot.

In the competitive binding experime nt, 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 IC 50 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 IC 50 for the natural ligand, 17 β -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 17 β -estradiol, 19-norethindrone and octyltriethoxysilane.

2,4-D was tested over a concentration range that fu lly 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.

ER α Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300)

In an estrogen receptor transcrip tional activation a ssay (MRID 48614304), hER α -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 (lucifer ase) 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,2, 4-D FINAL

Page 36 of 63 but these deviations do not affect the interpretation of this study. The RPC Max 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

PC Max was 10

− 4 M for runs 1-3 and 10 -5 M for run 4. Because the RPC Max < PC 10 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 concentra tions 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 25.2 o C and a pH of 7.02 to 7.76.

The single mortality observed durin g the assay occurred in a fema le in the high treatment group. There were no significant differe nces for male or female body weight or length relative the negative control. At test termination, observations of secondary se x characteristics were observed in the negative control and tr eated groups; no treatment-related e ffects were reported. Clinical signs included the loss of an eye, ascites, and sc oliosis (bent tail) which were observed in single fish in the negative cont rol 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 decr eased fecundity in the lower treatment groups compared to the negative control. There were no significant differences for fertility between the 2,4-D treatm ents and the negative control.

There were no significant diffe rences (p>0.05) betw een 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) fo r 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 (compare d to Stage 3 or 4) was reported for the 2,4-D treatments compared to the negative control. Plas ma sex steroid concentrations were not reported.2, 4-D FINAL

Page 37 of 63 Hershberger Assay; (OCSPP 890.1400)

The requirement for a Tier 1 Hershberger assay is satisfied by OSRI: an extended dietary onegeneration reproductive toxic ity 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 assa y is satisfied by OSRI: an extended dietary onegeneration reproductive toxic ity study (MRID 47972101, 2010). The results from this study are presented with the OSRI in Appendix 2.

Male Pubertal A ssay; (OCSPP 890.1500)

The requirement for a Tier 1 male pubertal assa y is satisfied by OSRI: an extended dietary onegeneration reproductive toxic ity 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 (M RID 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 experime nts. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration in the assay of 0.1%.

Testosterone and estradiol levels were meas ured using LC-APPI-MS/MS. A Quality Control (QC) plate was run concurrently with each i ndependent 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 a nd testosterone production.

Guideline acceptability recommendations and requirements were generally met, including lack of cytotoxicity, adequate producti on of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction an d inhibition with positive contro ls, with two exceptions. The required concentration for estrad iol 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 mini mum detection level (10 pg/mL) as required. Secondly, all three2, 4-D FINAL

Page 38 of 63 runs of 1 μ M 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 pr oduction at concentrations up to 10

− 4 M, and no effect on estradiol production at concentrations up to 10

− 5 M. At the highest c oncentration 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 re producible increases in estrad iol production. 2,4-D treatment did not result in statistically signi ficant and reproducible alteration s in testosterone production.

Uterotrophic Assay; (OCSPP 890.1600)

The requirement for an uterotrophic assay is satisfied by OSRI: an extended dietary onegeneration reproductive toxic ity study (MRID 47972101, 2010). The results from this study are presented with the OSRI in Appendix 2.2, 4-D FINAL

Page 39 of 63 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 studies consid ered relevant for dete rmining the potential interaction of 2,4-D with the endocrine system are summarized below. Given the breath of toxicity data available, the onl y Part 158 studies discussed in th is appendix are those that fulfill current guideline requirements and are directly relevant to asse ssing potential endocrine effects (i.e. reproductive and developmental studies, su bchronic and chronic toxi city studies, special mechanistic studies, etc.). In addition, recen t re-evaluations of 2,4-D by the Agency (USEPA, 2012) were considered in summari zing 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) affin ity using a standardized ER competitive-binding assay. The assay was tested using multiple ch emicals 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-Daw ley 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 furthe r assayed with a wide range of concentrations to
determine the IC 50 and relative binding affinity (RBA) values of the compounds.

Adult (~245 days old; retired breeders), non-pregnant female Sp rague-Dawley rats (NCTR:SDN, an outbred albino rat stock originating from CR L:CD [SD]BR rats) were ovariectomized (mean n = 14 rats/cytosol batch) 10 days prior to receptor preparation. After sacrifice by CO 2 asphyxiation, the uteri were excised, trimmed of excess fat and mesenter y, 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 pool ed uteri were homogenized at 4°C in fresh, ice-cold TEDG buffer (1 g tissue/10 mL buffer). The resul ting homogenate was cen trifuged at 105,000 × g for 60 min at 4°C, separated into 3-mL portions, a nd stored at –70°C until used in the competition

assays.

