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 THE ESTROG EN, ANDROGEN OR THYROID

PATHWAYS

CHEMICAL: GLYPHOSATE

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYGLYPHOSATE 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..........................................22

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................22

1. Tier 1 in vivo Assays.............................................................23

2. OSRI...............................................................................23

B. Estrogen Pathway...........................................................................25

C. Androgen Pathway.........................................................................26

D. Thyroid Pathway...........................................................................26

E. Conclusions.................................................................................26

V. EDSP Tier 2 Testing Recommendations....................................................26

VI. References.......................................................................................27

LIST OF TABLES: Table 1. Tier 1 Assays for Glyphosate................................................................7
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Glyphosate ..................................10
Table 3. Androgenic/Anti-Androge nic Pathway for Glyphosate ................................15
Table 4. Thyroid Pathway for Glyphosate .........................................................20

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................32
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................42

APPENDIX 3. References Not Utiliz ed in the Glyphosate WoE Analysis...........................62GLYPHOSATE 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 enerationGLYPHOSATE 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 ConcentrationGLYPHOSATE FINAL

v Abbreviation Terminology

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

OPP Office of Pesticide Programs

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

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

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

T4 Thyroxine (tetraiodothyronine)GLYPHOSATE 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-EvidenceGLYPHOSATE FINAL

Page 1 of 62 Executive Summary

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

In determining whether glyphosate interacts with E, A or T hormone pathways, the number and type of effects induced, the magnitude of respons es, and the pattern of responses observed across studies, taxa, and sexes were c onsidered. Additionally, the conditi ons under which effects occur were considered, in particular, whether or not endocrine-related responses occurred at dose(s) that also resulted in general syst emic toxicity or overt toxicity.

On September 17, 2014, the EDSP Tier 1 Assa y 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 glyphosate with the E, A or T hormone 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).

For the estrogen pathway, while glyphosate showed estrogen receptor (ER) antagonism in vitro with estrogen-dependent human br east cancer cells (Thongprakaisang et al ., 2013), there was no evidence of potential interacti on of glyphosate with the estrogen pathway in the EDSP Tier 1 in

vitro assays [ i.e, ER binding, ER transactivation assay (ERTA), aromatase and steroidogenesis assays]. Additionally, glyphosate was negative in the Tier 1 in vivo mammalian assays ( i.e., uterotrophic or female pubertal assays). In th e fish short-term reproduction assay (FSTRA), the non-treatment-responsive decrease [only significant at mid-treatment] in vitellogenin (VTG) was seen in isolation in the absence of any treatment-related effects in the other estrogen-related endpoints such as gonado-somatic index (GSI), g onadal staging, fecundity and fertilization. In addition, there were no notable gonadal histopat hology. There were no treatment-related effects on female reproductive parameters in the ex isting glyphosate Part 158 mammalian or wildlife studies (decreases in offspr ing body weight observed in one avian reproduction study). Therefore, there is no convincing evidence of a pot ential interaction with the estrogen pathway for glyphosate.

For the androgen pathway, there was no evidence of interaction of glyphos ate with the androgen pathway in the Tier 1 in vitro [ i.e , androgen receptor (AR) binding and steriodogenesis assaysGLYPHOSATE FINAL

Page 2 of 62 were negative] or Tier 1 in vivo FSTRA and mammalian assays ( i.e ., Hershberger and male pubertal assays were negative in the absence of overt toxicity). In addition, glyphosate was negative in an AR transactivation assay (Kojima et al. , 2004). The only treatment-related effects
observed in the Part 158 mammalian studies in the absence of overt toxicity were decreases in sperm count in the subchronic rat study (1678 mg /kg/day) and a delay in preputial separation (PPS)at 1234 mg/kg/day in the post-1998 two-ge neration reproduction study in rats (the EDSP Tier 2 study). Both effects were observed at a dose that was above the limit dose (1000 mg/kg/day) for those studies. No androgen-related effects were seen in the wildlife Part 158 studies (decreases in offspr ing body weight observed in one avian reproduction study).

For the thyroid pathway, there was no convincing evidence of potential inte raction of glyphosate. There were no treatment-related effects on thyr oid hormones (T4 and TSH) , thyroid weights or thyroid histopathology in the male pubertal assay in the absence of overt toxicity. There were no thyroid-related effects observed in the female pubertal assay. In the amphibian metamorphosis assay (AMA), there were no developmental effect s or alterations in thyroid histopathology. No thyroid-related effects were noted in any of the Part 158 studies.

Based on weight of evidence considerations, mamm alian or wildlife EDSP Tier 2 testing is not recommended for glyphosate since there was no c onvincing evidence of potential interaction with the estrogen, androgen or thyroid pathways.GLYPHOSATE FINAL

Page 3 of 62 I. Introduction

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

In determining whether a pesticide chemical in teracts with E, A or T hormone pathways, the number and type of effects induced, the magnit ude of responses, and the pattern of responses observed across studies, taxa and sexes were considered. Additionall y, the conditions under
which effects occur were consid ered, in particular, whether or not endocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On September 17, 2014 the EDSP Tier 1 Assa y 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 glyphosate with the E, A or T hormone 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).

Glyphosate has a vapor pressure of 9.8×10 -8 mm Hg at 25°C, with a water solubility of 10.5 g/L
at 20°C. The octanol/water partition coefficient is log K ow = –3.2 to

− 3.4 with fish bioconcentration factors rangi ng from 0.2-0.63. Based on its vapor pressure and low Henry’s Law constant, glyphosate has low potential to vola tilize from soils or from water. Glyphosate adsorbs strongly to soil and is stable to chemical and photochemical decomposition and hydrolysis; however, it is readil y degraded by soil microbes.

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

Page 4 of 62 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 Orde r 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), and a review of the available scientific liter ature conducted by Health Canada’s Pest Manageme nt Regulatory Agency (USEPA, 2014). The Agency’s review of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team on OPP (USEPA, 2010). Since then, the Ag ency has also conducted a more recent search of available scientific literature for any a dditional relevant information. Summ aries of the available OSRI are presented in Appendix 2. Additiona lly, literature/studies considered but not utilized for the WoE analysis are liste d 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.

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) otherGLYPHOSATE FINAL

Page 5 of 62 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 discerning whether or not the chemical has interacted with the endocrine syst em, are considered. The co llective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indi cation of potential interaction as measured by the study.

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

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

Page 6 of 62 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 ., organ weights or blood chemistry). The circumstances for which a dose/concentration was considered overtly toxic for a particular study are described in Section

IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/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 addres sing the three component s 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.

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,GLYPHOSATE FINAL

Page 7 of 62 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 glyphosate are shown below
in Table 1

.

Table 1 Tier 1 Screening Assays for Glyphosate

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied (MRID 48671305)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300;
OECD 455 Requirement Satisfied (MRID 48671307)

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied (MRID 48671301)
Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550;

OECD 456 Requirement Satisfied by OSRI ( Hecker et al ., 2011)

Aromatase Assay (human recombinant microsomes) OSCPP 890.1200 Requirement Satisfied (MRID 48671303)

Uterotrophic Assay (Rat) OSCPP 890.1600;

OECD 440 Requirement Satisfied (MRID 48677003)

Hershberger Assay (Rat) OSCPP 890.1400;
OECD 441 Requirement Satisfied (MRID 48617001)

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied (MRID 48671315)

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied (MRID 48671313)

Fish Short-term Reproduction Assay OSCPP 890.1350;
OECD 229 Requirement Satisfied (MRID 48671311)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100;

OECD 231 Requirement Satisfied (MRID 48671309)GLYPHOSATE FINAL

Page 8 of 62 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for glyphosate to inte ract 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-estrogenic or HPG axis effects. This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reporte d in the table and not considered further in the WOE assessment.

Glyphosate was negative (or not interactive) in the in vitro estrogen receptor (ER) binding, aromatase, and steroidogenesis assays. In the Tier 1 ER α transcriptional activation (ERTA), the data for the very weak agonist control chemical (17 α -methyltestosterone) did not respond appropriately in the assay, and th erefore, this study was not c onsidered reliable for evaluating potentially very weak agonists. However, it is unlikely that glyphosate is an estrogen receptor agonist because (1) there was no act ivity in an ERTA assay in wh ich the strong and weak agonist reference chemicals (17 β -estradiol and 17 α -estradiol, respectively) showed the expected activity, and (2) negative responses were observed in th e Tier 1 ER binding and uterotrophic assays, which also assess estrogen receptor-related activity. In a literature study by Thongprakaisang et
al. , 2013, glyphosate induced ERE activation in T47D -KBluc cells. Co-incubation of glyphosate with ICI 182780 (ER antagonist) re sulted in a decreased or inhi bited response in these cells.

The uterotrophic assay was negative up to the limit dose (1000 mg/kg/day) . No estrogen-related
effects were observed in the fe male pubertal rat assay up to th e limit dose (1000 mg/kg/day). No estrogen-related effects were seen in the mammalian Part 158 studies.

In the FSTRA, there was a significant decrease in plasma vitellogenin (VTG) concentrations in the medium high treatment group (6.2 mg a.i/L) as compared to the negative control. This reduction from control was 55%, w ith non-dose-dependent decreases in VTG concentrations of approximately 30-33% at all other treatment c oncentrations. Other female-related endpoints, including gonado-somatic index (GSI), gonadal st aging, fecundity, and fertilization success were not significantly affected. A dditionally, there were no notable histopathological diagnoses that were attributed to glyphosate exposure.

In available Part 158 avian repr oduction studies (two with the mallard duck and one with the bobwhite quail), there were no treatment-related e ffects on all reproductive parameters (tested up to 1000 mg/kg-diet). In another mallard duc k reproduction study, test up to 2250 mg/kg-diet, reductions in 14-d offspring body weight were observ ed at 501 mg/kg-diet as well as reductions in male body weight; there were no other effects observed.GLYPHOSATE FINAL

Page 9 of 62 In a OSRI fish study (Le Mer et al. , 2013) with wild larval three-spined sticklebacks ( Gasterosteus aculeatus ), there was no VTG induction observe d in fish treated with glyphosate up to 10 μg/L. Additionally, there were no effects on histology including pr oportions of intersex individuals. In a Part 158 fish life-cycl e toxicity study with fathead minnow, there was no treatment-related effect on any of the reproductive or growth parameters up to 25.7 mg/L.GLYPHOSATEFINALPage 10 of 62Table 2. Estrogenic/Anti-Estrogenic Pathway for GlyphosateLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Glyphosate1Study 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 Obsserved 2 Overt Toxicity Observed 3EDSP Tier 1 AssaysER Binding(MRID 48671305)NERTA(MRID 48671307)NAromatase(MRID 48671303)NSteroidogenesis(MRID 48617005)NUterotrophic(MRID 48617003)NNNFemale Pubertal Rat(MRID 48671315)NNNNNNX(M, H)4NFSTRA(MRID 48671311)NENNNNNNOSRIReceptorTransactivation Assay(Kojimaet al., 2004)NReceptorTransactivation Assay(Thongprakaisangetal., 2013)P590-Day SubchronicToxicity (Rat; MRID40559401)NEN NENNGLYPHOSATEFINALPage 11 of 62Table 2. Estrogenic/Anti-Estrogenic Pathway for GlyphosateLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Glyphosate1Study 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 Obsserved 2 Overt Toxicity Observed 390-Day SubchronicToxicity (Rat; NTP,1992)NEN NEX(H)N90-Day SubchronicToxicity (Mouse;MRID 00036803)NN NEX(H)N90-Day SubchronicToxicity (Mouse; NTP,1992)NEN NE NX(H)X(H)DevelopmentalToxicity (Rat; MRID00046362)NRNX(H)XDevelopmentalToxicity (Rabbit;MRID 00046363)NRNX(M, H)XThree-GenerationReproduction (Rat;MRIDs 00081674,00105995)NENNN NENNNTwo-GenerationReproduction (Rat;MRID 41621501)NENN NENX(H)X(H)Two-GenerationReproduction (Rat;MRIDs 48865101-48865105)NNNNNNX(H)NGLYPHOSATEFINALPage 12 of 62Table 2. Estrogenic/Anti-Estrogenic Pathway for GlyphosateLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Glyphosate1Study 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 Obsserved 2 Overt Toxicity Observed 3Chronic Toxicity(Dog; MRID00153374)NENNNN NCarcinogenicity(Mouse; MRID,00130406)NRNN NRN NChronic Toxicity/Carcinogenicity (Rat;MRIDs 00093879,00150564)NE N N NX(L, M)NChronic Toxicity/Carcinogenicity (Rat;MRID 41643801)NE NE N NEX(H)NAvian Reproduction(Duck; MRID00036328; 00111953)NN NAvian Reproduction(Duck; MRID48876602)P6N N7Avian Reproduction(Quail; MRID00108207)NN NEarly Life-StageToxicity (Sticklebacks;Le Meret al., 2013)NNFish Life CycleToxicity (MRID00108171)NNGLYPHOSATEFINALPage 13 of 621.Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatmentArrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that this parameter is not routinely evaluated or is not applicable in thisassay. Changes in weight are absolute unless otherwise indicated. I= Inconclusive; LW= Liver weight; I= inconclusive2. 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 anX in this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Rales was observed in medium- and high-dose animals (4/15 and 13-15, respectively) at approximately 4 hours post-dose.5. Glyphosate caused hormone-dependent T47D cells to proliferate, approximately 15-30% of control, in the absence of E2. Glyphosate also induced EREactivation by 5- to 13-fold in T47D-KBluc cells compared to control. Both responses were inhibited in the presence of ICI 182780.6. Reductions in 14-d offspring body weight at 501 mg/kg-diet. No effects on egg production or embryo viability were observed.7. Reduction in male body weight gain at 501 mg/kg-diet; no effects on female body weight gain or other clinical signs of toxicity.P Positive findingsN Negative (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedGLYPHOSATE FINAL

Page 14 of 62 2. Effects on Androgen Pathway

The potential for glyphosate to interact with the androgen pathway is summarized in Table 3. The various targets of the androgen pathway across the relevant Ti er 1 assays are delineated so as to facilitate determination of potential for a ndrogenic, anti-androgenic, or HPG axis effects.
This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

Glyphosate was negative (o r a non-binder) in the in vitro androgen receptor (AR) binding (rat prostate cytosol) and steroidogenesis assay.

Glyphosate was also classified as negative in the in vivo Hershberger assay as there were no
treatment-related effects in accessory sex tissue (AST) weights up to the limit dose (1000 mg/kg/day). Glyphosate had no effect on androgenic endpoints in the FSTRA, including GSI, gonadal histology and staging, as well as no effects on VTG.

In the male pubertal assay, decreases were observed in the weights of the ventral prostate, seminal vesicle, LABC, pituitary glands as we ll as a 2.1 day delay in PPS at the high dose (1000 mg/kg/day) but were concurrent with significant decreases in body weight (10%). Additionally, there was no treatment-related changes in serum testosterone concentrations or any histopathological changes in any tissues in at any dose.

In the Part 158 two-generation rat reproducti on study, a delay in attainment of PPS, and increased body weight at a ttainment, occurred in F 1 male pups at 1234 mg/kg/day. In the 90-day subchronic rat study, relative test es weight was increased 21 % at a dose of 3393 mg/kg/day which occurred in the presence of overt toxicity ( ↓ 18% body weight); no changes were observed in absolute weights. Additionally, sperm c ounts were decreased at 1678 and 3393 mg/kg/day; however, no changes in testes histopathology were observed. In the 90-day mouse study, relative testes weight were increased 10 and 18% at a dose of 4776 and 10,780 mg/kg/day, respectively, which was concurrent with decreases in body wei ght greater than 10%. In the carcinogenicity study in mice, relative testes weights were increased (17.5%; NS) at a dose (4500 mg/kg/day) which is approximately four-fol d higher than the limit dose.

