NR 481420-01

-2.32,

GLYPHOSATE:

A Comprehensive Review and Evaluation of Other

Scientifically Relevant Information (OSRI) that Addresses

the Requirements for Tier 1 Screening Under the Endocrine

Disrupter Screening Program

Prepared on behalf of the

Joint Glyphosate Task Force, LLC

June 22, 2010

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Executive Summary

Introduction and Background

In June 2007, EPA published in the Federal Register a Notice announcing the Draft List of Initial

Pesticide Active Ingredients and Pesticide Inert Ingredient to be Considered for Screening under

the Federal Food, Drug and Cosmetic Act [72 FR 33486]. The initial chemicals to be tested

under the EPA Endocrine Disrupter Screening Program (EDSP) were selected based on four

human exposure pathways described in detail in a September 2005 Federal Register notice,

namely food consumption, drinking water consumption, residential use exposure and

occupational exposure (contact with pesticide-treated surfaces) [70 FR 56449]. The highest

priority for inclusion on the initial list was for substances, such as glyphosate, having potential

exposure through all four pathways. Throughout the selection process, EPA clearly stated that

"[t]his list should not be construed as a list of known or likely endocrine disruptors. Nothing in

the approach for generating the initial list provides a basis to infer that by simply being on the

list these chemicals are suspected to interfere with the endocrine systems of human or other

species, and it would be inappropriate to do so."

The Office of Management and Budget stated in its "Terms of Clearance" related to EPA's

issuance of the first set of testing orders that "EPA should promote and encourage test order

recipients to submit OSRI in lieu of performing all or some of the Tier I assays, and EPA

should accept OSRI as sufficient to satisfy the test orders to the greatest extent possible"

(OMB, 2009).

This document provides a comprehensive overview of Other Scientifically Relevant Information

(OSRI) to assess endocrine-related endpoints that provide functionally equivalent information to

the proposed Tier 1 screening battery. OSRI is defined by EPA as information that informs the

determination as to whether the substance may have similar effect to a substance that interacts

with estrogen, androgen or thyroid systems. Functionally equivalent, in this context, refers to

data of a suitable nature and quality to provide the same essential predictive information on a

chemical even if different methods and procedures may have been used for obtaining the data.

Specifically, EPA has characterized OSRI as being either: (1) functionally equivalent to

information obtained from the Tier 1 assays or (2) data from assays that perform the same

function as EDSP Tier 1 Screens. OSRI for glyphosate is drawn from both of these categories.

Factors included in this evaluation of OSRI are functional equivalence of the endpoints, their

sensitivity, their specificity for a particular mode of action, confidence in the conclusions and

applicability across taxa. Factors also include route and duration/frequency of administration,* •

dose levels tested, age at exposure, species, number of test units, variability, arjd* fjhether tht'datS

provides a basis for conclusions for potential interaction with the endocrine syStern" i&

mammalian and non-mammalian systems. OSRI to provide information to bypasf»Tjer 1 ; \.€

screening can broadly include the following study types: structure activity relationsbfps, in

assays, metabolism, subchronic, chronic, reproductive and developmental studrasjG§RI for**'

glyphosate primarily includes information from toxicity tests carried out to inteirii^ikflial

regulatory guidelines that includes and assessment of reproduction, fertility and developmwitei*

benchmarks predictive of endocrine modulating potential. Additionally, in many of these la..,

these studies histopathology is typically carried out on:
• Endocrine tissues (pituitary, thyroid, parathyroid, pancreas, adrenal, ovary and testis)

• Hormone sensitive male tissues (prostate, epididymides, seminal vesicles)

• Hormone sensitive female tissues (mammary, uterus and reproductive tract)

Other relevant scientific information presented here for glyphosate is data obtained from study

protocols that focus on reproduction, fertility and development for mammalian species and other

taxa, such as birds and fish.

The Tier 1 screening battery is comprised of 11 assays, five of which are in vitro and 6 of which

are in vivo assays. The in vitro screening assays include the estrogen receptor (ER) competitive

binding assay, the ER transcriptional activation assay with stably transformed cell line, the

androgen receptor (AR) competitive binding assay, the aromatase inhibition assay and the

steroidogenesis assay in H295R cells. The in vivo screening assays include the uterotrophic

assay, the Hershberger assay, the male and female pubertal assays, the amphibian metamorphosis

assay, and the short-term fish reproduction assay.

Glyphosate (CAS number 1071-83-6) [N-(phosphonomethyl) glycine] is an herbicide belonging

to the phosphono amino acid class of pesticides. The herbicidal properties of glyphosate were

discovered by Monsanto in 1970, and the first commercial formulations were introduced in 1974

under the RoundupTM® brand name. Glyphosate is a foliar, non-selective, systemic herbicide

widely used to control weeds in agricultural crops and non-agricultural sites. Glyphosate is a

specific inhibitor of the enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS). This

enzyme is the sixth enzyme of the shikimate pathway and it is essential for the biosynthesis of

aromatic amino acids (i.e., tyrosine, tryptophan and phenylalanine) and the other aromatic

compounds in algae and higher plants, bacteria, fungi and some parasites. Inhibition of this

enzyme leads to plant cell death. It also important to point out that the shikimate pathway is

absent in animals. Because of its very specific mode of action, glyphosate exhibits low toxicity

to many taxonomic groups of non-target organisms including mammals, birds, fish, aquatic

invertebrates and terrestrial invertebrates.

Important considerations in evaluating potential to interact with the endocrine system include

absorption, distribution, metabolism and excretion (ADME). Low absorption, limited

distribution and metabolism, and rapid elimination greatly diminish the potential for a compound

to interact with the endocrine system. Glyphosate has been shown in multiple studies conducted

on multiple species to have low GI tract and dermal absorption (Ridley and Mirly, 1988; Bo

Nielsen et al., 2009), limited distribution (Colvin and Miller, 1973), almost no mammalian

metabolism (Ridley, 1983), and rapid excretion (Wester, 1991). Therefore, the potential for

systemic exposures to endocrine tissues is extremely low for glyphosate and transformation

products.

Glyphosate has undergone a comprehensive battery of in vivo toxicology and ecotoxicology

testing that cover a broad spectrum of endocrine endpoints. This testing includes reproductive,

developmental, subchronic and chronic tests that have robust experimental designs, follow

internationally accepted protocols, have a high level of replication and a long history of use in

hazard identification and risk assessment. These in vivo studies thoroughly assess endocrine

functions that are required for reproduction, development, and chronic health. It is noteworthy

that the two recent rat multigenerational studies have been conducted that conform to the 1998

US EPA guideline (one of which also conforms to the 2001 OECD 416 guideline), including
rigorous investigations of endocrine sensitive endpoints. In many of the chronic studies no

effects and/or no effects related to endocrine activity were detected at high concentrations and at

highest concentration tested. This provides strong evidence that glyphosate does not have the

potential to be an endocrine active compound. Additionally, the endocrine-modulating potential

of glyphosate has been evaluated in a variety of in vitro assays, which also demonstrate that

glyphosate does not have endocrine activity. An overview of the OSRJ to meet all of these Tier

1 screening requirements is summarized below.

ER binding and transcription activation: Assays that provide data that are functionally

equivalent to data generated by the ERa/p competitive binding assay using rat uterine cytosol

and the ERa transactivation assay have already been conducted. Peer reviewed articles by

Kojima et al. (2004) and Petit et al. (1997) present scientific information from in vitro test

systems that are very comparable in methodology, sensitivity and specificity to the two Tier 1

ER binding assays. The results from these assays consistently demonstrate that glyphosate is not

an ER agonist or antagonist. Furthermore, a structure activity relationship (SAR) analysis with

methodology developed by EPA's Office of Research and Development clearly shows that

glyphosate does not have the chemical structure of a typical molecule with estrogenic potential.

The in vitro data demonstrating that of glyphosate does not have ER binding potential is

consistent with the comprehensive in vivo data available for glyphosate. The in vivo data

demonstrates no effects on any endpoint that is sensitive and specific to compounds that have

estrogenic or anti-estrogenic activity. A detailed review of the in vivo data used to evaluate

estrogenicity is discussed in the sections for the uterotrophic, female pubertal assay and fish

screening assays. Based on the weight of evidence, it can be concluded that no additional Tier 1

in vitro screening is required to evaluate the potential for ER agonism and antagonism.

AR binding: Assays that provide data that is functionally equivalent to data generated by the AR

competitive binding assay using rat prostate cytosol have also already been conducted. The peer

reviewed article by Kojima et al. (2004) presents scientific data using an in vitro test system and

methodology that is functionally equivalent to the methodology for the AR binding assay in the

Tier 1 screening battery that demonstrates that glyphosate is not an AR agonist or antagonist.

Furthermore, the SAR analysis again indicates that glyphosate does not have the chemical

structure of a typical molecule that exhibits androgenic potential. Lack of AR binding is

confirmed by the comprehensive in vivo data available from chronic studies on glyphosate. The

in vivo data demonstrates no effect on endpoints that are sensitive and specific to compounds that

have androgenic or anti-androgenic activity. A detailed review of the in vivo data that confirms

glyphosate's is not androgenic or anti-androgenic is extensively reviewed in the sections for the

Hershberger, male pubertal assay and fish screening assay. Based on the weight of evidence it

can be concluded that no additional in vitro screening is required to evaluate the potential for AR

agonism and antagonism.

Aromatase Inhibition: Although in vitro data is not available to assess aromatase inhibition,

OSRJ of the same nature and quality is available from existing in vivo chronic studies. No

treatment related effects were observed in these studies on sensitive and specific endpoints that

would be indicative of aromatase inhibition, which provides strong and consistent evidence that

glyphosate in not an inhibitor of aromatase activity. Scientifically relevant information related to

steroidogenesis is comprehensively discussed in the following section and also applies to

aromatase inhibition. Consequently, the requirement for performing the in vitro aromatase assay
has been met by providing OSRI from standard regulatory in vivo studies that have the ability to

detect effects of inhibition of aromatase activity.

Steroidogenesis: In vitro data assessing potential impacts on the complete steroidogenic

pathway are not available for glyphosate; however, data that is functionally equivalent to the in

vitro Steroidogenesis assay is provided by endpoints included in rat multigenerational studies.

Specifically, it was confirmed in the most recent multigenerational study that there was no

treatment related effect at high dietary exposure levels on endpoints that would be impacted by

an alteration of Steroidogenesis, e.g., the weight of testes, cauda epididymides, epididymides,

seminal vesicles and coagulating glands, uterus, ovary and liver of the FO and Fl parent animals

(Moxon, 2000; Dhinsa et al., 2007). Additionally, there was no treatment related effect up to the

highest dose tested (HDT) on the histopathology of left testis, left epididymis, prostate, seminal

vesicles with coagulating gland, uterus, left ovary, vagina, cervix, and oviducts. Furthermore,

there was no treatment related effect up to the HDT on male and female reproductive indices that

included mating, fertility, conception, gestation, lactation age and weight at VO, age at PPS,

testicular descent, ovarian evaluation for follicles, parturition, normality of external genitalia and

number of implantation sites. The in vivo information from these studies provides compelling

evidence of no impact of glyphosate on Steroidogenesis.

Uterotrophic: A strong weight of evidence has been provided in Section 3.1 of this document,

with functionally equivalent information supporting a lack of estrogenic activity of glyphosate in

vivo for four multigenerational studies, subchronic and chronic studies in rats. The results of the

in vivo studies are consistent with results from in vitro assays that demonstrate glyphosate is not

an ER ligand. Functionally equivalent data (uterine weights) of the same nature and quality to

data generated with the Uterotrophic assay (uterine weights and histopathology) demonstrate that

glyphosate does not possess estrogenic activity.

Hershberger: A strong weight of evidence has been presented in Section 3.3 of this document,

using functionally equivalent information supporting a lack of androgen receptor agonism and

antagonism and 5a-reductase inhibition of glyphosate in vivo with four multigenerational studies,

subchronic and chronic studies in rats. This functionally equivalent data from multigenerational

studies, subchronic and chronic studies in rats demonstrate that glyphosate is not an androgen

receptor agonist or antagonist or a 5a-reductase inhibitor.

Female Pubertal: Functionally equivalent data to the female pubertal endpoints are described

in Section 3.3 of this document, with a comprehensive overview of endpoints addressed in the

multigenerational reproductive, subchronic and chronic rat studies. Endpoints in the female

pubertal assay are largely a subset of the multigenerational studies performed on glyphosate and

consequently provide data of the same nature and quality to data developed in the female

pubertal assay of the Tier 1 battery of the Endocrine Disrupter Screening Program.

Male Pubertal: Like the female pubertal assay, functionally equivalent data to the male

pubertal endpoints are described in Section 3.4, with a comprehensive overview of endpoints

addressed in the multigenerational reproductive, subchronic and chronic rat studies. Endpoints

in the male pubertal assay are also essentially a subset of endpoints from the multigenerational

studies performed for glyphosate and consequently provide data of the same nature and quality to

data developed in the male pubertal assay required to be conducted in the EDSP.

Amphibian Metamorphosis: The amphibian metamorphosis assay has been included in the Tier

1 battery as a generalized model for impacts on vertebrate thyroid function because the
amphibian thyroid system is regulated and responds in a similar manner to perturbation as that of

other tetrapods, including mammals. Therefore, combined results from existing glyphosate

studies provide information that is functionally equivalent to that generated in the amphibian

metamorphosis assay. Thyroid histopathology has been evaluated in standard mammalian

toxicology studies and demonstrates no impact on thyroid structure and function. Supporting

information is drawn from the fish full life-cycle study that shows no impact on growth and

development, which are sensitive measurement endpoints for impacts on thyroid structure and

function. Given all of this available information on glyphosate, the amphibian metamorphosis

screening assay does not offer any new relevant information to the toxicology data set for Tier 1

screening purposes.

Fish Short-term Reproduction: The methodology and information generated from a fish full

life-cycle study provides a comprehensive evaluation of potential modulation of endocrine

activity in fish and provides functionally equivalent information to information from the shortterm fish reproduction assay. The fish full life-cycle study is the highest Tier offish

ecotoxicology testing and typically performed with the fathead minnow (Pimphales promelas) as

a freshwater test species. In the study, the entire life-cycle is exposed; therefore, de facto the

most sensitive life-stage to any toxicant (including endocrine active compounds) is encompassed

by the test design. Although, endpoints that are potentially influenced by endocrine activity,

such as gonadal histopathology and steroid hormone production, are not typically measured in

the full life-cycle, a fish full life-cycle study provides a comparable investigation to the test

proposed in the higher Tier 2 testing for fish. The fish full life-cycle study for glyphosate

demonstrated no treatment-related treatment related effects on the survival, growth and egg

production of first generation fish or on hatchability, as well as survival and growth of secondgeneration eggs and fry over an 8 month exposure period to concentrations as high as 25.7 mg/L.

Furthermore, it has been shown in vivo that glyphosate does not induce production of

vitellogenin, which is in agreement with SAR analysis of glyphosate. The results from the fish

full-life cycle study are consistent with the results form subchronic and chronic test with

mammalian species, which demonstrate no changes in endocrine function, endocrine organ

histopathology, and all reproductive parameters. Additionally, the results from avian onegeneration reproduction studies demonstrate no effect on any of the measured endpoints at high

test concentrations. Based on this OSRI, there is sufficient information to meet the requirements

for performing a fish short term reproduction study.

Published Glyphosate Reviews for Endocrine Activity

Several in-depth reviews on the safety of glyphosate have been conducted by several regulatory

agencies and scientific institutions worldwide have concluded that there is no indication that

glyphosate has potential endocrine activity or will result in adverse effects on endocrine systems

in humans or other mammals. The following points summarize the conclusions of these reviews.

• The U.S. EPA (1998) reviewed the subchronic and chronic mammalian studies for

glyphosate and concluded that there was no evidence to suggest that glyphosate produces

endocrine-modulating effects.

• In a comprehensive review of the standard mammalian toxicology studies, Williams et al.,

(2000) concluded that glyphosate does not have the potential to produce adverse effects on

endocrine systems in humans or other mammals.
• A report prepared for the USDA forest service by Syracuse Environmental Research (SERA)

concluded that extensive testing in experimental animals and wildlife provided reasonably

strong evidence that glyphosate is not an endocrine disrupter (SERA, 2002).

• The list of purported endocrine disrupters compiled by the Institute of Environment and

Health (IEH, 2005) lists glyphosate as a substance with no evidence of potential endocrinedisrupting effects.

• Following a review of the standard mammalian and wildlife toxicology studies, it was

concluded in a recent ECETOC guidance document on identifying endocrine disrupting

effects that glyphosate is not an endocrine disruptor using the internationally accepted

Weybridge definition for endocrine disruption (ECETOC, 2009).

Summary and Conclusions

The endocrine-modulating potential of glyphosate has been evaluated in a variety of studies

including in vitro assays and standard in vivo toxicology studies that are functionally equivalent

to the data generated in the Tier 1 screening battery. The in vivo studies comprehensively assess

endocrine functions that are required for reproduction, development and chronic health and show

no indication of endocrine disruption in mammals and wildlife species. Glyphosate has

demonstrated no (anti-) estrogenic or (anti-) androgenic potential in in vitro assays, and there

was no indication of changes in endocrine function or endocrine organ histopathology in any of

the in vivo studies. Single effects were both minor and not consistent within or between studies

or were related to general toxicity. Additionally, structure activity models predict that

glyphosate does not have estrogenic or androgenic activity.

Based on this weight-of-evidence analysis from glyphosate's extensive toxicological database,

there is sufficient scientifically relevant information of the same nature and quality as that

developed in the Tier 1 screening battery to conclude that glyphosate does not have endocrinemodulating potential. Therefore, the requirements of Tier 1 testing have been satisfied and

additional testing under the Endocrine Disruption Screening and Testing Program is not

warranted.

1

 Weybridge (1996) "An endocrine disrupter is an exogenous substance that causes adverse health effects in an

intact organism, or its progeny, secondary (consequent) to changes in endocrine function. A potential endocrine

disrupter is a substance that possesses properties that might be expected to lead to endocrine disruption in an intact

organism."
Contents

Executive Summary 2

Introduction and Background 2

Published Glyphosate Reviews for Endocrine Activity 6

Summary and Conclusions 7

1.0 Introduction 11

1.1 Considerations for Dose Setting for Endocrine Screening and Testing 15

2.0 In Vitro Assays 16

2.1 Estrogen Competitive Receptor Binding Assay (OPPTS 890.1250) and ER

Transcriptional Activation Assay (OPPTS 890.1300) 16

2.1.1 Structure Activity Relationship 16

2.1.2 Estrogen Receptor (ER) Binding and Transactivation Assays 16

2.1.3 Conclusion -Estrogen Receptor Agonism and Antagonism 21

2.2 Androgen Receptor (AR) Binding Assay (OPPTS 890.1150) 22

2.2.1 Structure Activity Relationship (SAR) Prediction of AR Binding Potential 22

2.2.2 Androgen Receptor (AR) Binding Assay 22

2.2.1 Conclusion - Androgen Receptor Agonism and Antagonism of Glyphosate 24

2.3 Aromatase Assay (OPPTS 890.1200) 25

2.3.1 Conclusion - Aromatase Inhibition 26

2.4 Steroidogenesis Assay (OPPTS 890.1550) 27

2.3.1 Conclusion - Steroidogenesis 29

3.0 Mammalian In Vivo Assays 29

3.1 Uterotrophic Assay (OPPTS 890.1600) 30

3.1.1 Discussion 31

3.1.2 Conclusions - Uterotrophic Assay 34

3.2 Hershberger Bioassay (OPPTS 890.1400) 35

3.2.1 Prostate / Ventral Prostate 36

3.2.2 Seminal Vesicles 36

3.2.3 Levator Ani Bulbocavernosus (LABC) 37

3.2.4 Bulbourethral (Cowper's) Gland 37

3.2.5 Glans Penis Weight 38

3.2.6 Liver Weight 38

3.2.7 Luteinizing Hormone (LH) 39

3.2.8 Testosterone 39
3.2.9 Other endpoints of relevance to the Hershberger Assay 39

3.2.10 Conclusion - Hershberger Assay 40

3.3 Pubertal Development and Thyroid Function in Intact Juvenile / Peripubertal Female

Rats (OPPTS 890.1450) 40

3.3.1 Growth, Body Weight 41

3.3.2 Age at Vaginal Opening 42

3.3.3 Body Weight at Vaginal Opening 42

3.3.4 Uterus Weight and Histopathology 42

3.3.5 Estrus Cyclicity 43

3.3.6 Ovaries Weight and Histopathology 43

3.3.7 Thyroid Weight and Histopathology 43

3.3.8 Thyroid Stimulation Hormone (TSH) 44

3.3.9 Thyroxine (T4) 44

3.3.10 Kidney Weight and Histopathology 45

3.3.11 Liver Weight 45

3.3.12 Adrenals Weight 45

3.3.13 Pituitary Weight 46

3.3.14 Clinical Chemistry 46

3.3.15 Conclusion - Female Pubertal Assay 46

3.4 Pubertal Development and Thyroid Function in Intact Juvenile / Peripubertal Male

Rats (OPPTS 890.1500) 47

3.4.1 Growth, Body Weight 48

3.4.2 Age at Preputial Separation 49

3.4.3 Body Weight at Preputial Separation 49

3.4.4 Testes Weight and Histopathology 50

3.4.5 Epididymides Weight and Histopathology 50

3.4.6 Prostate Weight 50

3.4.7 Seminal Vesicles Weight 51

3.4.8 Testosterone 51

3.4.9 Thyroid weight and Histopathology 51

3.4.10 Thyroid Simulation Hormone (TSH) 51

3.4.11 Thyroxine (T4) 52

3.4.12 Kidney Weight and Histopathology 52

3.4.13 Liver Weight 52

3.4.14 Adrenals Weight 52
3.4.15 Pituitary Weight 53

3.4.16 Clinical Chemistry 53

3.4.17 Conclusion - Male Pubertal Assay 53

3.5 Summary of In Vivo OSRI for Mammalian Species 55

4.0 Non-Mammalian In Vivo Assays 55

4.1 Amphibian Metamorphosis Assay (AMA, OPPTS 890.1100) 55

4.1.1 Conclusion - Amphibian Metamorphosis Assay (AMA) 57

4.2 Fish Short-term Reproduction Assay (OPPTS 890.1350) 58

4.2.1 Results Summary-Fish Full Lifecycle Study 61

4.2.2 Structure Activity Relationship Indicates Lack of Estrogenicity 61

4.2.3 Glyphosate does not induce production of vitellogenin 61

4.2.4 Avian one-generation reproduction assays 62

4.2.5 Conclusion - Fish short-term reproduction assay 62

5.0 Conclusion - EDSP Tier 1 OSRI Overview 64

5.1 Glyphosate Reviews for Endocrine Activity 67

5.2 Summary and Conclusions 67

References 68

Appendix 1 75

Appendix 2 76

Appendix 3 81

10
1.0 Introduction

In June 2007, EPA published in the Federal Register a Notice announcing the Draft List of Initial

Pesticide Active Ingredients and Pesticide Inerts to be Considered for Screening under the

Federal Food, Drug, and Cosmetic Act [72 FR 33486]. The initial chemicals to be tested under

the EPA Endocrine Disrupter Testing Program were selected based on four human exposure

pathways described in detail in an earlier Federal Register notice, namely, food consumption,

drinking water consumption, residential use exposure, and occupational exposure (contact with

pesticide-treated surfaces) [70 FR 56449]. The highest priority for inclusion on the initial list

was for substances, like glyphosate, that have potential exposure through all four pathways.

Throughout the selection process, EPA clearly stated that "ftjhis list should not be construed as

a list of known or likely endocrine disruptors. Nothing in the approach for generating the initial

list provides a basis to infer that by simply being on the list these chemical are suspected to

interfere with the endocrine systems of human or other species, and it would be inappropriate to

do so."

Glyphosate (N-(phosphonomethyl)glycine, CAS number 1071-83-6) is an herbicide belonging to

the phosphono amino acid class of pesticides. Glyphosate is a foliar applied, non-selective,

systemic herbicide widely used to control weeds in agricultural crops and on non-agricultural

sites. Glyphosate is a specific inhibitor of the sixth enzyme of the shikimate pathway, 5-

enolpyruvyl-shikimate 3-phosphate synthase (EPSPS), which is essential for the biosynthesis of

aromatic amino acids (i.e., tyrosine, tryptophan and phenylalanine) and other aromatic

compounds in algae and higher plants, bacteria and fungi. Inhibition of this enzyme leads to

plant cell death. Since the shikimate pathway and, therefore, the EPSP synthase target site, is

found only in plants, bacteria and fungi, and not in animals, glyphosate generally exhibits low

toxicity to higher organisms, including mammals, birds, fish, aquatic invertebrates and terrestrial

invertebrates.

The Office of Management and Budget stated in its "Terms of Clearance" regarding EPA's

issuance of the initial set of test orders for the Endocrine Disruptor Screening Program (EDSP)

that "EPA should promote and encourage test order recipients to submit OSRI in lieu of

performing all or some of the Tier I assays, and EPA should accept OSRI as sufficient to satisfy

the test orders to the greatest extent possible" (OMB, 2009).

Other scientifically relevant information (OSRI) is defined by EPA as "information that informs

the determination as to whether the substance may have a similar effect to a substance that

interacts with estrogen, androgen and thyroid systems. " Functionally equivalent, in this context,

refers to data of a suitable nature and quality that provides the same essential predictive

information on a chemical even if different methods and procedures may have been used for

obtaining the data. Specifically, EPA has characterized OSRI as being either, (1) functionally

equivalent to information obtained from the Tier 1 assays, or (2) data from assays that perform

the same function as EDSP Tier 1 Screens.

This document provides scientific information regarding endocrine-related endpoints for

glyphosate that is functionally equivalent to the information proposed to be obtained by the Tier

1 Screening (T1S) battery of the EDSP. Factors included in this evaluation of other scientifically

related information were functional equivalence of the endpoints, sensitivity of the test,

specificity for a particular mode of action, confidence in the conclusions and applicability across

taxa. Other factors were route and duration/frequency of administration, dose levels, age at

11
exposure, species, number of test units, variability, and whether or not the data provided a basis

for conclusions related to the potential interaction of glyphosate with the endocrine system in

mammalian and non-mammalian systems.

It is plausible that OSRI for Tier 1 assays can broadly include a number of study types,

including, structure activity relationships (SAR) analyses, mechanistic in vitro assays and in vivo

metabolism, subchronic, chronic, reproductive and developmental studies. The majority of the

OSRI presented here for glyphosate is from toxicity tests conducted according to international

regulatory guidelines. This data primarily consists of developmental benchmarks predictive of

endocrine disruption potential and histopathology on key tissues, including:

• Endocrine tissues (pituitary, thyroid, parathyroid, pancreas adrenal, ovary and testis)

• Hormone sensitive male tissues (prostate, epididymides, and seminal vesicles)

• Hormone sensitive female tissues (mammary, uterus and ovaries)

Other relevant scientific information presented here for glyphosate is data obtained from study

protocols that focus on reproduction, fertility and development for mammalian species and other

taxa, such as birds and fish.