The test compounds 2,4-D (99% pur ity), atrazine (98%), carbaryl (99%), metolachlor (98.7%), and simazine (99%) were obtained from Supe lco. 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 in cubated at 4°C for 20 hours. After incubation,2, 4-D FINAL

Page 40 of 63 750 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 resu lting HAP pellet was resu spended 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 etha nol was added to extract the radiolabeled E2 from the HAP. Tubes were incubated on ice for 15 min, centrif uged at 4°C, and the resulting supernatant was decanted into vials containing 10 mL of scintillation cock tail. 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) a nd 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 quali ty 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). Radi oactivity 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 curv e were plotted as percent bound [ 3 H]-E2 versus molar concentration, and the IC 50 (50% inhibition of [ 3 H]-E2 binding) for eac h test compound was
determined. The RBA for the test co mpound was calculated by dividing the IC 50 of E2 by the

IC 50 of the competitor and was expre ssed as a percentage (E2=100).

Compounds with a log RBA of >0 were cons idered 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. 17 β -Estradiol had a mean

IC 50 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 EC 50 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 ch emical classes, for their potent ial to interact with two human estrogen receptor subtypes (hER α and hER β ) and one human androgen receptor (hAR) in transactivation assays with Chinese ha mster ovary cells (CHO-K1 cells).2, 4-D FINAL

Page 41 of 63 The human estrogen receptor (hER α ) and androgen receptor (hAR) expression vectors (pcDNAER α and pZeoSV2AR, respectively) were cons tructed as described previously (Kojima et al, 2003). The hER β expression vector was co nstructed by cloning the hER β cDNA with reverse transcriptase-polymerase chain reaction from human placental RNA. The sequence was
verified and inserted into the mammalia n expression vector pcDNA3.1Zeo(-) creating pcDNAER β . The construction of the estrogen or androgen responsive elements containing reporter plasmid (pGL3-tkERE and pIND-ARE, resp ectively) 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 hER α or hER β activity, CHO cells were transfected with pcDNAER α or pcDNAER β , 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
hER α , hER β and hAR, either 10 -11 or 10 -10 M 17 β -estradiol (E 2 , >97% purity) or 10 -10 M 5 α

dihydrotestosterone (DHT, 95% pur ity) was added to the cell cu ltures 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 experiment s were conducted for each compound.

Agonistic activities were evaluated by relative activity expr essed as 20% relative effective concentration (REC 20 ), the concentration of the compound showing 20% of the activity of 10 -10

M E 2 , 10 -9 M E 2 , or 10 -9 M DHT for ER α , ER β , and AR, respectively. Antagonistic activities were evaluated by relative activity expressed as 20% relative inhibito ry concentration (RIC 20 ), the concentration of the compound showi ng 20% inhibition of the activity of 10 -10 M E 2 , 10 -9 M

E 2 , or 10 -9 M DHT for ER α , ER β , 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 REC 20 the pesticide was judged to be positive for activity. When the activity of the test compound was higher than the RIC 20 the pesticide was judged to be positive for inhibito ry activity. The statis tical significance of
differences was evaluated with a two-tailed Student’s t -test with a nominal level of significance of p < 0.05.

E 2 demonstrated dose-dependent transactivation of ER α and ER β which indicated that both receptors can be activated at very low hormone concentrations. The maximal ER α activity was
achieved at 10 –10 M E 2 or more, exhibiting approximately 10-fo ld that of the control solvent.
The maximal ER β activity induced was 8.5-fold that of the solvent control at 10 –9 M E 2 or more. Thus, E 2 was more potent for ER α than for ER β . From the dose–response curves, REC 20 values
of E 2 for ER α and ER β were determined to be 2.5 × 10 –12 M and 5.3 × 10 –12 M, respectively. 5 α

DHT caused dose-dependent transactivati on of AR and was detectable from 10 –11 M DHT and2, 4-D FINAL

Page 42 of 63 plateaued at 10 –9 M DHT. The maximum induction was 21-fold that of the control solvent. The

REC 20 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 activ ity 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 ER α and ER β activation or inhibition in stable reporter cell lines, HELN ER α and ER β (HeLa cells stably tr ansfected with an EREdriven luciferase plasmid).