In the fish study by Le Mer et al. , 2013, there was no effect on the induction of spiggin (a protein secreted by males to construct nests) observed in any of the treatment groups. Additionally, while effects on male body weight gain and offspr ing body weight were obser ved in one Part 158 mallard duck reproduction study, there were no othe r androgen-related effects observed in the Part 158 avian reproduction studi es or fish life-cycle study.GLYPHOSATEFINALPage 15 of 62Table 3. Androgenic/Anti-Androgenic Pathway for GlyphosateLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Glyphosate1Study 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 1 AssaysAR Binding (MRID48671301)NSteroidogenesis(MRID 48617005)NHershberger (MRID48617001)NENN NMale Pubertal Rat(MRID 48671313)NNNNNNNNX4BW:↓10%(H)FSTRA(MRID 48671311)NENNNNN NE NOSRIReceptorTransactivationAssay (Kojimaetal., 2004)N90-Day SubchronicToxicity (Rat;MRID 40559401)N N N N NEN N90-Day SubchronicToxicity (Rat; NTP,1992)N N N N NEP5X(H)X(H)GLYPHOSATEFINALPage 16 of 62Table 3. Androgenic/Anti-Androgenic Pathway for GlyphosateLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Glyphosate1Study 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 390-Day SubchronicToxicity (Mouse;MRID 00036803)N N NE N NEX(H)N90-Day SubchronicToxicity (Mouse;NTP, 1992)N NN N NEX(M, MH, H)X(MH,H)Three-GenerationReproduction (Rat;MRIDs 00081674,00105995)NNNNN NTwo-GenerationReproduction (Rat;MRID 41621501)NNNNNNX(H)X(H)Two-GenerationReproduction (Rat;MRIDs 48865101-48865105)NN N N NN NAge:↑2.9 dWt:↑10%(H)6N NChronic Toxicity(Dog; MRID00153374)NN NE NR NN NCarcinogenicity(Mouse; MRID00130406)NN NR NR NRX(H)NGLYPHOSATEFINALPage 17 of 62Table 3. Androgenic/Anti-Androgenic Pathway for GlyphosateLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Glyphosate1Study 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 3Chronic Toxicity/Carcinogenicity(Rat; MRIDs00093879,00150564)NN NE N NN NChronic Toxicity/Carcinogenicity(Rat; MRID41643801)NN N N NEX(H)NAvian Reproduction(Duck; MRID00036328)NN NAvian Reproduction(Duck; MRID00111953)NN NAvian Reproduction(Duck: MRID48876602)P7N N8Avian Reproduction(Quail; MRID00108207)NN NEarly Life-StageToxicity(Sticklebacks; LeMeret al., 2013)N9NGLYPHOSATEFINALPage 18 of 62Table 3. Androgenic/Anti-Androgenic Pathway for GlyphosateLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Glyphosate1Study 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 3Fish Life CycleToxicity (MRID00108171)NN1.Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows (↓or↑)indicate the direction of the response. A shaded cell indicates that this parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate(DP), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP).; BW= Body weight; BWG: Bodyweight gain;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. Rales were observed in 9/15 and 14/15 males in the medium- and high-dose groups, respectively, approximately 4 hours post-dosing. This finding persistedduring the daily examinations in 7/15 high-dose males throughout the study.5.Sperm counts were decreased at 1678 mg/kg/day in the absence ofovert toxicity; however epididymal sperm motility, total spermatid head/testis, and totalspermatid heads/g caudal tissue in the treated animals were similar to control.6.Delay in attainment of PPS, and increased body weight atattainment, occurred in F1 male pups at 1234 mg/kg/day.7.Reductions in 14-d offspring body weight at 501 mg/kg-diet. Noeffects on egg production or embryo viability were observed.8.Reduction in male body weight gain at 501 mg/kg-diet; no effects on female body weight gain or other clinical signs of toxicity9.No induction of spiggin (a protein secreted by males to construct nests) in fishP Positive findingsN Negative (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedGLYPHOSATE FINAL

Page 19 of 62 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it
does include three in vivo assays that provide data to de tect changes in the HPT axis, i.e ., the pubertal female and male (rat) assays, and the AMA (frog).

The potential for glyphosate to interact w ith thyroid regulation is 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, there were no effects on median NF developmental st age and, no asynchronous development was observed. There was a signific ant decrease (p<0.05) in Day 7 normalized hind limb length at the middle treatment concentration (4.3 mg a.i/L) but this effect was not observed at Day 21 along with no significant (p>0.05) effect s at any other concentration for Days 7 and
21. There was no treatment-related effects on t hyroid histopathology. There was a significant increase (p<0.05) in Day 21 snout-vent-length (SVL) in the middle, middle-high and high treatment concentrations ( ↑ 5.2, 2.5 and 6.8%, respectively). Add itionally, there was a significant increase in Day 21 body we ight at the highest tr eatment concentration ( ↑ 17%). In a study examining the chronic effects of glyphosate and its formulations to four North American frog species (Howe et al, 2004), treatm ent with glyphosate alone did not reveal any effects on growth (length), tail morphology, percen t surviving to reach Gosner Stage 42, time to metamorphosis, gonadal development or thyroid gene expression rela tive to controls. It is noted that control mortality was relatively high in this study under chronic exposure (38%), and as such is not
included in table below.

There were no thyroid-rela ted effects observed in the female pube rtal assay. In the male pubertal assay, while total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) concentrations were decreased compared to control in all glyphosate treatment groups, they were not statistically significant and were not consider ed to be treatment-related. Additionally, there was a slight increase in the number of high-dose males with decreased colloid area that occurred in the presence of overt toxicity ( ↓ 10% body weight), and there were no treatment-related effects on follicular cell height at any dose compared to control. Furthermore, there were no effects on thyroid function observed in any of the mammalian Part 158 toxicity studies.GLYPHOSATE FINAL

Page 20 of 62 Table 4. Thyroid Pathway for Glyphosate

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Glyphosate 1

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmental stage(± or asynchronous,HLL)Growth (BW, BL,SVL)Systemic Toxicity2Overt Toxicity3

EDSP Tier 1 Assays

Female Pubertal
Rat (MRID 48671315) N N N N N P (M, H) 4 N

Male Pubertal Rat (MRID 48671313) N N N N N P (M, H) 5 BW: ↓ 10%

(H)

AMA (Frog; MRID 48671309) N N Day 21: SVL: ↑ 5, 3,7% (M, MH,

H) 6 ; BW: ↑ 17% (H) 7 N

OSRI 90-Day Subchronic Toxicity (Rat; MRID 40559401) NE N NE N N

90-Day Subchronic Toxicity (Rat; NTP, 1992) NE N NE X (H) X (H)

90-Day Subchronic Toxicity (Mouse; MRID 00036803) NE N NE X (H) N

90-Day Subchronic Toxicity (Mouse; NTP, 1992) NE N NE X (M, MH, H) X (MH,

H) Three-Generation Reproduction (Rat; MRIDs 00081674, 00105995) NE N N N N

Two-Generation Reproduction (Rat; MRID 41621501) NE NE NE X (H) X (H)

Two-Generation Reproduction (Rat;
MRIDs 48865101- 48865105) N N N X (H) N

Chronic Toxicity (Dog; MRID 00153374) N N N N NGLYPHOSATE FINAL

Page 21 of 62 Table 4. Thyroid Pathway for Glyphosate

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Glyphosate 1

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmental stage(± or asynchronous,HLL)Growth (BW, BL,SVL)Systemic Toxicity2Overt Toxicity3

Carcinogenicity (Mouse; MRID, 00130406) N N N X (H) N

Chronic Toxicity/ Carcinogenicity (Rat; MRIDs 00093879, 00150564) N N N X (L, M) N

Chronic Toxicity/ Carcinogenicity (Rat; MRID 41643801) NE N NE X (H) N

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows ( ↓ or ↑ ) indicate the direction of the re sponse. A shaded cell indicates that this parameter is not routinely evaluated or is not a pplicable in this assay. Changes in weight are absolute unless otherwise indicated. 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. Rales was observed in medium- and high-dose animals (4/15 and 13-15, respectively) at approximately 4 hours post-dose. 5. Rales were observed in 9/15 and 14/15 males in the medium- and high-dose groups, respectively, approximately 4 hours post-dosing. This finding persisted during the daily examinations in 7/15 high-dose males throughout the study. 6. Snout-vent length was increased in a non-dose-dependent manner in the top three dose groups on Day 21. 7. Body weight was increased 17% in the high-dose group on Day 21. P Positive findings N Negative findings NE Not examinedGLYPHOSATE FINAL

Page 22 of 62 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 glyphosate to interact with 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 sequelae of systemic effects. E ndocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence evalua tion of the results of the Ti er 1 assays 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).

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 and OSRI
studies are described below. Generally, one parameter (i.e ., death or >10% decrease in mammalian body weight) is 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).GLYPHOSATE FINAL

Page 23 of 62 1. Tier 1 in vivo Assays

In the Hershberger assay and the uterotrophic assays, all animals survived until scheduled termination, and no treatment-related clinical find ings were noted in any of the animals during the study. In the female pubertal rat assay, one animal in the control group was sacrificed in

extremis on PND 27 due to impairment of the right fore limb (due to possible mechanical injury);
all other animals survived until scheduled sacrific e. In the male pubertal rat assay, one high-dose male was found dead prior to dosing on PND 24; no significant clinical or macroscopic findings were observed in this animal, with all other rats surviving unt il scheduled sacrifice. Treatmentrelated clinical signs in the pube rtal studies were limited to ra les in 4/15 females and 9/15 males at the medium dose (approximately 4 hours post- dosing), and in 13/15 females and 14/15 males at the high dose. This finding di d not persist to the daily examin ations at the medium dose, but were present in the daily examinations in 7/ 15 males at the high dose throughout the study. No
other treatment-related clini cal signs were noted during th e 4-hour post-dosing or daily examinations at any dose level. Final body weight s were decreased by 7 and 10% in the mediumand high-dose males. Moderate d ecreases in liver weights (p<0.05; ↓ 10 and 15%) also were also noted in the medium- and high-dose males, but cons idered secondary to d ecreased body weights.

In the FSTRA, survival was 100% in the negati ve control and all trea tment groups, except the medium-dose treatment group (one male mortalit y). Glyphosate did not resu lt in any significant changes in weight or length for either sex at any treatment level. There were no observed effects on secondary sex characterist ics and clinical signs ( i.e. , behavioral and other sub-lethal effects) in male or female fish at any treatment group. Additionally, glyphosate exposure also did not affect the survival of tadpoles at any concentration in the AMA, and no clinical signs of toxicity
were observed.

2. OSRI

In a subchronic oral toxicity study in rats conducted by the National Toxicology Program (NTP; 1992), all animals survived to necropsy. Diarrhea was noted in the high-dose animals through Day 50 of the study. Body weight gain was reduc ed in the high-dose males (3393 mg/kg/day), and the final body weight was decreased 18%, but st atistical significance wa s not reported. Mean necropsy body weights were decreased in the 1678 and 3393 mg/kg/day males ( ↓ 12 and 21%, respectively). Body weight gain in high-dose females was decrea sed, but no effects on final or necropsy body weights were obser ved. Non-dose-dependent increase s in relative liver weights also were observed in males in all dose groups and in females in the two highest dose groups. These findings, combined with clinical chemistr y changes, are consistent with a hepatobiliary
effect that can be attributed to glyphosate administration.

In a subchronic oral toxicity study in mice, ther e were no treatment-relate d toxicological signs or mortalities during the study and no effects of treatment on food consumption. Body weight gains in the high-dose male and female groups were decreased compared to control.GLYPHOSATE FINAL

Page 24 of 62 In a subchronic oral toxicity study in mi ce conducted by NTP (1992), there were two nontreatment-related mortalities in the study. In addition, no clinical si gns of toxicity were reported. Final body weights were decreased in males a nd females in the two highest dose groups ( ↓ >10%
for males and ≤ 10% for females) with concomitant decr eases in body weight gain; statistical significance was not reported. Me an necropsy body weights were decreased for the medium-high (4776 and 5846 mg/kg/day, respectively) and high-dose (10,780 and 11,977 mg/kg/day, respectively) males and females. Non-dose-dependent increases in relative liver weight also were noted in males of all dose groups, except at the lo w dose. A decrease in absolute liver weight also was noted in high-dose females.

In a developmental toxicity study in rats, ther e were six deaths in the high-dose group, but none
of the deaths were determined to be related to treatment. At the high-dose, the majority of the dams (all but three) were observed at least once with diarrhea, soft stool s, breathing rattles, inactivity, and red matter in the region of nose, mouth, forelimbs, or dorsal head. No treatmentrelated clinical signs were obs erved in the low- and mid-dose groups; additionally, there were no
treatment-related effects on mate rnal body weight in these groups . At the high dose, maternal body weight gain was decreased during the test period (GDs 0-20) due to body weight losses during GDs 6-20 ( ≤ 10%). In a developmental toxicity study in rabbits, five rabbits aborted and were sacrificed. Additionally, rabbits at the me dium and the high dose died during the study; of these, five deaths were determined to not be re lated to treatment, but the cause of death for the remaining eight rabbits could not be determined at necropsy. Soft stool or diarrhea was noted in all groups, with a slight increase at the medium dose and at least one incidence in each rabbit of the high-dose group. In addition, an increase in nasal discharge was noted in the high-dose
animals. There were no toxicol ogically significant differences in maternal mean body weight among control and treated groups.

There were no treatment-related mortalities in a two-generation reproduc tion study with rats. Treatment-related clinical signs consisted of soft stools in high -dose males and females (both F 0 and F 1 ). Body weights of high-dose male and female F 0 rats were decrea sed (<10%) during the pre-mating growth period. Similarly, F 1 high-dose male and female adults weighed less at weaning compared to F 0 animals with continued decreases of 10-12%. The body weights of high-dose females remained decreased compared to control during gestati on and lactation of the

F 1 , F 2 a, and F 2 b litters, although the high-dose group body we ights were comparable to control by the end of lactation. Food consumption was ge nerally similar to control in high-dose males and females during most measurement periods; co nsumption was decreased in the medium- and high-dose F 1 females on pre-mating Days 0-14 ( ↓ 9 and 11%, respectively). Terminal body weights in the F 0 and F 1 a generations were decreased by 8-13 %. In a subsequent two-generation reproduction study with rats, there were no treat ment-related effects on clinical signs or mortality. No treatment-related effects were obs erved on mortality, clinic al signs, body weights, body weight gains, or food consumption. Absolute and relative (to body) liver weights wereGLYPHOSATE FINAL

Page 25 of 62 increased by 8-13% in both the P and F1 females at in the high-dose group with no histopathological findings.

In a chronic oral toxicity/car cinogenicity study in rats, surviv al was unaffected by treatment. Decreased body weight gains were observed in treated males beginning at Week 26 and continuing to Week 102, and decreased body weight gains were present in the low- and mediumdose female rats from Weeks 84 to 92. No dos e-response relationship was apparent in the decreased body weight gains in female rats, and the changes in body we ight did not affect survival. In a subsequent chronic oral toxicity /carcinogenicity study in rats, there were also no
treatment-related effects on c linical signs or mortality. Th e mean body weight of high-dose females was decreased by 13% at Week 81, with decreased a body weight gain of 23% compared to control. There were no decreases in body we ight or body weight gain in male rats. Liver weight (relative to body weight) was increased ( ↑ 13%) in high-dose males at interim sacrifice, and absolute liver weights were increased ( ↑ 13%) in high-dose males at terminal sacrifice. In a carcinogenicity study in mice, there were no trea tment-related toxic signs noted during the study, and mortality was low during the first eigh teen months of the study. Body weight was consistently decreased for males and, to a le sser extent, females at the high-dose (4500 mg/kg/day) during the study at se veral sampling intervals, with no treatment-related changes in body weight at the low- and mid-dose groups.

No treatment-related mortalities, clinical signs of toxicity, or body wei ght/body weight gain changes were observed in a subchr onic oral toxicity study in ra ts, a three-generation reproduction study in rats, a chronic toxicity study in dogs, or the avian reproduction studies in mallard ducks and bobwhite quail. In another mallard duck re production study, reductions in male body weight gain were observed at 501 mg /kg-diet and 2250 mg/kg-diet.