The endocrine-modulating potential of glyphosate has been evaluated in a variety of studies,

including in vitro assays and standard in vivo toxicology studies. Glyphosate has demonstrated

no estrogenic, anti-estrogenic, androgenic or anti-androgenic potential in any in vitro assays.

There were also no definitive findings of endocrine-modulating effects in the glyphosate

subchronic, chronic, developmental, or reproductive toxicity studies (see Tables 3 and 4 from

Williams et al. 2000; Moxon, 2000; Dhinsa et al, 2007).

Histopathological observations of endocrine and reproductive tissues from animals in chronic

and multigenerational toxicity studies indicate that glyphosate exposure had no adverse

histological consequence on any reproductive or endocrine tissue from either male or female rats

even at exaggerated dosage levels. Also, no adverse results were obtained in a dominant lethal

study conducted at very high doses of glyphosate. While this latter test is typically used to assess

genetic toxicity, substances that affect male reproductive function through endocrine modulating

mechanisms can also produce effects in this type of study; such reproductive indices include

mating, fertility and conception. Additionally, no effects indicative of endocrine activity were

detected in a fish full life-cycle study and several avian one-generation reproduction studies

performed at exaggerated dosage levels.

In summary, no endocrine-related effects were observed in four independent, multigenerational

reproduction studies conducted at several doses ranging from low to those exceeding expected

human glyphosate exposure by at least several orders of magnitude, a fish full life-cycle study

and several avian reproduction studies. It can be concluded, therefore, that a sufficient battery of

studies has already been conducted to evaluate the endocrine-modulating potential of glyphosate

and that the results from all these studies demonstrate that glyphosate is not a reproductive

toxicant and does not perturb the endocrine system.

In-depth reviews on the safety of glyphosate have been conducted by several regulatory agencies

and scientific institutions worldwide, and all have concluded that there is no indication that

glyphosate has potential endocrine activity or will result in adverse effects on endocrine systems

in humans or other mammals. The conclusions of these reviews are summarized below.

12
• The U.S. EPA (1998) reviewed the subchronic and chronic mammalian studies for

glyphosate and concluded that there was no evidence to suggest that glyphosate produces

endocrine-modulating effects.

• In a comprehensive review of the standard mammalian toxicology studies, Williams et al.,

(2000) concluded that glyphosate does not have the potential to produce adverse effects on

endocrine systems in humans or other mammals.

• A report submitted to the USDA Forest Service by Syracuse Environmental Research

(SERA) concluded that extensive testing in experimental animals and wildlife provided

reasonably strong evidence that glyphosate is not an endocrine disrupter (SERA, 2002).

• The list of purported endocrine disrupters compiled by the Institute of Environment and

Health (IEH, 2005) lists glyphosate as a substance with no evidence of potential endocrinedisrupting effects.

• Following a review of the standard mammalian and wildlife toxicology studies, it was

concluded in a recent ECETOC guidance document on identifying endocrine disrupting

effects that glyphosate is not an endocrine disrupter using the internationally accepted

Weybridge definition2 for endocrine disruption (ECETOC, 2009).

Important considerations in evaluating the potential of a chemical to interact with the endocrine

system include absorption, distribution, metabolism and excretion (ADME). Low absorption,

limited distribution and rapid excretion of a chemical from the body limits its potential to interact

with the endocrine system. Metabolites can have a greater or lesser potential to interact with the

endocrine system, depending on the nature of the metabolite relative to the parent molecule.

It has been shown in multiple studies conducted in multiple species that glyphosate exhibits low

GI tract and dermal absorption (Ridley and Mirly, 1988; Bo Neilsen et al, 2009), limited

distribution in the body (Colvin and Miller, 1973), almost no mammalian metabolism (Ridley,

1983), and rapid excretion from the body (Wester, 1991). Therefore, the potential for systemic

exposure of endocrine tissues to glyphosate metabolites is extremely low.

OECD member countries have long recognized the potential of structure activity relationships

(SAR) analysis as an aid in conducting initial hazard assessments for chemicals, including

potential endocrine activity. In August 2009, the EPA's midcontinent Division in Duluth

presented a structure activity model developed to evaluate estrogen receptor (ER) binding

potential. This model is an hypothesis-driven approach based on what is described as 'Expert

Rules' to assign ER binding potential and how the system provides information supporting the

assignment. EPA's FIFRA Scientific Advisory Panel, with the help of the Food Quality

Protection Act Science Review Board, endorsed the model as a tool to predict whether a

chemical can bind to the ER and eventually to help prioritize chemicals for Tier 1 screening

under the EDSP.

2

 Weybridge (1996) "An endocrine disrupter is an exogenous substance that causes adverse health effects in an

intact organism, or its progeny, secondary (consequent) to changes in endocrine function. A potential endocrine

disrupter is a substance that possesses properties that might be expected to lead to endocrine disruption in an intact

organism."

13
This model predicts that a chemical will have low potential for interaction with the ER if,

1. the chemical belongs to a group where testing showed no evidence of ER interaction (relative

binding affinity less than 0.00001%), as with acyclic molecules or cyclic molecules that do

not contain a likely H-bonding group, and

2. the log KOW ofthe chemical is less than 1.3.

Molecules that have been shown to have ER binding activity are cyclic or aromatic, having an

overall ring structure as described by Blair et al., 2000; Nishihara et al., 2000; Kojima et al,

2004. Glyphosate is an acyclic molecule, as illustrated below, and, therefore, does not contain

the core structure of compounds predicted to have estrogenic activity. Glyphosate also has a log

Kowof much less than 1.3 (log Kow = -3.2). Applying the expert rules of the model to

glyphosate, a relative binding affinity (RBA) of <0.00001% can be assigned, indicating

essentially no potential to interact with the ER.

1*

Glyphosate

log Kow = - 3.2

Common structure in estrogenic

compounds. R = hydrophobic moiety

and B = bulky moiety

Although SAR models are not developed to the same degree for androgens as they are for

estrogens, many of the same principals that apply to assessing estrogenic potential apply to

assessing androgenic and anti-androgenic potential. Based on its chemical structure, glyphosate

is predicted to not have androgenic activity (Devillers et al., 2009; Panaye et al., 2008).

The repeat dose in vivo toxicology studies required by the U.S. EPA and other regulatory

agencies worldwide detect modulation of endocrine system activity (Carney et al., 1997; Stevens

et al, 1997, 1998; Harvey and Johnson, 2002). These in vivo studies are more predictive than in

vitro screening assays as they assess a variety of endocrine-sensitive endpoints in animals that

are capable of metabolic activation and/or detoxification. These studies also use extended

exposure periods encompassing various stages of endocrine development in intact organisms.

Endocrine-active substances affecting a single or multiple endocrine target sites invariably

initiate direct or compensatory biochemical, cellular, and/or histopathological processes that will

14
be detected in standard toxicology studies required for pesticide registration in Canada, Europe,

Japan and the United States. A comprehensive histopathological assessment of endocrine tissues

combined with gross organ pathology and organ weight data allows detection of all adverse

endocrinopathies.

The standard toxicology and ecotoxicology studies that provide valuable information on

potential endocrine-modulating effects include subchronic, chronic, and developmental and

reproduction studies (Table 1). The multigenerational rat reproduction study is currently the most

comprehensive and definitive study for evaluating the potential of substances to produce

endocrine modulating effects in humans and other mammals. This study evaluates effects on

gonadal development and function, estrous cycles, mating behavior, fertilization, implantation, in

utero development, parturition, lactation, and the ability of offspring to survive, develop and

successfully reproduce. A histopathological assessment of relevant organ systems is a prominent

feature of these studies. Developmental toxicity studies evaluate effects on many of these same

processes, while subchronic and chronic studies incorporate numerous direct and indirect

evaluations of endocrine and reproductive tissues, such as target organ weights and a

comprehensive assessment of endocrine organ pathology.

Table 1: In vivo ecotoxicology guideline tests that provide endpoints capable of detecting endocrine effects

and a comparison to the rat multigenerational reproduction study

Endpoint

Fertilization Success

Hatchability

Birth or hatching weight

Morphological abnormalities

Viability

Behavior

Growth

Onset of maturity (or

puberty) or reproduction

Fecundity of offspring

Sex ratio of offspring

Intersex in offspring

Egg shell thinning

Fish Full Life

Cycle Test

(OPPTS 850.1500)

Yes

Yes

No

Yes

Yes

Yes (mating)

Yes

Yes

No

No

No

NA

Avian Reproduction Test

(OECD 206/OPPTS

850.2300)

Yes

Yes

Yes

Yes

Yes

No

Yes

Yes

No

No

No

Yes

Rat Multigenerational

Reproduction Test

Yes

NA

Yes

Yes

Yes

Yes (mating)

Yes

Yes

Yes

Yes

Yes

NA

1.1 Considerations for Dose Setting for Endocrine Screening and Testing

One of the most critical issues faced in endocrine testing is setting the appropriate dose levels or

concentrations at which to conduct the screening assays. Appropriate dose setting is critical to

not confuse systemic toxicity with genuine endocrine mediated effects. Therefore, dose setting

takes on considerably greater importance in the testing for adverse effects on the endocrine

15
systems than in traditional toxicity testing. The purpose of screening is to identify the potential

to interact with the endocrine system via an estrogenic, androgenic or thyrogenic mode of action,

not to merely identify adverse effects. Thus, it is critical that doses are set in the range where

effects on the measured endpoints are not indirectly impacted by general mechanisms of toxicity,

such as liver toxicity or body weight changes, or produce an indirect effect as the result of overt

toxicity or a general stress response.

An overview of each of the eleven assays in the Tier 1 battery is provided in the introduction to

each section below. Additionally, a brief tabular summary describing each of the assays is

presented in Appendix 1.

2.0 In Vitro Assays

2.1 Estrogen Competitive Receptor Binding Assay (OPPTS 890.1250) and ER

Transcriptional Activation Assay (OPPTS 890,1300)

Functionally equivalent data to that which can be obtained by performing the ER binding assay

and the ER transcriptional activation assay are available for glyphosate in three independent

studies reported in peer-reviewed journals. These studies clearly demonstrate that glyphosate

does not possess estrogenic or anti-estrogenic potential and were conducted using comparable

and functionally equivalent methodology to OPPTS guidelines for the Tier 1 ER competitive

binding assay and ER transcriptional activation assay. This OSRI demonstrates that glyphosate

does not possess estrogenic or anti-estrogenic potential. This section primarily reviews OSRI

that assesses the potential for direct interaction of glyphosate with the estrogen receptor using in

vitro test systems similar to those defined in the EDSP.

This section also includes a discussion on the use of structure activity relationship models to

evaluate a chemical's estrogen receptor binding potential and how it can be used to validate the

results of the estrogen receptor binding and transcriptional activation studies for glyphosate.

2.1.1 Structure Activity Relationship

There are two approaches to assess the potential for a chemical to interact with the estrogen

receptor. One approach is to utilize predictive structure activity relationship (SAR) models.

Member countries of the OECD, of which the U.S. is one, have long recognized the potential of

SAR analysis as an aid in conducting initial hazard assessments of chemicals. A summary of

EPA's recent approach to assessing potential endocrine activity based on structure was

summarized in the introduction of this document. The other approach to assess the potential for

a chemical to interact with the estrogen receptor is to conduct in vitro assays, which are

discussed in the following sections.

2.1.2 Estrogen Receptor (ER) Binding and Transactivation Assays

The two in vitro test systems that assess interaction with the estrogen receptor in the Tier 1

screening battery of the EDSP are the ERa/p competitive radioligand binding assay (OPPTS

890.1250) and the ERp transactivation assay (OPPTS 890.1300).

ERa mediates the classic estrogenic response and models currently being developed to measure

interaction with the estrogen receptor mainly use the ERa isoform. The ERa/p binding assay

utilizes rat uterine cytosol rather than a recombinant ER. EPA and OECD are currently

developing an ER binding assay that utilizes recombinant forms of the estrogen receptor rather

16
than rat uterine cytosol because the rat uterine cytosol preparation is unstable and a greater

potential for assay-to-assay variation is realized when using rat uterine cytosol. Binding assays

with recombinant endocrine receptors will yield more consistent results and eliminate the need to

use large numbers of animals to obtain the rat uterine cytosol substrate.

The ICso value for estradiol in ER competitive binding assays and transactivation assays are

comparable to the ICso value for the OPPTS 890.1250 ER competitive binding assay and the

OPPTS 890.1300 transcriptional activation assays, respectively. The measurement endpoints for

these two assays are highly correlated and demonstrate similar sensitivity and specificity

(Sonneveld et al., 2006; Takeyoshi, 2006; Laws et al., 2006). This result is plausible and not

unexpected since both assays are measuring the same functional endpoint, receptor-ligand

binding.

ER binding and transactivation assays have been shown to be good predictors of in vivo

estrogenic activity (Sonneveld et al., 2006; Takeyoshi, 2006). This is particularly true for

compounds where metabolism studies in rodents have shown that they are poorly metabolized,

are not substantially absorbed when ingested, are rapidly excreted, and do not accumulate in the

body (EPA, 1993; William et al., 2000, Ridley and Mirly, 1988), as is the case for glyphosate.

Competitive radioligand binding ER assays have been used for decades and are one of the "gold

standards" for identifying test chemicals that bind to the ER (Clark and Gorski, 1969; O'Keefe

and Handa, 1990; Laws et al. 2006). The ER binding assay, measures the ability of a

radiolabeled ligand (l?p-estradiol) to interact with the ER in the presence of increasing

concentrations of a test chemical. The methodology in EPA's estrogen receptor binding assay

OPPTS 890.1250 test guideline is essentially the same as the methodology outlined by Laws et

al. (2006). Rat uterine cytosol (RUC), containing the ER, is incubated in test tubes with

increasing concentrations of the test substance and an aliquot of radiolabeled [3H]-estradiol. If

the test substance interacts with the receptor's binding domain, less radioligand can bind, so an

active competitor produces a descending concentration-response plot. Similarly, test substances

that do not displace radiolabeled-estradiol from the estrogen receptor would not have an affinity

for estrogen-binding. The assay can, therefore, be used to determine the relative binding

affinities of environmental chemicals for the estrogen receptor by comparison with the ability of

IVp-estradiol to bind to the estrogen receptor. Although the ER binding assay detects both

agonists and antagonists, it cannot distinguish between them. The criterion for a positive

interaction with the ER receptors is >50% displacement of the radioligand in 2 of three replicate

assays.

The ERa transactivation assay utilizes ERa-HeLa-9903 immortal cell line in a method validated

by the Japanese Chemicals Evaluation and Research Institute through the OECD validation

process. There are also ongoing efforts to develop additional ER transactivation assays (e.g.,

LUMI-CELL) to assess agonism and antagonism. In agonism studies, cells are treated with a

test substance and the induction of the reporter gene product is utilized to measure the response.

For an assessment of relative potency, the response of the test substance can be compared to the

induction by a reference estrogen. Alternatively, when dose-response data are generated, the

EC50 for the test substance can be determined and compared with that for the reference estrogen.

In antagonism studies, the cells are exposed simultaneously to the reference estrogen and the test

substance, while control cells are exposed to the reference estrogen only. Usually, a constant

concentration of reference estrogen is used and the decline of its transcriptional activity with the

17
test substance is monitored. The difference in induction of the reporter gene product in the

presence and absence of the test substance is used as a measure of estrogen antagonism.

ER transact!vation assays demonstrate a high level of concordance for sensitivity and specificity

with the EDSP's ER binding assay and immature rat uterotrophic assay (Takeyoshi, 2006).

Therefore, results from stably transfected ER transactivation assays provide reliable information

about the biological effect of chemicals mediated by ER-ligand interactions (Takeyoshi, 2006).

The criterion for a positive interaction with the ER receptors in the ERa-HeLa-9903 immortal

cell line used in the OPPTS 890.1300 guideline is calculation of a PC 10 value, which represents

10% of the maximum inducing potential of 1 nM l?p-estradiol. The consensus of OECD's

independent peer review panel was that the PC10 value is not an appropriate criterion for a

positive response. The Peer-Review Panel's justification for this recommendation was that the

PC 10 represents a marginal response of doubtful predictive value and will likely generate an

unacceptably high rate of false positives (OECD, 2007). The fold-induction for 17-0 estradiol at

a concentration of 1 nM has been shown to range from 4- to 30-fold and typically result in

approximately a 10-fold induction. Four-fold induction represents a weak response with 1 nM

17-P estradiol and demonstrates the potential for a very small dynamic range for the assay on a

given day. With only a four-fold maximal induction, the PC50 would represent approximately a

2-fold induction and a PC 10 would be significantly less than a two-fold induction. It is routinely

understood by experts that less than a 2-fold induction in reporter gene assays is not considered a

positive response. Indeed, the results from the validation study have an unacceptably high falsepositive rate when assessing the PC 10 value as an endpoint. There were false positives observed

in 1 of the 3 replicate assays for 3 of the 9 test substances in 3 of the labs (see table 6.2 of the

validation report of Takeyoshi, 2006). Therefore, the results are equivocal for nearly one-third

of the substances tested in the validation when using the PC 10 endpoint. This result is a function

of the variability of the test system at low induction levels and demonstrates the lack of validity

of the PC 10 value as the criterion for a positive response. The Panel strongly recommended that

an intermediate value between the PC 10 and PC50 (e.g., PC25) should be used as the criterion

for a positive response (OECD, 2007; ICCVAM, 2007).

The EDSP's ER transactivation assay was developed by stably transforming the HeLa-9903 cell

line with a firefly luciferase reporter vector activated by a vitellogenin estrogen-responsive

element driven by a mouse metallothionein promoter TATA element. Stably transfected lines

have the obvious advantage over transient transfections of not having to continuously prepare

multiple batches of transiently transfected cells. However, transient transfections have the

advantage over stably transformed lines in that the level of ER at a higher level with transient

transfections and can provide greater sensitivity. The key functional difference between the

binding and transactivation assays is that the transactivation assay has the potential to

differentiate between agonism and antagonism. However, the ERa transactivation assay

currently in the Tier 1 screening battery does not provide methodology to detect ERa agonists.

The estrogenic and anti-estrogenic potential of glyphosate has been studied in vitro by

independent researchers and published in peer-reviewed journals. The methodology used in

these studies (Kojima et al. 2004; Petit et al. 1997; Xie et al. 2005) is functionally equivalent to

the ER binding and transactivation assays defined in the Tier 1 battery of the EDSP. Even

though the in vitro data were generated with different experimental methods, it is of suitable

nature and quality to provide the same essential information to evaluate potential estrogenic and

anti-estrogenic potential for glyphosate. The results from these three investigations provides

18
strong evidence that glyphosate is not an ERa/P agonist or antagonist. A comprehensive review

of each of these assays follows.

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

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

Chinese hamster ovary cells. Environ. Health Perspect. 1 12:524-531.

The ability of glyphosate to act as agonist and antagonist of the human ERq and human

isoforms was investigated by Kojima et al. using Chinese hamster ovary (CHO-K1) cells that

were transiently transfected with an estrogen responsive reporter vector. The estrogen

responsive element containing reporter plasmid was constructed as described by Kojima et al.,

(2003). For detection of ERq and ERp activity, cells were transfected with 5 ng of ERq or ERp

expression vectors and 50 ng of the ER reporter construct. Additionally, a pRL-SV40 construct

containing the Renilla luciferase gene was co-transfected to evaluate transfection efficiency and

to discriminate between specific down regulation from cytotoxicity.

Three hours post-transfection, cells were dosed for 24 hours with concentrations of glyphosate

ranging from 10"5 to 10"8 M (i.e., 0.01 uM to 10 uM). The 24-hour exposure duration for this

assay is the same as that required by the OPPTS 890.1 300 ER transactivation guideline.

Transfected cells were cultured in complete media, containing penicillin-streptomycin and 10%

charcoal-dextran-treated fetal bovine serum at 37°C in an atmosphere of 5% CO2/95% air under

saturated humidity. The approximate ECso values for ERq and ERp were 1 and 10 pM,

respectively, which are 6 to 7 orders of magnitude greater than the highest glyphosate

concentration tested. Parallel assays were performed to fully characterize the concentration

effect relationship with estradiol for ERq and ERp (Figure 1).

.s

S

*]

• ;

-11 -13 -12 -11 -10 -8 -7

Figure 1. Concentration-response curves for

estradiol in the ERq and ERp assays in Chinese

hamster ovary cells transiently transfected with hERa

or hERp, the ER luciferase reporter vector and a

constitutively active Renilla luciferase expression

reporter (transfection and toxicity control). Values

represent the mean ± SD of three independent

experiments and are presented as mean n-fold

induction over the vehicle control.

The sensitivity of this assay and dynamic range are comparable to the sensitivity and dynamic

range of other ER reporter assays reported in the open literature (Legler et al., 1999; Rogers et

al., 2000; Wilson et al., 2004; Gordon and Clark, 2005) and to that of the OPPTS 890.1300 ER

transactivation guideline (Takeyoshi, 2006). Also, the ICso value reported by Kojima et al. for

the transactivation assay for glyphosate is comparable to the ICso value reported by Laws et al.

using the OPPTS 890.1250 ER competitive binding assay (Laws et al., 2006). This is plausible

19
and not unexpected since both assays are measuring the same functional endpoint, receptorligand binding.

Agonist activity of glyphosate was evaluated by relative activity expressed as the 20% relative

stimulatory concentration (EC2o); that is, the concentration of glyphosate showing 20% of the

activity of maximally inducing concentrations of 10"10 M estradiol for hERa and 10~9 M estradiol

for ERp activity, respectively. Based on this criterion, glyphosate was not identified as a

chemical having ERa and ERp antagonist activity (Kojima et al, 2004).

Petit F., LeGoff P., Craved! J.P., Valotaire Y., F. Pakdel. (1997). Two complementary

bioassays for screening the estrogenic potency of xenobiotics: recombinant yeast for trout

estrogen receptor and trout hepatocyte cultures. J. Molecular Endocrinology. 19: 321-335.

Petit et al. (1997) screened glyphosate for estrogenic potency using an in vitro recombinant yeast

system expressing the rainbow trout estrogen receptor. Yeast cells containing a lacZ reporter

gene linked to two estrogen-responsive elements were treated in culture at 10"8 to 10"4 M (0.01 to

100 uM) glyphosate for four hours. For these assays, 17p-estradiol was used as the positive

control and produced good responsiveness with approximately a 100-fold induction of Pgalactosidase activity. To ensure that the absence of a response was not due to toxicity, cell

density measurements were made before and after treatment. The estradiol concentration

required to produce maximal activation was 10" M and was reported as 100% activity. Basal

induction represented 14 to 18% of the maximum activity obtained with the receptor in the

absence of ligand. The reported activity for glyphosate on estrogen dependent gene

transactivation was reported to be 15.84%, well within the range of basal activity, indicating no

evidence of estrogenic activity.

Xie L., Thrippleton K., Irwin M.A., Siemering G.S., Mekebri A., Crane D., Berry K.,

Schlenk D. (2005). Evaluation of estrogenic activities of aquatic herbicides and surfactants

using a rainbow trout vitellogenin assay. Toxicol Sci. 87:91-8.

In Xie et al. (2005), the estrogenic potency of glyphosate was evaluated using vitellogenin as a

biomarker of exposure for juvenile rainbow trout (standard length: 11.5 ± 2.2 cm) exposed to a

mean measured concentration of 0.11 mg/L glyphosate. The analytical method used for

measuring the concentration of glyphosate in water was EPA Method 547 with a limit of

detection of 0.0050 ug/L and recovery ranging from 80-87%.

Rainbow trout were exposed for 7 days at 16 ± 1°C in 20-L tanks under static-renewal and

received water exchanges daily. In addition, rainbow trout were exposed to five concentrations

of 17p-estradiol to characterize the concentration effect relationship for 17p-estradiol. Each

concentration of chemical (treatment or control) had three replicates with two fish in each tank.

The test water in each individual tank was monitored daily for water chemistry after every water

renewal. The hardness of the test water ranged from 142 to 162 mg/1 (as CaCOs); the free

chlorine was <0.2 mg/1. Alkalinity ranged from 148 to 180 mg/1, and ammonia (as N-NFh) was

<0.02 mg/1. Dissolved oxygen averaged 94.6% of the air saturation value, and pH values ranged

from 6.0 to 8.2 mg/1. Fish were fed trout chow equivalent to approximately 1% of their body

weight during the exposure and held under a 16 h:8 h (light:dark) photoperiod. The results of

this study demonstrate that glyphosate did not induce production of vitellogenin, providing

additional evidence that glyphosate does not have estrogenic activity (Figure 2).

20
O)

I

 90

 -

g> 80 -

7T 70 -

§ 60

0 50

| 40-

§>

 30

 '

0 20

I 10 -

> 0 -TMLJ» Pi s r

,._.,,,T,,O.i::,

o o o

o o

3. 3.

2. 2.

— * K)

Figure 2. Plasma vitellogenin

juvenile rainbow trout exposed

levels in

to glyphosate

(G) for 1 days. Control 1 and control 2

represent negative controls.

2.1.3 Conclusion - Estrogen Receptor Agonism and Antagonism

Data generated by Kojima et al. (2004), Petit et al. (1997) and Xie (2005) in peer-reviewed

journals clearly and consistently demonstrate that glyphosate does not have an ER binding

potential, either as an agonist or antagonist. The methodologies employed by Kojima and Petit

in these in vitro test systems are comparable to the overall approach of the two Tier 1 ER binding

assays of the EDSP—the ERa/P competitive radioligand binding assay using rat uterine cytosol

(OPPTS 890.1250) and the ERa transactivation assay (OPPTS 890.1300). Specifically,

transcriptional activation assays performed by Kojima et al. (2004) are functionally equivalent to

the ERa/P competitive radioligand binding assay (OPPTS 890.1250) and the ERp transactivation

assay (OPPTS 890.1300) for the following reasons:

• Kojima et al. (2004) evaluated the ability of glyphosate to agonize and antagonize ERa and

ERp binding, which is the purpose of the ERa/P competitive radioligand binding assay

(OPPTS 890.1250). The methodology of Kojima et al. was capable of differentiating

between ERa/p agonists and antagonists, which the ERa/P competitive radioligand binding

and transcriptional activation assays are not. Overall, ER transactivation assays demonstrate

a high level of concordance (sensitivity and specificity) with the EDSP's ER binding assay.

This is plausible and not unexpected since both assays are measuring the same functional

endpoint, ER-ligand binding. Therefore, results from ER transactivation assays provide

reliable and equivalent information to ER competitive radioligand binding assays.

• The ERa/p transactivation assay performed by Kojima et al. (2004) evaluated the same key

biochemical mechanisms as the ERp transactivation assay (OPPTS 890.1300). These steps

include ER ligand binding, nuclear translocation, binding to the DNA responsive element,

and reporter gene transactivation. Additionally, the sensitivity of the assay and dynamic

range of Kojima et al. are comparable to the sensitivity and dynamic range of other ER

reporter gene assays in the open literature and the OPPTS 890.1300 ER transactivation assay

used to develop the OPPTS guideline.