For the ER α and ER β competitive-binding assay, ER α and ER β cells were seeded at a density of

1.5×10 5 cells per well in 24-well tissue culture pl ates. Cells were labeled with 0.1 nM [ 3 H]-E 2 ([ 3 H]-17 β -estradiol, 41.3 Ci/mmol specific activity) in the presence or absence of increasing
concentrations of cold E 2 (as control) or each of the tested compounds in 400 μ l 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 assa y. Treatment with 2,4-D, as well as all other pesticides, did not result in cytotoxicity in the study.

To evaluate the estrogenic pot ential of pesticides, HELN ER α and HELN ER β were used to identify the active compounds and discriminate between ER α and ER β activities. To determine

ER α and ER β agonistic activity of pes ticides, the relative transact ivation activity on HELN ER cells was computed as the ratio of luciferase reporter gene induction values of each compound at
a concentration of 10 μ M to the luciferase reporter gene induction value of E 2 at 10 nM. Antagonistic effects of pesticides in the ER α and ER β transactivation assay were determined over a range of five concentrations from 10

− 7 to 10

− 5

.
To confirm the ER antagonist effects in the tran sactivation assays reflected an inhibition of E2 binding to ER α and/or ER β , whole cell competitive binding assays were conducted with the

HELN ER α and ER β cells. The binding affinity for ER of anti-estrogenic pesticides was assessed with 0.1 nM [ 3 H]-E 2 as a tracer. As expected, non-radiolabelled E 2 displaced [ 3 H]-E 2 by
50% at a concentra tion of 0.12 nM for ER α and 0.18 nM for ER β , and ICI 164,384 inhibited [ 3 H]-E 2 binding by 50% at the concen tration of 2.5±0.5 nM for ER α and 3.3±1.0 nM for ER β

.
2,4-D was reported negative with a percent activity in ER α and ER β of 8.4 ± 1.4% and 10.2 ± 2.1%, respectively, which was not statistically di fferent from the solvent DMSO (9.3 ± 1.3% and 10.8 ± 1.6%, respectively).2, 4-D FINAL

Page 43 of 63 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 agricultu ral 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 17 β -estradiol (E2).

The pesticide standards used in the study were ob tained from Dr. Ehrenstorfer (GmbH); purities ranged 95-99.5%. The yeast assay uses a geneti cally modified yeast strain in which hER- α has been integrated into the yeast genome, togeth er with expression plas mids carrying estrogenresponsive elements (EREs) which contro l 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 comb ined in microtitration plates with pesticide
standards dissolved in ethanol at 12 different concentrations (not re ported). A dilution series of E2, as the positive control and estrogenic standard, and an ethanol solvent blank were assayed along with the pesticide standard s. An estrogenic response wa s defined as an increase by the pesticide standard of at least 0.1 optical density (OD) units colo r (at 540 nm) in comparison with the solvent control. The estrogenic potency was re ported as the potency rela tive 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 demonstr ate 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 chemical s using the proliferative effect of estrogen and estrogenic chemical on and estrogen-sensitiv e target cell line. This quantitative in vitro assay compares the cell number achieved by similar inoc ula of human breast can cer estrogen-sensitive (MCF-7) cells in the absence of estrogens (n egative control), and in the presence of 17 β -estradiol (E 2 , 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 eff ect. The aim of the work was2, 4-D FINAL

Page 44 of 63 to: 1) validate the E-SCREEN A ssay, 2) screen a variety of chem icals, 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 compari ng the cell yield achieved by similar inocula harvested simultaneously during the late exponentia l 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 treatmen t and positive (E2) contro l were apparent after 4 days. Estrogenic activity was assessed by 1) rela tive proliferative poten cy (RPP) which is the
ratio between the minimal concentration of es tradiol 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 (R PE) which is 100 times the ratio between the highest cell yield
obtained with the chemical and with E2l. Th e 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 w ith a minimum of 5 replicated. Differences between groups were assessed by analysis of va riance and the a posteriori Shaffe’s test. A p
value ≤ 0.05 was regarded as signifi cant. 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 evalua te and compare estrogen receptor (ER), androgen receptor (AR) and thyroid hormone receptor (TR) activities of ta rget chemicals. The assay was tested using 2,4-dichlorophenoxyacetic acid (2,4-D), chlorpyrif os, di-2-ethylhexyl-phthalate, and bisphenol A; only data for 2,4-D ar e discussed herein.