B. Estrogen Pathway

While glyphosate showed ER antagonism in vitro with estrogen-dependent human breast cancer cells (Thongprakaisang et al., 2013), there was no evidence of potential interaction of glyphosate with the estrogen pathway in the Tier 1 in vitro assays ( i.e, ER binding, ERTA, aromatase and steroidogenesis assays). Additionally, glyphosate was negative in the Tier 1 in vivo mammalian assays ( i.e., uterotrophic or female pubert al assays). In the fish short-term reproduction assay (FSTRA), the non-treatment-responsive decrease [only significant at mid-treatment] in vitellogenin (VTG) was seen in isolation in the absence of any treatment-related effects in the other estrogen-related endpoints su ch as gonado-somatic index (GSI), gonadal staging, fecundity and fertilization. In addition, there were no notable gonadal histopat hology. There were no treatment-related effects on female reproductive parameters in the existing glyphosate Part 158 mammalian or wildlife studies (decreases in offspring body weight observed in one avian reproduction study). Therefore, there is no convincing evidence of a poten tial interaction with the estrogen pathway for glyphosate.GLYPHOSATE FINAL

Page 26 of 62 C. Androgen Pathway

There was no evidence of interaction of glyphos ate with the androgen pathway in the Tier 1 in

vitro ( i.e , AR binding and steriodogenesis a ssays were negative) or Tier 1 in vivo FSTRA and mammalian assays ( i.e ., Hershberger and male pubertal assays were negative in the absence of overt toxicity). In addition, gl yphosate was negative in an AR transactivation assay (Kojima et
al. , 2004). The only treatment-related effects obser ved in the Part 158 mammalian studies in the absence of overt toxicity were decreases in sperm count in the subchronic rat study (1678 mg/kg/day) and a delay PPS in the post 1998 2- generation reproduction study (1234 mg/kg/day)
in the rat. Both effects were observed at a dose that was above the limit dose (1000 mg/kg/day) for those studies. No androgen-related effect s were seen in the wildlife Part 158 studies (decreases in offspring body weight obser ved in one avian re production study).

D. Thyroid Pathway

There was no convincing evidence of potential interaction of glyphosat e with the thyroid pathway. There were no treatment-related eff ects on thyroid hormones (T4 and TSH), thyroid weights or thyroid histopathology in the male pubertal assay in the absence of overt toxicity. There were no thyroid-related e ffects observed in the female pubertal assay. In the AMA, there were no developmental effects or alterations in thyroid histopathology. No thyroid-related effects were noted in any of the Part 158 studies.

E. Conclusions

The conclusion of the WoE evaluation is that glyphosate demonstrates no convincing evidence of potential interaction with the es trogen, androgen or thyroid pathwa ys in mammals or wildlife.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations, mamm alian or wildlife EDSP Tier 2 testing is not recommended for glyphosate since there was no c onvincing evidence of potential interaction with the estrogen, androgen or thyroid pathways.GLYPHOSATE FINAL

Page 27 of 62 VI. References

Beavers, J. and Fink, R. (1978) Final Repor t: One-generation Reproduction study-Bobwhite Quail: Glyphosate Technical: Project N o. 139-141. (Unpublished study received Nov 13, 1978 under 524-308; prepared by W ildlife International, Ltd., submitted by Monsanto Co., Washington, DC; CDL: 235924-B). (MRID 00108207)

Beavers, J. and Fink, R. (1978) Final Report: One-generation Reproductio n Study-Mallard Duck: Glyphosate Technical: Project No. 139-143. (U npublished study received Nov 13, 1978 under 524-308; prepared by Wildlife International Ltd., submitted by Monsanto Co., Washington, DC; CDL:235924-A). (MRID 00111953)

Dhinsa, N.K.; Watson, P; Brooks, P.N. (2007) Glyphosate technical: dietary two generation reproduction study in the rat. Unpublished study conducted by Safepharm Laboratories Limited, Derbyshire, UK. SPL Project Number : 2060/0013, November 6, 2007. (MRID 48865101)

Dhinsa, N.K.; Watson, P; Brooks, P.N. (2012) Glyphosate technical: dietary two generation reproduction study in the rat; co rtical vacuolation historical data. Unpublished study conducted by Safepharm Laboratories Limited, Derbyshire , UK. SPL Project Number: 2060/0013, June 18, 2012. (MRID 48865105)

EG & G, Bionomics. (1975) Chronic Toxicity of Glyphosate to the Fathead Minnow ( Pimephales promelas , Rafinesque). (Unpublished study received Dec 27, 1978 under 524-308; submitted by Monsanto Co., Washi ngton, DC; CDL:097759-B). (MRID 00108171)

Frey, L.T. 2012. Glyphosate-acid: A Re production Study with the Mallard (Anas platyrhynchos). Unpublished study performed by Wildlife Intern ational Ltd., Easton, MD . Laboratory Study No. 123-187. Study sponsored by Syngenta Crop Protection, LLC, Greensboro. NC. Study initiated May 26, 1998 and completed January 11, 1999. (MRID 48876602).

Hecker, M., Hollert H., Cooper, R., et al . (2011) The OECD validati on program of the H295R steroidogenesis assay: phase 3. Final inte r-laboratory validation study. MRID 48617005.
Published: Environ. Sci. Po llut. Res. (2011) 18:503-515.

Howe, C. M, M. Berrill, B. D. Pauli, C. C. Helbing, K. Werry and N. Veldhoen. 2004. Toxicity of Glyphosate-based Pesticides to Four North American Frog Species. Society of Environmental Toxicology and Chemistry 23(8): 1928 - 1938.

Kojima, H., Iida, M., Katsura, E., Kanetoshi, A., Hori, Y., and Kobayashi, K. (2003) Effects of a diphenyl ether-type herbicide, chlornitrofen, and its amino de rivative on androgen and estrogen receptor activities. Environ. Health Perspect. 111:497–502.GLYPHOSATE FINAL

Page 28 of 62 Kojima, H., Katsura, E., Takeuchi, S., Niiyami, 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. Environ. Health Perspect . 112 (5): 524-531.

Knezevich, A. and Hogan, G. (1983) A Chr onic Feeding Study of Glyphosate (Roundup Technical) in Mice: Project No. 77-2061: BDN-77420. Final rept. (Unpub lished study received Aug 17, 1983 under 524-308; prepared by Bio/dyna mics, Inc., submitted by Monsanto Co., Washington, DC; CDL:251007-A; 251008; 251009; 251010; 251011; 251012;251013;251014). (MRID 00130406)

Lankas, G.R. and Hogan, G.K. (1981) A Li fetime Feeding Study of Glyphosate (Roundup Technical) in Rats: Project No. 772062. (U npublished study received Jan 20, 1982 under 524- 308; prepared by Bio/dynamics, Inc., submitted by Monsanto Co., Washington, D.C.; CDL:246617-A; 246618; 246619; 24 6620; 246621). (MRID 00093879)

Le Mer, C., Roy, R.L., Pellerin, J., Couillard, C.M ., and Maltais, D. (2013) Effects of chronic exposures to the herbicides at razine and glyphosate to larvae of the threespine stickleback ( Gasterosteus aculeatus ). Ecotoxicol. Environ. Saf. 89: 174-181.

McConnel, R. (1985) A Chronic Feeding St udy of Glyphosate (Roundup Technical in Mice): Pathology Report on Additional Kidney Sections: Addendum to Final Report Dated July 21, 1983: Project No. 77-2061A. Unpublished study pr epared by Bio/dynamics Inc. 59 p. (MRID 00150564)

National Toxicology Program. (1992) Technical Report on Toxicity St udies of Glyphosate (CAS No. 1071-83-6) Administered in Dosed Feed to F344/N Rats and B6C3Fl Mice , Toxicity Report Series Number 16, NIH Publication 92-3135, July 1992. U.S. Department of Health and Human Services, Research Triangle Park, NC.

Reyna, M. (1985) Twelve Month Study of Glyp hosate Administered by Gelatin Capsule to Beagle Dogs: Project No. ML-83-137: Study No. 830116. Unpublished study prepared by Monsanto Company Environmental Health. 317 p. (MRID 00153374)

Reyna M. (1990) Two Generation Reproduction Feeding Study with Glyphosate in Sprague Dawley Rats: Lab Project No: MSL-10387, Study No. ML 88-106. Unpublished study prepared by Monsanto Agricultural Co. (MRID 41621501)

Rodwell, D.E., Tasker, E.J., Blair, A.M. et al. (1980) Teratology Study in Rabbits: IRDC No. 401056, Study No. IR 79-018. (Unpublished study; pr epared by International Research and Development Corp., submitted by Monsanto C o., Washington, D.C.; CDL: 242516-B). (MRID 00046363)GLYPHOSATE FINAL

Page 29 of 62 Rodwell, D.E., Tasker, E.J. Blair, A.M. et al. (l980) Teratology Study in Rats: IRDC No. 401- 054, Study No. IR 79-016. (Unpublished study incl uding IRDC no. 999-021; received May 23, 1980 under 524-308; prepared by Inte rnational Research and Development Corp., submitted by Monsanto Co., Washington, D.C.; CDL:242516-A. (MRID 00046362)

Schneider, S.Z., Kendall, T.Z., and Kruege r, H.O. (2012) Glyphosate: Amphibian Metamorphosis Assay for the Detection of Thyroid Active Substances. Unpublished study conducted by Wildlife Interna tional, Ltd., Easton, Maryland 21601. Laboratory report number 707A-103. Study sponsored by Joint Glyphosate Task Force c/o Data Group Management, Inc., Raleigh, North Carolina 27615. Study co mpleted April 11, 2012. (MRID 48671309)

Schneider, S.Z., Martin, K.H., Kendall, T.Z., a nd Krueger, H.O. (2012) Glyphosate: Fish ShortTerm Reproduction Assay with the Fathead Minnow ( Pimephales promelas ). Unpublished study conducted by Wildlife Interna tional, Ltd., Easton, MD 21601. Laboratory report number 707A102A. Study sponsored by Joint Glyphosate Task Force c/o Data Group Management, Inc., Raleigh, NC 27615. Study completed April 11, 2012. (MRID 48671311)

Schroeder, R.E. and Hoga n, G.K. (1981) A Three-Generation Reproduction Study with Glyphosate in Rats: Project No. 77-2, Study No. BDN 77-417 (Unpublished study received Sep 22,1981 under 524-308; prepared by Bio/dynamics , Inc., submitted by Monsanto Co., Washington, D.C.; CDL: 245909-A). (MRID 00081674)

Stout, L. and Johnson, C. (1987) 90-day Study of Glyphosate Administered in Feed to Sprague Dawley Rats: Proj. ID ML-86-351/EHL 86128. Unpublished study prepared by Monsanto Agricultural Co. (MRID 40559401)

Stout, L. and Ruecker, F. (1990) Chronic Study of Glyphosate admi nistered in Feed to Albino Rats: Lab Project Number: MSL-I0495: R.D. 1014, Study No. ML 87-148. Unpublished study prepared by Monsanto Agricultural Co. (MRID 41643801)

Street, R. (1982) Letter sent to R. Taylor dated Jul 6, 1982: Roundup Herbicide: Addendum to pathology report for a thr ee-generation reproduction study in ra ts with glyphosate. (Unpublished study received Jul 7, 1982 under 524-308; submitted by Monsanto Co., Washington, DC; CDL:247793-A). (MRID 00105995)

Street, R.W., Conkin, R.A., Edwards, G.A., et al. (1980) A Three-Month Feeding Study of Glyphosate in Mice: Special Report# MSL- 1154. (Unpublished study received Jul 2, 1980 under 524308; submitted by Monsanto Co., Washington, D.C.; CDL:242799. (MRID 00036803)

Stump, D. G. (2012) A Hershberger Assay of Gl yphosate Administered Orally in Peripubertal Orchidoepididymectomized Rats. Unpublished st udy conducted by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Report No.: WIL-843003, January 6, 2012. (MRID 48617001)GLYPHOSATE FINAL

Page 30 of 62 Stump, D.G. (2012) A Pubertal Development and Thyroid Function Assay of Glyphosate Administered Orally in Intact Juvenile/Peripubertal Female Rats. Unpublished study conducted by WIL Research Laboratories, LLC, Ashla nd, OH, Laboratory Project ID: WIL-843007, April 10, 2012. (MRID 48671315)

Stump, D.G. (2012) A Pubertal Development and Thyroid Function Assay of Glyphosate Administered Orally in Intact Juvenile/Peripubertal Male Ra ts. Unpublished study conducted by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Project ID: WIL-843005, April 10, 2012. (MRID 48671313)

Stump, D. G. (2012) A Uterotrophic Assay of Glyphosate Administered Orally in Ovariectomized Rats. Unpublished study conduc ted by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Report No.: WIL-843002, January 6, 2012. (MRID 48617003)

Thongprakaisang, S., Thiantanawat, A., Rangkadilok, N., Suriyo, T., and Satayavivad, J. (2013) Glyphosate induces human breast cancer cells growth via estrogen receptors. Food Chem.
Toxicol . 59:129-136.

USEPA (2010) Glyphosate: Report of the Endocrine Disruptor Review Team; Test Order #: EDSP- 417300-229; 230; 240; 241; 244; 246; 247; and 248, December 16, 2010. Office of Chemical Safety and Pollution 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 th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention Science Coordina tion and Policy. U.S. Environmental Protection Agency, Washington, DC.

USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
held July 30 - August 1, 2013 on Weight-of

- Evidence: Evaluati ng Results of EDS P Tier 1 Screening. Office of Chemical Safety and Pollution Pr evention. U.S. Environmental Protection Agency, Washington, DC.

USEPA (2014) Glyphosate: Lite rature Search Review. March 7, 2014. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.

Wilga, P.C. (2012) Glyphosate: Human R ecombinant Aromatase Assay. Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI . Laboratory Study No.: 6500V-100334AROM, March 9, 2012. (MRID 48671303)

Willoughby, J.A. (2012) Estrogen Receptor Transcri ptional Activa tion (Human Cell Line (HeLa9903)) Screening Assay with Glyphosate. Unpu blished study conducted by CeeTox, Kalamazoo, MI. Laboratory Report No.: 6500V- 100334ERTA, March 8, 2012. (MRID 48671307)GLYPHOSATE FINAL

Page 31 of 62 Willoughby, J.A. (2012) Glyphosate: Androgen Receptor Binding (Rat Prostate Cytosol) Screening Assay. Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 6500V-100334ARB, March 8, 2012. (MRID 48671301)

Willoughby, J.A. (2012) Glyphosate: Estrogen Receptor Binding (Rat Uterine Cytosol). Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 6500V100364ERB, March 8, 2012. (MRID 48671305)

Willoughby, J.A. (2012) Supplemental Information - Laboratory Proficiency Data for ERTA assays and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. July 19, 2011. MRID 48843501)GLYPHOSATE FINAL

Page 32 of 62 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100)

The 21-day assay (MRID 48671309) of gl yphosate (85.14% purity) on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 per control and treatment group) were exposed to nominal concentrati ons of 0 (negative control), 0.16, 0.80, 4.0, 20, and 100 mg a.i./L. Mean-measured concentrations were <0.100 (<LOQ; control), 0.13, 0.79, 4.3, 20, and 90 mg a.i./L. The test system was maintain ed at 21.4 to 22.3°C and a pH of 7.0 to 8.3.

The survival of tadpoles exposed to glyphosate wa s not significantly affected (p>0.05) as it was 100% in the control group and 99% in each of the treatment concentrations. Tail curvature was the only observed clinical sign (i.e ., behavioral and other sublethal effects) at test termination and occurred in 46 to 57 (out of 80 exposed) tad poles in the control and treatment groups (64, 63, 65, 53, 53, and 78% of tadpoles in the negative control, 0.13, 0.79, 4.3, 20, and 90 mg a.i./L treatment groups, respectively), and did not appear to be treatment-related.

Glyphosate caused no significant acceleration or delay of median NF developmental stage throughout the test. Further, no asynchronous de velopment was observed. No tadpoles in the control and treatment groups developed beyond NF stage 57. Glyphosate exposure did not cause significant effects on Day 7 or 21 normalized (for snout-vent length) hind -limb length (HLL) at any concentration tested except a significant decrease (p<0.05) at the mid (4.3 mg a.i/L) concentration ( ↓ 15.6%) at Day 7. This effect was not obs erved in any treatment group at Day 21. Snout-vent length (SVL) was not si gnificantly affected at any treatment concentration at Day 7 but was significantly increased (p <0.05) in the 4.3, 20, and 90 mg a.i/L treatment concentrations at Day 21 ( ↑ 5.2, 2.5, and 6.7%, respectively) compared to the control. Additionally there was a significant increase in Day 21 body weight at 90 mg a.i./L ( ⇖ 17%).