• The in vitro ER transactivation assay performed by Petit et al. (1997) also provides data of

the same nature and quality as OPPTS 890.1300 ER transactivation guideline. This assay

21
assesses the key steps evaluated by OPPTS 890.1250 and OPPTS 890.1300 that include ER

binding and the other key steps of DNA binding and gene transactivation.

• The in vivo assay reported by Xie et al. (2005) provides another confirmatory and sensitive

mechanistic assessment of ER binding potential.

These in vitro and in vivo mechanistic findings that demonstrate no ER agonistic or antagonistic

binding potential are further supported by a SAR analysis using methodology developed by

EPA's Office of Research and Development. SAR analysis clearly shows no estrogenic potential

for glyphosate and the comprehensive in vivo data available for glyphosate demonstrate no

effects on any endpoint that is sensitive and specific to compounds that have estrogenic or antiestrogenic activity. A detailed review of the in vivo data used to evaluate estrogenicity is

reviewed in the sections for the uterotrophic, pubertal assays and fish screening assays.

The data generated by Kojima et al. (2004) and Petit et al. (1997) are functionally equivalent to

both the Estrogen Competitive Receptor Binding (OPPTS 890.1250) and the ER Transcriptional

Activation Assay (OPPTS 890.1300) defined in the EDSP Tier 1 test battery. Consequently, no

additional in vitro screening should be required to evaluate the potential for ER agonism and

antagonism.

2.2 Androgen Receptor (AR) Binding Assay (OPPTS 890.1150)

The EDSP Tier 1 screening battery includes one in vitro assay to assess the potential for a

chemical to interact with the androgen receptor (AR). An independent study that is functionally

equivalent to the AR Binding Assay of the EDSP (OPPTS Guideline 890.1150) and published in

Environmental Health Perspectives by Kojima et al. in 2004 clearly demonstrates the lack of any

antagonistic androgen receptor activity for glyphosate. This section reviews currently available

scientifically relevant information, generated using an in vitro test system similar to the AR

binding assay (OPPTS 890.1150) defined in the EDSP, that can be used to assess the potential

for glyphosate to directly interact with the androgen receptor.

This section also discusses the use of structure activity relationship (SAR) models to evaluate a

chemical's androgen receptor binding potential and how it can be used to validate the results of

the assay for glyphosate.

2.2.1 Structure Activity Relationship (SAR) Prediction of AR Binding Potential

Although SAR models are not developed to the same degree for androgens as they are for

estrogens, many of the same principals that apply to evaluating glyphosate for estrogenic

potential apply to an assessment of androgenic potential. Based on its structure, glyphosate is

predicted not to have androgenic activity (Devillers et al., 2009; Panaye et al., 2008). The

conclusion that glyphosate does not have androgenic potential has been empirically confirmed by

a number of in vitro assays and in vivo tests. A review of the in vitro and in vivo data that has

evaluated potential androgenic and anti-androgenic activity of glyphosate is discussed in the

following sections.

2.2.2 Androgen Receptor (AR) Binding Assay

The androgen receptor (AR) binding assay is very similar to the ER competitive ligand binding

assay in its general procedure. The AR binding assay in the Tier 1 battery of the EDSP utilizes

rat prostate cytosol from animal tissue (rat prostrate) rather than a recombinant androgen

receptor. EPA and OECD are, however, currently developing an AR binding assay that utilizes

22
recombinant forms of the androgen receptor because, as with the estrogen receptor binding

assay, the rat prostate cytosol preparation is unstable and its use exhibits a greater potential for

assay-to-assay variation. Binding assays with recombinant ARs demonstrate more consistent

results and eliminate the need to sacrifice large numbers animals to obtain the prostrate cytosol

preparation.

Overall, \C$Q values with androgenic compounds for AR binding and transactivation assays are

comparable to the ICso values for the OPPTS 890.1150 AR competitive binding assay

(Sonneveld et al., 2006; Beck et al., 2007). This is plausible and not unexpected since both

assays are measuring the same functional endpoint, AR-ligand binding. Additionally, AR

binding and transactivation assays have been shown to be good predictors of in vivo androgenic

activity (Sonneveld et al., 2006).

Competitive radioligand binding AR assays have been used for decades and are one of the

standards for identifying test chemicals that bind to AR (Kelce et al, 1995). The AR binding

assay measures the ability of a radiolabeled ligand ([3H]-R1881) to interact with the AR in the

presence of increasing concentrations of a test chemical. The methodology in EPA's AR binding

assay (OPPTS 890.1150) is essentially the same standard methodology outlined by Kelce et al.

(1995) and in a series of publications published by Kelce et al. Rat uterine prostate containing

AR is incubated in test tubes with increasing concentrations of the test substance and an aliquot

of radiolabeled [3H]-R1881. If the test substance interacts with the receptor's binding domain,

less radioligand can bind, so an active competitor produces a descending concentration-response

plot. Similarly, test substances that do not displace the radiolabeled ligand from AR would not

have an affinity for AR-binding. The assay can, therefore, be used to determine the relative

binding affinities of environmental chemicals for the androgen receptor, by comparison with the

ability of [3H]-R1881 to bind. Although the AR binding assay detects both agonists and

antagonists, it cannot distinguish between them. The criterion for a positive interaction with the

androgen receptor is >50% displacement of the radioligand.

The androgenic potential of glyphosate has been studied in vitro by independent researchers and

published in a peer-reviewed journal. The methodology used in this study (Kojima et al. 2004) is

functionally equivalent to the AR binding assay defined in the Tier 1 battery of the EDSP. The

conclusion of the researchers in this study is that glyphosate does not exhibit androgen receptor

antagonist activity. A comprehensive review of this assay follows.

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

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

Chinese hamster ovary cells. Environ. Health Perspect. 112:524-531.

The ability of glyphosate to act as agonist and antagonist of the human AR was investigated

using Chinese hamster ovary (CHO-K1) cells that were transiently transfected with an androgen

responsive reporter vector (Kojima et al., 2004). The androgen responsive element containing

reporter plasmid was constructed as described in Kojima et al., (2003). To evaluate agonist and

antagonist effects of AR activity, cells were transfected with 2.5 ng of androgen receptor

expression vectors and 50 ng of the AR reporter construct. Additionally, a pRL-SV40 construct

containing the Renilla luciferase gene was co-transfected to evaluate transfection efficiency and

to distinguish between specific down regulation from cytotoxicity.

23
At three hours post-transfection, cells were dosed for 24 hours with concentrations of glyphosate

ranging from 10"5 to 10~8 M. This 24-hour exposure is the same as that required by the AR

transactivation guideline OPPTS 890.1150. Transfected cells were cultured in complete media

containing penicillin-streptomycin and 10% charcoal-dextran-treated fetal bovine serum at 37°C

in an atmosphere of 5% CC>2/95% air under saturated humidity.

The approximate ICso value for the AR was approximately 6 orders of magnitude greater than

the highest glyphosate concentration tested. Parallel assays were performed to fully characterize

the concentration effect relationship with estradiol for AR (Figure 3).

I

-14 -13 -12 -11 -10 -8

LoglM! DHT

Figure 3. Concentration-response curve for 5adihydrotestosterone and androgen receptor (AR) in

Chinese hamster ovary cells transiently transfected

with the AR and the AR luciferase reporter vector

and a constitutively active Renilla luciferase

expression reporter (transfection and toxicity

control). Values represent the mean ± SD of three

independent experiments and are presented as mean

n-fold induction over the vehicle control.

The sensitivity of this assay and dynamic range is comparable to the sensitivity and dynamic

range of other standard AR reporter assays in the open literature (Beck et al., 2008).

Additionally, the \C$o value for the transactivation assay conducted by Kojima et al. is

comparable to the ICso value for the AR competitive binding assay conducted according to

OPPTS guideline 890.1150 (Araki et al., 2005; Sonneveld et al., 2005; Beck et al., 2008). This

is plausible and not unexpected since both assays are measuring the same functional endpoint,

receptor-ligand binding.

Agonist activity was evaluated by relative activity expressed as the 20% relative stimulatory

concentration (ICio); that is, the concentration of glyphosate showing 20% of the activity of

maximally inducing concentrations of 10"'° M dihydrotestosterone. Based on this criterion,

glyphosate was not identified as a chemical having androgen receptor antagonist activity

(Kojima et al, 2004).

2.2.1 Conclusion - Androgen Receptor Agonism and Antagonism of Glyphosate

Data generated by Kojima et al. (2004) clearly and consistently demonstrate that glyphosate is

not an androgen receptor agonist or antagonist. This peer-reviewed article describes an in vitro

test system and methodology that is comparable and functionally equivalent to the methodology

for the AR binding assay in the Tier 1 screening battery described in OPPTS Guideline

890.1150. The rationale for functional equivalence is provided below.

• Specifically, Kojima et al. evaluated the ability of glyphosate to agonize AR binding or

antagonize radiolabeled [3H]-R188 binding to the AR, which is the sole purpose of the

AR competitive radioligand binding assay.

24
• The methodology of Kojima et al. was capable of not only detecting AR agonists and

antagonists but also to differentiate between AR agonists and antagonists. The AR

competitive radioligand binding assay is not capable of making this differentiation.

• Overall, ICso values with androgenic compounds for AR binding and transactivation

assays are comparable to the ICso values for the OPPTS 890.1150 AR competitive

binding assay. This is plausible and not unexpected since both assays are measuring the

same functional endpoint, AR-ligand binding.

• The transactivation assay, which has already been conducted on glyphosate, provides

data of the same nature and quality as the competitive binding assay.

This finding of no androgen binding potential is further supported by a structural activity

relationship analysis that indicates that glyphosate does not have androgenic potential.

Additionally, comprehensive in vivo data obtained from chronic studies with glyphosate

demonstrate no effects on endpoints that are sensitive and specific to compounds that have

androgenic or anti-androgenic activity. A detailed review of the in vivo data that confirms the in

vitro data that shows that glyphosate is not androgenic or anti-androgenic is extensively

reviewed in the sections on the Hershberger, male pubertal and fish screening assays presented

later in this document.

The data generated by Kojima et al. (2004), which demonstrates that glyphosate is not an

androgen receptor agonist or antagonist, is substantially equivalent to the Androgen Receptor

Binding Assay (OPPTS 890.1150) defined in the EDSP Tier I test battery and no additional in

vitro screening is needed or should be required to evaluate glyphosate's potential for androgen

receptor agonism and antagonism.

23 Aromatase Assay (OPPTS 890,1200)

The Tier 1 in vitro aromatase assay (OPPTS 890.1200) of the EDSP uses recombinant human

aromatase (CYP19) and cytochrome P450 reductase to detect inhibition of aromatase activity.

Aromatase catalyzes the conversion of androgen to estrogen. In this Tier 1 in vitro assay,

aromatase activity is evaluated using the tritiated androstenedione 'water-release' method.

Briefly, radioactive substrate (3H-androstenedione) and NADPH are added to microsomes

containing the aromatase and reductase complex. Tritiated-water (3HiO) is released during the

conversion of androstenedione to estrone over a 15 minute incubation period and is quantified as

a direct measurement of aromatase activity per unit reaction time.

Although an in vitro study designed and conducted as described in OPPTS 890.1200 is not

available to evaluate the potential inhibition of aromatase activity by glyphosate, considerable

data is available from the rat subchronic and chronic toxicity studies of glyphosate to make the

same evaluation. Information of particular significance and relevance to evaluate the potential of

glyphosate to inhibit aromatase activity is obtained from the rat multigenerational study.

Inhibition of aromatase activity would produce a diagnostic pattern in a number of sensitive and

specific endpoints that are evaluated in the rat multigenerational study. With females, aromatase

inhibition can result in increased age at vaginal opening (VO) and increased body weight at

vaginal opening, along with a significant decrease in uterine weights and/or ovarian weights

(O'Connor et al., 2002; Marty et al., 1999). In male rats, inhibition of aromatase activity can

25
increase the time of pre-putial separation (PPS), increase prostate weight and increase seminal

vesicle weights (O'Connor et al., 2002).

The rat multigenerational study is able to effectively and unequivocally identify patterns of

aromatase inhibition and has been shown to be more sensitive than the pubertal assays for this

effect. Many of the same endpoints that indicate aromatase inhibition in the pubertal assay are

also evaluated for in the rat multigenerational study. For example, fenarimol was negative for

aromatase inhibition activity in the female pubertal study, yet in a multigenerational assay it

produced the expected effects on parturition associated with lowered estrogen levels after

aromatase inhibition (EPA, 2007).

In recent rat multigenerational studies, there was no evidence of potential aromatase inhibition

by glyphosate. No adverse effect of glyphosate was observed on endpoints indicative of

aromatase inhibition (Moxon, 2000; Dhinsa et al., 2007).

Evaluating the key diagnostic endpoints in the female pubertal assay that indicate aromatase

inhibition, there was no impact on age at vaginal opening (VO) or body weight at vaginal

opening, and no decrease in uterine weights or ovarian weights in the Fl parent animals at the

highest doses tested (HDT) of 10,000 ppm (Moxon, 2000) and 15,000 ppm (Dhinsa et al., 2007).

Additionally, there was no significant effect of glyphosate on estrous cyclicity, pre-coital

interval, duration of gestation, number of primordial follicles, corpora lutea, sex distribution,

anogenital distance or histopathology of the female reproductive tissues (uterine and ovary) in

the multigeneration studies (Moxon, 2000; Dhinsa et al. 2007). These findings are consistent

with findings in earlier chronic and subchronic studies on glyphosate (Schroeder and Hogan

1981; Stout and Johnson, 1987). Dhinsa et al. (2007) noted irregular estrus cycling in Fl

females at both 5,000 and 15,000 ppm, but considered this finding incidental and of no

toxicological significance since no change was noted in preciotal intervals or mating performance.

In terms of key diagnostic endpoints in the male pubertal assay to detect aromatase inhibition,

there was no impact on the time of pre-putial separation (PPS) and no increase in prostate and

seminal vesicle weights (Moxon, 2000; Dhinsa et al., 2007). A finding of no effect on these

sensitive and specific endpoints provides strong and consistent evidence that glyphosate is not an

inhibitor of aromatase activity. Additionally, there was no significant effect on weight or

histopathology of male reproductive tissues (testicular and accessory sex organs), testicular

descent, sperm count and motility, and external genitalia in the multigeneration studies (Moxon,

2000; Dhinsa et al., 2007). This finding is consistent with findings in earlier chronic and

subchronic studies (Schroeder and Hogan 1981; Stout and Johnson, 1987).

2.3.1 Conclusion - Aromatase Inhibition

Although in vitro data is not available to assess aromatase inhibition potential of glyphosate,

other scientifically relevant information of a equivalent nature and unquestionable quality is

available from several existing in vivo chronic studies. A number of male and female endpoints

(pathologies) in the two generation rat studies are predictive of aromatase inhibition and

considered to be functionally equivalent to the data developed in the in vitro assay in the Tier 1

battery of the EDSP. These endpoints include:

• testicular/uterine/ovary weights and histopathology

• the size, function and histopathology of sex organs

26
• fertility

• anogenital distance

• sperm count and mobility

• ovarian evaluation for follicles and corpa lutea

• age and weight at preputial separation and vaginal opening

• number of implantation sites and lactation

No treatment related effects were observed in these studies on any of these sensitive and specific

endpoints that would be indicative of aromatase inhibition and provides strong and consistent

evidence that glyphosate is not an inhibitor of aromatase activity. Information on the potential

effect of glyphosate on steroidogenesis, which is also indicative of aromatase inhibition, is

comprehensively discussed in the next section. The scientific information presented here from

existing standard regulatory in vivo studies should be considered as unequivocal proof that

glyphosate does not inhibit aromatase activity in the male or female, and, consequently, the

requirement for performing the in vitro aromatase assay (OPPTS 890.1200) of the EDSP should

be waived.

2.4 Steroidogenesis Assay (OPPTS 890,1550)

The steroidogenesis assay uses the H295R human adrenocortical carcinoma cell line to detect

interference with the body's production of male and female steroid sex hormones. The endpoints

measured in this assay are testosterone and estradiol levels.

The biosynthesis of all steroid hormones begins with the transfer of cholesterol across inner

mitochondria! space from the outer mitochondrial membrane to the inner mitochondrial

membrane; a process that depends on the action of the steroidogenic acute regulatory (StAR)

protein (Stocco, 2001; Christenson et al, 2001). It has been proposed that movement of

cholesterol to the mitochondrial membrane by the StAR protein is the rate-limiting step in

steroidogenesis (Stocco, 2001). In an investigation of the potential effect of glyphosate on StAR

protein function and steroidogenesis in MA-10 Leydig cells, it was demonstrated that glyphosate

does not affect steroidogenesis in MA-10 Leydig cells at the maximum tested concentration of

100 ug/mL (-590 uM) (Walsh et al., 2000). However, Walsh et al. (2000) demonstrated that

inhibition of progesterone production resulted from a component other than glyphosate in the

formulation. Levine et al. (2007) demonstrated that Roundup® branded formulation

steroidogenesis reported by Walsh et al. (2000) resulted from exposing cells in culture to

unrealistically high concentrations of the surfactant. Steroidogenesis in MA-10 cells stops at the

biosynthesis of progesterone from pregnenolone. Consequently, assays with MA-10 Leydig

cells only allow for a partial evaluation of the steps responsible for androgen and estrogen

biosynthesis. However, assays with this cell line by Walsh et al. and Levine et al. demonstrated

that glyphosate does not affect the initial steps and rate-limiting steps of steroidogenesis.

Typically, inhibitors of steroidogenesis would inhibit through a direct interaction with the

cytochrome P450 enzymes involved in steroid hormone synthesis (Taxvig et al, 2008). The

profile for inhibition of steroidogenesis in vivo is characterized by a relatively specific and

diagnostic array of in vivo effects at the organ level. For example, inhibition of steroidogenesis in

female rats would result in an increase in age and weight at vaginal opening (VO), a decrease in

27
uterine weight and an increase in ovarian weight. In male rats, expected responses for inhibition

of steroidogenesis is dependent on whether there is inhibition of testosterone production,

inhibition of 5ct-reductase or aromatase activity.

With inhibition of testosterone biosynthesis, the expected organ level profile would include an

increase in age at pre-putial separation (PPS) and decrease in testes, epididymides, prostate, and

seminal vesicles weights. Additionally, there would typically be a related increase in liver

weight in males and females (non-reproductive organ weight).

With inhibition of 5a-reductase, the expected organ level profile would include a decrease in

epididymides, prostate, and seminal vesicles weights. Additionally, there would typically be a

related increase in liver weight in males and females (non-reproductive organ weight).

With inhibition of aromatase activity, the expected organ level profile would include a decrease

in body weight at the time of PPS, and increases in prostate and seminal vesicles weights.

Additionally, there would typically be a related increase in liver weight in males and females

(non-reproductive organ weight).

The rat multigenerational study along with the other chronic and subchronic studies provides the

scientifically relevant information that is required to perform an assessment for potential effects

of glyphosate on steroidogenesis. A rat multigenerational study that followed the OPPTS

870.3800 (August, 1998) and OECD 416 guidelines, provides all of the critical endpoints

required to assess the potential for detecting inhibition of steroidogenesis. The rat multigenerational study is able to effectively detect aromatase inhibition and has been shown to have

greater sensitivity than the pubertal assays, which includes endpoints capable of detecting

aromatase inhibition. For example, fenarimol was negative in the female pubertal study, yet in a

multigenerational assay produced the expected effects on parturition associated with lowered

estrogen levels after aromatase inhibition (EPA, 2007).

Overview of Rat Multi Generation Study as it Applies to an Assessment of Steroidogenesis

There were no effects in the multigenerational rat studies by Moxon (2000) and Dhinsa et al.

(2007) indicative of potential effects of steroidogenesis. A comprehensive review of the

endpoints that would be impacted by an affect on steroidogenesis is discussed in the following.

There were no effects on a range of endpoints indicative of an effect on steroidogenesis in rats

treated at up to the highest doses tested of 10,000 ppm and 15,000 ppm glyphosate in these two

multigenerational studies. These endpoints include the weight of testes, cauda epididymides,

epididymides, seminal vesicles and coagulating glands, uterus, ovary and liver of the FO and Fl

parent animals (Moxon, 2000; Dhinsa et al., 2007). Glyphosate-related reductions in body

weight were recorded only for Fl males treated at the HOT, but there is no evidence that this

reduction is an endocrine-mediated effect.

There were no effects recorded following treatment at up to the HDT's of 10,000 ppm and

15,000 ppm glyphosate on the histopathology of testis, epididymes, prostate, uterus, left ovary,

vagina, cervix, and oviducts. No effects on sperm motility parameters and sperm morphology

were noted at all doses (Moxon, 2000; Dhinsa et al., 2007).

There were no effects on male and female reproductive indices that included mating, fertility,

conception, gestation, lactation, age and weight at VO, age at PPS, testicular descent, ovarian

evaluation for follicles, parturition, normality of external genitalia and number of implantation

28
sites following treatment at up to the HDT's of 10,000 ppm and 15,000 ppm glyphosate.

Additionally, there was no impact on the number of sperm, sperm motility parameters and sperm

morphology for FO and Fl males (Moxon, 2000; Dhinsa et al., 2007).

2.3.1 Conclusion - Steroidogenesis

In vitro data assessing potential impacts on the complete steroidogenic pathway are not available

for glyphosate; however, data that is functionally equivalent to the in vitro Steroidogenesis assay

is provided by endpoints included in rat multigenerational studies. Specifically, it was confirmed

in the most recent multigenerational studies that there were no treatment related effects at high

dietary exposure levels on endpoints that would be impacted by an alteration of Steroidogenesis

which include:

• testicular/uterine/ovary weights and histopathology

• size, function and histopathology of sex organs

• fertility

• anogenital distance

• sperm count and mobility

• ovarian evaluation for follicles and corpa lutea

• anogenital distance

• age and weight at preputial separation and vaginal opening

• number of implantation sites and lactation.

Glyphosate-related reductions in body weight were recorded only in Fl males at the HOT, but

there is no evidence that this reduction is an endocrine-mediated effect. Additionally, there was

no effect on several endpoints that would be impacted by inhibition of Steroidogenesis following

treatment at up to the HOT. The information from these in vivo studies with glyphosate provides

compelling evidence that glyphosate has no effect on Steroidogenesis.

3.0 Mammalian In Vivo Assays

Glyphosate is one of the most extensively tested agricultural chemicals being used today.

Multiple regulatory toxicology data sets exist for glyphosate, which have been reviewed by

panels of international experts, including, most recently, a toxicological evaluation in a Joint

review by the FAO/WHO in 2004. In 2009, ECETOC published Guidance on Identifying

Endocrine Disrupting Effects, Technical Report 106. In this guidance document, glyphosate was

reviewed as an example, with mammalian and wildlife toxicology studies evaluated, of a

chemical that poses no endocrine disruption concerns. There is substantial in vivo mammalian

data supporting the positions that glyphosate is not an endocrine active compound and that no

further animal testing is necessary to characterize its lack of endocrine disrupting potential.

In OPPTS Guidelines 890.1400 (Hershberger) and 890.1600 (Uterotrophic), section (d) of each

guideline discusses "Initial Considerations and Limitations" to be considered before proceeding

with either assay. For the first consideration, in vitro evaluations, a distinct absence of potential

for estrogen binding and transcriptional activation (also evaluated in the Uterotrophic assay), and

androgen receptor binding and 5a-reductase inhibition (also evaluated in the Hershberger assay)

29
are discussed in Sections 2.1 and 2.2. Other considerations mention that, in both OPPTS

890.1400 and 890.1600, metabolic activation/deactivation, distribution to target tissues and

clearance from the body are distinctly addressed in multiple metabolism and ADME studies.

These ADME studies include single and repeat dose oral studies in multiple species (rat, rabbit

and monkey), and single dose studies via multiple routes of administration, and are summarized

in Appendix 2. These studies show that glyphosate is not readily metabolized, is not

substantially absorbed when ingested, is rapidly excreted, and does not accumulate in the body.

Glyphosate offers low systemic exposure when administered orally, and, similarly, has very low

dermal absorption. Given the low potential for systemic exposure to glyphosate, the value of

further animal testing in the EDSP program should be critically evaluated before conducting the

assays.

Considerable value is found in multigenerational reproduction studies for the evaluation of the

potential for a substance to potentially interact with the endocrine system. At least four such

studies have been conducted to evaluate glyphosate. Multigenerational studies cover a range of

specific and apical endpoints, which may be used to evaluate endocrine activity, including

behavioral changes, signs of difficult or prolonged parturition, ability to become pregnant,

duration of gestation, sex ratio of the offspring, feminization or masculinization of offspring,

number of pups, stillbirths, and gross pathology and histopathology of the vagina, uterus,

ovaries, testes, epididymis, seminal vesicles, prostate, and any identified target organs (Stevens

etal., 1997).

The rat multigenerational study is the most comprehensive of the current tests for reproductive

and endocrine toxicity, providing an enormous amount of information that is of the same nature

as a significant portion of the data intended to be generated in many of the Tier 1 screening

assays. The revised 1998 U.S. EPA guideline (OPPTS 870.3800) for a multiple generation

reproductive and developmental toxicity assay includes many developmental benchmarks

predictive of endocrine disruption potential, including:

• day of vaginal opening

• mean estrus cycle length and pattern

• a diverse set of female organ weights and histopathology (ovaries, uterus, vagina, and

mammary glands)

• mating and fertility indices

• number of implantation sites

• male tissue weights and histopathology (testes, epididymis, prostate, and seminal

vesicles)

• male gamete parameters (e.g. sperm number, motility and morphology)

3.1 Uterotrophic Assay (OPPTS 890,1600)

The Uterotrophic assay is an in vivo assay in rats designed to detect estrogenic activity of a

chemical based solely on an increase in uterine weight due to estrogen-induced water imbibitions

and hypertrophy, and is intended to complement the in vitro ER Competitive Receptor Binding

assay (OPPTS 890.1250) and the ER Transcriptional Activation assay (OPPTS 890.1300) of the

EDSP. There are already a total of seven in vivo studies on glyphosate, conducted at high dose

30
levels in two species (rats and rabbits), where uterine and other organs and tissues that can be

evaluated as indicators of estrogenic potential have not shown any statistical difference in weight

or any other treatment related macroscopic or microscopic findings that would indicate any

estrogenic effects of glyphosate. This collection of data, along with the in vitro ER binding and

transactivation assays by Petit et al., Kojima et al., and Xie et al., discussed in Section 2.0, that

demonstrate that glyphosate does not interact with the ERa and the ERp, and which this in vivo

study is intended to complement, provides more evidence as to the lack of estrogenic activity of

glyphosate than this one additional uterotrophic assay could provide.

3.1.1 Discussion

The uterotrophic assay, which is based solely on the ability of ER agonists to induce an increase

in uterine weight due to hypertrophy and/or uterine fluid imbibition, is widely accepted as a

useful assay for identifying estrogenic compounds (Clark and Mani, 1994; Reel et al., 1996). It

should be noted, however, that the uterus predominantly contains the ERa receptor subtype, with

very little ER|3. The uterotrophic assay is designed to complement the in vitro ER Competitive

Receptor Binding assay and the ER Transcriptional Activation assay of the EDSP Tl test battery.