All the assays utilized Vero (African green monkey kidney) cells wh ich do not contain the endogenous receptors. For the ER Reporter Gene Assay, the cells were transfected with pERETATA-Luc, rER α /pCl, and phRL-tk (used as internal cont rol 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 (REC 20 ), the
concentration at which the tested chemical show ed 20% of the maximum activity of the positive control (E 2 , testosterone, or T 3 ). Antagonistic activity was reported as the 20% relative inhibitory concentration (RIC 20 ), the concentration at which th e tested chemical induced a 20% reduction of activity in E 2 , testosterone, or T 3 . The REC 20 and RIC 20 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 were2, 4-D FINAL

Page 45 of 63 then analyzed by one-way ANOVA followed by D uncan’s multiple comparisons test when appropriate. Statistical signifi cance 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 (E 2 , testosterone, or T 3 ) group.

The cytotoxicity of the test chemicals was dete rmined prior to conducting the receptor assays. The test chemicals did not affect the viability or proliferation of the Vero cells with or without

E 2 , testosterone, or T 3 . All three positive controls exhibite d appropriate concentration-dependent responses in the respective assays. Based on the concentration-re sponse curves, the EC 50 values

were 1.78×10 –6 mg/L for E 2, 3.32×10 –4 mg/L for testosterone, and 2.09×10 –3 mg/L for T 3

.
2,4-D showed no ER, AR or TR agonist or antag onist 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, signi ficantly (p<0.01) enhancing the effects of testosterone.

AR Activation

In a published, non-guideline study, Kim et al ., 2005 , characterized the a ndrogenic activity of 2,4-D (>98% pure) and its metabolit e 2,4-dichlorophenol (DCP) with 5 α -dihydrotestosterone (DHT) using an in vitro mammalian system with prostate cancer cell lines. Additionally, the molecular mechanisms of phenoxy compound action re lated AR activation we re investigated.

Two human prostate cancer cell lines were used in the st udy: AR expressed 22Rv1 (derived from a relapsed tumor) and androgen-independen t PC3 (androgen-insensiti ve 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 assa y, the 22Rv1 cells were seeded in 96-well plates (3 × 10 3 cells/well) and DHT was added to the experimental medi um (phenol red-free RP MI 1640 containing 10% dextran charcoal-stripped FBS) 24 hours later. The bioassay was terminated on the fourth day
and the cells were fixed and st ained with sulforhodamine-B (S RB). Briefly, the cells were
treated with 10% trichloroacetic acid and incubated at 4°C fo r 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 dr ied, and the bound dye solubilized with 10 mM Tris base (pH 10.5). The amount of SRB product in each well was dete rmined with a plate reading spectrophotometer at 570 nm; the assays were performed in triplica te for each cell line.

In the transactivation assays, the 22Rv1 and PC3 cells (1.2 × 10 5 ) were seeded in 24-well plates and transfected with mouse mammary tumor viru s luciferase vector (MMTV-Luc) and pCMVhAR expression vector or with MMTV-Luc al one using ExGene 500. After transfection (24 hours), DHT and the test chemical were added at specified concentr ations with the experimental2, 4-D FINAL

Page 46 of 63 medium. After another 24-hour incubation, the ce lls 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]5 α -DHT for 2 hours at 37°C in the presence and absence of increasing concentrations of the test compounds. Nonspecific binding of [ 3 H]5 α -DHT was determined by adding a 100-fold molar excess of
unlabeled 5 α -DHT. Cells were washed in PBS, harv ested 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 cu ltured 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% dextra n 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 conf ocal laser system connected to a fluorescence microscope. For the evaluation of GFP-AR local ization, the relative nuc lear and cytoplasmic fluorescence in >100 cells per condition from th ree 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 exceed ing nuclear fluorescence (1); and exclusive cytoplasmic fluorescence (0).

The study authors concluded that 2,4-D and DC P in combination with DHT have androgenic activity in cell proliferation a nd androgen-induced transactivati on, 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 synerg istic androgenic activities. Co-treatment of 2,4-D or DCP with DHT stimulat ed cell proliferation by 1.6-fold, compared to
DHT alone. In transient tran sfection 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 eff ect on either mRNA or protein levels of the AR. In a competitive AR binding assay, 2,4-D a nd DCP inhibited androgen binding to the AR (up to 50% at concentrations of approximately 0.5 mM for bo th compounds). The nuclear translocation of green fluorescen t protein-AR fusion protein in the presence of DHT was further promoted with the addition of 2,4-D and DCP. Th e authors therefore concluded that the results2, 4-D FINAL

Page 47 of 63 indicate that 2,4-D and DCP enha ncement of DHT-induced AR tran scriptional activity may be attributable, at least in part, to the promotion of AR nucl ear translocation.