There were no treatment-related effects on thyroi d gland histopathology at any treatment level, with comparable incidence and severity of thyroid gland atrophy and hypertrophy, and follicular cell hypertrophy and hyperplasia in the control and treatment concentrations. While there appeared to be an increased in cidence of mild thyroid gland hypertrophy at the highest treatment concentration, the same incidence was observed at the lowest treatment concentration and the effect was not concentration responsive. Similar findings were observed for follicular cell height increase: an apparent increase in mild severity at the top concentration with a similar incidence at
the lowest treatment concentration and no c oncentration-responsive pattern. Finally, the pathologist report indicated that there were no treatment-related ch anges in the thyroid glands of tadpoles exposed to glyphosate when compar ed to those in the negative control.GLYPHOSATE FINAL

Page 33 of 62 Androgen Receptor (AR) Bindi ng Assay; (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48617301), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct saturation and competitive binding experiments. The saturation binding experiment was conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonab le numbers and was functioning with appropriate affinity for the radio-labeled reference a ndrogen (R1881). The competitive binding assay measured the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (logarithmic increase from 10

− 10 to 10

− 3 M) of glyphosate (95.93% glyphosate acid, 85.14% calculated glyphosate co ntent, Batch # GLP-1103-21149-T). Low-salt TEGD buffer was used as the vehicle for glyphosate. A total of 3 runs were performed, and each run included dexamethasone as a weak positive control, and R1881 as the ligand reference
standard.

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. The dissociation constant (K d ) for [ 3 H]-R1881 was
0.613±0.041 nM and the estimated B max was 0.817±0.049 fmol/100 μg protein for the single batch of prostate cytosol that was prepared. The mean and individual K d values were below the range reported in the EPA validation program (0.685 to 1.57 nM). Confidence in these numbers is high according to the goodness of fit (R 2 = 0.957-0.984) and the small variation among runs.

In the competitive binding experiment, the estimated mean log IC 50 s for R1881 and the weak positive control (dexamethasone) were

− 9.0 and

− 4.6 M, respectively, and the mean relative binding affinity (RBA) for the weak positive co ntrol, dexamethasone, was 0.004%. Confidence in the numbers for the reference standards is high as the values as variation between runs was small. All performance criteria were met.

At glyphosate concentrations of 10

− 10 to 10

− 3 M, specific binding of [ 3 H]-R1881 was 92.4- 101.3% with the exception of one concentration (10

− 9 M) in Run 1, which had an average binding of 66.5%. Review of the data indicated that this value was a result of a single replicate with a specific binding of 7.5%. Excluding this value yielded a mean specific binding of 96.0%, which concurs with the other runs. As the specific [ 3 H]-R1881 binding was >75% at all concentrations of glyphos ate in all runs, an IC 50 and RBA could not be calculated for glyphosate.

Based on the results from the three runs, glyphos ate is classified as a Non-Binder in the Androgen Receptor Binding Assay.GLYPHOSATE FINAL

Page 34 of 62 Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRI D 48671303), glyphosate (95.93% glyphosate acid; 85.14% calculated glyphosate content in sample; Lot # GLP-1103-21149-T) was incubated
with human recombinant aromatase and tritiated androstenedione ([1 β

3 H(N)]-androst-4-ene3,17-dione; [ 3 H]ASDN) at log concentrations of 10

− 10 to 10

− 3 M for 15 minutes to assess the potential of glyphosate to inhi bit aromatase activity. The so lvent vehicle was 0.1 M phosphate buffer for glyphosate, ethanol for ASDN, and dime thyl sulfoxide (DMSO) for 4-OH ASDN, with a final assay volume of ≤ 1% DMSO.

Aromatase activity was determined by measuring the amount of tritiated water produced at the
end of a 15-minute incubation for each concentration of chemical. Tritia ted water was quantified using liquid scintillation counting (LSC). Four independent runs were conducted; however, the
first run was not used because of incorrect standa rd preparation. The remaining three runs were
conducted and each run included a full activity control, a backgr ound activity control, a positive control series (10

− 10 to 10

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

− 10 to 10

− 3 M) with three repetitions per concentration.

Aromatase activity in the full activity controls was 0.676 ± 0.072 nmol·mg-protein

− 1 ·min

− 1 . The response of each full activity c ontrol within a run was between 90 to 110% of the average full activity. Activity in the bac kground controls ranged 0.23 to 0.38% and averaged 0.30% of the full activity control. The response of the fu ll activity controls and background controls was acceptable for each run.

For the positive control substance (4-OH ASDN), aromatase activity results were within the recommended ranges for the performance criteria. The estimated log IC 50 for 4-OH ASDN averaged

− 7.29 M and the Hill slope was

− 0.96.

For glyphosate, aromatase activity averaged 0.673 ± 0.066 nmol·mg-protein

− 1 ·min

− 1 at the lowest
tested concentration of 10

− 10 M and 0.741 ± 0.100 nmol·mg-protein

− 1 ·min

− 1 at the highest tested
concentration of 10

− 3 M. The average aromatase activity was ≥ 99.67% of the control at all tested glyphosate concentrations for all runs.

Based on the data from the average response curve, glyphosate is classified as a Non-inhibitor of aromatase activity in this assay.GLYPHOSATE FINAL

Page 35 of 62 Estrogen Receptor (ER) Binding; (OCSPP 890.1250)

In an estrogen receptor (ER) binding assay (MRID 48671305) for glyphosate (95.93% glyphosate acid, 85.14% calculated glyphosate co ntent, Batch # GLP-1103-21149-T), uterine cytosol from Sprague Dawley rats was used as the source of ER to conduct saturation and competitive binding experiments. A satura tion binding experiment was conducted to demonstrate that the ER in the rat uterine cyto sol was present in reasonable numbers and was functioning with appropriate affin ity for the radio-labeled refere nce estrogen prior to conducting ER competitive binding experiments. The compe titive binding experiment measured the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in th e presence of increasing concentrations (10

− 10 to 10

− 3 M) of glyphosate. TEGD buffer was used as the solvent vehicle for glyphosate. A total of 3 r uns were performed, and each r un included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17- β -estradiol as the natural ligand reference material.

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. The protein concentrations used in the saturation binding runs varied between each run, and were approxima tely 3- to 6-fold greater than recommended (160 to 320 μg versus 50±10 μg). The K d for [ 3 H]-17 β -estradiol was 0.331 ± 0.061 nM and the estimated Bmax was 74.55 ± 3.03 fmol/100 μg protein for the prepared rat uterine cytosol. The

K d for each run was within the expected Guideline range of 0.03 to 1.5 nM.

In the competitive binding experiment, the estimated mean log IC 50 s for 17 β -estradiol and 19-
norethindrone were

− 9.0 and

− 5.5 M, respectively. The mean relative binding affinity (RBA) was 0.032% for 19-norethindrone, compared to the natural ligand.

Glyphosate was tested over a concentration range (10

− 10 to 10

− 3 M) that fully defined the top of the curve. The percent binding at the top plateau (101.2-116.9%) was within 25 percentage points of the lowest concentra tion of the estradiol standard (98.6-101.8%). Across all runs, the lowest average percent radiolab eled estradiol binding in the presence of glyphosate was >81% ( i.e. showed less than 25 % displacement) at concentrations up to 10

− 3 M indicating that gylphosate was not competing with the na tural ligand for binding to the ER.

Based on the results from the th ree runs, glyphosate is classified as Not Interactive in the Estrogen Receptor Binding Assay.GLYPHOSATE FINAL

Page 36 of 62 ER α Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48617307), hER α -HeLa-9903
cells cultured in vitro were exposed to glyphosate (85.14% a.i. Batch # GLP-1103-21149-T) at logarithmically increasing concentrations from 10

− 10 to 10

− 3 M in cell culture media for 24 hours in three independent runs. The experiments were performed using 96- well plates and each glyphosate concentration was tested in 6 wells/plate in each run. The solvent vehicle was culture media for glyphosate and DMSO (0.1%) for the refere nce chemicals. Cells were exposed to the test agent for 24±2 hr to induce reporter (luciferase) gene produc ts. Luciferase expression in response to activation of the estrogen receptor was measured using a proprietary luciferase assay.

Glyphosate was tested up to the limit dose, with no precipitation or cytoto xicity observed at any tested concentration. At concentrations up to 10

− 3 M, the relative transcriptional activation of glyphosate was ≤ 2.4%. In the main assays, the responsiv eness of the cells to the very weak positive control 17 α -methyltestosterone was lower than the expected values, indicating a decreased sensitivity of the assay to very weak agonists. Alt hough the conditions of this assay were not optimal to detect very weak activity, glyphosat e responses were similar to those of the negative control corticosterone and no t comparable to the responses of 17 α -methyltestosterone,
which was able to reach a maximum of 40.8-42.6% PC. Glyphosate was only able to reach a maximum of 0.8-2.4% PC when tested up to the highest concentra tion possible based on cytotoxicity. Because the RPC Max < PC 10 in both assay runs, glyphosat e 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 48671311) of glyphosate with Fathead minnow ( Pimephales promelas ) was conducted under flow-through conditions. Adult fish (2 males and 4 females in each group, 4 groups/treat ment, 5.5 months of age) were exposed to glyphosate (85.14% purity) at nominal concentr ations of 0 (nega tive control), 0.048, 0.24, 1.2, 6.0, and 30 mg a.i./L. Mean-measured concentrat ions were <0.03 (<LOQ; negative control), 0.046, 0.23, 1.2, 6.2, and 33 mg a.i./L. The high test c oncentration was based on 1/3 of a 96-hr LC50 value. The test system was maintain ed at 24.3 to 29.1°C and a pH of 8.0 to 8.3.

Survival was 100% in the negative control, 0.046, 0.23, 6.2 and 33 mg a.i./L treatment groups and 95.8% in the 1.2 mg a.i./L treatment group. Glyphosate did not result in any significant increases or decreases in weight or length for either sex at an y treatment level. There were no observed effects on secondary sex characteristics or clinical si gns (i.e., behavioral and other sublethal effects) in males or females in any treatment group.GLYPHOSATE FINAL

Page 37 of 62 Spawning and mean fecundity in th e negative control were at least every 4 days in each replicate and 23.5 eggs/female/day/replicate (range: 23.2-23.9 eggs/female/day), respectively; fertilization success in the negative control was 97.3%. F ecundity and fertilization success were not significantly different from the negative control for any treatment group.

Plasma vitellogenin (VTG) was si gnificantly decreased 55% (p<0.05) in female fish at the mid high-treatment level (6.2 mg a.i./L) compared to the negative control; male VTG was unaffected by treatment. There were no effects in gonado-soma tic index (GSI) or nuptia l tubercle scores for male or females (none noted in females) in any treatment group relative to the negative control. Although there were gonadal hist opathology observations reported for both males and females (e.g. minimal to mild granulomatous inflammatio n, oocyte atresia, increased mature oocytes),
there were no treatment-rela ted patterns in gonadal histopathology for males or females.

Hershberger Assay; (OCSPP 890.1400)

In a Hershberger Assay (MRID 48617001) screeni ng for androgenic activity, glyphosate (85.1% a.i., Batch/lot# GLP-1103-21149-T) in 0.5% methyl cellulose (w/v) was ad ministered daily via oral gavage (5 mL/kg) to groups of six 54- or 55-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300, or 1,000 (lim it dose) mg/kg/day. The androgenic positive control group consisted of 6 castrated rats expo sed to 0.2 mg/kg/day of testosterone propionate (TP) by subcutaneous (s.c.) injection.

To screen for potential anti-a ndrogenic activity, glyphosate in 0.5% methylcellulose (w/v) was administered daily via oral gavage to groups of six 54- or 55-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300, or 1,000 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.2 mg/ kg/day by s.c. injection. The anti-androgenic positive control group consisted of 6 castrated rats exposed to 0.2 mg/kg/day TP by s.c. injection and 3 mg/kg/day flutamide (FT) via oral gavage . TP alone was used as the anti-androgenic negative control.

For both components of the assay, body weights were determined da ily. The animals were dosed for 10 consecutive days and terminated approxi mately 24 hours after the final dose. At necropsy, the five androgen-dependent tissues were collected and weighed.

All animals survived until scheduled termination. No animals exhibited an y dose-related clinical signs of toxicology and there were no treatment-related gross pat hological findings.

In the androgen agonist assay, there were no tr eatment-related effects on body weights, overall body weight gains, or the weights of accessory sex organs for any glyphosate dose group. Animals in the positive TP control group had incr eased (p<0.01) accessory sex organ weights asGLYPHOSATE FINAL

Page 38 of 62 follows: 437% in seminal vesicles; 728% inventral prostate; 200% in levator anibulbocavernosus (LABC); 361% in Cowper’s gland; and 45% in glans penis. The performance criteria indicated that this assay was performing as expected.

In the anti-androgen assay, there were no treatme nt-related effects on body weights, overall body weight gains, or the weights of accessory sex organs for any glyphosate dose group. Animals dosed with TP + FT (positive control) had decr eased (p<0.01) accessory sex organ weights as follows: 76% in seminal vesicles; 80% in vent ral prostate; 63% in LABC; 70% in Cowper’s gland; and 29% in glans penis. The performa nce criteria indicated that this assay was performing as expected.

Statistically significant changes were not seen in two or more of the five androgen sensitive tissue weights. Glyphosate was negative for an drogenicity and anti-androgenicity in the Hershberger assay.

Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48671315), 15 Crl:CD(SD) Sprague-Dawley rats/dose group were treated daily via oral gavage with glyphosate technical (95.93% a.i., Lot #: GLP-1103- 21149-T) in 0.5% methylcellulose at doses of 0, 100, 300 or 1000 mg/kg/day (limit dose) from post-natal day (PND) 22 to 42. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainment were recorded. Following sacrifice on PND 42, blood was collected for cl inical chemistry analyses, including total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels, which were analyzed using an electrochemiluminescent immunoassay (T 4 ) and a magnetic [ 125 I]rTSH gamma counter immunoassay (TSH). Liver, adrenal glands, th yroid, pituitary, and ur ogenital organ weights were recorded, and microscopic examinations we re performed on the thyr oid, kidneys, ovary and

uterus.

One animal in the control group was sacrificed in extremis on PND 27 due to impairment of the right forelimb (due to possible mechanical injury ). All other animals survived until scheduled
sacrifice. Treatment-related clin ical signs were limited to rales in 4/15 and 13/15 females in the 300 and 1000 mg/kg/day groups, respectively, at approximately 4 hours post-dosing. This finding did not persist to the dail y examinations. No other treatme nt-related clinical signs were noted during the 4-hour post-dosing or daily examinations at any dose level. There were no
treatment-related differences in age of attainment of VO, body we ight at VO, final body weights, or body weight gains in the treated groups relative to controls. One female each in the control and 300 mg/kg/day groups failed to attain VO. Th ere were no statisticall y significant differences in mean age at first vaginal estrus, mean cycle length, or percent cycli ng. The cycle status at necropsy was similar among all groups. Serum T 4 and TSH were not affected by treatment, andGLYPHOSATE FINAL

Page 39 of 62 no adverse treatment-related effect s on any clinical chemistry pa rameter were observed at any
dose. There were no treatment-related microsc opic findings in the thyr oid, ovaries, uterus, or kidneys at any dose.

The dose levels tested were adequate since the high dose was the Limit Dose (1000 mg/kg/day).