According to OPPTS 890.1600, immature, or preferably ovariectomized rats, (6 per group) are

dosed via gavage or subcutaneous injection for 3 days and then necropsied, and ER agonists are

detected by an increase in wet or blotted uterine weight. However, as noted in the test guideline,

the uterotrophic response is not always of estrogenic origin and compounds other than agonists

or antagonists of estrogens can also provide a response. For example, relatively high doses of

testosterone can lead to a stimulative response.

As previously discussed in Section 2.0, existing studies functionally equivalent to the in vitro ER

binding and transactivation assays of the EDSP, already demonstrate that glyphosate does not

interact with the ERa and the ER0 receptors (Petit et al. 1997, Kojima et al., 2004, Xie et al.,

2005). Glyphosate has also been shown to not possess estrogenic or androgenic properties in

several in vivo studies.

Four multigenerational studies in rats, each individually, provide consistent evidence that

glyphosate does not exhibit any estrogenic activity.

Schroeder, R.E.; Hogan, G.K. (1981) A Three-Generation Reproduction Study with

Glyphosate in Rats: Project No. 77-2 , Study No. BDN 77-417 (Unpublished study; prepared by

Bio/dynamics, Inc., submitted by Monsanto Co., Washington, D.C.; CDL:245909-A); MRID's

00081674,00105995

In this three-generation reproduction study, where 12 male and 24 female Sprague-Dawley rats

per group received dietary exposure to glyphosate at 0, 3, 10 and 30 mg/kg/day, the highest dose

tested (HDT) was considered the no-effect level. Endpoints that can be evaluated as indicators

of estrogenic potential in this study included mating and fertility indices, ovary weights,

microscopic evaluations of uterus and ovaries in FO, Fib and F2b parents and F3b offspring

(control and high doses), and litter sex ratios, all of which had no significant findings, which

includes no findings of any estrogenic effects.

31
Reyna, M. (1990). Two Generation Reproduction Feeding Study with Glyphosate in SpragueDawley Rats: Lab Project No: MSL-10387, Study No. ML 88-106. Unpublished study prepared

by Monsanto Agricultural Co.; MRJD 41621501

In compliance with OPP Guideline 83-4, a multigenerational study (Reyna, 1990) was conducted

with Sprague-Dawley rats (30 rats/sex/group) dosed at 2,000, 10,000 and 30,000 parts per

million glyphosate in the diet. Only a slight, transient body weight change was noted at 10,000

ppm (approximately 777 mg/kg/day for males and 666 mg/kg/day for females), which was

considered to be the NOAEL for reproductive and parental toxicity. Endpoints relevant to the

uterotrophic assay in FO and Fla parents included ovary weights, macroscopic evaluation of

uterus and ovary, and microscopic evaluation of vagina, uterus and ovary, all of which showed

no glyphosate-related effects.

Dhinsa, N. K., Watson, P. and Brooks, P. N. (2007) Glyphosate Technical: Dietary Two

Generation Reproduction Study in the Rat. Unpublished study to be submitted to EPA by

Nufarm Americas.

Dhinsa et al. (2007) conducted a multigeneration reproduction study in Sprague-Dawley Crl:CD

(SD) IGS BR rats (FO 28 rats/sex/group, Fl 24 rats/sex/group), which is in compliance with the

current 2001 OECD Test Guideline 416. The oral administration of glyphosate technical to rats

by dietary admixture at a maximum dose level of 15,000 ppm for two successive generations

resulted in possible treatment related changes at 15,000 ppm. The effects, however, were

considered not to represent an adverse health effect; therefore, the No Observed Adverse Effect

Level (NOAEL) was considered to be 15,000 ppm for adult toxicity for both the FO and Fl

generations. The NOEL for reproductive and developmental toxicity for both generations and

offspring was also considered to be 15,000 ppm. Endpoints included:

• day of age when vaginal opening occurred in the Fl females

• absolute and relative weights of the ovaries and uterus (including oviducts and cervix) of

the FO and Fl parent animals

• absolute and relative weight of the uterus of the Fl and F2 offspring animals

• in follicle numbers from the ovaries from treated Fi females

• macroscopic or microscopic findings observed in any tissue from the FO or Fl animals,

including uterus with oviducts and cervix, ovaries and vagina

• matings in all treatment groups

• ratio of male to female pups born

• litter size

• viability of offspring

Moxon, M. E. (2000) Glyphosate Acid: Multigeneration Reproduction Toxicity Study in Rats;

Report No. CTL/P/6332. Unpublished study to be submitted by Syngenta Crop Protection.

32
Moxon (2000) conducted a multigenerational reproduction study in Wistar derived rats (26

rats/sex/group), which is compliant with the current 1998 US EPA OPPTS 870.3800 guideline.

Dietary exposure to glyphosate at 0, 1000, 3000 and 10000 parts per million (ppm) had no

adverse effect on reproductive performance, including:

• day of age when vaginal opening occurred in the Fl females

• unadjusted weight and organ to body weight ratio of the ovaries or uterus (including

cervix) of the FO and Fl parent animals

• number of primordial and small growing follicles of the left ovary of the Fl parent

females

• macroscopic or microscopic findings observed in any tissue from the FO or Fl animals,

including uterus, cervix, ovaries and vagina

• matings in all treatment groups

• ratio of male/female pups born

• litter size during lactation

In addition to the four multigenerational studies, there are three other studies that provide

evidence for the lack of estrogenic activity—a teratology study in the rat, a teratology study in

the rabbit and a 90-day feeding study in the rat. In the two teratology studies, the uterine weights

were measured and showed no statistical difference between the control and the highest dosed

test group. Each of these studies is discussed below.

Rodwell, D.E.; Tasker, E.J.; Blair, A.M.; et al. (1980a) Teratology Study in Rats: IRDC No.

401-054, Study No. IR 79-016. (Unpublished study including IRDC no. 999-021; prepared by

International Research and Development Corp., submitted by Monsanto Co., Washington, D.C.;

CDL:242516-A); MRID 00046362

Rodwell, D.E.; Tasker, E.J.; Blair, M.; et al. (1980b) Teratology Study in Rabbits: IRDC No.

401-056, Study No. IR 79-018. (Unpublished study under 524-308; prepared by International

Research and Development Corp., submitted by Monsanto Co., Washington, D.C.; CDL:242516-

B); MRID 00046363

Glyphosate was shown to not be a selective developmental toxicant in two teratology studies,

one in rats dosed at 0, 300, 1000 and 3500 mg/kg/day (Rodwell et al., 1980a) and one in rabbits

dosed at 0, 75, 175 and 350 mg/kg/day (Rodwell et al., 1980b). In the rat study, with a maternal

and developmental NOAEL of 1,000 mg/kg/day, a reduced gain in mean maternal body weight

was noted over the treatment period at 3,500 mg/kg/day, as well as an increase in early

resorptions, a slight increase in postimplantation loss and decreases in mean fetal body weight

and the mean number of viable fetuses at this maternally toxic dose. In the rabbit study, with a

maternal NOAEL of 175 mg/kg/day and developmental NOAEL of 350 mg/kg/day, there were

no biologically meaningful differences in mean number of viable fetuses, early or late

resorptions, total implantations, corpora lutea, or the fetal sex distribution.

In both these studies, uterine weights were measured after excision and no statistically significant

trends were noted across this wide range of doses.

33
Stout, L.; 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 and Johnson (1987) conducted a 90-day rat feeding study with glyphosate at 0, 1000, 5000

and 20000 ppm in the diet. The high dose was considered the NOAEL. Microscopic evaluation

of the ovaries, skin (with mammary gland) and uterus (corpus and cervix), in the control and

high dose group were conducted. All the tissues relevant to the uterotrophic assay, showed no

glyphosate-related effects, which provides further evidence that there was no estrogenic activity

as well.

Lastly, there is already available a study where microscopic evaluations of uterine tissues had

been conducted following chronic exposure to high levels of glyphosate.

Stout, L.; Ruecker, F. (1990) Chronic Study of Glyphosate Administered in Feed to Albino

Rats: Lab Project Number: MSL-10495: R.D. 1014, Study No. ML 87-148. Unpublished study

prepared by Monsanto Agricultural Co.; MRID 41643801.

Stout and Rucker (1990) conducted a chronic dietary rat study dosing at 0, 2000, 8000 and 20000

ppm in the diet for two years. No glyphosate related effects were noted at any dose for any

microscopic evaluations of uterus, ovaries or mammary glands in the 1-year interim sacrifice, 2-

year terminal sacrifice or any unscheduled deaths.

3.1.2 Conclusions - Uterotrophic Assay

Owens and Ashby (2002) conducted a thorough critical review of the uterotrophic assay, and the

predictive value of the uterotrophic assay itself was determined by comparison with observations

made in the multigenerational reproductive and developmental toxicity assays. They concluded

that "labeling or classification action should not be based on in vitro or in vivo screens (e.g.,

transcriptional assays or the uterotrophic assay), but await the results of robust, definitive testing

protocols for adverse effects, such as the multigenerational reproductive and developmental assay,

so that both the data on mechanism and the occurrence of an adverse effect are available." Most

importantly, this paper provided a comparison of no observed effect levels (NOEL) and lowest

observed effect levels (LOEL) for uterotrophic and rat multigeneration studies, and concluded

high concordance for NOEC and LOEC values for estrogenicity.

There is consistent evidence showing a lack of estrogenic activity of glyphosate in four

multigenerational studies, a subchronic study, and a chronic study, all in rats. These equivalent

data provide strong grounds for the uterotrophic assay being considered duplicative and

unnecessary to characterize glyphosate potential as a non-estrogenic compound in an in vivo

assay.

End Point in Uterotrophic assay Functionally Equivalent Information from Existing Studies

Uterine Weight • No impact on uterine weights with cervix at the highest dose tested of

10,000 ppm in diet (Moxon, 2000) of the FO and Fl parent animals.

• No impact on uterine histopathology which is consistent with no effect on

uterine weight at 10,000 ppm in diet (Moxon, 2000).

• No impact on uterine weights with cervix and oviducts at the highest dose

tested of 15,000 ppm in diet (Dhinsa et al., 2007) of the FO and Fl parent

34
End Point in Uterotrophic assay Functionally Equivalent Information from Existing Studies

animals.

• No impact on uterine histopathology which is consistent with no effect

uterine weight at 15,000 ppm in diet (Dhinsa et al, 2007).

• No impact on uterine weights at the highest dose tested of 15,000 ppm

diet (Dhinsa et al., 2007) of the Fl and F2 offspring animals.

on

in

3.2 Hershberger Bioassay (OPPTS 890.1400)

The Hershberger assay (OPPTS 890.1400) is widely accepted as a useful test that utilizes

measurements of five androgen-dependent accessory sex tissue weights from castrated male rats

following oral gavage exposure to the test substance for identifying potential AR agonists, AR

antagonists, and 5<x-reductase inhibitors. These tissues are the ventral prostate (VP), seminal

vesicle (SV) (plus fluids and coagulating glands), levator ani-bulbocavernosus (LABC) muscle,

paired Cowper's glands (COW) and the glans penis (GP).

O'Connor et al. (2002) evaluated a number of the EDSP Tier 1 assays, and, in the evaluation of

the Hershberger assay, they summarized the relative correlations between changes in weight (as

a percentage of total body weight) of the various target organs of the assay and the potential

endocrine activity. Those correlations (from Table 2 in O'Connor et al.) are summarized here in

Table 2. As an example, this table illustrates that if a chemical is a strong agonist of androgen

receptor activity, you would see an increase in the relative weight (relative to total body weight)

of the prostate, seminal vesicles, levator ani-bulbocavernosus muscle and bulbourethral

(Cowper's) glands, and may or may not see an increase in the relative weight of the liver. On the

other hand, a weak antagonist would result in an increase in relative liver weight and maybe or

maybe not a decrease in relative weights of the other four tissues.

None of the results from the four multigenerational, one subchronic and one chronic study with

glyphosate in rats indicate any androgenic activity when evaluated against the effects correlation

matrix of O'Connor et al. These data are functionally equivalent to data expected to be generated

from the Hershberger assay, as described below in each subsection for the endpoints noted in OPPTS

890.1400. Therefore, the requirement to run this assay should be waived.

Table 2: Correlation of Effects on Hershberger Target Tissues (Adapted from O'Connor et al., 2002, Table 2)

Endocrine Activity

Androgen Receptor,

strong agonist

Androgen Receptor,

strong antagonist

Androgen Receptor,

weak antagonist

Antiandrogen

5a-reductase inhibitor

PorVP

(% b.wt.)

t

i

-/i

-/|

4

SV

(% b.wt.)

T

i

-l{

-/l/r

1

LABC

(% b.wt.)

T

-/I

-/I

-/i

-

BU

(% b.wt.)

t

1

-/I

-/4/T

1

Liver

(% b.wt.)

-/T

-/t

T

T

-

LH

(ng/mL)

T

T

(ng/mL)

-

P = prostate; VP = ventral prostate; SV = seminal vesicles; LABC = levator anibulbocevernosus muscles; BU =

bulbourethral (Cowper's) glands; L = liver; LH = lutenizing hormone; T = testosterone, b.wt. = body weight

35
A more complete discussion of OSRI related to the Hershberger assay is presented by

tissue/endpoint in the following subsections. Many of the findings are discussed in relation to

the effects matrix in Table 2.

3.2.1 Prostate / Ventral Prostate

All six of the glyphosate feeding studies below included weight measurement and/or microscopic

examination of the prostate. No differences in absolute or relative weights of the prostate or any

treatment-related effects regarding the prostate were noted in any of these studies.

In the multigenerational reproduction study on glyphosate by Moxon (2000), there were no

differences noted in the absolute weight or relative weight of the prostrate gland between the

control and dose groups in the FO parents or Fl parents.

In the most recent multigenerational reproduction study on glyphosate by Dhinsa et al. (2007),

there were no differences noted in the absolute weight or relative weight of the prostrate gland

between the control and dose groups in the FO parents or Fl parents. No treatment related

histopathological differences of the prostate were noted between the control and high dose male

groups.

In the three-generation reproduction study on glyphosate by Schroeder and Hogan (1981),

microscopic examination of the prostate from the FO parents, Fib parents, F2b parents and F3b

offspring of the high dose group noted no treatment-related effects relative to the study controls.

The two-generation reproduction study by Reyna (1990) included microscopic examination of

the prostate of the FO parents and Fla parents in the control and high glyphosate dose groups.

There were no treatment-related effects noted in any of the groups.

Both the glyphosate subchronic rat feeding study of Stout and Johnson (1987) and the chronic

study of Stout and Rucker (1990) included microscopic examination of the prostate at 90 days

and at 12 and 24 months, respectively. As with the multigenerational studies, there were no

treatment-related effects noted on the prostate in either study.

3.2.2 Seminal Vesicles

Three of the four multigenerational reproduction toxicity studies on glyphosate included weight

measurements or microscopic examination of the seminal vesicles. No difference in absolute or

relative weights or any treatment related effects were noted in the seminal vesicles in the three

studies.

In the multigenerational rat reproduction study on glyphosate conducted by Moxon (2000), there

was no difference in either the absolute or relative weights of the seminal vesicles in FO parents

or Fl parents from the control and dose groups. Microscopic examination of the seminal

vesicles of the FO parents and Fla parents from the control and high dose groups in the twogeneration glyphosate reproduction toxicity study by Reyna (1990) also showed no treatment

related effects of any kind, including any androgenic effects.

In the most recent multigenerational reproduction study on glyphosate by Dhinsa et al. (2007),

there were no differences noted in the absolute weight or relative weight of the seminal vesicles

between the control and dose groups in the FO parents or Fl parents. No treatment related

histopathological differences of the seminal vesicles were noted between the control and high

dose male groups.

36
3.2.3 Levator Ani Bulbocavernosus (LABC)

LABC weight was not evaluated in any of the glyphosate studies. However, the effects

correlation matrix described by O'Connor (2002) (Table 2) shows that, in a number of instances

of androgenic effect, a difference in the relative weight of the LABC between controls and test

groups are also accompanied by changes in the relative weights of the prostate and seminal

vesicles. No changes in prostate or seminal vesicle weights were seen in the 90-day rat feeding

study on glyphosate conducted by Stout and Johnson (1987).

A change in the relative weight of the LBAC can also be accompanied by an increase in the

relative weight of the liver when it is the result of androgenic activity. The multigenerational rat

reproduction study on glyphosate conducted by Moxon (2000) showed a statistically significant

(p<0.01) higher relative mean liver weight over the control in the FO males, as well as a

statistically significant (p<0.05) lower absolute mean liver weight compared to the control in the

Fl males, both at the high dose level. Dhinsa et al. (2007) noted an increase in liver weights for

females treated at the high dose of 15,000 ppm from both generations. No such effect was noted for

males treated with 15,000 ppm or for animals of either sex treated with 5,000 ppm or 1,500 ppm.

According to the effects correlation matrix, an increase in relative liver weight can be reflective

of (i) a strong androgen receptor agonist when accompanied by increased prostate and seminal

vesicles weight, or (ii) a strong androgen receptor antagonists when accompanied by decreased

prostate and seminal vesicle weights. As indicated in Sections 3.2.1 and 3.2.2 above, these

multigenerational rat reproduction studies on glyphosate showed no difference in either the

absolute or relative weight of either the prostate or the seminal vesicles; therefore, there is no

evidence that glyphosate is either a strong androgen receptor agonist or antagonist. Also

according to the effects correlation matrix, androgenic activity only causes an increase in relative

liver weight, never a decrease; therefore, the decreased relative liver weight in the Fl male

indicates that the changes are totally unrelated to endocrine activity. This conclusion is

reasonable since liver weight changes are common in high dose groups of toxicological

investigations, and the high dose males in this study exhibited compound related reductions in

body weight and food consumption, which would explain the increase in liver weight relative to

total body weight.

3.2.4 Bulbourethral (Cowper's) Gland

Bulbourethral gland weights were not measured in any of the glyphosate studies. However, the

effects correlation matrix of the Hershberger assay (Table 2) notes that in a number of instances,

a difference in bulbourethral gland % body weight between controls and test groups are also

accompanied by changes in relative weights of the prostate, seminal vesicles, and liver. As

discussed in section 3.2.1, the 90-day rat feeding study on glyphosate conducted by Stout and

Johnson (1987) did not find any effects following microscopic examination of the prostate. The

two-generation rat reproduction toxicity study on glyphosate conducted by Moxon (2000), as

discussed in Section 3.2.2, did not find any change in the absolute or relative weight of the

seminal vesicles, and as discussed in Section 3.2.3, and in the next section, the increase in

relative liver weight in the FO male and decrease in the relative liver weight in the Fl male, along

with the no effect findings on the prostate and seminal vesicles, is strong evidence that these

liver changes were not related to endocrine activity and simply due to other mechanisms in high

dose test groups. The most recent multigenerational rat study (Dhinsa et al., 2007) noted no

difference in the absolute or relative weights, or any histopathological differences, of either the

37
prostate glands or seminal vesicles of the high dose animals when compared to those of the

control groups.

3.2.5 Glans Penis Weight

In the O'Connor et al. (2000) evaluation of the then proposed EDSP Tier I screening approaches,

data on glans penis weight were not available for evaluation. However, given the considerable

substantiation of no effects on fertility and male reproductive organ weights in four multigenerational reproduction studies, this endpoint may be considered redundant.

3.2.6 Liver Weight

Measurement of liver weight is an optional endpoint in the Hershberger assay (OPPTS

890.1400). While collection of liver weight would provide insight into general toxicity, for

endocrine evaluation purposes, liver weight would only be useful as supportive if there were

other data that suggested possible endocrine activity.

The multigenerational reproductive study on glyphosate conducted by Schroeder and Hogan

(1981) showed no differences in the absolute or relative weights of the liver in male FO adults,

Fl adults, F2 adults and F3 offspring between the control and dose groups. Also, no treatment

related effects were identified in the microscopic examinations of the livers of the parental FO,

Fib, F2b and weanlings from F3b animals. In the F2 treated females, a non-dose-related, albeit

statistically significant, decrease in mean liver/body weight ratio was evident; liver/brain weight

ratios for these females showed a similar reduction; however, these differences from control

were not statistically significant.

The multigenerational rat reproduction study on glyphosate conducted by Moxon (2000) showed

a statistically significant (p < 0.01) higher relative mean liver weight over the control in the FO

males, as well as a statistically significant (p < 0.05) lower absolute mean liver weight compared

to the control in the Fl males, both at the high dose level. Dhinsa et al. (2007) noted an increase

in liver weights for females from both generations treated at the high dose of 15,000 ppm. No such

effect was noted for males treated with 15,000 ppm or for animals of either sex treated with 5,000 or

1,500 ppm. According to the effects correlation matrix, an increase in relative liver weight can

be reflective of (i) a strong androgen receptor agonist when accompanied by increased prostate

and seminal vesicles weight, or (ii) a strong androgen receptor antagonist when accompanied by

decreased prostate and seminal vesicles weights. As indicated in Sections 3.2.1 and 3.2.2, this

multigenerational rat reproduction study on glyphosate showed no difference in either the

absolute or relative weight of either the prostate or the seminal vesicles; therefore, there is no

evidence that glyphosate is either a strong androgen receptor agonist or antagonist. Also

according to the effects correlation matrix, androgenic activity only causes an increase in relative

liver weight, never a decrease; therefore, the decreased relative liver weight in the Fl male

indicates that the changes are totally unrelated to endocrine activity. Liver weight changes are

common in high dose groups of toxicological investigations and the high dose males in this study

exhibited compound related reductions in body weight and food consumption, which would

explain the increase in liver weight relative to total body weight. Stout and Rucker (1990), in the

chronic rat study on glyphosate, found statistically significant increases in liver-to-body weight

ratios in males at 12 months, and absolute liver weight and liver-to-brain weight ratios at 24

months at the maximum tolerated dose (high dose). As mentioned earlier, liver weight changes

are common in high dose groups of toxicological investigations, especially at maximum tolerated

doses.

38
Stout and Johnson (1987) found no changes in either absolute or relative liver weight after 90

days of oral dosing in rats and microscopic examination of control and high dose livers noted no

treatment-related effects.

3.2.7 Luteinizing Hormone (LH)

Measurement of luteinizing hormone (LH) serum concentration (ng/mL) is optional in the

Hershberger assay (OPPTS 890.1400). Increased LH levels provide additional weight of

evidence of strong androgen receptor antagonism (Table 2) and would be measured only if there

were other data that suggested that the test substance was an androgen receptor antagonist.

Given the volume of data that supports the conclusion that glyphosate is not an androgen

receptor antagonist, this optional endpoint provides no added value.

3.2.8 Testosterone

Measurement of testosterone serum concentration (ng/mL) is optional in the Hershberger assay

(OPPTS 890.1400). Since no male reproductive effects were noted in the four multigenerational

rat studies (Dhinsa et al., 2007; Moxon, 2000; Reyna, 1990; Schroeder and Hogan, 1981), the

measurement of testosterone serum concentrations would provide no significant value to the

understanding of the endocrine activity of glyphosate.

3.2.9 Other endpoints of relevance to the Hershberger Assay

Overall, in six in vivo studies conducted on glyphosate, including four multigenerational

reproduction studies, a subchronic feeding study and a chronic feeding study, no treatmentrelated effects were ever seen on the number of sperm produced, sperm motility or sperm

morphology, or mating, pregnancy or fertility indices. Four of these six studies showed no

changes in the absolute or relative weight of the testes or epididymides, or any treatment-related

effects on the microscopic examination of the testes. Only in the two-generation reproduction

study conducted by Reyna was there a slight increase in the relative weight of the testes (weight

of testes relative to total body weight) and only in the Fla adult in the highest treatment group

where the dosing level was in excess of the maximum tolerated dose for glyphosate. This effect,

however, was attributed to the lower body weight of the rats, since the absolute weight of the

testes was unchanged from that of the control group. The findings of each of these studies are

summarized below.

In the multigenerational reproduction studies on glyphosate conducted by Moxon (2000) and

Dhinsa et al. (2007), there were no differences noted in the absolute or relative mean weights of

the testes or epididymides between control and dose groups of the FO parents or Fl parents. No

effect of glyphosate on the number of sperm, sperm motility parameters or sperm morphology

were noted for either the FO or Fl males.

Schroeder and Hogan (1981) found no effects on the absolute or relative mean weight of the

testes of rats over three generations of continual dosing with glyphosate. Microscopic

examination of the testes from the control and high dose groups noted no treatment-related

effects.

Significantly decreased terminal body weights were noted for both sexes at the highest exposure

level in the two-generation reproduction study on glyphosate conducted by Reyna (1990). The

only statistically significant organ weight change was a slight increase in the mean testes-to-body

weight ratios in the Fla adult high dose group, but the mean absolute testes weight remained

unchanged. This finding was attributed to their lower terminal body weights at this dose level,

39
which exceeded the maximum tolerated dose. No differences in the mean relative testes weights

were noted in the FO at all dose levels or in the Fla low and mid-dose groups. Microscopic

examination of testes from the control and high dose groups, and of the epididymides of the FO

and Fla parents, noted no treatment related effects. Mating, pregnancy and fertility indices were

not affected by glyphosate in either the FO or Fl animals.

Microscopic examination of the testes and prostate from the control and high dose groups noted

no treatment-related effects after 90 days of oral dosing in the subchronic study conducted by

Stout and Johnson (1987).

In the chronic feeding study on glyphosate conducted by Stout and Rucker (1990), microscopic

examination of testes from the control and high dose groups noted no treatment-related effects

after 12 and 24 months in the study.

3.2.10 Conclusion - Hershberger Assay

Clear and consistent results from four multigenerational reproduction studies and two repeatdose toxicity studies on glyphosate provide strong scientific evidence that glyphosate is not an

AR agonist, AR antagonist or a 5a-reductase inhibitor, and go beyond the five male sex gland

weight endpoints assessed in the Hershberger assay. Based on this information, the Hershberger

assay conducted according to OPPTS 890.1400 guideline is considered duplicative and

unnecessary to characterize the potential for glyphosate to interact with androgen receptors or

inhibit 5a-reductase in vivo.

Table 3: Summary of Findings Relative to the Hershberger Assay for Glyphosate

End Point in Hershberger Assay

Prostate/ventral weight

Seminal Vesicles weights

LABC weights

Bulbourethral (Cowper's) Gland

Glans Penis weight

Functionally Equivalent Information from Existing Studies

• No differences in absolute or relative weights of the prostate or any

treatment related effects regarding the prostate were noted in a subchronic

rat study, a chronic rat study, four multigenerational rat studies.

• No difference in absolute or relative weights or any gross or microscopic

treatment related effects were noted in the seminal vesicles (where

evaluated) in multigenerational studies.

• LABC weight was not evaluated in any of the glyphosate studies.

However, the absence of fertility issues and corroborative male

reproductive organ weight data provides a substantial weight of evidence

across multiple studies, suggesting no effect on LABC weights.