ER/AR Activation and O varian Steroidogenesis

In a published study, Orton et al. (2009) , investigated eleven pestic ides (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 re sponse and ovarian steroidogenesis in Xenopus oocytes. In the yeast (anti-) estrogen screen (YES) and y east (anti-) androgen screen (YAS), activation of transfected human estrogen or androgen recepto r results in a color change in the media as measured by absorbance at 540 nm. Turbidity was m onitored 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 gonadot ropin (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 ma ximum 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 T oxicity 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 th e available copy of the DER.

All rats survived until schedul ed termination. Although body weights and food consumption were recorded throughout the st udy, achieved doses were not repor ted. No overt toxicity was observed, but one male in the high-mid-dose gr oup (100 mg/kg/day) lost weight on two days mid-study (Days 55 and 62), with subsequent we ight 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 te rmination. Decreased mean body weight also was observed beginning on Day 59 in the low mi d- and high-mid-dose males, and continuing through study termination. Body weight gains we re decreased (NS) in the high-dose animals, and food consumption was occasionally decreased in the high-dose animals throughout the2, 4-D FINAL

Page 48 of 63 study. Terminal body weights were decreased in th e high-dose animals, with lesser decreases (NS) in the high-mid-dose group.

Concentrations of T 4 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 weight s were affected in the high- dose animals, but these changes were secondary to decrea sed terminal body weights.

Histopathology was conducted on 10 animals/sex/gr oup. 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 wa s more pronounced in the two highest dose groups. The original pathology report of this stu dy stated that there was an increased epithelial cytoplasmic homogeneity of the c onvoluted 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 α 2u-globulins which are sex-dependent urinary proteins of hepatic origin. D ecreased amounts of protein droplets could represent changes in the nutritional status of the rats, a metabo lic change in the liver cells where α 2u-globulins are produced, or a direct effect on the kidneys.

The dose of 60 mg/kg/day exceeded the maximu m-tolerated dose as evidenced by cellular alterations in the kidneys of both sexes. Th e 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 T oxicity in Rodents (Rat)

In a subchronic oral toxicity study (MRI D 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 d eaths. Clinical signs of toxic ity 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-re lated increase in the incidence of pale/opaque eyes in both sexes. Decreas ed (p<0.05) body weights were observed during the study in the high-dose males and females, with th e magnitude of the deficit increasing with time (males ↓ 15%/23% and females ↓ 21%/28% at Weeks 6/13, respectiv ely). 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-we ight gain (Overall: males ↓ 9%/females ↓ 11%) were observed.2, 4-D FINAL

Page 49 of 63 A corresponding decrease in food consumption wa s observed in both sexes at the two highest
doses.

Decreased (p<0.05) T 3 and T 4 concentrations were observed at the high-mid- and high-doses at both intervals, except for T 3 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 ab solute 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 primar ily at the high-dose in both se xes, and many of these changes may be attributable to decrea sed body weight. Gross findings, mainly in the high-dose group, included small testes and epid idymis and opaque eyes (females). Treatment-related histopathological changes were observed primarily in the hi gh-dose animals and included centrilobular hepatocellular hype rtrophy, atrophy of the testes , hypertrophy of the zona glomerulosa (adrenal cortex, both sexes)’ and fo llicular cells (thyroid, females). Many of the
lesions correlated well with th e 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 T 3 (females) and T 4 (both sexes)] parameters, and cataract formation in females. The NOAEL is 15 mg/kg/day.

90-Day (Subchronic) Oral Toxi city 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/Bat ch #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 inta ke 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 eff ects on survival, body wei ght, body weight gain, food consumption, and gross pathology up to th e high intermediate dose (100 mg/kg/day).
Treatment-related effects at th e high-mid dose included decreased glucose (p<0.05; females) and

T 4 concentrations (statistical an alysis confounded; males), and in creases in mean absolute and relative kidney weights (females). Treatment-rela ted 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

T 4 concentrations (statistical an alysis confounded; males); and hi stopathological changes in the liver characterized by nuclear hyperchromatism and decrea sed glycogen in periportal hepatocytes. Under the condi tions 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 to xicity of 2,4-D to male and female mice. The LOEL is based on decreased glucose and T 4 concentrations, and incr eased mean and relative kidney weights, observed at 100 mg/kg/day.2, 4-D FINAL

Page 50 of 63 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 concentra tions 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 expos ure, 2,4-D was associated with portal-of-entry effects that consisted of squamous metaplas ia and epithelial hyperp lasia with increased mixed inflammatory cells within the larynx. The incide nce and severity of the effects were increased in a dose-related manner, and the effects persisted after the 4-we ek recovery period, although the incidence and severity were reduced. Clinical signs associ ated 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 increa sed (p<0.05 and 0.01, respectively) in the mid-high and high-dose females, and aspa rtate aminotransferase concentrations were increased (p<0.01) in the high-dose females, but no correlating microsco pic 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) weight s; organ weights were comparable among the male groups.