Male Pubertal A ssay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48671313), 15 Crl:CD(SD) Sprague-Dawley rats/dose group were treated daily via oral gava ge (5 mL/kg) with glyphosate t echnical (95.93% a.i., Lot #: GLP1103-21149-T) in 0.5% methylcellulose at doses of 0, 100, 300 or 1000 mg/kg/day (limit dose) from post-natal day (PND) 23 to 53. Animals we re examined for preputial separation (PPS) daily beginning on PND 30, and age and weight at day of attainme nt were recorded. Following sacrifice on PND 53, blood was taken for total thyroxine (T 4 ), testosterone, thyroid stimulating hormone (TSH), and clinical chemistry an alysis. The hormones were analyzed by radioimmunoassay (RIA) or chemiluminescence. Weights were recorded for the liver, adrenal glands, thyroid, pituitary, and ur ogenital organs, and microscopic examinations were performed on the thyroid, kidneys, right testis and epididymides.

One male in the 1000 mg/kg/day group was f ound dead prior to dosing on PND 24; no significant clinical or macroscopic findings were observed in this an imal. All other rats survived until scheduled sacrifice. Treatment-related clinical findings were limited to rales in 9/15 and 14/15 males in the 300 and 1000 mg/kg/day groups, respectively, approximately 4 hours postdosing. This finding persisted in the daily examinations in 7/15 males at 1000 mg/kg/day throughout the study.

Treatment-related decreases in overall (PND 23-53) body weight gain s were observed at 300 mg/kg/day ( ↓ 8%, not significant; NS) and 1000 mg/kg/day ( ↓ 12%, p<0.01). On PND 53, final body weights in the 300 and 1000 mg/kg/day gr oups were decreased (p<0.05) by 7-10%. A treatment-related delay in the mean age at attainment of complete PPS was noted at 1000 mg/kg/day (48.0 days) compared to controls (45.9 days). Howeve r, it was determined that the delay in attainment of complete PPS at this dose was a result of th e treatment-related decrease in body weight, rather than a di rect anti-andr ogenic effect.

No compound-related effects on or gan weights were observed at a ny dose. No treatment-related
effects on T 4 , TSH, or testosterone levels were observed at any dose. T 4 and TSH levels were lower than the control group in all treated gr oups and testosterone was lower at 300 and 1000 mg/kg/day. However, these changes were not stat istically significant and were not associated with any histopathological findings . At 1000 mg/kg/day, the increas es (p<0.01) in ALT (also atGLYPHOSATE FINAL

Page 40 of 62 300 mg/kg/day), sodium, albumin, ALP, AST, ch loride, phosphorous, and total protein, and the decrease in urea nitrogen were consider ed to be related to treatment.

At 1000 mg/kg/day, there was a slight increase in the number of animals with colloid area Grade 4 (5 treated vs. 1 control) and Gr ade 5 (1 treated vs. 0 controls). There were no treatment-related effects on follicular cell height at any dose compared to controls. There were no treatmentrelated findings in the testes , epididymides or kidneys.

The highest dose tested showed evidence of overt toxicity based on the decreases in terminal body weight, clinical signs, and mo rtality at 1000 mg/kg/day; the dos e concentrations used in the study are considered adequate.

Effects on androgen-related endpoints included d ecreased ventral prostate, seminal vesicle, LABC, and pituitary glands weights, as well as a 2.1 day delay in PPS at the high dose but these occurred with significant decreases in body weight . There were no treatment-related effects on thyroid-related endpoints in this assay.

Steroidogenesis Assay; (OCSPP 890.1550)

The purpose of this study was to validate the us e of a standardized st eroidogenesis assay as
detailed in OECD Guideline for the Tes ting of Chemicals: Draft Proposal for a New Guideline 4XX – The H295R Steroidogenesis Assay (available on-line at http://www.oecd.org/dataoecd/56/11/44285292.pdf). In this validation study, 28 chemicals were selected as a screen for potential effects of endocrine-disrupting chemi cals on the production of testosterone (T) and 17 β -estradiol (E2). These chemicals we re selected based on their known or suspected endocrine activity, or lack thereof, a nd included inhibitors a nd inducers of different potencies as well as positive and negative controls . These chemicals were selected and approved by the OECD Validation and Management Gr oup for Non-Animal Testing (VMG NA). Glyphosate was one of the chemicals evaluated. A total of seven laboratories from the USA, Denmark, Germany, Japan, Hong Kong, and Canada, each with different levels of experience in conducting the H295R steroidogenesis assay, were invited to particip ate in this validation study.
Inclusion of laboratories with different levels of proficie ncy in conducting the assay was essential to evaluate the completeness of the te st protocols and their transferability. Each laboratory was assigned a random code number (1–7) as part of the study. However, part way through the study, two of the seve n laboratories decided to ceas e their participation in the validation studies. Thus, with the exception of the QC exposure data, only the data for the remaining five laboratories that completed the validation studies are presented (Labs 1, 2, 3, 4, and 6). One laboratory evaluated all 28 chemicals, and one other laboratory (#4) also evaluated glyphosate. The laboratorie s were not identified.GLYPHOSATE FINAL

Page 41 of 62 In this steroidogenesis assay (MRID 48617005), H295R cells cultured in vitro in 24-well plates were incubated with glyphosate (purity and lot # not provided) at seven concentrations between
0.0001 and 100 μ M (specific concentrations not reported) for 48 hours in triplicate for three independent experiments. The test chemical’s vehicle was not identif ied. The presence or absence of precipitation and/or cytotoxicity was not reported. A Quality Control (QC) plate was run concurrently with each indepe ndent run of a test chemical plat e to demonstrate that the assay responded properly to positive control agents at two concentrations; positive controls included the known inhibitor (p rochloraz) and inducer (f orskolin) of estradiol a nd testosterone production. T and E2 levels were measured using radioimm unoassays or ELISA; responses of the QC plates measured by these assays were c onfirmed by LC-MS (at Lab 1).

The report stated that with a few exceptions, all of the laboratories met the key quality performance parameters for conducting the H295R assay protocol. The report stated that two
laboratories demonstrated that glyphosate exposure does not affect testosterone or estradiol levels in this assay; however, data were not presented.

Uterotrophic Assay; (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48617003) conducted to screen for potential estrogenic activity, glyphosate (85.1% a.i., Batch/lot# GLP-1103-21149- T) in 0.5% methylcellulose (w/v) was administered daily via oral gavage to groups of six ovariectomized female Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300, or 1,000 (limit dose) mg/kg/day on post-natal days (PND) 66/67 to 68/69. The positive control group was treated with a daily dose of 17 α -ethynyl estradiol (EE) at 3 μ g/kg/day by oral gavage. B ody weights were determined daily. All animals were terminated and necropsied on PND 69/70 a pproximately 24 hours after the final dose
administration to determine wet a nd blotted uterin e weights.

All animals survived until scheduled termination and no treatment-related c linical findings were observed in glyphosate dosed animals. Body weight s, body weight gains, and uterine weights in the glyphosate groups were compar able to the vehicle control.

In the positive control (EE) group, mean body weights decreased on Days 3 and 4 (not significant, NS), leading to an overall body weight loss during the study of 5.6 g (p<0.01) compared to a gain of 11.3 g in the controls. Absolute wet and blotted uterus weights for the positive control (EE) group were increased (p<0.01) by 758% and 256%, respectively, as expected.

No statistically si gnificant changes were seen in uterine weight in this assay. Glyphosate is negative in the uterotrophic assay.GLYPHOSATE FINAL

Page 42 of 62 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 ge neral toxicity studies considered relevant for determining the potential interaction of glyphosate with the endo crine system are summarized below. Given the breadth of toxicity data ava ilable on glyphosate, the only Part 158 studies discussed in this appendix are those that fulfill current guideline requirements and are directly relevant to assessing potential endocrine effects ( i.e. , multiple-generation rodent reproductive studies, development studies, chronic toxicity studies, etc.).

ER Binding and Tran sactivation Assays

In a published, non-Guideline study ( Kojima et al ., 2004) , the potential of glyphosate (>95% purity) to interact with two human ER subtypes (hER α and hER β ) and one human androgen receptor (hAR) in transactivati on assays with Chinese hamster ovary cells (CHO-K1 cells) was investigated. The human ER (hER α ) and AR (hAR) expression vectors (pcDNAER α and pZeoSV2AR, respectively) were cons tructed as described previously (Kojima et al. , 2003) . The
hER β expression vector was cons tructed by cloning the hER β cDNA with reverse transcriptasepolymerase chain reaction from human placental R NA. The sequence was verified and inserted into the mammalian expression ve ctor pcDNA3.1Zeo(-) creating pcDNAER β . The construction of the estrogen or androgen responsive elements containing re porter plasmid (pGL3-tkERE and pIND-ARE, respectively) was described previously (Kojima et al. , 2003) . pRL-SV40 containing
the Renilla luciferase gene was used as an inte rnal control for transfection efficiency.

For detection of hER α or hER β activity, cells were tr ansfected with pcDNAER α or pcDNAER β

, pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were transfected with pZeoSV2AR, pIND-ARE, and pRL-SV40. Flutolanil was tested over a concentration range from

10

− 8 to 10

− 5 M, and the responses were compared to the activity of 17 β -estradiol (E 2 , >97% purity) or 5 α -dihydrotestosterone (DHT, 95% purity) for the ER or AR assays, respectively. For measurement of antagonistic activity to hER α , hER β and hAR, either 10

− 11 or 10

− 10 M E 2 or 10

− 10 M DHT was added to the cell cultures al ong with the test compound, respectively. Agonistic activities were evaluated by the relative activity ex pressed as 20% relative effective concentration (REC 20 ), the concentration of the compound de monstrating 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 similarly by the relative activity expressed as 20 % relative inhibitory concentration (RIC 20 ), the concentration of the compound showing 20% in hibition of the activity
of E 2 or DHT against ER α , ER β , and AR, respectively. Glyphosat e did not have agonist or antagonist activity for ER α and ER β , or agonist activity for AR in this study.

Thongprakaisang et al. ( 2013 ) evaluated the estrogenic and/or anti-estrogenic effects of glyphosate (>98% purity) at concentrations that ha ve been reported in environmental conditions and exposed humans. Effects were investigated in estrogen-dependent human breast cancer cells, T47D; a stably ERE (estrogen response element) -luc construct-transfected, hormone-dependentGLYPHOSATE FINAL

Page 43 of 62 breast cancer, T47D-KBluc; and a hormone -independent human breast cancer, MDA-MB231. To differentiate the effects of endogenous estr ogen, cells were studied both in completed (standard) medium (medium containing fetal bovine serum) and in estrogen withdrawal medium (medium containing dextran-charcoal treated fetal bovine serum).

Cells were seeded in 96-well luminometer plat es and allowed to attach overnight. Media was then replaced with dosing media in which the final concentration of glyphosate ranged from

10

− 12 to 10

− 6 M. 17 β -estradiol (E2) at the same range of concentrations was used as the positive control agonist for ER activation. The media was used as the negative control. Cells were allowed to incubate 24 hours. Cell growth and viab ility were tested with the MTT reagent assay.

In the cell viability assay, glyphosate caused hor mone-dependent T47D cells to proliferate, approximately 15-30% of control, in the ab sence of E2; the eff ect was significant (p ≤ 0.05) at

10

− 9 and 10

− 8 M and was approximately half the E2 response in T47D cells. Glyphosate had no effect on the growth of hormone-independent MD A-MB231 cells in the absence or presence of E2. Because the proliferation effect of glyphosate on T47D cells was only observed in the absence of E2, the possibility that ER si gnaling may be involved was investigated by co-incubating glyphosate or E2 with the ER an tagonist, ICI 182780, in T47D cells. ICI 182780 at a concentration of 1 nM mitigated the proliferativ e effect of glyphosate and E2 in T47D cells; the proliferative effects of glyphos ate on T47D cells were comple tely inhibited at an ICI 182780 concentration of 10 nM. The results suggest th at the proliferative effects of glyphosate may
involve the ER.

The estrogenic activity of gl yphosate on ERE transcription activ ity was investigated with
T47D-KBluc cells. In th e concentration range 10

− 12 to 10

− 6 M, glyphosate induced ERE activation 5- to 13-fold compared to control; the effects were <50% compared to E2 induction. Co-incubation of glyphosate with 1 nM ICI 182780 resulted in a decreased response, with complete inhibition in the presence of 10 nM ICI 182780. When cells were co-incubated with glyphosate and E2, the E2-induced ERE activatio n was suppressed, indicating that in the presence of endogenous agonist (E2) , glyphosate acted as an antagonist.

90-Day (Subchronic) Oral Toxicity in Rodents (Rat; OSCPP 870.3100)

In a subchronic oral toxicity study in rats ( MRID 40559401 ), glyphosate (95.21% purity) was administered to SD rats (12/sex/group) in th e diet at nominal concentrations of 0, 1000, 5000, or 20,000 ppm for three months. Mean act ual intakes were 950, 4600, and 19,000 ppm corresponding to 63, 317, and 1267 mg/kg/day fo r males and 84, 404, and 1623 mg/kg/day for females (based on actual body weight s, food consumption, and analyses of feed). Body weights and food consumption were measured weekly, and hematology, clinical chemistry, and urine analyses were conducted at the e nd of the study. Organs that were weighed included the liver, kidneys, and testes (including epididymides). For control and high-dose animals, gross and microscopic pathological examinations were co nducted on these tissues as well as adrenals,GLYPHOSATE FINAL

Page 44 of 62 ovaries, mammary gland, prostate, pituitary, semi nal vesicles, thyroid, urinary bladder, and uterus (corpus and cervix). The kidneys and livers of the low- and medium-dose animals were also examined microscopically.

There were no mortalities or treatment-related toxic signs duri ng the study, and there were no effects of treatment on body weight or food cons umption. Possibly treatment-related findings in male rats were increased (p<0.05) serum phosphorus (low and high dose) and potassium (medium dose) concentrations; increased se rum glucose concentrations at 317 and 1,267 mg/kg/day at the medium and high doses; a nd increased (NS) serum BUN and alkaline phosphatase concentrations at the high dose appa rently due to a single animal. In addition, histopathological lesions observed in the pancreas of high-dose male rats may have been
treatment-related. Possible treatment-related fi ndings in female rats were increased (p<0.05) serum phosphorus concentrations and increased (p <0.05 or NS) potassium concentrations in all treated females. No other histopathological findings were noted. Because clinical chemistries were conducted only once during the entire study, and as the pancreas was not examined histologically in the low- and medium-dose gr oups, it could not be determined whether or not these findings were attributable to the test material on the basis of this study alone.

In a subchronic oral toxicity study in rats conducted by the National Toxicology Program ( NTP; 1992 ), glyphosate (99% purity) was administ ered in the diet to F344/N rats (10/sex/group) at nominal concen trations of 0, 3125, 6250, 12,500, 25,000, or 50,000 ppm (mean-measured doses of 205, 410, 811, 1678, or 3393 mg/kg/day for males and 213, 421, 844, 1690 or 3393 mg/kg/day for females, respectively, based on food consumption and body weight measurements) for 90 days. Ten additional rats/s ex were included at each dose level for evaluation of hematology and clinical chemis try parameters. Body weights were measured weekly. Samples for hematology and clinical ch emistry determinations were collected from additional study animals on Study Days 5 and 21, and from the regular study animals at the end of the study. Organs that were weighed included kidney (right), liver, an d testis (including left caudal and left epididymal tissue for the control and 12,500, 25,000, and 50,000 ppm dose groups). For control and high dose animals, gro ss and microscopic pathological examinations
were conducted on these tissues as well as adrenals, mammary gla nd, ovaries, pituitary, preputial/clitoral glands, prosta te, seminal vesicles, thyroid, uter us, and vagina; histopathological examinations of the salivary gland were conducted for rats in all dose groups. In addition, for rats in the control and three highe st dose groups, sperm morphology (s perm motility, sperm count per gram caudal tissue, and spermatid head count) evaluations were conducte d at the end of the study, and vaginal cytology evaluations were conduc ted during the 12 days prior to sacrifice.