• Bulbourethral gland weights were not measured in any of the glyphosate

studies. However, the absence of fertility issues and corroborative male

reproductive organ weight data provides a substantial weight of evidence

across multiple studies, suggesting no effect on Bulbourethral weights.

• Glans penis weights were not measured in any of the glyphosate studies.

However, the absence of fertility issues and corroborative male

reproductive organ weight data provides a substantial weight of evidence

across multiple studies, suggesting no effect on glans penis weights.

3.3 Pubertal Development and Thyroid Function in Intact Juvenile /

Peripubertal Female Rats (OPPTS 890.1450)

O'Connor et al. (2002) states in their review of the Tier I female pubertal assay that "this assay

has redundant endpoints with the Tier II multigenerational reproduction study, which allows

confirmation or refutation of screening results under more robust test conditions." There are

40
already four multigenerational rat reproduction studies conducted on glyphosate. To require

another in vivo study where more test animals would be sacrificed to obtain more of the same

information already available would waste valuable resources and time of both the registrants

and the Agency. The female pubertal assay should be waived for glyphosate.

The female pubertal assay measures discrete endpoints to ascertain effects on pubertal development

and thyroid function in the intact juvenile/peripubertal female rat. These endpoints include

developmental milestones, organ weights and histopathology, estrous cyclicity, hormone serum

levels and clinical chemistry parameters. All of these endpoints are either specifically addressed

or captured in downstream apical endpoints in more comprehensive in vivo studies, such as

multigenerational reproduction, developmental toxicity, subchronic toxicity and chronic toxicity

studies. The multigenerational reproduction study, which is a key Tier 2 EDSP assay, provides

functional equivalence to OPPTS 890.1450 and renders the female pubertal assay redundant

(O'Connor etal., 2002).

Correlations between the various endpoint effects of the female pubertal assay and endocrine

activity were assessed by O'Connor et al. (2002). Those correlations (from Table 3 in O'Connor

et al., 2002) are reproduced here in Table 4. To illustrate, if a chemical is a strong estrogen

receptor agonist, you would expect to see a decrease in the age at vaginal opening (VO), a

decrease in body weight at vaginal opening, and a decrease in the weight of the ovaries. On the

other hand, evidence of a neuroendocrine mechanism would be an increase in age and body

weight at vaginal opening along with a decrease in the weight of the uterus and ovaries.

A more complete discussion of OSRI related to the female pubertal assay is presented by

endpoint in the following subsections. Many of the findings are discussed in relation to the

effects matrix in Table 4.

Table 4: Correlation of Effects in Female Pubertal Assay (Adapted from O 'Connor et al., 2002, Table 3)

Endocrine Activity

Strong estrogen receptor

agonist

Weak estrogen receptor

agonist

Neuroendocrine

mechanism

Broad steroid biosynthesis

inhibitor

Highly specific aromatase

inhibitor

Thyroid active

compounds

Age at

VO

1

-/I

T

T

T

-

B.wt at

VO

1

-/i

T

T

T

i

Uterus

weight

-

1

1

4

-

Ovaries

weight

4

I

-

-

4

Thyroid

weight

-

-

-

-

T

TSH

-

T

T4

-

1

VO = vaginal opening; B.wt = body weight, TSH = thyroid stimulating hormone; T4 = thyroxine

3.3.1 Growth, Body Weight

Measurements of growth and body weight were captured in four glyphosate multigenerational

reproduction studies in rats.

41
In the multigenerational reproduction studies conducted by Moxon (2000) and Dhinsa et al.

(2007), no differences in female body weight or in pup weight at birth for F1A and F2A litters

between controls and dosed groups were noted.

In the two-generation rat reproduction study of glyphosate conducted by Reyna (1990), reduced

body weights and soft stools were noted in adult animals in the 30,000 ppm (high-dose) group.

Body weights of pups in this high-dose group decreased during the last week of lactation. In the

10,000 ppm (mid-dose) group smaller decreases in body weight were noted and only on lactation

day 21. These body weight effects toward the end of lactation occurred when pups began to

supplement their milk intake with treated-food. The ingestion of available ground rat chow

begins after the eyes of the pups open (generally day 13) and as they begin to explore their caged

environment. Therefore, the body weight decreases observed in the mid- and high-dose pups on

lactation day 21 were considered to be primarily due to the reduced relative consumption of

treated diet by the high dose offspring due to palatability issues and is not considered an

endocrine-mediated effect.

In the three-generation rat reproduction study of Schroeder and Hogan (1981), no treatmentrelated effects were evident in adult mortality data, body weight or food consumption data

(growth and rest periods), or in in-life physical observation data throughout the study for the FO,

Fl and F2 generations.

3.3.2 Age at Vaginal Opening

No effect on the day of age when vaginal opening occurred in the Fl parent animals in the two

multigenerational rat reproduction studies (Moxon, 2000; Dhinsa et al., 2007).

3.3.3 Body Weight at Vaginal Opening

No difference in body weight at vaginal opening was noted in two multigenerational rat

reproduction study conduced on glyphosate.

Initial body weights were recorded for Fl females at selection on day 29postpartum in the

Moxon (2000) multigenerational rat reproduction study. The body weight of each rat was then

weighed at weekly intervals throughout the premating period. Mean body weights of Fl females

at day 29 postpartum were comparable to those in the control group. Measured and adjusted

mean body weights for weeks 2 and 3 of the Fl premating period align with the time of vaginal

opening and were comparable between controls and all dose groups.

In the most recent multigenerational study by Dhinsa et al. (2007), all selected Fi offspring were

observed for sexual development. For females the day of appearance of vaginal opening (separation

of the labia) was recorded. The bodyweight for each individual at the time of sexual maturation was

also recorded, noting no effects on body weight on the day of vaginal opening.

3.3.4 Uterus Weight and Histopathology

As explained in this section for each individual study, there were no treatment-related effects on

the uterus found in the four rat multigenerational reproduction and the chronic toxicity studies.

No difference was noted between control and dose groups for uterus with cervix weights

(absolute or % relative to body weight) in FO parents or Fl parents in the Moxon (2000)

multigenerational rat reproduction study. Similarly, no differences were noted between control

and dose groups for uterus with oviducts and cervix weights (absolute or % relative to body

42
weight) in FO parents or Fl parents in the Dhinsa et al. (2007) multigenerational rat reproduction

study.

Microscopic examination of uteri of the FO parents, Fib parents, F2b parents and F3b offspring

from the control and high dose groups were conducted in the three-generation rat reproduction

study on glyphosate (Schroder and Hogan, 1981) with no treatment-related effects noted.

No treatment related effects were noted following microscopic examination of control and high

dose uteri for FO parents, or F1 a parents in the two-generation rat reproduction study conducted

byReyna(1990).

Stout and Rucker (1990) conducted microscopic examination of uteri from the control and high

dose groups in this chronic toxicity study and noted no treatment related effects at the 12- and

24-month time points.

3.3.5 Estrus Cyclicity

No effect of glyphosate on mean cycle length or number of cycles in the 3 week period prior to

mating was seen in the multigenerational reproduction study (Moxon, 2000). Slightly higher

number of cycles for FO females dosed with 10,000 ppm and a slightly lower mean cycle length

in Fl females relative to the control groups in this study were considered marginal and

inconsistent across generations and not treatment related.

3.3.6 Ovaries Weight and Histopathology

There was no difference in absolute or relative ovary weights and no treatment related effects

noted following microscopic examination of the ovaries in the four multigenerational rat

reproduction studies conducted on glyphosate.

In the multigenerational studies conducted by Moxon (2000) and Dhinsa et al. (2007), no

differences were noted in the absolute or relative ovary weights between control and dose groups

of the FO or Fl parents.

Schroeder and Hogan (1981) noted no differences in absolute or relative ovary weights across

FO, Fib and F2b female adults and weanlings in F3b in this 3-generation rat reproduction study,

and microscopic examination of the ovaries noted no treatment-related effects.

In the reproduction study conducted by Reyna (1990), microscopic examination of ovaries of FO

and Fla parents noted no treatment-related effects between the control and high dose.

3.3.7 Thyroid Weight and Histopathology

No treatment-related thyroid weight effects were noted in the multigenerational study by Dhinsa et

al. (2007). In the FO female mid-dose of 5,000 ppm the mean absolute thyroid weight was

greater than the controls, while the FO male high and mid-doses of 15,000 ppm and 5,000 ppm

the mean absolute thyroid weight was lower than the controls. These findings were considered

contradictory and unrelated to treatment.

If histological lesions are not observed in the thyroid glands, then the chemical is considered to

be thyroid inactive (OPPTS 890.1110). No treatment related effects were noted following

microscopic examination of the thyroid in three in vivo studies conducted on glyphosate.

43
Schroeder and Hogan (1981) conducted microscopic examination of the thyroid and parathyroid

of the FO, Fib and F2b parents and F3b offspring in the three-generation rat reproduction study

and noted no treatment-related effects.

Microscopic examination of the thyroid after 90 days of dosing in the glyphosate subchronic

study (Stout and Johnson, 1987) and in the high dose group at 12 and 24 months in the

glyphosate chronic study in rats (Stout and Rucker, 1990) also noted no treatment related effects.

3.3.8 Thyroid Stimulation Hormone (TSH)

TSH is secreted by the anterior pituitary gland, which regulates the endocrine function of the

thyroid gland. As described in sections 3.3.1, 3.3.2, 3.3.3, and 3.3.13, no differences in growth

and development or the absolute and relative weights of the pituitary were noted in rat

reproduction studies conducted with glyphosate, nor were there any treatment-related effects

noted upon microscopic examination of the gland. If no developmental delays or histological

lesions are observed in the thyroid glands, then the chemical is considered to be thyroid inactive.

The rationale for is that the thyroid glands are under the influence of TSH and any chemical that

alters circulating thyroid hormone sufficiently to alter TSH secretion will result in

histopathological changes in the thyroid glands.

The effects correlation matrix of O'Connor et al. (2002) (Table 4) for the female pubertal assay

notes that an increase in TSH is supporting evidence for thyroid active compounds along with a

decrease in body weight at vaginal opening and ovary weight, and an increase in thyroid weight.

Multigenerational reproduction studies and/or subchronic and chronic studies on glyphosate have

shown no decrease in body weight at vaginal opening or in ovary weight (Dhinsa et al., 2007;

Moxon, 2000; Schroeder and Hogan, 1981; Reyna, 1990) and no increase in thyroid weight

(Schroeder and Hogan, 1981; Stout and Johnson, 1987; Stout and Rucker, 1990).

Therefore, measurements on the thyroid stimulation hormone would not provide additional

information on the potential endocrine activity of glyphosate. Furthermore, thyroid weight may

be considered one of the most sensitive parameters in response to xenobiotic disturbances

(Wilson et al., 1996), as thyroid hormones may equilibrate to normal serum levels due to

homeostasis in repeat dose studies.

3.3.9 Thyroxine (T<t)

Although alteration thyroid hormone levels are indicative of a thyroid-related effect, it is insufficient

to determine which mode or mechanism of action is related to the response. Any chemical that alters

circulating thyroid hormone sufficiently to alter TSH secretion will result in histopathological

changes in the thyroid glands. Just like for an alteration in thyroid stimulating hormone, the

effects correlation matrix of O'Connor et al. (2002) (Table 4) for the female pubertal assay notes

that a decrease in thyroxine levels is supporting evidence for thyroid active compounds, along

with a decrease in body weight at vaginal opening and ovary weight, and an increase in thyroid

weight. As discussed in Section 3.3.7, histopathology of the thyroid in several studies is

diagnostic of no effects of glyphosate on the thyroid structure and function. Furthermore, as

discussed in Section 3.3.8, several studies also show none of the other associated indicators for a

thyroid active compound. Data on thyroxine levels would, therefore, also provide no additional

information on the endocrine modulating activity of glyphosate beyond what is already provided

by existing histopathology data. As discussed above, thyroid weight may be considered one of

the most sensitive parameters in response to xenobiotic disturbances (Wilson et al., 1996) as

44
thyroid hormones may equilibrate to normal serum levels due to homeostasis in repeat dose

studies.

3.3.10 Kidney Weight and Histopathology

Three multigenerational rat reproduction studies already conducted on glyphosate showed no

difference in the absolute or relative weight of the kidneys of the control and treated test

subjects, as well as no microscopic effects.

The multigenerational reproduction study on rats conducted by Dhinsa et al. (2007) noted FO

females treated with 15,000 ppm displayed an increase in kidney weights, both absolute

(pO.OOl) and relative to terminal bodyweight (p<0.01) and FO females treated with 5,000 ppm

showed statistically significant increases in absolute kidney weights. These findings were not

noted in the FO and Fl males and Fl generation females and were, therefore, considered

unrelated to treatment.

The multigenerational reproduction study on rats conducted by Moxon (2000) showed no

difference in the absolute and relative kidney weights of FO and F1A adult females between the

control and dose groups, and no test related microscopic kidney findings were noted for the FO

and Fl females.

Schroeder and Hogan (1981) saw no differences in absolute or relative kidney weights of FO,

Fib and F2b adult females and the F3b offspring between the control and dose groups in the

three-generation rat reproduction study.

3.3.11 Liver Weight

There was no change in the absolute or relative weight of the liver of the FO and Fl females in

the rat reproduction study conducted on glyphosate by Moxon (2000).

No differences in absolute or relative liver weights of FO and Fib adult females, and of F3b

offspring were noted between controls and dose groups in the three-generation rat reproduction

study conducted on glyphosate by Schroeder and Hogan (1981). The absolute liver weights of

the F2b females were lower than controls in a non-dose-response manner. This decreased

relative liver weight was attributable to slightly higher absolute liver weights in the control

group.

Dhinsa et al. (2007), however, noted FO females treated with 15,000 ppm displayed statistically

significant increases in liver weights, both absolute and relative to terminal bodyweight

(pO.OOl). An increase in liver weight was also noted for Fl females treated with 15,000 ppm

(absolute: p<0.05, relative: p<0.01).

3.3.12 Adrenals Weight

The rat reproduction studies on glyphosate conducted by Moxon (2000) and Dhinsa et al. (2007)

found no difference in the absolute or relative weights of the adrenal gland and no test item

related microscopic findings in the gland of FO and Fl females.

Schroeder and Hogan (1981) found no difference in the absolute and relative weights of the

adrenal gland between control and dose groups of FO and F2b adult females and F3b offspring.

Slightly lower absolute and relative weights of the adrenal gland in F2b females at the mid-dose

level were considered incidental, as it was not seen at the high dose level or noted in the

offspring.

45
3.3.13 Pituitary Weight

No differences in the absolute and relative weights of the pituitary were noted in two rat

reproduction studies conducted on glyphosate, nor were there any treatment related effects noted

upon microscopic examination of the gland.

In the multigenerational studies conducted by Moxon (2000) and Dhinsa et al. (2007), there were

no effects of glyphosate on the absolute or relative weights of the pituitary of the FO and Fl

parents. Importantly, no treatment related microscopic findings were detected in the pituitary,

which synthesizes and secretes important endocrine hormones, such as TSH, FSH, and LH, of

the FO and Fl parents.

No differences in absolute and relative pituitary weights were noted in the FO, Fib and F2b adult

females and F3b offspring relative to controls in the three-generation rat reproduction study

conducted by Schroeder and Hogan (1981).

3.3.14 Clinical Chemistry

In the 90-day dietary study in rats (Stout and Johnson, 1987), some small changes in clinical

chemistry values that were not dose-related were noted. After careful consideration, these

changes in clinical chemistry values were considered to be either unrelated to treatment or

biologically insignificant.

Stout and Rucker (1990) measured clinical chemistry parameters at months 6, 12 (interim

sacrifice), 18, and 24 (study termination). There were various changes in hematology and serum

chemistry parameters noted; however, the changes were not consistently noted at multiple time

points, were within historical control ranges, small in magnitude, and/or did not occur in a doserelated manner. They were, therefore, considered to be either unrelated to treatment or

lexicologically insignificant.

3.3.15 Conclusion - Female Pubertal Assay

The lack of potential for glyphosate to interact with the female endocrine system has already

been comprehensively addressed in four multigenerational rat reproduction studies conducted on

glyphosate, with additional weight-of-evidence provided by subchronic and chronic toxicity

studies on glyphosate. The EDSP Tier 1 assay, "Pubertal Development and Thyroid Function in

Intact Juvenile / Peripubertal Female Rats," is capable of detecting estrogenic/anti-estrogenic

activity of chemicals, or agents that alter pubertal development, via changes in steroidogenesis or

hypothalamic-pituitary regulation of the ovary and thyroid homeostasis. The key endpoints

evaluated in this study are also evaluated in the multigenerational reproduction, subchronic

and/or chronic studies. A summary of the findings from these studies as they relate to the

endpoints evaluated in the Female Pubertal Assay is presented in the Table 5.

46
Table 5: Summary of Findings Relative to the Female Pubertal Assay for Glyphosate

End Point

Age at VO

Body Weight at VO

Uterus

Estrus Cyclicity

Ovaries

Thyroid

Kidney

Liver

Adrenal

Pituitary

Clinical Chemistry

Observations (

No change in age

No change in body weight

No change in absolute or relative weight

Slight marginal changes in number and

mean cycle length at the high dose level

(10,000 ppm); inconsistent across

generations; considered not treatment

related

No change in absolute or relative weight

n/a

No change in absolute or relative weight

No difference in absolute or relative weight

in FO and Fib adults and F3b offspring.

Slight decrease in relative weight in the

F2b females attributed to slightly higher

weight in the control group; non-doserelated response

No difference in absolute or relative weight

in one study, and only slightly lower in

mid-dose level of another; considered

incidental

No change in absolute or relative weight

Small changes in some values; not

considered to treatment related or

biologically significant

Microscopic Evaluation

n/a

n/a

no treatment related effects

no treatment related effects

no treatment related effects

no treatment related effects

n/a

no treatment related effects

no treatment related effects

The results from the multigenerational studies, which demonstrate no effects on pubertal

development and thyroid function in the intact juvenile/peripubertal female rats, offer compelling

evidence that glyphosate does not interact with the endocrine system. To require further testing

on animals via the Female Pubertal assay to evaluate the endocrine interaction potential of

glyphosate would provide no new evidence and be clearly redundant.

3,4 Pubertal Development and Thyroid Function in Intact Juvenile /

Peripubertal Male Rats (OPPTS 890,1500)

The male pubertal assay measures discrete endpoints to ascertain effects on pubertal development

and thyroid function in the intact juvenile/peripubertal male rat. These endpoints include

developmental milestones, organ weights and histopathology, hormone serum levels and clinical

chemistry parameters. All of these endpoints are either specifically addressed or captured in

downstream apical endpoints in more comprehensive in vivo studies, such as multigenerational,

developmental toxicity studies, subchronic toxicity and chronic toxicity studies. The

multigenerational reproduction study, which is a key Tier 2 EDSP assay, provides functional

equivalence to OPPTS 890.1500 and renders the male pubertal assay redundant (O'Connor et al.,

2002).

47
A more complete discussion of OSRI related to the male pubertal assay is presented by endpoint

in the following subsections. Many of the findings are discussed in relation to the effects matrix

in Table 6.

Table 6: Correlation of Effects in the Male Pubertal Assay (Adaptedfrom O'Connor et al., 2002, Table 5)

Endocrine

Activity

Strong estrogen

receptor agonist

Weak estrogen

receptor agonist

Androgen

receptor

antagonist

Testosterone

biosynthesis

inhibitor

5-areductase

biosynthesis

inhibitor

Aromatase

biosynthesis

inhibitor

Direct thyroid

antagonist

Indirect thyroid

antagonist

D2 agonist

D2 antagonist

Neuroendocrine

mechanism

Age

at

PPS

T

-/t

T

-/t

-

-

t

-

t

-

T

BWt

at

PPS

-/T

-

-

-

1

4

-

-

4

1

Testes

wt

1

-/I

-/I/t

-

-/I

-

4

1

-

-

-

Epidid.

Wt

1

-/i

1

1

1

-

1

-

-

-

-

P

wt

1

-/I

-/|

I

4

-/t

^

1

-

1

sv

wt

4

4

4

4

4

-/t

4

4

-

-

T

T

-/T

4

-/t

-

4

-

-

-

-

DH

T

-

-

4

-

4

-

-

-

Thy.

Wt

-

-

-

T

t

-

4

TSH

4

T

-

-

-

T4

4

4

4

-

PPS = preputialseparation; BWt = body weight; Epidid. = epodidymides; P = prostate; SV = seminal vesicles; T =

testosterone; DHT = dihydrotestosterone; Thy. = thyroid; TSH = thyroid stimulating hormone; T4 = thyroxine, D2 =

dopamine receptor.

3.4.1 Growth, Body Weight

There was no adverse effect on bodyweight change for males throughout the treatment period in the

most recent multigenerational study in rats (Dhinsa et al., 2007).

In the multigenerational rat reproduction study conducted by Moxon (2000), reductions in body

weight related to the test substance were limited to F1A males at day 29postpartum from the

10,000 ppm dietary dose group as a result of reduced food consumption due to reduced food

palatability. However, no effect on pup weight at birth for Fl A and F2A litters were noted.

In the two-generation rat reproduction study of glyphosate conducted by Reyna (1990), reduced

body weights and soft stools were noted in adults in the 30,000 ppm (high dose) dietary dose

48
group, and body weights of pups decreased during the last week of lactation. In the 10,000 ppm

(mid-dose) group, smaller decreases in body weight were noted and only on lactation day 21.

These body weight effects towards the end of lactation occurred when pups began to supplement

their milk intake with treated food. The ingestion of available ground rat chow begins after the

animals' eyes open (generally day 13) and as they begin to explore their caged environment.

Therefore, the body weight decreases observed in the mid- and high-dose pups on lactation day

21 were considered to be primarily due to the consumption of treated diet by the offspring and

not due to any endocrine effects.

In the three-generation rat reproduction study of Schroeder and Hogan (1981), no treatmentrelated effects were evident in adult mortality, body weight and food consumption data (growth

and rest periods), or in the in-life physical observation data throughout the study for the FO, Fl

and F2 generations.

3.4.2 Age at Preputial Separation

In Dhinsa et al. (2007), the mean Fl day of preputial separation of the prepuce from the glans

penis was statistically higher for the 15,000 ppm high dose than the controls, being recorded as

45.9 days and 43.0 days, respectively. This high dietary dose substantially exceeds the EPA

limit dose of 1,000 mg/kg/day for repeat dose studies for the lactating FO females (lactation days

1-4, 1740 mg/kg/day; lactation days 4-7, 2375 mg/kg/day; lactation days 7-14, 2749 mg/kg/day)

and Fl pups up to sexual maturity (pre-pairing week 1, 2127 mg/kd/day to pre-pairing week 5,

1278 mg/kg/day). In addition to substantially exceeding the limit dose in the maturing pups, no

concomitant endocrine-related responses associated with increased age at PPs, such as decreased

weight of the testes, epididymides, prostate or seminal vesicles were noted in the high dose Fl

males, supporting this finding was not endocrine mediated.

No effect on the day of mean age when preputial separation was noted in the Fl male parents in

the multigenerational rat reproduction studies (Moxon, 2000) at dietary doses up to 10,000 ppm,

even though the doses to these young Fl male rats exceeded the limit dose of 1,000 mg/kg/day

(up to 1,800 mg/kg/day) over the first six weeks of the pre-mating period.

3.4.3 Body Weight at Preputial Separation

In the multigenerational rat reproduction study by Moxon (2000), initial body weights were

recorded for Fl males at selection on day 29postpartum and then again at weekly intervals

through the pre-mating period. Mean body weights of Fl males at day 29 post partum were

lower in comparison to the control group. Adjusted body weights for weeks 2 and 3 of the Fl

pre-mating period noted a statistically significant (p < 0.05) lower body weight for the high dose

males. Therefore, it is assumed that high dose mean male body weights at the day of preputial

separation was lower (p < 0.05) than that of the control Fl males.

The non-endocrine mediated increase in age at preputial separation in Fl high dose males in Dhinsa

et al. (2007), resulted in statistically higher mean bodyweights at the time of sexual maturation for

the 15,000 ppm dose group due to these rats being, on average, 2.9 days older than the control group

at sexual maturing.

Referring back to the effects correlation matrix of O'Connor et al. (2002) (Table 6), a decrease in

body weight at preputial separation is an indicator of (i) aromatase inhibition, when accompanied

by prostate and seminal vesicle weight increases; (ii) direct acting thyroid antagonism, when

accompanied by testes and epididymides weight decreases and thyroid weight decrease; (iii) D2

49
receptor antagonism, when accompanied by thyroid weight decrease; or (iv) neuroendocrine

mechanism, when accompanied by prostate weight decrease. An increase body weight at

preputial separation is an indicator of a weak estrogen receptor agonist. As discussed further in

the following sections, no change in weight of the prostate, seminal vesicles, testes, epidiymides

and thyroid between the control and treated groups was seen in the three rat reproduction studies

on glyphosate (Dhinsa et al., 2007; Moxon, 2000; Schroeder and Hogan, 1981; Reyna, 1990);

therefore, the weight of evidence strongly suggests that the decreased mean body weight at

preputial separation of the Fl males at 10,000 ppm (Moxon, 2000) and the increased mean body

weight at preputial separation of the Fl males at 15,000 ppm (Dhinsa et al., 2007) were not

related to an endocrine-mediated pathway.

3.4.4 Testes Weight and Histopathology

No difference was noted in the absolute or relative weight of the testes of the FO or Fl parents

from the control and dose groups in the multigenerational rat reproduction studies conducted by

Moxon (2000) and Dhinsa et al., (2007), or of the FO, Fib and F2b adult males and F3b

weanlings in the three-generation rate reproduction study conducted by Schroeder and Hogan

(1981). Microscopic examination noted no treatment-related effects on the testes.

In the two-generation rat reproduction study on glyphosate conducted by Reyna (1990), a

statistically significant decrease in terminal body weights were noted for both sexes at the

highest exposure level tested (30,000 ppm), which exceeded the maximum tolerated dose. The

only statistically significant organ weight change, however, was a slight increase in mean testesto-body-weight ratio (not mean absolute weight) of the Fla adult in the high dose group and this

finding was attributed to a lower mean terminal body weight of the Fla male at this highest dose.

No differences were noted in the mean absolute weights of the testes or the relative weights of

the FO males at all doses or in the Fla males at the low- and mid-doses. Microscopic

examination of the testes for FO and Fla parents from the control and high-dose groups noted no

treatment-related effects. Mating, pregnancy and fertility indices were not affected in either the

FO or Fl animals.

3.4.5 Epididymides Weight and Histopathology

No difference in the absolute or relative weights of the epididymides weights of the FO or Fl

parents from the control and treated groups were noted in the rat reproduction studies conducted

by Moxon (2000) and Dhinsa et al., (2007). Microscopic examination of the epididymides of the

FO and Fl parents from the control and high-dose group noted no treatment-related effects.

Mating, pregnancy and fertility indices were not affected in either the FO or Fla animals.