The systemic toxicity LOAEL is 1.0 mg/L/da y, based on increased alkaline phosphatase and aspartate aminotransferase con centrations, 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 infl ammatory 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/Batc h #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 gr oups throughout the study, but dams at the highdose displayed a decrease (NS) in body-weight ga in during the dosing peri od (79% of control for2, 4-D FINAL

Page 51 of 63 GD 6-15; 57% of control for GD 6-10). The co rrected 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 comp arable among the groups, and there were no dead fetuses. The number of resorptions, as well as pre- and post-implant ation 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 we re 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 dos e 75 mg/kg/day) in a 2-genera tion reproduction study (MRIDs 00150557 and 00163996) in the same strain of rat. Additionally, skeletal findings [2nd wavy ribs, lumbar ribs] and missing st ernebrae were observed in anot her 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 fo r 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 administ ered 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 th e 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 gr oups throughout the study, but body-weig ht 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-wei ght gain, and the high-dose group displayed a negative bodyweight gain. On GDs 15-19, the high-dose gr oup displayed no body-weight gain, and corrected body-weight gain was decr eased 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. One2, 4-D FINAL

Page 52 of 63 control doe had 100% resorptions, and the number of resorption, and pre- and post-implantation losses, were comparable among the groups. Grav id uterine weights also were comparable among the groups.

No differences were observed in mean fetal body we ight due to treatment. At the high-dose, there was an increase (p<0.05) in the percent of live male fetu ses (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, de creased body-weight gain, and c linical signs of toxicity. The developmental toxicity NOAEL is 30 mg/kg/da y, based on abortions at the developmental toxicity LOAEL of 90 mg/kg/day.

Extended One-Generation Repr oduction 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/s ex/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 pr ior to mating and continuing through mating (up to 2 weeks), gestation, and l actation. P1 males were exposed for a minimum of 11 weeks including 7 weeks from the in itiation of the mating phase. P1 females were exposed until lactation day 22 (LD22). A satel lite group of P1 females (12/dose) were subject to the same exposures as the P1 females on the main st udy (exposure for 4 weeks during the pre-mating, up to 2 weeks during the mating period and during gestation until termina tion 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 analys is of toxicity and t oxicokinetic (TK) data collected from the 2,4-D range-finding/TK st udy (MRID 47417901) and titration studies (MRID 47417902). The high dose was set at or slightly a bove the threshold for nonlinear kinetics. The range-finding study confirmed a gender-based diffe rence in the renal clearance of 2,4-D in adult rats, and different high-dose levels were sele cted for the sexes. The male high dose was 40 mg/kg/day (dietary concentrati on of 800 ppm), slightly higher than the inflection point for nonlinear TK in male pups from PND 35 to a dulthood. The female high dose was 30 mg/kg/day (600 ppm), which was clearly higher than the in flection point in female pups and adults throughout the entire (r ange-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,2, 4-D FINAL

Page 53 of 63 mating performance, conception, gestation, parturit ion and lactation, as well as survival, growth and development of the offspring. Selected systemic toxicity parameters we re 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, clin ical chemistry/hematology, thyroid hormone levels, thyroid weights, plasma 2, 4-D levels, hist opathology, and selected re productive parameters during gestation (corpora lutea and implantation numbers).

F1 Generation: F1 offspring were evaluated for potential effect s on the nervous system, immune system, reproductive and endocrine systems, t hyroid function, and other systemic toxicity parameters. 2, 4-D plasma levels were also assesse d 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 offspr ing 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): assessmen t of general systemic and thyroid toxicity, which included clinical chemistry/hematology pa rameters, thyroid hormone assessment, and urinalysis (males only). Post-mortem evaluations in Set 1a (PND 70) included gross path ology, 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), moto r activity and acoustic startle response (ASR). On PND 60, Set 1b animals were perfused for central nervous sy stem (CNS) and peripher al 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 sh eep red blood cell (SRBC) antibody-forming cell (AFC) assay on PND 70-74.

Set 2b (10/sex/dose): assessment of potential de velopmental immunotoxicity (DIT): examination
of innate cellular immun ity using the natural killer cell (NK) assay on PND 87-93.

Set 3 (23-27/sex/dose): assessmen t of reproductive/endocrine toxi city, 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.2, 4-D FINAL

Page 54 of 63 In addition, selected pups cull ed on PND 4 were used to assess thyroid hormone levels. Additional data were gathered from F1 offs pring 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 toxici ty, which included thyroid hormone assessment, organ weights, and post-mortem examinati ons (gross pathology and histopathology) in
10/sex/dose.