All animals survived to necropsy. Diarrhea wa s noted in the high-dose animals (males and females) through Day 50 of the study. Body wei ght gain was reduced in the high-dose males ( ↓ 25%; 3393 mg/kg/day) and the final body weight was decreased by 18%, but statistical significance was not reported fo r final body weights or body weight gains. Mean necropsy bodyGLYPHOSATE FINAL

Page 45 of 62 weights were decreased (p<0.05 and 0.01, respec tively) for the 1678 and 3393 mg/kg/day males ( ↓ 12 and 21%, respectively). Body weight gain in high-dose females (3393 mg/kg/day) was decreased ( ↓ 10%), but no effects on final or necr opsy body weights were observed. Clinical chemistry results showed increases in ALP and ALT in some or all of the dose groups; ALP
concentrations in the control animals was vari able over the sampling days (Days 5, 21, and 90). Increases (p<0.01) in ALP in males were genera lly dose-dependent in the top three dose groups on Days 5 and 21, but were similar to control by Day 90. Increases (p<0.0 1 or 0.05) in ALP in females were dose-dependent in the top three do se groups on Days 5 and 21, and still present on Day 90 in the top two dose groups. Increases inALT in both sexes were generally non-dosedependent, and seen in all but the lowest dose gr oup in males (except for Day 90) and in all dose groups in females (except for Day 90). In addition, effects on bile acids were noted in both sexes. On Day 5, variable increases (NS) were noted wi th a clear dose-depende nt response in the two highest dose groups. A stronger, dose-depende nt response was observed on Day 21 with increases (NS, p<0.05, and p<0.01) in males ( ↑ 2-70%) and females ( ↑ 23-102%). Concentrations of bile acids on Day 90 were more variable, w ith a generally non-signifi cant, but dose-dependent response at the two highest doses in both sexes. Non-dose-dependent incr eases (p<0.01 or 0.05) in relative liver weights also were ob served in males in all dose groups ( ↑ 6-15%) and in females in the two highest dose groups ( ↑ 10 and 6%). Taken together, these findings are consistent with a hepatobiliary effect that can be attr ibuted to glyphosate administration.

In the 1678 and 3393 mg/kg/day males, there were in creases (p<0.01 or 0.05) in the relative right kidney ( ↑ 8% and 13%, respectively) a nd right testis weights ( ↑ 7% and 21%, respectively); relative right kidney weight also was in creased (p<0.05) in high-dose females ( ↑ 7%). Absolute weights for these organs in treat ed rats were similar to contro l. Left caudal, epididymal, and testicular weights of treated males (811, 1678, and 3393 mg/kg/day groups) were similar to
control.

A dose-related increase in the incidence and sever ity of cytoplasmic alteration of the parotid and submandibular salivary glands wa s noted in the microscopic eval uations, beginning at the lowest
dose in both sexes. These lesions were diagnosed as “cytopl asmic alteration” and consisted of basophilic change and hypertrophy of acinar cells. No other treatment-related histopathological findings were reported.

Sperm counts were decreased (p<0.01) in th e two high-dose groups ( 1678 and 3393 mg/kg/day; ↓ 20%). Epididymal sperm motility, total spermatid head/testis, and total spermatid heads/g
caudal tissue in the treated animals were similar to control. A delayed (p<0.05) estrous cycle was observed in the 3393 mg/kg/day group (5.4±0.21 da ys) compared to control (4.9±0.10 days).

The LOAEL is the low dose (205/213 mg/kg/day fo r males and females, respectively) based on
dose-related increases in the inci dence and severity of cytoplasmi c alterations in the parotid and submandibular salivary glands of male a nd female rats. A NOAEL is not determined.GLYPHOSATE FINAL

Page 46 of 62 90-Day (Subchronic) Oral Toxicity in Rodents (Mouse; OSCPP 870.3100)

In a subchronic oral toxicity study in mice ( MRID 00036803 ), glyphosate (98.7% purity) was administered to CD-1 albino mice (15/sex/group) at dietary concentrations of 0, 5000, 10,000, or 50,000 ppm (0, 750, 1500, or 7500 mg/kg/day, based on st andard conversion) for three months.
The mice were sacrificed at th e end of the study and complete gross necropsies were conducted. Organs that were weighed included the gona ds, kidneys, and liver. Histopathological examinations included these organs, as well as adrenals, epididymis , pituitary, thyroid, parathyroid, urinary bladde r, uterus, and prostate.

There were no treatment-related toxicological signs or mortalities during the study, and there were no effects of treatment on food consumpti on. Body weight gains in the male and female high-dose groups were decreased ( ↓ approximately 24% and 18%, respectively) compared to control. There were no changes in absolute or re lative organ weights at a ny dose, and there were no treatment-related histopatholog ical findings in any organ. Th e LOAEL for the study is 50,000 ppm (7500 mg/kg/day) based on reduced body weight gain and the NOAEL is 10,000 ppm (1500 mg/kg/day).

In a subchronic oral toxicity study in mice conducted by NTP (1992 ), glyphosate (99% purity) was administered in the diet to B6C3F1mice ( 10/sex/group) at nominal concentrations of 0, 3125, 6250, 12,500, 25,000, or 50,000 ppm (equivale nt to 0, 507, 1065, 2273, 4776, or 10,780 mg/kg/day for males and 753, 1411, 2707, 5846, 11,977 mg /kg/day for females, respectively, based on food consumption and body weight meas urements) for 90 days. Body weights were measured weekly. Organs that were weighed in cluded kidney (right), liver , and testis (including left caudal and left epididymal tissue for the control and 12,500, 25,000, and 50,000 ppm dose groups). For control and high-dose animals, gro ss and microscopic pathological examinations
were conducted on these tissues as well as adrenals, mammary gla nd, ovaries, pituitary, preputial/clitoral glands, prosta te, seminal vesicles, thyroid, uter us, and vagina; histopathological examinations of the salivary gland were conduc ted for mice in all dose groups. In addition, for mice in the control and three highest dose groups, sperm morphology (sperm motility, sperm count per gram caudal tissue, and spermatid head count) evaluations were conducted at the end of the study, and vaginal cytology evaluations were conducted during the 12 days prior to
sacrifice.

There were two mortalities in the study; neither was related to tr eatment. In addition, no clinical signs of toxicity were reported. Final body weight s were decreased in males and females in the two highest dose groups ( ↓ 11% and 17% for males and 6% and 10% for females) with concomitant decreases in body weight gain ( ↓ 35% and 63% for males and 14% and 27% for females); statistical significan ce was not reported. Mean necrop sy body weights were decreased for the 4776/5846 mg/kg/day (p<0.05; ↓ 8 and 6%, respectively) and 10,780/11,977 mg/kg/dayGLYPHOSATE FINAL

Page 47 of 62 (p<0.01; ↓ 15 and 11%) males and females. Average daily food consumption was comparable across the treated and control groups.

Relative right testis weight s were increased (p<0.01) at the two highest doses ( ↑ 10% at 4776 mg/kg/day and ↑ 18% at 10,780 mg/kg/day); absolute weig hts were comparable to control. Absolute left caudal, epididymal, and testic ular weights of treated males (2273, 4776, and 10,780 mg/kg/day groups) were similar to control. Th e relative weights of the right kidney were increased (p<0.05), in a non-dose-related manner, in males in all dose groups except the lowest ( ↑ 11%, 15%, 23%, and 16%, respectively); absolute kidney weights were increased only in the medium-dose males ( ↑ 15%). Non-dose-dependent increases (p< 0.05) in relative liver weight also
were noted in males of all dos e groups except the lowest ( ↑ 9%, 4%, 8%, and 9%, respectively). A decrease (p<0.01) in the absolute liver weight was noted in high-dose females ( ↓ 14%).

Dose-related increases in the incidence and severity of lesions in the paro tid salivary glands of male and female mice were observed, begi nning at the second lowest dose (1065 and 1411 mg/kg/day, respectively); no lesions were observed in mice in the low-dose group. These lesions were diagnosed as “cytoplasmic alteration” and consisted of basophilia of the acinar cells. In more severely affected glands, the cells and acini also appeared enlarged with an associated
relative reduction in the numb er of ducts. This finding was considered adverse.

Histopathology examination of control and high dos e animals did not reveal any other related findings. There were no effects of treatment on sperm counts, sperm motility, total spermatid head/testis, and total spermatid heads/g caudal tissu e in males, or on length of estrous cycle in
females.

Prenatal in Rodents (Rat; OCSPP 870.3700)

In a developmental toxicity study in rats ( MRID 00046362 ), glyphosate (98.7%) was administered by gavage to mated female SD rats (25/group) at doses of 0 (0.5% aqueous methocel), 300, 1000, or 3500 mg/kg/day on GDs 6 to 19, inclusive. Individual dam body weights were recorded on GDs 0, 6, 9, 12, 16, a nd 20; food consumption was not determined. The surviving rats were sacrific ed on GD 20 and the uterus was wei ghed; the locations of viable and nonviable fetuses, early and late resorptions, and the tota l number of implantations and corpora lutea were recorded. All fetuses were weighed, sexed, and examined for gross external
malformation. One-half of the fe tuses in each litter were examined for visceral soft tissue alterations by the Wilson technique. The remaini ng fetuses in each litter were examined for skeletal alterations by the Da wson method. This study was conducte d prior to the publication of
EPA GLP Guidelines.

There were six deaths in the high-dose group (one each on GDs 10 and 17, and two each on GDs 11 and 12); the causes of death were not determin ed. At the high-dose, al l of the dams (except three) were observed at least once with diarrhea, soft stools, breathing rattles, inactivity, and redGLYPHOSATE FINAL

Page 48 of 62 matter in the region of nose, mouth, forelimbs, or dorsal head; no treatment-related clinical signs were observed in the low- and mid-dose groups . There were no treatment-related effects on maternal body weight in the low- and mid-dose groups. At the high dose, maternal body weight gain was decreased ( ↓ 23%) during the test period (GDs 0-20), due to a body weight losses during GDs 6-20 ( ↓ 6-10%). It is not known if statistical analyses we re conducted on body weights/body weight gains, as no statistical differen ces were reported for these decreases.

At 300 and 1000 mg/kg/day, there were no effects in m ean number of viable fetuses, late or early resorptions, postimplantation loss, co rpora lutea, the fetal sex dist ribution, or fetal body weight. Decreases in viable fetuses/dam (11.9 ± 4.36; p<0.05) and total implantations/dam (12.1 ± 4.45; p<0.01) were noted at the low dose comp ared to control (14.4 ± 1.26 and 15.0 ± 1.67, respectively), but were not considered to be rela ted to treatment because the decreases were not dose-related. At the high dose, decreases in viable fetuses/dam (11.5 ± 4.12; p<0.05), total implantations/dam (12.8 ± 3.77; p<0.05), and mean fetal body weight (3.2 ± 0.34 g vs. 3.0 ± 0.21 g in control; p<0.01) were observed. There were no fetal malformations in low- and mid-dose groups. The number of malformed fetuses in th e high-dose group (10) was increased compared to control (3). However, because the number of litters with malformed fetuses was the same for both groups, this was not considered to be an effect. There was an increase in the number of
litters and fetuses with unossified sternebrae observed at the high dose. The maternal LOAEL is 3500 mg/kg/day, based on mortal ity, toxic signs, and decrease d body weight gain, and the maternal NOAEL is 1000 mg/kg/day. The develo pmental LOAEL is 3500 mg/kg/day based on decreases in fetal body weight, increases in the number of litters and fetuses with unossified sternebrae, decreases in viable fetuses/dam, and decreases in total implantations/dam; the developmental NOAEL is 1000 mg/kg/day.

Prenatal Development in No n-Rodents (Rabbit; OCSPP 870.3700)

In a developmental toxicity study in rabbits ( MRID 00046363 ), glyphosate (98.7% purity) was administered to artificially-inseminated, female Dutch Belted rabbits (16/dose group) by gavage at doses of 0 (0.5% aqueous methocel), 75, 175, or 350 mg/kg/day from GDs 6-27, inclusive. Individual doe body weights were recorded on GDs 0, 6, 12, 18, 24, and 28; food consumption was not determined. On GD 28, all surviving does were sacrificed, and the uterus was examined and weighed; the number and location of viable fetuses and early and la te resorptions, and the total number of implantations and corpora lu tea were recorded. Each fetus was weighed, examined for external malformations, dissected, se xed, examined for visceral abnormalities, then eviscerated, stained with Alizarin Red S, and examined for skeletal abnormalities by the Dawson method. This study was conducted prior to th e publication of EPA GLP Guidelines.

One rabbit in the low-dose group died apparen tly of pneumonia on GD 26. Two rabbits in the control group, one in the low-dose group, and one in the high-dose group aborted and were sacrificed. Additionally, two rabbits at the medi um dose and ten rabbits at the high dose diedGLYPHOSATE FINAL

Page 49 of 62 during the study; of these, death was attributed to gastroenteritis for two rabbits, to enteritis for one rabbit, and to respiratory di sease for one rabbit. The cause of death for the remaining eight rabbits could not be determined at necropsy. Soft stool or diarrhea was note d in all groups with a slight increase at the medium dose and at least once in each rabbit of the high-dose group; in addition, an increase in nasal discharge was noted in the high-dose animals. There were no toxicologically significant differences in matern al mean body weight among control and treated groups; however, a non-dose-dependent decrease in overall body weight gain was observed in the medium-dose group ( ↓ 135%). No treatment-related changes were seen in the number of viable fetuses, early or late resorptions, total implantations, corpora lutea, or fetal sex distributions at any dose. A sli ght decrease was noted for mean fetal body weight of all of the treated groups compared to the control group. However, mean fetal body weights for all groups were comparable to historical control mean fetal body weight values. There were no treatmentrelated malformations in fetuses from litters of treated rabbits in comparison to control. A few
malformations were noted in each of the three treated groups. Because these did not occur in a dose-related pattern and their fre quency was comparable to that of the historical control group, they were not considered treatment-related.

The maternal LOAEL is 350 mg/kg/day, based on in creased incidences of soft stool, diarrhea, nasal discharge, and death, and the maternal NOAEL is 175 mg/kg/day. The developmental LOAEL is not determined (>350 mg/kg/day), an d the developmental NOAEL is 350 mg/kg/day.

Multi-Generation Reproduction in Rodents (Rat; OCSPP 370.3800)

In a three-generation reproduction study in rats ( MRID 00081674 ), glyphosate (98.7% purity; assumed to be 100% for dosing preparations) was administered in the diet to three consecutive generations of CD (SD) rats ( 12 males and 24 females/generation/ dose) at nominal doses of 0, 3, 10, or 30 mg/kg/day. Each parent generation was mated to produce two litters. Offspring from
the second litters of the F 0 and F 1 parents (F 1b and F 2b litters, respectively) were selected to be parents for the subsequent pair of generati ons. Parameters evaluated for each generation included: mortality, body weight and food consumption data, in-life physical observation data, maternal body weights (gestation/lactation), re production-fertility indi ces (mating, pregnancy and fertility indices), litter data at parturition, and organ weight data. Offspring from each litter interval were evaluated during a 21-day lactation period for grow th, survival, sex distribution data, and gross postmortem observations including organ weight data (F 3b offspring only). All parents were sacrificed after w eaning of the second litters and randomly selected offspring from
the second litters of the second generation (21-day F 3b weanlings) were sacr ificed; the organs that were weighed at sacrifice included the adrenals, gonads, kidneys, liver, and pituitary. Microscopic examinations of these tissues as we ll as mammary glands, thyroid and parathyroid, prostate, and uterus were conducted for 10 rats/s ex from the control and high-dose groups of F 0 , F 1 , and F 2 parents and F 3b weanlings.GLYPHOSATE FINAL

Page 50 of 62 There were no treatment-related effects on adult mortality, clinic al signs, body weight, or food consumption. Although there were some differences between control and treated groups, there were no consistent, dose-related effects on mating, fertility, or pregnancy indices over the entire study that would indicate an adverse effect of tr eatment. There were no consistent adverse effects of treatment in maternal body weight or body weight gain, gestation length , parturition data, or
litter survival indices throughout the study. No consistent trea tment-related effects were found in sex distribution data, body weight s, survival, or gross postmorte m findings in offspring. There
were no effects of treatment on absolu te or relative organ weights in F 3b offspring or F 0 , F 1 , or F 2 adult males. Gross postmortem evaluations of the tissues from randomly selected F 0 , F 1 , and

F 2 control and high-dose animals, and F 3b control and high-dose offspring, revealed a high
incidence of focal tubular dilati on in the kidneys of the male F 3b high-dose offspring. In a subsequent submission ( MRID 00105995 ), the results of examination of the kidneys from the
low- and mid-dose F 3b offspring were reported, which showed that the incidence of renal focal
tubular dilation in the low- and mid-dose F 3b offspring was similar to the study control, as well as, historical control data.