3.4.6 Prostate Weight

No difference in the absolute or relative weight of the prostate gland of the FO or Fl parents was

noted between the control and high-dose groups in the multigenerational rat reproduction studies

conducted by Moxon (2000) and Dhinsa et al., (2007).

In the three-generation rat reproduction study conducted by Schroeder and Hogan (1981),

microscopic examination of the prostate gland of the FO, Fib and F2b parents and the F3b

offspring from the control and high-dose groups noted no treatment-related effects.

50
3.4.7 Seminal Vesicles Weight

No difference in the absolute or relative weight of the seminal vesicles of the FO or Fl parents

was noted between the control and treated groups in the multigenerational rat reproduction

studies conducted by Moxon (2000) and Dhinsa et al., (2007).

Microscopic examination of the seminal vesicles of the FO and Fl a parents from the control and

high dose groups of the two-generation rat reproduction study conducted by Reyna (1990) noted

no treatment related effects.

3.4.8 Testosterone

The effects correlation matrix for the male pubertal assay proposed by O'Connor (2002) (Table

6) notes that for a number of endocrine effects, any differences in testosterone levels between

controls and test groups is also reflected in changes in age at preputial separation and weights of

testes, epididymides, prostate and seminal vesicles. However, as discussed previously in this

Section, data from the multigenerational rat reproduction studies conducted by Moxon (2000)

and Dhinsa et al., (2007) show that changes in these endpoints between the controls and treated

groups were not seen. Therefore, functionally equivalent information for this endpoint in the

male pubertal assay is provided with the current comprehensive in vivo data set on glyphosate.

3.4.9 Thyroid weight and Histopathology

Although some thyroid weight effects were noted in the multigenerational study by Dhinsa et al.

(2007), the findings were considered contradictory and unrelated to treatment. For the FO

females in the mid-dose treatment of 5,000 ppm, the mean absolute thyroid weight was greater

than the controls, while for the FO male in the high- and mid-doses of 15,000 ppm and 5,000

ppm, the mean absolute thyroid weight was lower than the controls. As previously stated, these

findings were considered contradictory and unrelated to treatment.

Further evidence as to glyphosate's lack of effects on the thyroid is found in the findings of

microscopic examination of the gland. If histological lesions are not observed in the thyroid

gland, then the chemical is considered to be thyroid inactive (OPPTS 890.1110), and

microscopic examination of the thyroid in the three-generation rat reproduction study and the

subchronic and chronic toxicity studies on glyphosate noted no treatment-related effects.

In the three-generation rat reproduction study conducted by Schroeder and Hogan (1981),

microscopic examination of the thyroid and parathyroid of the FO, Fib and F2b parents and F3b

offspring from control and high-dose groups noted no treatment-related effects.

The same conclusion of no treatment-related effects was reached following microscopic

examination of the thyroid of the control and high-dose groups following 90 days of oral dosing

in the subchronic toxicity study conducted by Stout and Johnson (1987) and at 12 and 24 months

in the chronic toxicity study on glyphosate conducted by Stout and Rucker (1990).

3.4.10 Thyroid Simulation Hormone (TSH)

The effects correlation matrix of the male pubertal assay (Table 6) indicates that for a number of

endocrine effects, differences in TSH levels between controls and test groups may also be

reflected in changes in age and weight at preputial separation and weights of testes,

epididymides, prostate, seminal vesicles and thyroid. However, as discussed previously in this

Section, data from the multigenerational rat reproduction studies conducted by Moxon (2000)

and Dhinsa et al. (2007) show that changes in these sex organ weights between the controls and

51
treated groups were not seen. Therefore, functionally equivalent information for this endpoint in

the male pubertal assay is provided with the current comprehensive in vivo data set on

glyphosate.

3.4.11 Thyroxine (T4)

The effects correlation matrix of the male pubertal assay (Table 6) indicates that for a number of

endocrine effects, a decrease in thyroxine levels of treated groups compared to the controls may

also be reflected in changes in age and weight at preputial separation and weights of testes

epididymides, prostate, seminal vesicles and thyroid function. However, as discussed previously

in this Section, data from the multigenerational rat reproduction studies conducted by Moxon

(2000) and Dhinsa et al. (2007) show that changes in these endpoints between the controls and

treated groups were not seen. Therefore, functionally equivalent information for this endpoint in

the male pubertal assay is provided with the current comprehensive in vivo data set on

glyphosate.

3.4.12 Kidney Weight and Histopathology

No effects on male kidney weights were noted in the multigenerational rat study by Dhinsa et al.

(2007). Also, in the three-generation rat reproduction study conducted by Schroeder and Hogan

(1981) no differences in absolute or relative weight of the kidneys of the FO, Fib and F2b adult

males and F3b offspring of the control and treated groups were noted.

In the multigenerational rat reproduction study conducted by Moxon (2000), a statistically

significant (p < 0.01) increase in the mean kidney weight adjusted for body weight of the FO

male over the controls was noted at the high dose only and not in the Fl males.

3.4.13 Liver Weight

No effects on male liver weights were noted in the multigenerational rat study by Dhinsa et al.

(2007). Also in the three-generation rat reproduction study conducted by Schroeder and Hogan

(1981) no differences in the absolute and relative liver weights of the FO, Fib and F2b adult

males and F3b offspring in the control and treated groups were noted.

A statistically significant (p < 0.01) increase in the mean liver weight adjusted for body weight

of the FO male over controls at the high dose level was seen in the multigenerational rat

reproduction study conducted by Moxon (2000), and statistically significant (p < 0.05) decrease

in the absolute liver weight of the Fl male at the high dose level relative to the controls. It was

noted in the study report that the high-dose males exhibited test substance-related reductions in

body weight and food consumption. These inconsistent liver weight findings were considered

incidental and unrelated to endocrine effects.

3.4.14 Adrenals Weight

No differences in the absolute or relative weights of the adrenal glands were noted in three rat

reproduction studies on glyphosate.

In the multigenerational rat reproduction studies conducted by Moxon (2000) and Dhinsa et al.

(2007), there were no differences in the absolute and relative weights of the adrenal gland in the FO

and Fl males. In the three-generation rat reproduction study conducted by Schroeder and Hogan

(1981), no difference in the absolute and relative weight of adrenal gland of the FO, Fla and F2b

adult males and F3b offspring in the control and treated groups were noted.

52
No treatment-related microscopic findings were detected in the adrenal glands of the FO and Fl

males in the Moxon (2000) rat reproduction study. Dhinsa et al. (2007), however, noted that

while there were no treatment-related histopathological changes in the adrenal glands of FO

generation animals, treatment-related changes in the Fl generation male adrenal glands were

noted as lower incidences and severities of cortical vacuolation of the adrenal glands when

compared to controls, but these findings were not considered to be an adverse finding.

3.4.15 Pituitary Weight

No differences in the absolute or relative weights of the pituitary glands and no treatment-related

microscopic findings were noted in three rat reproduction studies on glyphosate.

In the multigenerational rat reproduction studies conducted by Moxon (2000) and Dhinsa et al.

(2007), there were no differences in the absolute or relative weights of the pituitary gland of the

FO and Fl parents from the control and treated groups. Also for the FO and Fl parents, no

treatment-related microscopic findings were detected in the pituitary, which synthesizes and

secretes important endocrine hormones, such as TSH, FSH, and LH.

In the three-generation rat reproduction study conducted by Schroeder and Hogan (1981), no

differences in the absolute and relative weight of the pituitary glands and no treatment-related

microscopic findings of the pituitary glands in the FO, Fib and F2b adult males and F3b

offspring were noted.

3.4.16 Clinical Chemistry

In a 90-day dietary study conducted by Stout and Johnson (1987), some changes in clinical

chemistry values were noted; however, these changes were small and/or not dose-related, and,

therefore, considered to be unrelated to treatment or biologically insignificant.

In the chronic toxicity study conducted by Stout and Rucker (1990), clinical chemistry

parameters were measured at months 6, 12 (interim sacrifice), 18, and 24 (study termination).

There were various changes in hematology and serum chemistry parameters considered to be

either unrelated to treatment or lexicologically insignificant.

3.4.17 Conclusion - Male Pubertal Assay

The lack of potential for glyphosate to interact with the male endocrine system has already been

comprehensively addressed in four multigenerational rat reproduction studies conducted on

glyphosate, with additional weight-of-evidence provided by subchronic and chronic studies on

glyphosate. The EDSP Tier 1 assay, "Pubertal Development and Thyroid Function in Intact

Juvenile / Peripubertal Male Rats," is capable of detecting antithyroid, androgenic, or

antiandrogenic (androgen receptor or steroid-enzyme-mediated) activity of chemicals, or agents

that alter pubertal development via changes in gonadotropins, prolactin, or hypothalamic

function. All of the endpoints evaluated in this EDSP assay are also evaluated in the

multigenerational reproduction, subchronic and/or chronic studies, or can be evaluated based on

other information from these studies. A summary of the findings from these studies as they

relate to the endpoints evaluated in the Male Pubertal assay is presented in Table 7.

53
Table 7: Summary of Findings Relative to the Male Pubertal Assay for Glyphosate

End Point

Growth, Body Weight

Age at Preputial

Separation

Body Weight at Preputial

Separation

Testes Weight

Epididymides Weight

Prostate Weight

Seminal Vesicles Weight

Testosterone

Thyroid Weight

Thyroid Stimulation

Hormone (TSH)

Thyroxine (T4)

Kidney Weight

Liver Weight

Adrenal Weight

Pituitary Weight

Clinical Chemistry

Observations

Reduction in body weight limited to high dose

group as a result of reduced food consumption

due to reduced palatability, and not due to

endocrine effects

No glyphosate related change in age at preputial

separation for doses at or below the limit dose of

l,000mg/kg/day

Mean body weight decrease at 10,000 ppm and

increase at 15,000 ppm. Both doses exceeded

the limit dose of 1,000 mg/kg/day during sexual

maturation; however, no other related endocrine

effects were noted in sex organ weight

differences, therefore the weight of the evidence

is that body weight changes at sexual maturation

were not related to endocrine effects

No change in absolute or relative weight in one

rat reproduction study, and only a slight increase

in relative weight (not absolute weight) of the

30,000 ppm test group (high dose) due to a

lower mean terminal body weight

No change in absolute or relative weight

No change in absolute or relative weight

No change in absolute or relative weight

No other related endocrine effects observed in

the multigenerational reproduction studies

n/a

No other related endocrine effects observed in

the multigenerational reproduction studies

No other related endocrine effects observed in

the multigenerational reproduction studies

Increase in relative weight only in the FO male at

the high dose; no change in the Fl male or in

any adult males or offspring in the threegeneration reproduction study

Inconsistent findings (decrease in absolute and

increase in relative weights) in one reproduction

study were considered incidental and unrelated

to endocrine effects; no changes in the threegeneration reproduction study in males

No changes in absolute or relative weight

No changes in absolute or relative weight

Small changes in some values; not considered to

be treatment related or biologically significant

Microscopic Evaluation

n/a

n/a

No treatment related effects

No treatment related effects

No treatment related effects

No treatment related effects

n/a

No treatment related effects

n/a

n/a

n/a

n/a

No treatment related effects

No treatment related effects

n/a

The results from the multigenerational studies, which demonstrate no effects on pubertal

development and thyroid function in the intact juvenile/peripubertal male rats, offer compelling

evidence that glyphosate does not interact with the endocrine system. To require further testing

54
on animals via the Male Pubertal assay to evaluate the endocrine interaction potential of

glyphosate would provide no new evidence and clearly be redundant.

3,5 Summary of In Vivo OSRI for Mammalian Species

The multigenerational rat reproduction studies conducted by Moxon (2000) and Dhinsa et al.

(2007) are currently proposed as Tier 2 EDSP guideline studies. These studies clearly negate the

need for much of the Tier 1 screening battery evaluation. With the preponderance of other

supporting data in multiple repeat dose studies discussed throughout this document, the Tier 1

screening battery adds little if any information to the currently available scientific body of

knowledge for glyphosate with respect to its potential to interact with the endocrine system.

In addition to the "other scientifically relevant information" presented here, the FAO/WHO 2004

review of glyphosate (FAO/WHO, 2005) evaluated four, 4-week studies in rats, mice and dogs,

two subchronic rat studies, three subchronic mouse studies, one 90-day dog study, three 1-year

dog studies, a 1-year chronic toxicity study in rats, two 2-year chronic toxicity studies in rats,

one 2-year mouse study, an additional 2-generation rat reproduction study and two additional

developmental toxicity studies of glyphosate in rats and rabbits. Many of these studies were also

considered in the 2009 ECETOC document "Guidance on Identifying Endocrine Disrupting

Effects, Technical Report 106," in which glyphosate is highlighted as an example of a nonendocrine disrupting compound based on the extensive toxicology data available.

4.0 Non-Mammalian In Vivo Assays

4.1 Amphibian Metamorphosis Assay (AMA, OPPTS 890,1100)

The amphibian metamorphosis screen was included in the Tier 1 screening battery as a

generalized vertebrate model to detect thyroid system-disrupting compounds. Through the

hypothalamus-pituitary-thyroid gland axis, thyroid hormone regulates many processes in

organisms, including development, somatic growth, metabolism, and reproduction.

Metamorphosis in anuran amphibians is a well studied model for thyroid hormone function and

is applicable and functionally equivalent to information generated in standard regulatory studies

with other vertebrate classes. Extensive scientifically relevant information on thyroid structure

and function is collected in standard subchronic, chronic mammalian studies and reproduction

studies. This information is functionally equivalent to data collected in the amphibian

metamorphosis assay. Additionally, data on growth and development offish and birds in the fish

full life cycle study and avian reproduction study provide additional relevant scientific

information to address the requirement for performing the amphibian metamorphosis assay.

The amphibian thyroid system is regulated and responds in a similar manner to

perturbation as that of other tetrapods, including mammals (Fort et al., 2007). Therefore,

data from mammalian assays can be used to make a robust assessment of potential impacts on

thyroid structure and function. The common endpoint in all three Tier 1 assays that evaluate

potential effects on the thyroid is thyroid gland histology, which screens for direct toxicity at the

gland itself. Developmental retardation, in the absence of overt toxicity, is a strong indicator of

anti-thyroid activity, but amphibian developmental stage analysis by itself is not considered a

diagnostic indicator of anti-thyroidal activity (OPPTS 890.1100). If no developmental delays or

treatment related histological lesions are observed in the thyroid glands, then the chemical is

considered to be thyroid inactive (OPPTS 890.1100). The rationale is that the thyroid glands are

55
under the influence of TSH and any chemical that alters circulating thyroid hormone sufficiently

to alter TSH secretion will result in histopathological changes in the thyroid glands (OPPTS

890.1100). However, it should be emphasized that the ability of a chemical to cause a reduction

in serum T4 by any mechanism would deprive the fetus in a reproduction study of essential

hormone. Thus, any observed reduction in serum T4 would be detected in a mammalian

reproduction study as an impact on developmental endpoints and can be evaluated in a weightof-evidence interpretation using all of the data from the repeat dose studies that have evaluated

thyroid histopathology and/or growth and development.

While a few different species have been evaluated in this screen, the South African clawed frog

(Xenopus laevis) was chosen as the test organism. Metamorphosis can be induced or accelerated

by agonists of the thyroid system, whereas anti-thyroidal agents can delay metamorphosis

(Degitz et al. 2005, Opitz et al. 2005, 2006; Tietge et al. 2005). The protocol currently in use

includes a 21-day treatment of tadpoles, with evaluations occurring on Day 7 and Day 21. The

test is initiated at developmental stage 51, which can be reached approximately 14 days postfertilization. Four concentrations of the test substance and a dilution water control and four

replicate tanks per concentration are employed in a flow-through system. At test initiation, a

total of 20 stage 51 Xenopus laevis tadpoles are placed in each of the four replicate tanks per

treatment group. Environmental conditions of 22 ± 1°C, pH of between 6.5-8.0, and a 12:12

lightdark photoperiod are employed. Five tadpoles from each treatment tank are randomly

removed at day 7 for evaluation of wet weight, whole body length, hind limb length, and snoutvent length. On Day 21, the remaining tadpoles are evaluated for these same endpoints.

Histological evaluation of thyroid tissue in a subsample of five tadpoles from each replicate tank

is also performed in this screen in order to link any observed changes in the rate of

metamorphosis to morphological changes in the primary thyroid synthesis organ.

No treatment-related thyroid weight effects were noted in the multigenerational study by Dhinsa et

al. (2007). In the FO female from the mid-dose treatment group of 5,000 ppm, the mean absolute

thyroid weight was greater than the controls, while the mean absolute thyroid weight of the FO

males from the high- and mid-dose groups of 15,000 ppm and 5,000 ppm was lower than the

controls. These findings were considered contradictory and unrelated to treatment.

Histopathology in a three generation reproduction study (Schroeder and Hogan, 1981), a 90-day

subchronic study (Stout and Johnson, 1987) and a chronic rat study (Stout and Rucker, 1990) did

not identify any glyphosate treatment-related effects to the thyroid glands.

Glyphosate showed no evidence of effects on growth and development through an endocrine

mechanism in four multigenerational studies in rats (Schroeder and Hogan, 1981; Reyna, 1990;

Moxon, 2000; Dhinsa et al., 2007) and no evidence of being a selective developmental toxicant

in developmental toxicity studies in rats (Rodwell et al., 1980a) and rabbits (Rodwell et al.,

1980b).

No effect on growth of the FO and Fl minnows was observed in the fish full-life cycle study

(Figures 4 and 5; EG&G Bionomics, 1975). This data provides consistent and strong evidence of

a no effect finding for glyphosate on thyroid structure and function. Additionally, in four avian

reproduction studies, no effects on growth were observed at dietary concentrations of greater

than 1000 ppm glyphosate (see summary in section 4.2.4).

56
Total Length

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glyphosate for 30 and 60 days. Means are shown with the standard error of the mean (EG&G

Bionomics, 1975).

Total Length

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Figure 5: Growth of Fl fathead minnows (Pimephalespromelas) continuously exposed to glyphosate

for 30 days. Means are shown with the standard error of the mean (EG&G Bionomics, 1975).

4.1.1 Conclusion - Amphibian Metamorphosis Assay (AMA)

The amphibian metamorphosis assay has been included in the Tier 1 battery as a generalized

model for impacts on vertebrate thyroid function because the amphibian thyroid system is

regulated and responds in a similar manner to perturbation as that of other tetrapods, including

mammals (Fort et al., 2007). Therefore, combined results from existing glyphosate studies

provide sensitive and diagnostic information on thyroid structure, function, growth and

development that is functionally equivalent to that generated in the amphibian metamorphosis

assay. Thyroid histopathology has been evaluated in a three-generation reproduction study

(Schroeder and Hogan, 1981), a 90-day subchronic study (Stout and Johnson, 1987) and a

chronic rat study (Stout and Rucker, 1990), which did not identify any glyphosate treatmentrelated effects to the thyroid glands.

57
Confirmatory information was developed for an aquatic vertebrate in the fish full life-cycle study

that shows no impact on growth and development, which are sensitive measurement endpoints

for impacts on thyroid structure and function. Additionally, information of no measureable

effects on thyroid function can be evaluated with data from avian reproduction studies. No

effects on growth were observed in four avian reproduction studies that tested glyphosate dietary

concentrations greater than 1000 ppm.

Table 8: Summary of Findings Relative to the Amphibian Metamorphosis Assay (AMA)for Glyphosate

End Points in AMA

Thyroid gland histopathology

Growth and development

Functionally Equivalent Information from Existing Studies

• Three generation rat reproduction study, a 90-day rat study and a chronic

rat study did not identify any treatment-related microscopic findings of the

thyroid glands.

• Growth and development in rat multigenration studies including

developmental landmarks demonstrated no indication of treatment related

effects.

• No treatment related effects on FO and Fl growth and development in fish

full life cycle study and four avian reproduction studies.

Given all of this available information on glyphosate, the amphibian metamorphosis screening

assay does not offer any new relevant information to the toxicology data set for Tier 1 screening

purposes.

4,2 Fish Short-term Reproduction Assay (OPPTS 890,1350)

Information from several guideline studies for glyphosate provides data of the same nature and

quality as data developed by the short-term fish reproduction study. Several of these studies have

been reviewed by EPA in support of the continued registration of glyphosate and provide OSRI

that fully addresses the requirement for the fish short-term reproduction study. Additionally, the

potential for glyphosate to induce vitellogenin was evaluated in peer-reviewed publications. The

following studies provide scientifically relevant information equivalent to data generated in the

fish short-term reproduction study:

1. Fish full life-cycle toxicity with the fathead minnow (Pimephales promelas) (MRID

00108171).

2. Assessing rainbow trout ER activation and vitellogenin production (Petit, 1997; Xie et al.

2005).

3. Fish bioconcentration study (MRID 41228301; MRID 41228302)

4. Avian reproduction studies (MRID 00108207; MRID 00111953)

5. Rat multigenerational reproduction studies (MRID 00081674; MRID 00105995; MRID

41621501; Moxon 2000; Dhinsa et al., 2007)

The fish short-term reproduction screen (OPPTS 890.1350) is initiated with mature male and

female fish with over a 21-day exposure period. The screen examines abnormalities associated

with survival, reproductive behavior, secondary sex characteristics, gonadal histopathology, and

fecundity (i.e., number of spawns, number of eggs per spawn, fertility, and development of

offspring) offish exposed to test chemicals (See Table 1 for endpoints that assess endocrine

activity and a comparison with the 2-generation rat reproduction test). Data that is functionally

equivalent to the data generated in the fish short-term reproduction screen with the fathead

58
minnow (OPPTS 890.1350) is provided by the fish full life-cycle study (OPPTS 850.1500). The

fish full life-cycle study is the highest Tier offish ecotoxicology testing and typically performed

with the fathead minnow (Pimphalespromelas] as a freshwater test species.

In the fish full life-cycle study, the entire life-cycle is exposed; therefore, de facto the most

sensitive life-stage to any toxicant (including endocrine active compounds) is encompassed by

the test design. However, endpoints that are potentially influenced by endocrine activity, such as

gonadal histopathology, are not typically measured in the full life-cycle. A standard fish full

life-cycle study provides a comparable investigation to the test proposed in Tier 2 testing for fish

in the EDSTP, even though a comprehensive assessment of the impacts of glyphosate on

reproductive organs (organ weight and histopathology) has already been evaluated using

information from the rat multigeneration studies and the results of these studies demonstrate no

treatment-related impact on all measured reproductive and developmental endpoints.

In the first or parental generation (Fo) the following endpoints are evaluated: embryo hatching

success, embryo time-to-hatch, survival at various time points after hatch, length and wet weight

as an indicator of growth at various time points after hatch, and reproductive success (fecundity)

as measured by number of eggs per female per day, number of spawns and number of eggs per

spawn. In the second generation (Fi) the endpoints include embryo hatching success, embryo

time-to-hatch, survival, and development as measured by length and wet weight at specified time

points.

In the glyphosate study, fathead minnows (Fo + FI) were continuously exposed to mean

measured glyphosate concentrations of 0.7, 2.8, 7.0, 13.0 and 25.7 mg/L for 255 days. The

current guidance in OPPTS 890.1350 states "to test the highest concentration that does not result

in increased mortality or signs of overt morbidity compared to controls; or 1/3 the derived 96-hr

LC50 will serve as the highest exposure concentration in the 21-day test." In a static 24-hour

acute study run in advance of the full life-cycle study, using ~1.5 g fathead minnows, the 24-

hour LCso of glyphosate was determined to be 84.4 mg/L and mortality was shown to result from

the low pH at the highest test concentrations. Therefore, the highest tested concentration in the

full life-cycle study was approximately 1/3 the acute LCso value as recommended by OPPTS

890.1350. This conclusion is generally supported by the range of acute toxicity values for other

fish species reported in the Glyphosate RED (1993). Additionally, glyphosate has been shown to

not bioconcentrate in fish, with a bioconcentration factor in whole fish of less than IX (MRID

41228301; MRID 41228302).

The full life-cycle study with fathead minnows exposed to glyphosate began with two groups of

thirty fertilized eggs incubated in each test aquarium at 25°C. Post-hatch, fish were randomly

divided into two groups of twenty and distributed to growth chambers until sexual maturity was

reached. When secondary sexual characteristics were well developed (circa day 134) the number

offish in each tank was reduced initially to 4 males and 4 females, and subsequently to 2 males

and 4 females. This ratio of 2 males to 4 females is required by the OPPTS 890.1350 guideline.

When spawning began, eggs were removed from the underside of spawning tiles daily and eggs

of each spawn were counted. Fifty eggs from each of the first ten spawning in each tank were

then oscillated in their respective test waters by means of the egg cup and rocker arm apparatus

until hatching was completed (3-5 days at 25°C). After 30 days exposure, fry groups were

terminated and total lengths determined by the photographic method. Total wet weight and

percent survival were also determined at this time for each fry group. Total length, wet weight,

59
sex and gonadal conditions were determined for each adult fish at the termination of the

experiment.

Continuous exposure to concentrations of glyphosate as high as 25.7 mg/L had no statistically

significant effects on any of the parameters studied during 254 days of continuous exposure.

Hatchability of eggs was excellent (>94%) in all concentrations of glyphosate and controls.

Percent survival and total length of fathead minnows after 30 and 60 days exposure to

concentrations of glyphosate as high as 25.7 mg/L did not differ significantly from the control.

Survival of the fifteen original fathead minnows placed in each spawning chamber was excellent

in all concentrations of glyphosate and controls through day 134, when fish were thinned to a

ratio of 4 males and 4 females. At termination, total length and wet weight of the female fathead

minnows exposed to all concentrations of glyphosate were similar to controls.

Spawning began in many tanks around day 112 of exposure and had occurred in all tanks by day

148. The number of spawnings, eggs per female and eggs per spawn did not differ significantly

between controls and fish exposed to concentrations of glyphosate as high as 25.7 mg/L. One

spawn of 33 eggs was recovered from the B replicate of 25.7 mg/L before the accidental death of

fish due to a diluter malfunction early in the spawning period. Prior to that time, all fish

appeared healthy and had reached sexual maturity. Percentage of live fry hatching in

concentrations of glyphosate as high as 25.7 mg/L was similar to that which was observed in the

controls. Although gonadal histopathology was not evaluated in the fish full life cycle study,

absence of effects on the number of spawns, eggs per female and eggs per spawn provides strong

evidence of no impact on gonadal structure and function.

Survival and total length and wet weight of second generation (Fi) fathead minnows was similar

to controls for fish exposed 30 days to concentrations of glyphosate as high as 25.7 mg/L. Based

on the absence of any significant effect to fathead minnows chronically exposed to glyphosate,

the no observed effect concentration (NOEC) for this species was determined to be >25.7 mg/L,

the highest concentration tested.

Figure 6: Glyphosate does not effect spawning and egg hatchability of fathead minnows (Pitnephales

promelas) continuously exposed to glyphosate.

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4.2.1 Results Summary - Fish Full Lifecycle Study

Fathead minnows (Fo + FI) continuously exposed to high concentration of glyphosate for 255

days demonstrated no treatment-related effects on the survival, growth and egg production of

first generation fish or on hatchability, survival and growth of second-generation eggs and fry.