Reproductive and selected data from the F1 gene ration were used to assess whether a second generation would be produced. None of the criter ia 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 toxi city in either sex of P1 adults. Body weights and body-weight ga ins were comparable among the groups during the pre-mating and mating phases (both sexes) a nd 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 displaye d 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 attr ibuted to the increase in the actual dose ( ≈ 65 mg/kg/day) above the targeted le vel (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 treatm ent-related effects on hematol ogy, differential white blood cell counts, and prothrombin time, and clinical chemis try and urinalysis parameters were comparable among the groups (both sexes). P1 males displa yed 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) a nd are consistent with previous findings that the kidney is a target or gan. 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 histor ical control range. P1 females at 600 ppm displayed increased uterine weights ( ↑ 17%, both absolute and relative), a lthough 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.2, 4-D FINAL

Page 55 of 63 GD 17 Satellite Females : All P1 satellite females survived to scheduled sacrifice, and body weights were comparable among the groups. Hemato logy 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 vari ability (standard devi ations exceed the means). There was a slight increase in post-im plantation loss at 600 ppm (9.2 vs 5.5). It should
be noted that this observati on 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 in crease in thyroid weight ( ↑ 9%), but there wa s no dose-response. There were no statistically signifi cant, 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 rena l 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, wh ich 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 se x and no differences in nipple/a reolae retention between control and high-dose groups in either sex. F1 males at 800 ppm displayed a 1.6 da ys delay in preputial separation (well within normal variability), whic h 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 statistica lly-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 wa s not dose-related and none of the differences in thyroid hormone levels were sta tistically 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.2, 4-D FINAL

Page 56 of 63 F1 Unselected Offspring (PND 22 weanlings): There were no effects on survival of the unselected weanlings used for systemic toxicity (non-perfused). All tr eated 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 de ficit of 10%. Organ weights were comparable among the groups of females. There were no signif icant 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 we re 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 sc heduled sacrifice. Males at 800 ppm displayed decreased body weight ( ↓ 11%-17%) and body-w eight 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 orga n weights observed in th e 800 ppm males may be
attributed to the 10% decreas e in body weight at termina tion, 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 fi nding 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, alth ough statistical signifi cance 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-re sponse and kidney weights were comparable among the male groups. Decreased thymus weights ( ↓ 12% absolute and ↓ 10% relative) were observed in females at 600 ppm a nd in Set 3 females at 600 ppm ( ↓ 14% absolute and ↓ 13% relative). An increased incidence of degeneration of the proxim al 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 diffe rences 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 neurotoxic ity results were not applicable to EDSP evaluation.2, 4-D FINAL

Page 57 of 63 F1 Offspring Set 3 (PND 90 or 139): Reproductive Toxicity: Ther e were no treatment-related
deaths or clinical si gns of toxicity. Terminal body weights were comparable among the groups (both sexes). No significa nt differences were observed in m ean 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 gla nds. 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 signi ficance 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 se xes 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 pp m (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 rele vant effects were identified in P1 females or in the GD 17 satellite female groups at the highe st 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 t hyroid 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 increa sed kidney weights and increased incidence of degenera tion of the proximal convoluted tubules. The offspring NOAEL is 300 ppm (20.9/ mg/kg bw/day in male s and 23.3 mg/kg/day in females).

The F1 offspring (PND 22) LOAEL is 800/600 pp m, based on decreased body weight observed throughout lactation. The offspr ing NOAEL is 300 ppm. The dose on a mg/kg/day basis for the PND 22 F1 offspring wa s 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 orga n histopathology). The reproductive N OAEL is2, 4-D FINAL

Page 58 of 63 800/600 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 administer ed 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 d eaths or clinical signs of to xicity. 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 term ination, 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 term ination 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 decrea sed body-weight gains were decreases in food consumption in the same groups.

There were dose-related decreases (p<0.05) in T 4 values throughout the st udy 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 T 3 concentration at any dose. Thyroid weights were increased (p<0.05) in the mid-dose females and in both se xes at the high-dose at the 12-month interim sacrifice. At study terminati on, thyroid weights were increase d (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 generall y 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). Simila rly, decreased (NS at 12 months; p<0.05 at 24 months) ovarian weights were observed at th e high-dose at both sacrifice times and in the mid-dose females at study termination. The de creases in testes and ovarian weights are consistent findings in other studie s on 2,4-D and its salts/esters.