The reproductive LOAEL is 30 mg/kg/day based on increased focal tubul ar dilation in the kidneys of male F 3b offspring. The reproductive NOAEL is 10 mg/kg/day. The systemic NOAEL is 30 mg/kg/day, and the LOAEL is not determined (>30 mg/kg/day).

In a two-generation reproduc tion study with rats ( MRID 41621501 ), glyphosate (97.67% purity)
was administered in the diet to SD rats (30/sex/group) at 0, 2000, 10,000, or 30,000 ppm (0, 100, 500, or 1500 mg/kg/day, based on standard conversi on) for eleven weeks. After Week 11, the rats were mated to produce the F 1 generation. Litters were culled to eight pups and weaned on Day 21, after which 30 rats/sex/group were rand omly selected to continue as the second generation. The F 1 rats were mated after approxim ately 14 weeks to produce the F 2 a and F 2 b litters. Rats were maintained on dosing diet s throughout pre-mating, mating, gestation, and
lactation until sacrifice. Parameters evaluated for each gene ration included: mortality, clinical signs, body weight, and food consumption, maternal body weights (gestation/lactation), reproduction-fertility indices (pre-c oital interval, gestation length, litter size, dead pups/litter, and pup survival), and organ weight (ovaries a nd testes with epididymides). Microscopic
examinations of tissues of F 0 and F 1 adults were conducted for sele cted tissues: kidneys, ovaries, pituitary (F 1 only), prostate, seminal vesicles, ma mmary gland, testes, epididymides, and uterus/vagina. In addition, kidneys from F 2 b weanlings (1/sex/litter) were examined microscopically.

There were no treatment-related mortalities during the study. Treatment-related clinical signs
consisted of soft stools in hi gh-dose males and females (both F 0 and F 1 ). Body weights of highdose male and female F 0 rats were decreased (p<0.01, ↓ 8%) during the prema ting growth period. Similarly, F 1 high-dose male and female adults wei ghed less at weaning compared to F 0 animals with continued decreases (p<0.01 or 0.05) of 10-12%. The body weights of high-dose femalesGLYPHOSATE FINAL

Page 51 of 62 remained decreased (p<0.01 or 0.05) compared to control during gestation and lactation of the

F 1 , F 2 a, and F 2 b litters, although high-dose body weights we re comparable to control by the end of lactation. Food consumption wa s generally similar to control in high-dose males and females during most measurement periods; consumption was decreased in the medium- and high-dose F 1 females on Days 0-14 ( ↓ 9 and 11%, respectively). There were no treatment-related effects on mating and fertility in the F 0 to F 1 generation, or in the F 1 a to F 2 a or F 1 a to F 2 b generations. At Days 14 and 21 in the F 2 b litter

, and Day 21 in F 1 and F 2 a litters, the mean body weights of highdose pups were decreased (p<0.01 or 0.05, ↓ 6-14%) and body weight gain of high-dose pups was decreased 15-20%. There were no consistent, dos e-related effects on litte r size at Day 0 or on pup survival during lactation. Ther e were no treatment-related fi ndings in gross necropsy or histopathology in examined treated rats compar ed to control. Terminal body weights in the F 0 and F 1 a generations were decreased (p<0.01) by 8-13%. Increased rela tive weights of testes of high-dose F 0 (NS, ↑ 6%) and F 1 (p<0.01, ↑ 12%) rats were noted; however, this effect was
considered to be due to the decreased terminal we ights, and not be of t oxicological significance as absolute weights were not a ffected by treatment. There were no treatment-related gross or microscopic findings in the kidneys of F 2b weanlings.

The reproductive NOAEL is 30,000 ppm (1500 mg /kg/day). The systemic LOAEL is 30,000 ppm based on increased incidences of soft stool, and decreased food consumption and body weight during growth; the systemic NOAEL is 10,000 ppm (500 mg/kg/day). The developmental LOAEL is 30,000 ppm based on decreased pup body weight on LDs 14 and 21; the developmental NOAEL is 10,000 ppm.

In a subsequent two-generation reproduction study with rats ( MRIDs 48865101 through

48865105 ), glyphosate (95.7% purity) was administered to Crl:CD(SD) IGS BR rats (28/sex/dose) in the diet at nominal doses of 0, 1500, 5000 and 15,000 ppm (equivalent to 121/126, 408/423, and 1234/1273 mg/kg/day in male s/females during pre-mating) for two successive generations with one litter per genera tion. The P generation animals were fed the test diets for ten weeks prior to mating to produce the F 1 litters. The F 1 litters were not standardized; litter sizes ranged from 4 to 21 pups. On PND 21, 24 pups/sex/dose were selected and fed the same test diet as their parents for te n weeks prior to ma ting to produce the F 2 litters. Organs that were weighed in parental animals included ad renals, epididymides, ki dneys, liver, ovaries, prostate, pituitary, seminal vesicles, testes, thymus , thyroid, and uterus; of these tissues, adrenals, epididymides, ovaries, prostate, pituitary, seminal vesicles, testes, and uterus of the control and high-dose P and F 1 group were subjected to microscopic examination.

No treatment-related effects were observed on mortality, clinical signs, body weights, body weight gains, food consumption, food efficiency, or gross or microscopic pathology in either the

P or F 1 generation.GLYPHOSATE FINAL

Page 52 of 62 Absolute and relative (to body) liver weights were increased(p<0.05, 0.01, or 0.001) by 8-13% in both the P and F1 females at in the high- dose group with no hist opathological findings. Additionally, at this dose, abso lute and relative kidney weights were increased (p<0.01 or 0.001) by 7-11% in the P females only.

The LOAEL for parental toxicity was not observed. The NOAEL is 15,000 ppm (equivalent to 1234/1273 mg/kg/day in males/fe males during pre-mating).

There were no effects of treatment on the mean nu mbers of corpora lutea and implantations, preand post-implantation loss, num bers of pups born, litter size on PND 1, 4, 7, 14, and 21, live birth, viability, and lactation indi ces, and sex ratio at birth and on PND 1 and 21, clinical signs of toxicity, litter weights, pup body weights, developmental landma rks or reflexes, ano-genital distance, or brain, spleen or thymus weights in either the F1 or F2 offspring.

There was a delay (p<0.01) of 2.9 days in at taining complete PPS (PND 45.9 treated vs. PND 43.0 control) in the 15,000 ppm F1 male pups, along with a 10% increase (p<0.01) in body weight at attainment.

The LOAEL for offspring toxicity is 15,000 ppm (equivalent to 1234/1273 mg/kg/day in males/females during pre-mating), based on delayed age and increased weight at attainment of PPS. The NOAEL is 5000 ppm (equivalent to 408/423 in males/females during pre-mating).

The number of males impregnating their female pa rtner, and the number of females not pregnant, with no litters observed, with total litter loss, a nd rearing their litters to weaning was similar in all groups. The pre-coital intervals were similar across all groups with the majority of pairs mating during the first four days of the mating period. Gestation duration, mean estrous cycle length and periodicity, follicle number, and sper m parameters were unaffected by treatment.

The LOAEL for reproductive toxicity was not observed. The NOAEL is 15,000 ppm (equivalent to 1234/1273 mg/kg/day in male s/females during pre-mating).

Chronic Oral Toxicity/Carcinogenici ty in Rodents (Rat; OCSPP 870.4300)

In a chronic oral toxicity/ carcinogenicity study in rats ( MRIDs 00093879 and 00150564 ), glyphosate (98.7% purity) was administered to rats (50/sex/group) at nominal concentrations of 0, 30, 100, or 300 ppm (3, 10, or 30 mg/kg/day based on standard conversion) for the first week of the study; rats were then dosed at 3.05, 10.30 and 31.49 mg/kg/day for the males and 3.37, 11.22 and 34.02 mg/kg/day for the females for the remainder of the study. Animals were terminated at 26 months, when survival had decr eased to 30% in one group per sex. All rats were observed twice daily for mortality and toxic signs and given a detailed physical examination each week throughout the study. Body weights and food consumption were determined at pretest, weekly through 14 weeks, and biweekly for the remainder of the study. Clinical andGLYPHOSATE FINAL

Page 53 of 62 hematological parameters were determined ( 10 rats/sex/group) at 4, 8, 12, 18, and 24 months. Complete necropsies were performed on all rats that died or were sacrificed during or at the end of the study. Organ weights determined at sacrifice included the adre nals, kidneys, liver, testes/ovaries, pituitary, and thyroid. Microscopic examinati on was conducted on these tissues, as well as epididymides, mammary gland, para thyroid, prostate, seminal vesicles, urinary bladder, and uterus for all treated and control rats.

Survival was unaffected by treatment. Decreas ed (NS) body weight ga ins were observed in treated males beginning at Week 26 and contin uing to Week 102, and decreased (significance not reported) body weight gains ( ↓ 10-15%) were present in the low- and medium-dose female rats from Weeks 84 to 92. No dose-response relationship was present in the decreased body weight gains in female rats and the changes in body weight did not affect survival. Overall food consumption was unaffected by treatment. Hematol ogy, clinical chemistry, and urinalyses values for treated male and female rats were comparable to control values. Absolute and relative organ weights of treated rats also were comparable to control. No treatment-related findings were observed during gross necropsies. Microscopic ex aminations revealed lymphocytic hyperplasia of the thymus occurring at incr eased (significance not reported) incidences in the mid- and highdose female rats. In addition, focal vacuolation of liver was found with increased (significance not reported) incidence in high-dose male rats. Test icular interstitial cell tumors were seen in 3/50, 1/50 and 6/50 rats at the low- , mid-, and high-dose groups when compared to control (0/50) with the 12% incidence at the high dose reachi ng statistical significan ce (p=0.013). However, it
was concluded that the increased incidence was similar to the incidence observed in control groups of male rats in other c oncurrent studies and th erefore was not related to treatment. C-cell
carcinomas were seen in the th yroid of 5/47 females at the hi gh dose (11%; p<0.001) compared to 1/47 control females (2%). No increases were seen in adenomas, and the incidences of C-cell hyperplasia were comparable betwee n the treated and control groups.

The LOAEL for chronic toxicity is 10.30/11.22 mg /kg/day (males/females) based on increased lymphocytic hyperplasia. The NOAEL for chronic toxicity is 3.05/3.37 mg/kg/day (males/females). The oncogeni c potential is negative.

In a subsequent chronic oral toxi city/carcinogenicity study in rats ( MRID 41643801 ), glyphosate (96.5% purity) was administered to SD rats (6 0/sex/group) at nominal concentrations of 0, 2000, 8000, or 20,000 ppm (89, 362, and 940 mg/kg/day in males and 113, 457, and 1183 mg/kg/day in females) for 24 months; 10 rats/s ex/group were assigned for interi m sacrifice after 12 months of treatment. All rats were observed twice daily fo r mortality and toxic signs and given a detailed physical examination each week throughout th e study. Body weights were determined once weekly for 13 weeks and then monthly therea fter. Ophthalmological examinations were conducted pretest and twice prior to sacrifice. Clinical and he matological parameters were determined (10 rats/sex/group) before treatment and at 6, 12, 18, and 24 months; urinalyses also were conducted at 6, 12, 18, and 24 months. The orga ns that were weighed at sacrifice includedGLYPHOSATE FINAL

Page 54 of 62 liver, kidneys, testes, epididymides, and pros tate. Gross and micros copic pathological
examinations were conducted on these and other tissues, including the seminal vesicles, ovaries, uterus, urinary bladder, mammary glands, pitu itary, adrenals, and pa rathyroids/thyroids.

There were no treatment-related e ffects on clinical signs or mort ality. The mean body weight of high-dose females was decreased (p<0.05) by 3% at Day 51, 13% (p<0.01) at Week 81, and 3% (NS) at Week 104; at Week 81, body weight ga in was decreased by 23% in high-dose females compared to control. There were no decreases in body weight or body weight gain in male rats.
The incidence of cataracts and lens abnorma lities in male rats in the high-dose group was increased (p<0.05) compared to control accordin g to two examiners, and not significantly different according to two other examiners. There were no treatment-related hematology or clinical chemistry findings that were c onsidered toxicologi cally significant.

Liver weight (relative to body we ight) was increased (p<0.05, ↑ 13%) in high-dose males at interim sacrifice, and absolute liv er weights were increased (p<0.05, ↑ 13%) in high-dose males at terminal sacrifice. No treatment-related change s were seen in absolute or relative weights of the testes, epididymides, and prostate glands, and no treatment-related histopathological lesions were observed in the testes, epididymides, se minal vesicle, prostate, ovaries, uterus, and mammary, adrenal, and pituitary gl ands. The incidences of the panc reatic islet call adenomas in the low-, medium- and high-dose males exceeded the historical control range (1.8-8.5%); however, because there was no posit ive dose-related trend in the oc currence of these tumors in males, no progression to carcinoma, and the inci dence of hyperplasia was not dose-related, the pancreatic islet cell tumors were not considered to be treatment-related. The incidences of thyroid C-cell adenomas were increased in animals in the medium- and high-dose groups compared to control; however, these increases were not considered to be treatment-related
because none of the increases reached statistica l significance, there was no dose-response in either sex, there was no increase in severity of grade or incide nce in hyperplasia or progression to malignancy, and the incidences were within the historical control ranges.

The NOAEL is 8000 ppm (362 and 467 mg/kg/day in males and females, respectively) and the LOAEL is 20,000 ppm (940 and 1183 mg/kg/day in males and females, respectively) due to decreased body weight and body wei ght gain in females, cataracts in males, and increased relative liver weight (to body) at 12 months and increased abso lute liver weight at 24 months in
males.

In addition, the HED Carcinogenicity Peer Review Committee evaluated the carcinogenic potential of glyphosate in June 1991. Glyphosate was classified in to Group E (evidence of noncarcinogenicity in humans) based on a lack of convincing evidence of carcinogenicity in adequate studies in two species.GLYPHOSATE FINAL

Page 55 of 62 Carcinogenicity in Rodents (Mouse; OCSPP 870.4200)

In a carcinogenicity study in mice ( MRIDs 00130406 and 00150564 ), glyphosate (99.7% purity) was administered in the diet to CD-1 mice ( 50/sex/group) at nominal concentrations of 0, 1000, 5000, or 30,000 ppm (0, 150, 750, or 4500 mg/kg/day, ba sed on standard conversion) for 24 months. The parameters evaluated were clini cal signs, mortality, body weight, food and water consumption, and hematology at 12, 18, and 24 mont hs. At the end of the study, all animals were necropsied and selected organs (n ot specified) were weighed; tissues (not specified) were stained and examined microscopically.

There were no treatment-related toxic signs noted, and mortality was low during the first eighteen months of the study. Body weight was consistently decrea sed for males and, to a lesser extent, females in the 30,000 ppm dose group dur ing the study at several sampling intervals (extent and significance not re ported). Changes in body weight at the low- and mid-dose group were variable and not dose-related. Food consumption showed no trea tment-related or doserelated effect. Hematological values, although si gnificant in some instances, did not show a consistent dose-related response. Necropsy did not show treatment-related lesions. There was good correlation between gross a nd microscopic findings. The re lative (to bodyweight) testes weight was non-significantly increased (17.5%) at the high dose; however there were no significant changes in absolute or relative-to-brain weights for the testes. Absolute and relative ovarian weights were increased at 30000 ppm; however, the ch ange was not statistically significant and attributed to a single animal in the group. There were no histopathological findings in the ovaries.

Renal tubule adenomas were found in ma le mice in the 5000 and 30,000 ppm dose groups; however, it was concluded that the renal tumors we re not dose-related. Trends, and effects in the high-dose group (p<0.01), were observed in non- neoplastic lesions. The lesions considered treatment-related were hepatocy te hypertrophy, central lobular hepatocyte n ecrosis, and chronic interstitial nephritis in hi gh-dose males, and proximal t ubule epithelial basophilia and hypertrophy in high-dose females.