Observing no effects on any of these endpoints over this concentration range, provides strong

evidence that glyphosate does not have estrogenic, anti-estrogenic, androgenic or anti-androgenic

activity. Furthermore, as discussed in detail in section 4.3.2, the structure activity relationship of

glyphosate predicts no estrogenic activity nor is glyphosate able to transactivate an ER

responsive report gene. Also, an absence of any detectable effects on growth, development and

reproduction demonstrates a lack of an effect on steroidogenesis, gonadal function and the

hypothalamic-pituitary-gonadal (HPG) axis function. Glyphosate has been shown not to induce

vitellogenin in a separate study, as discussed in section 4.2.3.

Additional relevant information that supports the findings of the fish full-life cycle study is

reviewed in the following sections. This includes information from in vitro and in vivo studies

examining the potential for estrogenicity and a review of the avian one-generation studies.

4.2.2 Structure Activity Relationship Indicates Lack of Estrogenicity

As previously discussed in the Introduction section of this document, glyphosate does not have

the structure of a molecule predicted to have estrogenic or androgenic activity. Although SAR

models are not developed to the same degree for androgens, many of the same principals that

apply to evaluating glyphosate for estrogenic potential apply to an assessment of androgenic

potential. Based on its chemical structure, glyphosate is predicted to not have androgenic

activity (Devillers et al., 2009; Panaye et al., 2008). The conclusion that glyphosate does not

have estrogenic and androgenic potential has been empirically confirmed in a number of in vitro

assays and in vivo tests. A full review of the in vitro and in vivo data is provided in the

presentation of the OSRI for each of the assays that evaluate the potential for estrogenic activity.

4.2.3 Glyphosate does not induce production of vitellogenin

Vitellogenin is an egg yolk precursor protein expressed in females offish, amphibians, reptiles

(including birds) and insects. In fish, liver vitellogenin production is stimulated by estradiol and

vitellogenin production in male fish has a history of use to assess exposure to estrogenic

compounds (Sumpter and Jobling, 1995). Production of vitellogenin in adult females is

61
mediated by estrogenic activity; thus vitellogenin expression is thought to serve as a biomarker

for chemicals likely to alter estrogenic activity in fish and other animals.

The estrogenic potency of glyphosate was evaluated in vivo with rainbow trout by measuring

vitellogenin levels post-glyphosate exposure. As previously discussed in section 2.1.2, seven

days of exposure to 0.11 mg/L glyphosate had no effect on vitellogenin levels, indicating that

glyphosate is not estrogenic (Xie et al, 2005). Consistent with this result, Petit et al. (1997)

demonstrated that glyphosate does not bind to and transactivate a rainbow trout ER receptor

(study summarized in section 2.1.2).

4.2.4 Avian one-generation reproduction assays

Additional supporting information to evaluate the potential for endocrine activity of glyphosate,

particularly the fish short term reproduction study, is supported by four separate one-generation

avian reproduction studies that followed EPA OPPTS 805.2300, EPA Sub-division E

guideline? 1-4 and OECD 206 guidelines (see Table 1). In these studies, birds were fed three diet

concentrations containing glyphosate throughout a chronic exposure period. Birds are induced by

photoperiod manipulation to lay eggs and the eggs are collected over a ten-week period. Eggs

are incubated and hatched, and the young maintained for 14 days. Mortality of adults, egg

production, cracked eggs, egg shell thickness, viability, hatchability and effects on young birds

are observed during the study. The endpoints that are assessed and evaluated in the avian

reproduction study and the fish full life-cycle study are summarized in the table below. From this

comparison it is clear that the avian reproduction study provides significant and relevant

information to evaluate the potential for reproductive effects in birds. Reading across to the

mammalian multigenration study provides an additional list of endpoints capable of detecting

endocrine activity that are evaluated.

The original reproduction studies with northern bobwhite and mallard duck included a test

concentration at the highest nominal level to test in OECD 206 of 1,000 mg glyphosate/kg feed.

Two newer reproduction studies with northern bobwhite and mallard duck were conducted with

the highest tested dietary concentration of 2,250 mg glyphosate/kg feed (ECETOC, 2009). In all

four of these studies, no effects were observed on measured endpoints that included the numbers

of eggs laid, eggs damaged/laid, eggs set, viable embryos, live 3 week embryos, hatchlings, 14-

day survivors, eggs laid/female, eggs laid/female/day, 14-day survivors/female and egg shell

thickness. The results from these avian reproduction studies are consistent with the results from

the fish full-life cycle study and further support the conclusion that no evidence of potential

endocrine activity exists for glyphosate even at extremely high concentrations and in long-term

continuous feeding studies.

4.2.5 Conclusion - Fish short-term reproduction assay

The methodology and information generated from a fish full life-cycle study provides a

comprehensive evaluation of potential endocrine activity and provides information functionally

equivalent to information obtained from the short-term fish reproduction assay. A summary of

the relevant existing functionally equivalent data is summarized in the following table.

62
Table 9: Summary of Findings Relative to the Fish Short-Term Reproduction Assay for Glyphosate

End Points in Fish Short-Term

Reproduction Assay with the

Fathead Minnows

Functionally Equivalent Information from Existing Studies

Reproductive behavior • No effect on spawning behavior was observed in the glyphosate fathead

full life-cycle study. However, no impact on spawning behavior is

consistent with no impact on reproduction in the life cycle study.

Secondary sexual characteristics • There were no reported observations of an impact on male and female

secondary sexual characteristics in the glyphosate fish full life cycle

study. However, no impact on secondary sexual characteristics is

consistent with no impact on reproduction in the life cycle study.

Fecundity (gonadal output) No effect on fecundity was evident in the glyphosate fathead full lifecycle study. No treatment related effects were evident for the number of

spawns, number of eggs spawned, number of eggs per spawn and percent

hatchability. In the glyphosate rat multigenerational studies fecundity

was unaffected in the FO and Fl groups.

Fertility (fertilization success) • No effect on fertility was evident in the glyphosate fathead full life-cycle

study. No treatment related effects were evident for number of spawns,

number of eggs spawned, number of eggs per spawn and percent

hatchability. No fertility issues were observed in multiple rat

reproduction studies and no effects were observed for sperm count and

motility

Gonadal histopathology • Gonadal histopathology was not assessed in the glyphosate fathead full

life cycle study. However, histopathology was performed on testicular,

uterine, ovary and accessory sex organs in rat multigenerational studies

and demonstrated no treatment related effects.

Gonadosomatic index Gonadosomatic index was not assessed in the glyphosate fathead full life

cycle study but there was no effect on gonadal output in the fathead full

life cycle study performed for glyphosate. However, absolute and

relative reproductive organ weights were comprehensively assessed in

the multigenerational studies and showed no treatment related impact.

Plasma concentration of

vitellogenin (VTG)

Plasma concentrations of VTG which is a specific measure of

estrogenicity were not measured in the fathead full life cycle study.

However, glyphosate has been shown not to induce VTG production by

Xie et al. (2005) in an intact organism nor impact the reproductive

endpoints measured in a life cycle study. Additionally, the ER responsive

reporter gene assay performed by Petit et al. (1997) and summarized in

section 2.1.1 demonstrates that glyphosate is not a ligand for the rainbow

trout estrogen receptor. This result is consistent with the assays of Xie et

al and Kojima et al. which demonstrate that glyphosate is not an ER

ligand and shows no potential for estrogenicity.

The fish full life-cycle study with glyphosate demonstrated no treatment-related effects on the

survival, growth and egg production of first generation fish, or on hatchability, as well as

survival and growth of second-generation eggs and fry, over an 8 month exposure period to

concentrations as high 25.7 mg/L. Additionally, it has been shown in vivo that glyphosate does

not induce production of vitellogenin nor induce an estrogen responsive reporter gene is a trout

cell line, which is consistent with SAR analysis of glyphosate. The results from the fish full-life

cycle study are also consistent with the results from subchronic and chronic tests with

mammalian and avian species, which demonstrate no changes in endocrine function and all

reproductive parameters. Based on these studies, sufficient information already exists to negate

63
the need to perform the fish short-term reproduction study. An additional fish reproduction

screening with glyphosate is not necessary.

5.0 Conclusion - EDSP Tier 1 OSRI Overview

Glyphosate has undergone a comprehensive battery of in vivo toxicology and ecotoxicology

testing that cover a broad spectrum of endocrine endpoints. This testing includes

multigenerational reproduction, subchronic and chronic tests that have robust experimental

designs, follow internationally-accepted protocols, have a high level of replication and a long

history of use in hazard identification and risk assessment. These in vivo studies

comprehensively assess endocrine functions that are required for reproduction, development, and

chronic health. It is important to point out that in many of the chronic studies no effects and/or

no effects related to endocrine activity were detected at high concentrations and at the highest

concentration tested. Additionally, the endocrine-modulating potential of glyphosate has been

evaluated in a variety of in vitro assays, negating the need for further in vitro evaluation.

An overview of the OSRI addressing the eleven assays of the EDSP Tier I Screening follows.

ER binding and transcription activation: Assays that provide data that are functionally

equivalent to data generated by the ERa/p competitive binding assay using rat uterine cytosol

and the ERa transactivation assay have already been conducted. Peer-reviewed articles by

Kojima et al. (2004), Petit et al. (1997) and Xie et al. (2005), present scientific information from

in vitro test systems that are very comparable in methodology, sensitivity and specificity to the

two Tier 1 ER binding assays. These assays by Kojima et al., Petit et al., and Xie et al. measured

the same mechanistic endpoints of ligand binding to assess agonism and antagonism, nuclear ER

translocation, DNA binding to responsive ER elements upstream of the reporter gene and

transactivation of the reporter gene. The results from these assays consistently demonstrate that

glyphosate is not an ER agonist or antagonist. Furthermore, a structure activity relationship

(SAR) analysis with methodology developed by EPA's Office of Research and Development

clearly shows that glyphosate does not have the chemical structure of a typical molecule with

estrogenic potential. The in vitro data demonstrating that glyphosate does not have ER binding

potential is consistent with the comprehensive in vivo data available for glyphosate. The in vivo

data demonstrates no effects on any endpoint that is sensitive and specific to compounds that

have estrogenic or anti-estrogenic activity. Based on the weight of evidence, it can be concluded

that no additional Tier 1 in vitro screening is required to evaluate the potential for ER agonism

and antagonism.

AR binding: Assays that provide data that are functionally equivalent to data generated by the

AR competitive binding assay using rat prostate cytosol have also already been conducted. The

peer-reviewed article by Kojima et al. (2004) presents scientific data that demonstrates that

glyphosate is not an AR agonist or antagonist using an in vitro test system and methodology that

is functionally equivalent to the methodology for the AR binding assay in the Tier 1 screening

battery. Kojima et al. evaluated the same mechanistic endpoint of ligand binding to assess

agonism and antagonism as the OPPTS AR binding guideline. Again, the SAR analysis

indicates that glyphosate does not have the chemical structure of a typical molecule that exhibits

androgenic potential. Lack of AR binding is confirmed by the comprehensive in vivo data

available from chronic studies on glyphosate. The in vivo data demonstrates no effect on

endpoints that are sensitive and specific to compounds that have androgenic or anti-androgenic

activity. A detailed review of the in vivo data that confirms glyphosate's is not androgenic or

64
anti-androgenic is extensively reviewed in the sections for the Hershberger, male pubertal assay

and fish screening assay. Based on the weight of evidence it can be concluded that no additional

in vitro screening is required to evaluate the potential for AR agonism and antagonism.

Aromatase Inhibition: Although in vitro data is not available to assess aromatase inhibition,

OSRI of the same nature and quality is available from existing in vivo chronic toxicity studies

with glyphosate. No treatment-related effects were observed in these studies on sensitive and

specific endpoints that would be indicative of aromatase inhibition by glyphosate, which

provides strong and consistent evidence that glyphosate in not an inhibitor of aromatase activity.

Scientifically relevant information related to steroidogenesis is comprehensively discussed in this

document and also applies to aromatase inhibition. Consequently, the requirement for

performing the in vitro aromatase assay has been met by providing OSRI from existing standard

regulatory in vivo studies that have the ability to detect effects of inhibition of aromatase activity.

Steroidogenesis: In vitro data assessing potential impacts on the complete steroidogenic

pathway are not available for glyphosate; however, data that is functionally equivalent to the in

vitro steroidogenesis assay is provided by endpoints included in multigenerational rat studies.

Specifically, it was confirmed in the most recent multigenerational study that there were no

treatment-related effects at high dietary exposure levels on endpoints that would be impacted by

an alteration of steroidogenesis, e.g., the weight of testes, cauda epididymides, epididymides,

seminal vesicles and coagulating glands, uterus, ovary and liver of the FO and Fl parent animals

(Moxon, 2000; Dhinsa et al., 2007). Additionally, there were no treatment-related effects up to

the highest dose tested (HOT) on the histopathology of left testis, left epididymis, prostate,

seminal vesicles with coagulating gland, uterus, left ovary, vagina, cervix, and oviducts.

Furthermore, there were no treatment-related effects up to the HDT on male and female

reproductive indices that included mating, fertility, conception, gestation, lactation age and

weight at VO, age at PPS, testicular descent, ovarian evaluation for follicles, parturition,

normality of external genitalia and number of implantation sites. The in vivo information from

these studies provides compelling evidence of no impact of glyphosate on steroidogenesis.

Uterotrophic: A strong weight of evidence has been provided with functionally equivalent

information supporting a lack of estrogenic activity of glyphosate in vivo from four existing

multigenerational studies, and a subchronic and chronic toxicity study in rats. The results of the

in vivo studies are consistent with results of in vitro assays that demonstrate glyphosate is not an

ER ligand. Functionally equivalent data of the same nature and quality to data expected to be

generated with the Uterotrophic assay (uterine weights and histopathology) demonstrate that

glyphosate does not possess estrogenic activity.

Hershberger: A strong weight of evidence has been presented using functionally equivalent

information supporting a lack of androgen receptor agonism and antagonism and 5a-reductase

inhibition of glyphosate in vivo from four multigenerational studies, and a subchronic and

chronic toxicity study in rats. This functionally equivalent data from multigenerational studies,

subchronic and chronic studies in rats demonstrate that glyphosate is not an androgen receptor

agonist or antagonist or a 5a-reductase inhibitor.

Female Pubertal: Functionally equivalent data to the female pubertal endpoints are described

in Section 3.3 of this document, with a comprehensive overview of endpoints addressed in the

multigenerational reproductive, subchronic and chronic rat studies. Endpoints in the female

pubertal assay are largely a subset of the multigenerational studies performed on glyphosate and

65
consequently provide data of the same nature and quality to data developed in the female

pubertal assay of the Tier 1 battery of the Endocrine Disrupter Screening Program.

Male Pubertal: Like the female pubertal assay, functionally equivalent data to the male

pubertal endpoints are described in Section 3.4, with a comprehensive overview of endpoints

addressed in the multigenerational reproductive, subchronic and chronic rat studies. Endpoints

in the male pubertal assay are also essentially a subset of endpoints from the multigenerational

studies performed for glyphosate and consequently provide data of the same nature and quality to

data developed in the male pubertal assay required to be conducted in the EDSP.

Amphibian Metamorphosis: The amphibian metamorphosis assay has been included in the Tier

1 battery as a generalized model for impacts on vertebrate thyroid function because the

amphibian thyroid system is regulated and responds in a similar manner to perturbation as that of

other tetrapods, including mammals. Therefore, combined results from existing glyphosate

studies provide information that is functionally equivalent to that generated in the amphibian

metamorphosis assay. Thyroid histopathology has been evaluated in standard mammalian

toxicology studies and demonstrates no impact on thyroid structure and function. Supporting

information is drawn from the fish full life-cycle study that shows no impact on growth and

development, which are sensitive measurement endpoints for impacts on thyroid structure and

function. Given all of this available information on glyphosate, the amphibian metamorphosis

screening assay does not offer any new relevant information to the toxicology data set for Tier 1

screening purposes.

Fish Short-term Reproduction: The methodology and information generated from a fish full

life-cycle study provides a comprehensive evaluation of potential modulation of endocrine

activity in fish and provides functionally equivalent information to information from the shortterm fish reproduction assay. The fish full life-cycle study is the highest Tier offish

ecotoxicology testing and typically performed with the fathead minnow (Pimphalespromelas) as

a freshwater test species. In the study, the entire life-cycle is exposed; therefore, de facto the

most sensitive life-stage to any toxicant (including endocrine active compounds) is encompassed

by the test design. Although, endpoints that are potentially influenced by endocrine activity,

such as gonadal histopathology and steroid hormone production, are not typically measured in

the full life-cycle, a fish full life-cycle study provides a comparable investigation to the test

proposed in the higher Tier 2 testing for fish. The fish full life-cycle study for glyphosate

demonstrated no treatment-related effects on the survival, growth and egg production of first

generation fish or on hatchability, as well as survival and growth of second-generation eggs and

fry over an 8 month exposure period to concentrations as high as 25.7 mg/L . Furthermore, it

has been shown in vivo that glyphosate does not induce production of vitellogenin, which is in

agreement with SAR analysis of glyphosate. The results from the fish full-life cycle study are

consistent with the results form subchronic and chronic test with mammalian species, which

demonstrate no changes in endocrine function, endocrine organ histopathology, and all

reproductive parameters. Additionally, the results from avian one-generation reproduction

studies demonstrate no effect on any of the measured endpoints at high test concentrations.

Based on this OSRI, there is sufficient information to meet the requirements for performing a

fish short term reproduction study.

3

 Chronic aquatic exposure testing under the fish early life-stage study (OECD 210) does not require testing at

concentrations that exceed 10 mg/L of active substance. Additionally, the highest concentration tested of 25.7

mg/L represents approximately 1/3/ the 96-hr LC50 value for this species.

66
5.1 Glyphosate Reviews for Endocrine Activity

Several in-depth reviews on the safety of glyphosate have been conducted by several regulatory

agencies and scientific institutions worldwide and have concluded that there is no indication that

glyphosate has potential endocrine activity or will result in adverse effects on endocrine systems

in humans or other mammals. The points below summarize the conclusions of these reviews.

• The U.S. EPA (1998) reviewed the subchronic and chronic mammalian studies for

glyphosate and concluded that there was no evidence to suggest that glyphosate produces

endocrine-modulating effects.

• In a comprehensive review of the standard mammalian toxicology studies, Williams et al.,

(2000) concluded that glyphosate does not have the potential to produce adverse effects on

endocrine systems in humans or other mammals.

• A report prepared for the USDA forest service by SERA concluded that extensive testing in

experimental animals and wildlife provided reasonably strong evidence that glyphosate is not

an endocrine disrupter (SERA, 2002).

• The list of purported endocrine disrupters compiled by the Institute of Environment and

Health (IEH, 2005) lists glyphosate as a substance with no evidence of potential endocrinedisrupting effects.

• Following a review of the standard mammalian and wildlife toxicology studies, it was

concluded in the recent ECETOC guidance document on identifying endocrine disrupting

effects that glyphosate is not an endocrine disrupter using the internationally accepted

Weybridge definition4 for endocrine disruption (ECETOC, 2009).

5.2 Summary and Conclusions

Based on this weight-of-evidence analysis from glyphosate's extensive toxicological database,

there is sufficient OSRI that is functionally equivalent and of the same nature and quality as that

developed in the Tier 1 screening battery to conclude that glyphosate does not have endocrinemodulating potential. Therefore, the requirements of Tier 1 testing have been satisfied and

additional testing under the Endocrine Disruption Screening and Testing Program is not

warranted.

The endocrine-modulating potential of glyphosate has been evaluated in a variety of studies

including in vitro assays and standard in vivo toxicology studies that are functionally equivalent

to the data generated in the Tier 1 screening battery. The in vivo studies comprehensively assess

endocrine functions that are required for reproduction, development and chronic health.

Glyphosate has demonstrated no estrogenic, anti-estrogenic, androgenic and anti-androgenic

potential in in vitro assays, and there was no indication of changes in endocrine function or

endocrine organ histopathology in any of the in vivo studies. Additionally, structure activity

models predict that glyphosate does not have estrogenic or androgenic activity.

4

 Weybridge (1996) "An endocrine disrupter is an exogenous substance that causes adverse health effects in an

intact organism, or its progeny, secondary (consequent) to changes in endocrine function. A potential endocrine

disrupter is a substance that possesses properties that might be expected to lead to endocrine disruption in an intact

organism."

67
References

Araki N, K Ohno, M Takeyoshi, M lida. (2005). Evaluation of a rapid in vitro androgen receptor

transcriptional activation assay using AR- EcoScreenTM cells, Toxicol. In Vitro. 19:335-352.

Beck V, E Reiter, A Jungbauer. (2008). Androgen receptor transactivation assay using green

fluorescent protein as a reporter. Anal Biochem. 373:263-71.

Blair RM, Fang H, Branham WS, Hass BS, SL Dial, CL Moland, W long, L Shi, R Perkins, DM

Sheehan. (2000). The estrogen receptor relative binding affinities of 188 natural and

xenochemicals: structural diversity of ligands. Toxicol Sci. 54:138-53.

BoNileson J, et al. (2009). The Usual Suspects - Influence of Physicochemical Properties on Lag

Time, Skin Deposition, and Percutaneous Penetration of Nine Model Compounds, J Toxicology

Environ Health, Part A, 72:315-323.

Carney EW, AM Hoberman, DR Farmer, RW Kapp, AI Nikiforov, M Bernstein, ME Hurtt, WJ

Breslin, SZ Cagen, and GP Daston. (1997). Estrogen modulation: tiered testing for human hazard

evaluation. Reprod. Toxicol. 11, 879-892.

Christenson LK, Strauss I, and F Jerome. (2001). Steroidogenic Acute Regulatory Protein; An

Update on Its Regulation and Mechanism of Action. Arch Med Res. 32:576-586.

Clark, JH, and J. Gorski. (1969). Estrogen receptors: An evaluation of cytoplasmic-nuclear

interactions in a cell-free system and a method for assay. Biochem Biophys Acta. 192:508-515.

Clark, JH and SK Mani. (1994). Actions of ovarian steroid hormones. In The Physiology of

Reproduction (J. D. Neill, ed., pp. 1011-1059. Raven Press, New York.

Colvin L, J Miller, J. Marvel. (1973). Final Report on CP 67573 Residue and Metabolism: Part

9: The Gross Distribution CP 67573-14C (N-phosphonomethylglycine-'4C) in the Rabbit:

Agricultural Research Report No. 298. (Unpublished study received Nov 12, 1973 under

4G1444; submitted by Monsanto Commercial Products Co., St. Louis, MO; CDL: 093849-D).

MRID 00108099.

Degitz SJ, GW Holcombe, KM Flynn, PA Kosian, JJ Korte, JE Tietge. (2005). Progress towards

development of an amphibian-based thyroid screening assay using Xenopus laevis. Organismal

and thyroidal responses to the model compounds 6-propylthiouracil, methimazole, and

thyroxine. Toxicol Sci. 87:353-64.

Devillers J, JP Doucet, A Panaye, N Marchand-Geneste, JM Porcher. (2009). Structure-activity

modeling of a diverse set of androgen receptor ligands. In: Endocrine Disrupter Modeling ed.

James Devillers. CRC Press, Boca Raton, FL, pages 335-356.

Dhinsa, NK, P. Watsonand NP Brooks. (2007). Glyphosate Technical: Dietary Two Generation

Reproduction Study in the Rat. Unpublished study to be submitted to EPA by Nufarm Americas.

68
ECETOC Technical Report, No. 106. Guidance on Identifying Endocrine Disrupting Effects.

http://www.ecetoc.org/technical-reports. Available upon request.

EG & G, Bionomics. (1975). Chronic Toxicity of Glyphosate to the Fathead Minnow

(Pimephalespromelas, Rafmesque). (Unpublished study received Dec 27, 1978 under 524-308;

submitted by Monsanto Co., Washington, DC; CDL:097759-B). MRJD 00108171.

Environmental Protection Agency (1993). Registration Eligibility Decision (RED). EPA 738-R93-014.

Environmental Protection Agency (1998a). Glyphosate: Pesticide tolerance, Final Rule—40

CFR, Part 180 [OPP-300736; FRL 6036-1]. Fed. Reg. 63(195), 54058-54066.

Environmental Protection Agency. (2007). Meeting minutes from the FIFRA Scientific

Advisory Panel Meeting on: A Set of Scientific Issues Being Considered by the Environmental

Protection Agency Regarding: Status of the In Utero Through Lactational Assay in the

Endocrine Disruptor Screening Program (EDSP).

http://www.epa.gov/scipoly/sap/meetings/2007/february/sap-2007-04-report-f.pdf

Environmental Protection Agency. (2008). Meeting minutes from the FIFRA Scientific

Advisory Panel Meeting on the Endocrine Disruptor Screening Program (EDSP) Proposed Tier 1

Screening Battery.

http://www.epa.gov/scipolv/SAP/meetings/2008/march/minutes2008-03-25.pdf

Environmental Protection Agency. (2009). Meeting minutes from the FIFRA Scientific

Advisory Panel Meeting on The Use of Structure Activity Relationships of Estrogen Binding

Affinity to Support Prioritization of Pesticide Inert Ingredients and Antimicrobial Pesticides for

Screening and Testing, http://www.epa.gov/scipoly/sap/meetings/2009/082509meeting.html

FAO/WHO (2004) Pesticides residues in food - 2004. Report of the Joint Meeting of the FAO

Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core

Assessment Group on Pesticide Residues (JMPR). Rome, Italy, 20-29 September 2004. FAO

Plant Production And Protection Paper 178. World Health Organization and Food and

Agriculture Organization of the United Nations. Rome, Italy.

http://www.fao.orR/ag/agp/aRpp/Pesticid/JMPR/DOWNLOAD/2004 rep/report2004impr.pdf

Fink R and J Beavers. (1978) Final Report: One-generation Reproduction study—Bobwhite

Quail: Glyphosate Technical: Project No. 139-141. (Unpublished study received Nov 13, 1978

under 524-308; prepared by Wildlife International, Ltd., submitted by Monsanto Co.,

Washington, DC; CDL:235924-B). MRID 00108207.

Fink R and J Beavers. (1978) Final Report: One-generation Reproduction Study—Mallard Duck:

Glyphosate Technical: Project No. 139-143. (Unpublished study received Nov 13, 1978 under

524-308; prepared by Wildlife International Ltd., submitted by Monsanto Co., Washington, DC;

CDL:235924-A). MRID 00111953.

69
Forbis A. (1989). Uptake, Depuration and bioconcentration of Carbon 14-Glyphosate to Bluegill

Sunfish (Lepomis macrochirus): Project ID MSL-9304. Unpublished study prepared by

Analytical Biochemistry Laboratories, Inc. 425 p. MRID 41228301.

Fort DJ, S Degitz, J Tietge, and LW Touart. (2007). The hypothalamic-pituitary-thyroid (HPT)

axis in frogs and its role in frog development and reproduction. Crit Rev Toxicol.

37:117-161.