Microscopically, there were increased incidences of lesions in the li ver (altered tinctorial properties in mid-dose females, and both sexes at high dose), and thyroi d (hyperplasia in highdose males; hypertrophy and epithelia l cells in high-dose females) at the interim sacrifice. At study termination, there was an increased inciden ce of increased size of the hepatocytes with altered tinctorial properties in both sexes at the high-dose. Tu mor incidence was not affected by

treatment.2, 4-D FINAL

Page 59 of 63 The LOAEL is 75 mg/kg/day based on multiple endpoints, including decreased body-weight gain and food consumption in females, altera tions in hematology and clinical chemistry parameters (including decreased T 4 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 sexe s), 62 (males)/150 (females), and 120 (males)/ 300 (females) mg/kg/day. Additionally, 10 mice/ sex/group were sacrifi ced (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 slight ly lower body weight at the 3- and 6-month intervals (p<0.05; ↓ 4%). Body-weight gains were decrease d (p<0.05) at all doses in the females at the 3-month interval ( ↓ 7%, ↓ 6%, and ↓ 14% at the low-, mid-, a nd high-dose, respectively), and the high-dose females had decreased (p<0.0 5) 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 re productive 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 sexe s in the mid- and highdose groups. There was no treatment-related in crease in any tumor type in either sex.

The LOAEL of 62 mg/kg/day (males)/150 mg/kg/da y (females) is based on increased absolute and/or relative kidney weights, and an increased incidence of re nal microscopic lesions. There
was no treatment-related increase in the in cidence 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 contai ning 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 f eed consumption, or gross abnormalities observed upon necropsy. In addition, there were no treatment -related effects on reproductive parameters.2, 4-D FINAL

Page 60 of 63 (The reviewer detected a significan t reduction in eggs set/eggs laid at the highest treatment dose, 962 ppm. Despite being statisti cally 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 dimethyl amine salt (2,4-D DMA; 99.3%) (MRID 41767701). Hatching success, larval survival, and growth we re 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 to xicity 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 va riability in measured
test concentrations were observed.2, 4-D FINAL

Page 61 of 63 APPENDIX 3: References Not Utilized in the 2, 4-D WoE Analysis

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

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

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

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., Fitc h T.G., James L.M., McCleam A.B., and Adams M.J. Jr. (1984) Vietnam veterans' risks fo r 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 . 11:563-572.2, 4-D FINAL

Page 62 of 63 Evangelista de Duffard, A.M., Bortolozzi, A. and Duffard, R. (1995) Altered behavioral responses in 2,4-Dichlorophenoxyacetic acid tr eated and amphetamine challenged rats. NeuroToxicology . 16:470-488.

Hwang, U-G. (2002) Effect of 2,4-dichlorophenoxy acetic 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 dete rmination 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-te trachlorodibenzo-p-dioxin. J Toxicol. Environ. Health . 8(5- 6):825-34.

Lamb, J.C., Moore, J.A., Marks, T.A., and Hase man, J.K. (1981) Development and viability of offspring of male mice treated with chlorina ted 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 a nd Environmental Sciences, The Dow Chemical Company, Midland, MI, 48674. Study sponsor: Tec hnical 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 form ulation containing 2,4-D and pi cloram 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, a nd breast by 2,4-dichlorophenoxyacetic acid in preand postnatal exposed rats. E nviron Toxicol. 28(1):1-10.

Saghir, S.A., Zablotny, C.L., Bus, J.S., Mart y, 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 offspri ng in preparation for a subsequent F1-extended one-generation toxicity study in rats. Unpub lished Report of The Dow Chemical Company, Midland, MI. Report Number HET K-002372-130. (MRID 47417901)2, 4-D FINAL

Page 63 of 63 Saghir, S.A., Perala, A.W., and Clark, A. J. (2008) A dietary titration study of 2,4- dichlorophenoxyacetic acid (2,4-D) pharmacokinetic s in female CRL:CD(SD) rats. Laboratory Project Study ID 071210. Unpublishe d Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Co mpany, Midland, MI. (MRID 47417902)

Schreinemachers, D. M. (2010). Perturbation of lipids and glucose metabolism associated with previous 2,4-D exposure: a cross-s ectional study of NHANES III data, 1988-1994.
Environmental health : a global access sc ience source. 9:11.

Van den Berg, K.J., van Raaij, J.A., Bragt, P.C., and Notten, W.R. (1991) Interactions of halogenated industrial ch emicals with transthyretin and eff ects on thyroid hormone levels in
vivo. Arch Toxicol . 65(1):15-9.