The NOAEL for systemic effects was 5000 ppm (750 mg/kg/day). At the LOAEL, 30,000 ppm (4500 mg/kg/day), there were in creased incidences of hepa tocyte hypertrophy, hepatocyte necrosis, and interstitial nephr itis in male mice and increase d incidences of proximal tubule epithelial basophilia and hypertrophy in female mice. Addition ally, there were treatment-related decreases in body weights in male and female mice at 30,000 ppm.

Chronic Oral Toxicity in Non-Rodents (Dog; OCSPP 870.4100)

In a chronic toxicity study in dogs ( MRID 00153374 ), glyphosate (96.13% purity) was administered to Beagle dogs (6/sex/group) at concentrations of 0, 20, 100, or 500 mg/kg/day for one year. Criteria evaluated in cluded clinical signs, morta lity, body weight, food consumption,GLYPHOSATE FINAL

Page 56 of 62 ophthalmologic evaluation, hematology, clinical chemistry, urinalysis, organ weights (including adrenals, kidneys, liver, ovaries, testes, pituitary, and thyroid/pa rathyroid), and histopathology of selected tissues (not report ed) and all gross lesions.

All animals survived the duration of the study. Th ere were no dose-related, clinical signs of toxicity, and body weight and food consumption in treated dogs were comparable to control. There were no dose-related change s in hematological or clinical chemistry values. Decreased pituitary weights (absolute and relative) we re observed in mid-and high-dose male dogs, however, these were not considered to be tr eatment-related because there were no underlying histopathological changes in that organ and there was no dose relationship in the weight changes. No treatment-related changes were seen in absolute or relative weights of the other organs, including testes, ovaries, thyr oid, and adrenal glands. Ther e were no treatment-related histopathological findings, and th e incidence and grade of micros copic lesions were comparable between treated and control dogs for all organs including testes, ovaries, thyroid, adrenals, and pituitary.

The NOAEL for the study is the high dose of 500 mg/kg/day; a LOAEL was not identified.

Avian Reproduction (OCSPP 850.2300)

In an avian reproduction study ( MRID 00111953 ), mature Mallard ducks were administered glyphosate (83% purity) at nominal concentratio ns of 0, 50, 200, or 1000 ppm (mg/kg) in the diet for 17 weeks (nine weeks prior to the start of egg production and eight weeks of egg production). Each treatment group was comprised of five pens , with five drakes a nd two hens per pen. Body weights were taken four times: at initiation, after five weeks, prior to onset of egg laying, and at termination of the study. Food consumption was recorded biweekly. Eggs were cleaned at weekly intervals and placed in an incubator. On Days 22 or 23 of incubation, the eggs were placed in a hatcher, and on Days 26 or 27, hatchl ings were housed according to the appropriate parental grouping and maintained on control diet until 14 days of age. For the purposes of measuring egg weight and egg shell thickness, one egg from each pen was randomly selected on a weekly basis.

Ducks treated with glyphosate up to 1000 ppm (limit dose) showed no symptoms of toxicity or
behavioral abnormalities for the duration of the study. There was one mortality on study in the high-dose group during Week 12. Because the mort ality occurred during the stress of egg production and no gross abnormalities were not ed upon gross necropsy, the death was not
considered to be treatment-related. There were no treatment-related effects on the reproductive parameters that were evaluated: eggs laid, eggs cracked, viab le embryos, live 3-week embryos, normal hatchlings, 14-day-old survivors, repres entative hatchlings body weight, representative 14-day-old survivors body weight , egg weight, and eggshell thickness. The NOAEC is 1000 ppm and the LOAEC is >1000 ppm.GLYPHOSATE FINAL

Page 57 of 62 In another avian reproduction study with the mallard duck ( MRID 00036328 ), test animals were exposed to concentrations of control, 3, and 30 ppm. There were five pens per treatment group with 2 males and 5 females per pen. Birds re ceiving glyphosate at 3 a nd 30 ppm in the diet showed no signs of toxicity or be havioral abnormalities during the course of the study. All test and control birds appeared norma l throughout the study and there were no observed mortalities. Additionally, there were no trea tment related effects on any reproductive parameter assessed.

In a third mallard duck reproduction study (M RID 48876602), the one-generation reproductive toxicity of Glyphosate-acid to 16 pairs per level of 21-week old Mallards ( Anas platyrhynchos ) was assessed over 21 weeks. Glyphosate-acid was admini stered to the birds in the diet at nominal concentrations of 0 (nega tive control), 500, 1000, and 2250 mg ae/kg diet. Mean-measured concentrations were 0 (control), 501, 987, a nd 2160 mg ae/kg diet. No treatment-related mortalities or signs of toxicity occurred during th e test. One incidental mortality was observed in each of the control, 501, and 987 mg ae/kg groups. Significant reductions were detected at the low and high treatment levels for 14-day surv ivor body weight and adult male weight gain (Dunnett’s, p<0.05); no effects were detected at the 987 mg ae/ kg diet level for any endpoint (p>0.05). Reductions in 14-day su rvivor body weights at the low and high dose were 10-11% of control body weight values and a dult male birds did not gain we ight at the low dose and lost weight during the study at the high dose. Additio nally, the reviewer’s analysis detected a suggestively dose-dependent, significant 6% reduc tion from control in ha tchling body weights at the high dose level (William’s, p<0.05). No other significant treatment-related differences were observed between the control group and any of the treatment groups. Based on the significant reductions from control in offspring survivor body weight and adult male body weight gain at the 501 mg ae/kg diet level, the revi ewer concluded that a NOAEL coul d not be determined in this study (i.e., <501 mg ai/kg diet).

In an avian reproduction study ( MRID 00108207 ), mature Bobwhite quail were administered glyphosate (83% purity) at nominal concentra tions of 0, 50, 200, or 1000 ppm diet for 17 weeks (nine weeks prior to the star t of egg production and eight w eeks of egg production). Each treatment group was comprised of 12 pens, w ith one male and two females per pen. Body weights were recorded at the initiation of the st udy, after five weeks, i mmediately prior to the onset of egg laying, and at study terminati on. Food consumption was recorded biweekly throughout the study. Day 19 eggs were placed in a hatcher. All eggs on day 21 or 22 were removed from the incubator. Hatchlings were housed according to the appropriate parental grouping and maintained on a contro l diet until 14 days of age. One egg from every other pen was taken weekly for egg weight and eggshell measurements.

Quails treated with glyphosate up to 1000 ppm (limit dose) showed no symptoms of toxicity or
behavioral abnormalities for the duration of the study. There were three mortalities during the study in the control group at Weeks 13, 14, and 17. Because the mortalities occurred during the stress of egg production and no gross abnormaliti es were noted upon gross necropsy, the deathsGLYPHOSATE FINAL

Page 58 of 62 were not considered to be treatment-related. There was no effect of treatment on the following reproductive parameters: eggs laid, eggs cracked , viable embryos, live 3-week embryos, normal hatchlings, 14-day-old survivors, and repres entative hatchlings body weight. Decreases in eggshell thickness (50 ppm group; ↓ 5%, p<0.05) and egg we ight (1000 ppm group; ↓ 8%, NS)
were not considered to be biol ogically meaningful, as there was no dose response observed for eggshell thickness decreases and no effect on overall reproductive success. The NOAEC is 1000 ppm and the LOAEC is >1000 ppm.

Fish Early Life-Stage Toxici ty (Threespine Stickleback)

In a published non-Guideline study ( Le Mer et al. , 2013 ), the effects of glyphosate ( ≥ 96% purity) exposure on larval threespine sticklebacks ( Gasterosteus aculeatus ) was determined according to growth, survival, the reproductive biomarkers vitellogenin (VTG) and spiggin (SPG), and sexual differentiation as endpoints in exposed larvae. The tested concentrations of 0.1, 1, and 10 μ g/L were considered to be representati ve of environmental concentrations found
in estuarine and coastal waters of east ern North America; a higher pharmacological
concentration of 100 μ g/L also was included.

Adult threespine sticklebacks were collected from an uncontaminated site on the Gaspé Peninsula (Rivière Saint-Jean, QC) and mature adult males and females displaying secondary sexual characteristics were placed separately in 105-L tanks supp lied with a constant flow of
filtered seawater and maintained at 18±1°C a nd salinity 15±1%, with a 16:8 hour light:dark photoperiod. After fish were acclimated for ten days, 40 males and females were placed in 45-L tanks containing nesting material s. Mating trials were conducte d, and fertilized egg clutches were transferred to 1 L beakers with aerated IML seawater, at 18-19°C, until hatching. Newly hatched larvae, at Day 7 after fe rtilization, were exposed to the tr eatments in 1-L glass beakers with 500-mL portions of aerated seawater at 18°C with a 16:8 hour light:dark photoperiod. The tested concentrations were 0.1, 1, 10, and 100 μ g/L glyphosate, and the te sts included a negative (seawater) control, a positive control of 17 α -ethinylestradiol (EE2; 0.05 μ g/L), and a positive control of dihydrotestosterone (DHT; 3 μ g/L). Each treatment included three replicates, with 9- 10 fish per replicate for glyphosate. Fish we re exposed throughout the embryo-larvae and juvenile life stages (from hatching to Day 42 post-hatching) until fish had a mean length of ~20 mm. Larvae were fed once per day with Artemia nauplii ; water was changed every day (>80% replacement) before feeding. Dissolved oxygen, temp erature, and fish mortality were monitored daily. Experiments were conducted during the su mmer of 2007 and repeated during the summer
of 2008.

At the end of the 42-day exposure period, survivi ng fish were sacrificed and total body lengths (L; mm) and wet weights (W; mg) were determin ed; a condition factor (CF) was calculated as CF = (W/L 3 )100. Half of the fish from each replicate, selected at random, were frozen at –80°C for VTG and SPG measurements, and the remaining fish were preserved fo r intersex evaluations;GLYPHOSATE FINAL

Page 59 of 62 a subset of these fish were processed for histol ogical evaluation. Vitellog enin was isolated from the whole-body homogenates of juvenile stickl ebacks with a previously described method and measured with an indirect competitive ELISA. Stickleback SPG was determined with a competitive ELISA that included an antibody ag ainst a synthetic peptide specific to SPG sequence. For histological evaluations, the gonads were examined by light microscopy; all sections were examined for phenotypic sex determination.

In 2007, mean glyphosate concentrations could no t be determined due to possible seawater interference. In 2008, mean glyphosate con centrations were 0.08, 1.0, 8, and78 mg/L ( ≤ 22% of nominal).

There was <11% mortality in the fish exposed to glyphosate over the 42-day exposure period. There were no significant differe nces in wet weight, body length, or condition of juvenile fish between glyphosate-exposed fish an d control in either test year . The study authors noted that there was a significant difference in the size of fish in the 2007 trials comp ared with those of the 2008 trials, even though fish came from the same field site; the 2008 fish were smaller (up to 12% less in body length) and weighe d less (up to 32% less in wet we ight) than the 2007 fish. For the 2007 trial, the condition factor for fish treated with EE2 was similar to all other treatments. In 2008, the condition factor for EE2-treated fish was decreased (p<0.05) compared to the DHT
treatment and the 1.0 and 100 μ g/L glyphosate treatments. Treatment with EE2 resulted in significant (p<0.05) induction of whole-body VTG compared to other treatments. There was no
VTG induction observed in fish treated with DHT or glyphosate, or in fish in the negative control groups. No induction of SPG was observed in any of the treatment groups, including the positive DHT control.

Glyphosate treatment had no effect on the proporti on of mixed sex individuals. A single mixed sex individual was found in the group exposed to 10 μ g/L glyphosate. The proportion of mixed sex individuals was higher in the positive controls (EE2, DHT) compared to the negative control. The proportion of female individu als did not differ significantly among treatments, but tended to be higher in the fish exposed to EE2 and the lowest dose of glyphosate, and lower in fish exposed to DHT. For histological evaluations, all sections with gonad tissue were examined for phenotypic sex determination. The nu mber of gonad sections per in dividual and the proportion of individuals without gonad sect ions did not differ among treatme nts. It was concluded that glyphosate exposure had no effect on larval survival or growth, a nd that glyphosate did not exert estrogenic or androgenic effects on early life-stag es of sticklebacks at environmentally-realistic
concentrations.

Toxicity to Amphibians (Non-guideline)

In a published non-guideline study (Howe et al, 2004), the effects of glyphosate (reported as technical grade) to leopard fr ogs (Rana pipeins) was investig ated at 0.6 and 1.8 mg glyphosate free acid equivalents (FAE).GLYPHOSATE FINAL

Page 60 of 62 The study reports on a series of studies conducte d over roughly a seven-year period examining the acute and chronic effects of glyphosate alone , the surfactant polyeth oxylated tallowamine (POEA) and glyphosate based product s on four aquatic-phase amphibi an (anuran) species. It is noted that only the effects of glyphosate alone were considered in the weight of evidence for endocrine related(decreases in offspring body we ight observed in one avian reproduction study) effects.

Treatment with glyphosate alone did not reveal any effects on growth (length), tail morphology, percent surviving to reach St age 42, time to metamorphosis, gonadal development or thyroid hormone gene expression relative to controls ; however, treatment with POEA alone or glyphosate formulations containing POEA reduced th e percentage of larvae surviving to reach Stage 42, decreased length at metamorphosis, in creased time to metamorphosis, and resulted in mixed-sex gonads.

Mortality was relatively hi gh in chronic study control gr oups (38%) and suggests that environmental conditions were not ideal for prom oting survival of leopard hogs. This may have been due to relatively high loading rates coupled with partial water changes every 96 hours. Although effects on metamorphosis and gonadal deve lopment appear to be treatment related, poor environmental conditions could affect development.

Fish Life Cycle Toxicity (Fathead Minnows) (OPPTS 850.1500)

In a fish full life- cycle toxicity study ( MRID 00108171 ), Fathead minnows ( P. promelas ) were continuously exposed to glyphosat e (87.3% purity) at nominal concentrations of 0, 1.6, 3.2, 6.3, 12.5, and 25.0 mg/L; mean-measured concentrations were 0.7, 2.8, 7.0, 13.0, and 25.7 mg/L. In duplicate aquaria for each treatmen t, two groups of thirty eggs were incubated; dead eggs were removed and counted each day until hatching was co mpleted (4 days at 25°C). Percent hatch was based on the number of live fry from 60 eggs. Fort y fish, divided into two groups of twenty each, were randomly selected and distributed to growth chambers in each aquarium. Two growth chambers were used to facilitate handling of fry for 30- and 60-day measurements by a photographic method. Percent survival based on cu mulative mortality was also determined at these intervals. After the Day 60 measurements, the number of fish released to each spawning chamber was impartially reduced to fifteen after combining fish from the growth chambers. On Day 64, five spawning sites were placed in the spawning chamber. After secondary sexual
characteristics were well deve loped (approximately Day 134), the number of fish in each tank was reduced initially to four males and four fema les, and finally to two males and four females on Day 179. Eggs were removed from the spaw ning sites daily and counted after spawning began (approximately Day 112). Fifty eggs from each of the first ten spawnings per tank were oscillated in their resp ective test waters by means of an e gg cup, and dead eggs were removed and counted each day until hatching was completed (3-5 days at 25°C). Percent hatch was based on the number of live fry from 50 eggs. Twenty fr y from the first two spawns in each tank in
which at least 80% live hatch was observed were placed in their respective growth chambers. After thirty days of exposure, fry groups were terminated and total le ngths determined by theGLYPHOSATE FINAL

Page 61 of 62 photographic method. Total wet weight and percent su rvival were also determined at this time for each fry group.

There was no treatment effect on any of the pa rameters measured: surv ival, growth and egg production of first generation fish ; and hatchability, survival, a nd growth of second generation eggs and fry. The NOAEC is 25.7 mg/L and the LOAEC is ≥ 25.7 mg/L.GLYPHOSATE FINAL

Page 62 of 62 APPENDIX 3: References Not Utilized in the Glyphosate 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 Glyphosate 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 data on invertebrates.

Forgacs, A. L., Ding, Q., Jaremba, R. G., Huhtanie mi, I. T., Rahman, N. A., and Zacharewski, T. R. (2012) BLTK1 murine Leydig ce lls: a novel steroidogenic model for evaluating the effects of reproductive and developmental toxicants. Toxicol. Sci. 127(2):391-402