Gordon, JD and CG Clark. (2005). ICCVAM Evaluation of In Vitro Test Methods for Detecting

Potential Endocrine Disrupters: Estrogen Receptor and Androgen Receptor Binding and

Transcriptional Activation Assays. (2003). NIH Publication No. 03-4503.

Harvey PW, and I Johnson. (2002). Approaches to the assessment of toxicity data with endpoints

related to endocrine disruption. J Appl Toxicol. 22:241-7.

Hershberger, LG, EG Shipley, and RK Meyer. (1953). Myotrophic activity of 19- ortestosterone

and other steroids determined by modified levator ani muscle method. Proc Soc Exp Biol Med

83:175-180.

Howe, R, R Chott, and R McClanahan. (1988). Metabolism of Glyphosate in Sprague-Dawley

Rats. Part II. Identification, Characterization, and Quantitation of Glyphosate and Its Metabolites

after Intravenous and Oral Administration: Laboratory Project No. MSL-7206: R.D. No. 877.

Unpublished study prepared by Monsanto Co. MRID 40767102.

ICCVAM. (2007). ICCVAM Comments on the draft OECD STTA Assay Test Guideline.

http://iccvam.niehs.nih.gov/methods/endocrine/OECDdocs/STTA/STTATG14Aug07.pdf

IEH (Institute for environment and Health). 2005. Chemicals purported to be endocrine

disrupters - a compilation of published lists. IEH Web report W20.

www.cranfield.ac.uk/health/.. ./ieh/ieh%20publications/w20.pdf

Kelce WR, CR Stone, SC Laws, LE Gray, JA Kemppainen. (1995).Wilson EM. Persistent DDT

metabolite p,p'-DDE is a potent androgen receptor antagonist. Nature. 375:581-5.

Kojima H, M lida, E Katsura, A Kanetoshi, Y Hori, and K Kobayashi. (2003). Effects of a

diphenyl ether-type herbicide, chlornitrofen, and its amino derivative on androgen and estrogen

receptor activities. Environ Health Perspect. 111:497-502.

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

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

hamster ovary cells. Environ. Health Perspect. 112:524-531.

Laws SC, S Yavanhxay , RL Cooper, JC Eldridge. (2006). Nature of the binding interaction for

50 structurally diverse chemicals with rat estrogen receptors. Toxicol Sci. 94:46-56.

Legler et al. (1999). Development of a stably transfected estrogen receptor-mediated luciferase

reporter gene assay in the human T47D breast cancer cell line. Toxicol Sci. 48:55-66.

70
Levine SL, Z Han, J Liu, DR Farmer, V Papadopoulos. (2007). Disrupting mitochondrial

function with surfactants inhibits MA-10 Leydig cell steroidogenesis. Cell Biol Toxicol. 6:385-

400.

Marty MS, JW Crissman, EW Carney. (1999). Evaluation of the EDSTAC female pubertal assay

in CD rats using 17beta-estradiol, steroid biosynthesis inhibitors, and a thyroid inhibitor. Toxicol

Sci. 52:269-277.

Moxon, ME. (2000) Glyphosate acid: multigenerational reproduction toxicity study in rats.

Unpublished study to be submitted to EPA by Syngenta Crop Protection, Inc.

Nishihara T, J Nishikawa, T Kanayama, F Dakeyama, K Saito, M Imagawa, S Takatori, Y

Kitagawa, S Hori, and H Utsumi . (2000). Estrogenic activities of 517 chemicals by yeast twohybrid assay. J. Health Science 46:282-298.

O'Connor JC, M Cook, S Marty, LG Davis, AM Kaplan, EW Carney. (2002). Evaluation of Tier

I screening approaches for detecting endocrine-active compounds (EACs). Crit Rev Toxicol.

32:521-49.

OECD (2007). Series on Testing and Assessment Number 83. Summary Report of the Peer

Review Panel on the Stably Transfected Transcriptional Activation Assay for Detecting

Estrogenic Activity of Chemicals, and Agreement of the Working Group of the National

Coordinators of the Test Guidelines Programme on the Follow-Up of this report

http://www.olis.oecd.org/olis/2007doc.nsf/LinkTo/NT000046BA/$FILE/JT03235654.PDF

O'Keefe, JA and RJ Handa. (1990). Transient elevation of estrogen receptors in the neonatal rat

hippocampus. Develop. Brain. Res. 57:119-127.

OMB, OIRA Action, Terms of Clearance: Tier 1 Screening of Certain Chemicals Under the EDSP. Oct.

2,2009. http://www.reginfo.gov/public/do/PRAOMBHistory?ombControlNiimber=2070-0176.

Opitz R, I Lutz, NH Nguyen, TS Scanlan, W Kloas. (2006). Analysis of thyroid hormone

receptor betaA mRNA expression in Xenopus laevis tadpoles as a means to detect agonism and

antagonism of thyroid hormone action. Toxicol Appl Pharmacol. 212:1-13.

Opitz R, T Braunbeck, C Bogi, DB Pickford, G Nentwig, J Oehlmann, O Tool, I Lutz, W Kloas.

(2005). Description and initial evaluation of a Xenopus metamorphosis assay for detection of

thyroid system-disrupting activities of environmental compounds. Environ Toxicol Chem.

24:653-64.

Owens, JW, and J Ashby. (2002). Critical review and evaluation of the uterotrophic bioassay for

the identification of possible estrogen agonists and antagonists: in support of the validation of the

OECD uterotrophic protocols for the laboratory rodent. Organisation for Economic Co-operation

and Development. Crit Rev Toxicol 32:445-520.

Panaye A, JP Doucet, J Devillers, N Marchand-Geneste, and JM Porcher. (2008). Decision trees

versus support vector machine for classification of androgen receptor ligands. SAR QSAR

Environ Res. 19:129-51.

71
Petit F, P LeGoff, J-P Cravedi, Y Valotaire, and F Pakdel. (1997). Two complementary

bioassays for screening the estrogenic potency of xenobiotics: recombinant yeast for trout

estrogen receptor and trout hepatocyte cultures. J. Mol Endocrin. 19:321-335.

Reel JR., JC Lamb-IV, and BH Neal. (1996). Survey and assessment of mammalian estrogen

biological assays for hazard characterization. Fundam. Appl. Toxicol. 34:288-305.

Reyna M. (1990) Two Generation Reproduction Feeding Study with Glyphosate in SpragueDawley Rats: Lab Project No: MSL-10387, Study No. ML 88-106. Unpublished study prepared

by Monsanto Agricultural Co. MRID 41621501.

Ridley W, M Dietrich, and R Folk. (1983). A Study of the Plasma and Bone Marrow Levels of

Glyphosate following Intraperitoneal Administration in the Rat: Study No. 830109.

(Unpublished study received Nov 15, 1983 under 524-308; submitted by Monsanto Co.,

Washington, DC; CDL:251737-F). MRID 40767101.

Ridley W and K Mirly. (1988). The Metabolism of Glyphosate in Sprague Dawley Rats-Part I.

Excretion and Tissue Distribution of Glyphosate and Its Metabolites following Intravenous and

Oral Administration: Laboratory Project No. 86139 (MSL-7215): R.D. No. 877. Unpublished

study prepared by Monsanto Co. MRID 40767101.

Rodwell, DE, EJ Tasker, AM Blair et al. (1980a) Teratology Study in Rats: IRDC No. 401-054,

Study No. IR 79-016. (Unpublished study including IRDC no. 999-021; received May 23, 1980

under 524-308; prepared by International Research and Development Corp., submitted by

Monsanto Co., Washington, D.C.; CDL:242516-A). MRID 00046362.

Rodwell, DE, EJ Tasker, AM Blair et al. (1980b) Teratology Study in Rabbits: IRDC No. 401-

056, Study No. IR 79-018. (Unpublished study; prepared by International Research and

Development Corp., submitted by Monsanto Co., Washington, D.C.; CDL:242516-B). MRID

00046363.

Rogers, JM and MS Denison. (2000). Recombinant cell bioassays for endocrine disrupters:

development of a stably transfected human ovarian cell line for the detection of estrogenic and

anti-estrogenic chemicals. In Vitro Mol Toxicol. 13:67-82.

Schmieder PK, GT. Ankley O, Mekenyan, JD Walker, and S Bradbury. (2003a). Quantitative

structure-activity relationship models for prediction of estrogen receptor binding affinity of

structurally diverse chemicals. Environ Toxicol and Chem. 22:1844-1854.

Schmieder PK, O Mekenyan, S Bradbury and GD Veith. (2003b). QSAR prioritization of

chemical inventories for endocrine disrupter testing. Pure Appl Chem. 75:2389-2396.

Schmieder PK, MA Tapper, JS Denny, RC Kolanczyk, BR Sheedy, TR Henry, and GD Veith.

(2004). Use of trout liver slices to enhance mechanistic interpretation of estrogen receptor

binding for cost-effective prioritization of chemicals within large inventories. Environ Sci

Technol. 38:6333-42.

72
Schroeder RE and GK Hogan. (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, 00105995.

SERA (Syracuse Environmental Research Associates). 2002. Neurotoxicity, Immunotoxicity,

and Endocrine Disruption with Specific Commentary on Glyphosate, Triclopyr, and Hexazinone:

Final Report, Prepared for the USDA, forest service by Patrick Durkin and Gary Diamond. Final

Report Syracuse Environmental Research Associates, Inc. USDA Order No. 43-3187-2-6002

Sonneveld E, H. Jansen, JAC Riteco, A Brouwer, B van der Burg. (2005). Development of

androgen- and estrogen-responsive bioassays members of a panel of human cell line-based

highly selective steroid responsive bioassays. Toxicol. Sci. 83:136-148.

Sonneveld E, JA Riteco, HJ Jansen, B Pieterse, A Brouwer, WG Schoonen, B van der Burg.

(2006). Comparison of in vitro and in vivo screening models for androgenic and estrogenic

activities. Toxicol Sci. 89:173-87.

Stevens, JT, W Gfeller, L Machemer, and , KH Leist. (1998). Adequacy of required regulatory

hazard testing for the detection of potential hormonal activity of crop protection chemicals. J

Toxicol Environ Health B 1:59-79.

Stevens, JT, A Tobia, JC Lamb, IV, C Tellone, and F O'Neal. (1997). FIFRA Subdivision F

testing guidelines: Are these tests adequate to detect potential hormonal activity for crop

protection chemicals? J. Toxicol. Environ Health. 50: 415-431.

Stocco DM. (2001). Related StAR protein and the regulation of steroid hormone biosynthesis.

Annu Rev Physiol. 63:193-213.

Stout L and C Johnson. (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 F Ruecker. (1990) Chronic Study of Glyphosate administered in Feed to Albino

Rats: Lab Project Number: MSL-10495: R.D. 1014, Study No. ML 87-148. Unpublished study

prepared by Monsanto Agricultural Co. MRID 41643801.

Sumpter JP and S Jobling. (1995). Vitellogenesis as a biomarker for estrogenic contamination of

the aquatic environment. Environ Health Perspect. 103 Suppl 7:173-8.

Takeyoshi M. (2006). OECD Validation Report of Pre-validation and Inter-laboratory

Validation For Stably Transfected Transcriptional Activation (TA) Assay to Detect Estrogenic

Activity - The Human Estrogen Receptor Alpha Mediated Reporter Gene Assay Using hERHeLa-9903 Cell Line - Chemicals Evaluation and Research Institute (CERI), Japan.

Taxvig C, AM Vinggaard, U Hass, M Axelstad, S Metzdorff, and C Nellemann. (2008).

Endocrine-disrupting properties in vivo of widely used azole fungicides. Int J Androl. 31:170-7.

73
Tietge JE, GW Holcombe, KM Flynn, PA Kosian, JJ Korte, LE Anderson, DC Wolf, SJ Degitz.

(2005). Metamorphic inhibition of Xenopus laevis by sodium perchlorate: effects on

development and thyroid histology. Environ Toxicol Chem. 24:926-33.

Walsh LP, C McCormick, C Martin, DM Stocco. (2000). Roundup inhibits steroidogenesis by

disrupting steroidogenic acute regulatory (StAR) protein expression. Environ Health Perspect.

108:769-776.

Wester, RC, J Melendres, R Sarason, J McMaster, and H. Maibach. (1991). Glyphosate skin

binding, absorption, residual tissue distribution, and skin decontamination. Fundam Appl

Toxicol. 16:725-732.

Williams GM, R Kroes, 1C Munro. (2000). Safety evaluation and risk assessment of the

herbicide Roundup and its active ingredient, glyphosate, for humans. Regul Toxicol Pharmacol.

31(2 Ptl):l 17-65.

Wilson AG, D Thake, WE Heydens, DWBrewster and KJ Hotz. (1996). Mode of Action of

Thyroid Tumor Formation in the Male Long-Evans Rat Administered High Doses of Alachlor.

Fundam Appl Toxicol, 33:16-23.

Wilson VS, K Bobseine, and LE Gray. (2004). Development and characterization of a cell line

that stably expresses an estrogen-responsive luciferase reporter for the detection of estrogen

receptor agonist and antagonists. Toxicol Sci. 81:69-77.

Xie L, K Thrippleton, MA Invin, GS Siemering, A Mekebri, D Crane, K Berry, and D Schlenk.

(2005). Evaluation of estrogenic activities of aquatic herbicides and surfactants using an

rainbow trout vitellogenin assay. Toxicol Sci. 87:91-8.

74
Appendix 1

EPA's EDSP Tier I Screening Battery

Tier 1 In Vitro Assays

Estrogen Receptor binding assay

ER transcriptional activation assay

Androgen receptor binding assay

Aromatase assay

Steroidogenesis assay

An ER binding assay that utilizes rat uterine cytosol to examine

the ability of a test chemical to bind with estrogen receptors

An ER binding assay that uses a human cell line to examine the

ability of a test chemical to bind with one of the estrogen

receptors

An AR binding assay that utilizes rat prostate cytosol to examine

the ability of a test chemical to bind with androgen receptors

Aromatase is an enzyme responsible for estrogen biosynthesis

that converts androgens into estrogens, estradiol, and estrone. The

aromatase assay uses human recombinant form of the protein and

focuses on this portion of the steroidogenic pathway to detect

substances that inhibit aromatase activity.

The Steroidogenesis assay utilizes H295R human adrenocortical

carcinoma cell line to detect interference with the body's

production of male and female steroid sex hormones (estrogen

and testosterone).

Tier 1 In Vivo Assays

Uterotrophic assay

Hershberger assay

Male pubertal assay (rat)

Female pubertal assay (rat)

Fish short term reproduction assay

Amphibian metamorphosis assay

(frog)

The Uterotrophic assay involves the use of female rats to screen

for estrogenic effects. In this in vivo assay, uterine weight

changes are measured in ovariectomised or immature female rats.

The Hershberger assay is designed to detect androgenic and antiandrogenic effects. In this assay, accessory sex gland weights,

including several androgen-dependent tissues, are measured in

castrated or immature male rats

The Pubertal Male assay involves the use of rats to screen for

androgenic, anti-androgenic, and thyroid activity in males during

sexual maturation. This assay examines abnormalities associated

with sex organs and puberty markers, as well as thyroid tissue

The Pubertal Female assay involves the use of rats to screen for

estrogenic and thyroid activity in females during sexual

maturation. This assay examines abnormalities associated with

sex organs and puberty markers, as well as thyroid tissue.

The Fish Screen assay screens for estrogenic and androgenic

effects. The assay examines abnormalities associated with

survival, reproductive behavior, secondary sex characteristics,

histopathology, and fecundity (i.e., number of spawns, number of

eggs/spawn, fertility) offish exposed to test chemicals.

The Amphibian Metamorphosis assay involves the use of

tadpoles to determine if chemicals affect the thyroid during

metamorphosis and consequently result in developmental effects.

75
Appendix 2

Summary of Glyphosate Adsorption, Distribution, Metabolism and Excretion

From the Glyphosate RED (EPA, 1993): Two metabolism studies with rats are available.

In the first study, single or repeated doses of radiolabeled

 14C-glyphosate were administered

orally to male and female Sprague-Dawley rats. Following a single oral dose of 14C-glyphosate,

30 to 36% of the dose was absorbed and less than 0.27% of the dose was eliminated as COi.

Ninety-seven point five percent of the administered dose was excreted in the urine and feces as

the parent compound, glyphosate. Amino methyl phosphonic acid (AMPA) was the only

metabolite found in urine (0.2-0.3% of the administered dose) and feces (0.2-0.4% of the

administered dose). Less than 1.0% of the absorbed dose remained in tissues and organs,

primarily in bone tissue. Repeated dosing at 10 mg/kg did not significantly change the

metabolism, distribution or excretion of glyphosate (MRIDs 40767101 and 40767102).

In a second study, male and female Sprague-Dawley rats received single intraperitoneal

injections of radiolabeled ' C-glyphosate. The dose level of glyphosate used for male and female

rats was 1150 mg/kg. Blood samples were collected 0.25, 0.50, 1, 2, 4, 6 and 10 hours after

injection. Femoral bone marrow samples were collected from one third of the male and female

rats sacrificed at 0.5, 4, or 10 hours after injection. Thirty minutes after injection of glyphosate,

the concentration of radioactivity in the bone marrow of male and female rats was equivalent to

0.0044% and 0.0072%, respectively, of the administered dose. Assuming first order kinetics, the

decrease in radioactivity in bone marrow occurred with a half-life of 7.6 and 4.2 hours for males

and females respectively. Similarly, the half-lives of the radioactivity in plasma were

approximately 1 hour for both sexes. These findings indicate that very little glyphosate reaches

bone marrow, that it is rapidly eliminated from bone marrow and that it is even more rapidly

eliminated from plasma. (MRID 00132685).

Table Al: (Ridley and Mirly, 1988). Distribution of radioactivity in rats dosed orally with 10 mg/kg of glyphosate

and sacrificed 24 hours after dosing (treatment group 1)

Sample

Urine

Faeces

Expired CO2

Cage wash

Total recovery*

Males

% of Dose

12.2

68.6

0.137

0.0539

81

Females

% of Dose

11.1

70.4

0.154

0.526

82.2

* Total recovery is the mean of individual animal data.

76
Table A2: (Ridley and Mirly, 1988). Distribution of radioactivity in rats dosed intravenously with 10 mg/kg of

glyphosate and sacrificed 7 days after dosing (treatment group 3)

Sample

Urine

Faeces

Organs/Tissues

Residual carcass

Tail

Gastrointestinal contents

Cage wash

Total recovery*

Males

% of Dose

79.0

4.65

0.0941

1.18

0.172

0.0394

0.890

86.0

Females

% of Dose

74.5

8.30

0.0521

1.04

0.161

0.0388

1.30

85.3

* Total recovery is the mean of individual animal data.

Table A3: (Ridley and Mirly, 1988). Distribution of radioactivity in rats dosed orally with 1000 mg/kg of

glyphosate and sacrificed 7 days after dosing (treatment group 4)

Sample

Urine

Faeces

Organs/Tissues

Residual carcass

Gastrointestinal contents

Cage wash

Total recovery*

Males

% of Dose

17.8

68.9

0.0355

0.248

0.0258

3.86

90.9

Females

% of Dose

14.3

69.4

0.0266

0.208

0.0429

8.00

92.1

* Total recovery is the mean of individual animal data.

Table A4: (Ridley and Mirly, 1988). Distribution of radioactivity in rats dosed orally with 10 mg/kg of glyphosate

and sacrificed 7 days after dosing (treatment group 5)

Sample

Urine

Faeces

Organs/Tissues

Residual carcass

Gastrointestinal contents

Cage wash

Total recovery*

Males

% of Dose

28.6

62.4

0.0460

0.395

0.0226

1.30

92.8

Females

% of Dose

22.5

69.4

0.0194

0.286

0.0145

1.96

94.2

* Total recovery is the mean of individual animal data.

77
Table AS: (Ridley and Mirly, 1988). Distribution of radioactivity in rats dosed orally for 15 days with 10 mg/kg of

glyphosate and sacrificed 7 days after dosing (treatment group 6)

Sample

Urine

Faeces

Organs/Tissues

Residual carcass

Gastrointestinal contents

Cage wash

Total recovery*

Males

% of Dose

30.9

61.0

0.0473

0.497

0.0138

0.820

93.3

Females

% of Dose

23.1

70.9

0.0313

0.315

0.0095

1.96

96.3

* Total recovery is the mean of individual animal data.

Ridley (1983) conducted a metabolism study of the plasma and bone marrow levels of

glyphosate following intraperitoneal administration in the rat and determined that

 l4C-glyphosate

reached maximum plasma and bone marrow concentrations within 0.5 hours following

intraperitoneal injection in rats. The elimination half-life times of glyphosate in plasma and bone

marrow were 1 hour and 4.2-7.6 hours, respectively.

Colvin and Miller (1973) evaluated the gross distribution of

 14C- N-phosphonomethylglycine in

rabbits and determined that

 14C-glyphosate was predominantly excreted in the faeces (80-97% of

the dose) rabbits over a period of 120 hours following oral administration by gavage. Only a

very limited fraction of the radioactivity was retained in the tissues (0.1-1.2% of the dose).

Wester (1991) evaluated the elimination of

 14C-GIyphosate in rhesus monkeys and determined

that 94.9% to 98.8% of the radioactivity was excreted in the urine over a period of 7 days

following intravenous injection of

 14C-glyphosate - nearly all of the glyphosate excretion

occurred within 24 hours.

Howe, Chott and McClanahan (1988) evaluated the metabolism of glyphosate in SpragueDawley rats at several doses via both oral and intra venous routes of administration. They

determined that the metabolism of glyphosate in the rat following oral or intravenous

administration was very minor. AMPA, the sole metabolite, appeared to be formed to the extent

of less than 0.5% by in vivo metabolism of glyphosate in rats orally dosed at 10 mg/kg, either in

single or multiple doses.

Colvin and Miller (1973) evaluated the dynamics of accumulation and depletion of orally

ingested

 14C labeled glyphosate. Data comparing tissue uptake and depletion are summarized

below in tables A6 and A7 respectively.

78
Table A6: (Colvin and Miller, 1973): Peak radioactivity expressed as glyphosate acid eq. In rat tissues during

chronic oral dosing with

 4C-glyphosate

Tissue

Liver

Kidney

Muscle

Fat

Gut

Spleen

Heart

Brain

Gonads

Blood

1 ng/g or ng/mL

Male

0.004

0.012

0.002

0.003

0.506

0.007

0.002

0.001

0.001

0.001

Female

0.004

0.008

0.001

0.006

0.370

0.011

0.004

0.001

0.009

0.001

10 ng/g or ng/mL

Male

0.041

0.136

0.018

0.065

3.494

0.076

0.038

0.014

0.014

0.013

Female

0.034

0.109

0.013

0.060

5.286

0.122

0.032

0.013

0.180

0.013

100 (ig/g or (ig/mL

Male

0.319

0.916

0.195

0.710

37.305

0.696

0.404

0.179

0.114

0.092

Female

0.291

0.779

0.125

0.681

26.903

0.636

0.209

0.116

0.590

0.090

The tissue radioactivity was clearly reversibly bound. As soon as the exogenous supply of

 I4Cglyphosate was removed, the radioactivity in the tissues immediately began to decrease.

Residues in tissues remaining after ten days withdrawal on a fresh-weight basis are shown in

Table A7.

Table A7: (Colvin and Miller, 1973): Concentration of radioactivity (expressed as glyphosate acid eq.) remaining

in rat tissues after 10 days withdrawal following chronic oral dosing with C-glyphosate

Tissue

Liver

Kidney

Muscle

Fat

Gut

Spleen

Heart

Brain

Gonads

Blood

1 (ig/g or u,g/mL

Male

0.001

0.001

0.001

0.002

0.001

0.001

0.001

0.001

0.001

0.001

Female

0.001

0.001

0.001

0.001

O.001

O.001

<0.001

<0.001

0.000

O.001

10 (ig/g or u.g/mL

Male

0.008

0.013

0.013

0.016

0.008

0.008

0.012

0.006

0.007

0.006

Female

0.008

0.012

0.009

0.015

0.008

0.008

0.009

0.005

0.010

0.006

100 (ig/g or jig/mL

Male

0.069

0.102

0.097

0.112

0.059

0.067

0.084

0.050

0.057

0.059

Female

0.065

0.098

0.090

0.128

0.076

0.060

0.078

0.043

0.062

0.046

Colvin and Miller (1973) concluded that upon continuous administration of C-glyphosate in

feed, 8.3-10.5% of the daily intake was excreted in urine. The combined excretion of

radioactivity in urine and faeces at the end of the 14-day dosing period was in equilibrium with

the daily intake of

 14C-glyphosate at the three feed concentrations tested. Upon withdrawal from

treatment the excretion of radioactivity dropped rapidly, but plateaued after four days due to

79
redistribution of previously established tissue stores. Most tissues reached maximum

radioactivity levels during the dosing period in 10 days or less. The radioactivity that was

present in the tissues was clearly not irreversibly bound.

Highlights from the above data addressing OPPTS 890.1400 and 890.1600 considerations of

ADME and metabolism, negating the need for in vivo screening with the Hershberger and

uterotrophic assays include the following.

• Low Gl tract absorption of glyphosate,

• Very low dermal absorption (Bo Neilsen et al., 2009)

• Very little systemic metabolism /activation of glyphosate, and

• Very rapid clearance from the body predominantly via urine.

80
Appendix 3

Cited Studies Submitted to EPA

Study EPA Classification MRID#

Rodwell, D.E.; Tasker, E.J.; Blair, A.M.; et al. (1980) Teratology

Study in Rats: IRDC No. 401-054. (Unpublished study including

IRDC no. 999-021; received May 23, 1980 under 524-308;

prepared by International Research and Development Corp.,

submitted by Monsanto Co., Washington, D.C.; CDL:242516-A)

Data Supporting Guideline

Requirements for the

Reregistration of GlyphosateReregistration Eligibility

Decision (RED)

00046362

Rodwell, D.E.; Tasker, E.J.; Blair, M.; et al. (1980) Teratology

Study in Rabbits: IRDC No. 401-056. (Unpublished study

received May 23, 1980 under 524-308; prepared by International

Research and Development Corp., submitted by Monsanto Co.,

Washington, D.C.; CDL:242516-B)

Data Supporting Guideline

Requirements for the

Reregistration of GlyphosateReregistration Eligibility

Decision (RED)

00046363

Schroeder, R.E.; Hogan, O.K. (1981) A Three-Generation

Reproduction Study with Glyphosate in Rats: Project No. 77-2

(Unpublished study received Sep 22, 1981 under 524-308;

prepared by Bio/dynamics, Inc., submitted by Monsanto Co.,

Washington, D.C.; CDL:245909-A)

Data Supporting Guideline

Requirements for the

Reregistration of GlyphosateReregistration Eligibility

Decision (RED)

00081674

00105995

Colvin, L.; Miller, J.; Marvel, J. (1973) Final Report on CP

67573 Residue and Metabolism: Part 9: The Gross Distribution

CP 67573-14C (N-phosphono-methylglycine-14C) in the Rabbit:

Agricultural Research Report No. 298. (Unpublished study

received Nov 12, 1973 under 4G1444; submitted by Monsanto

Commercial Products Co., St. Louis, MO; CDL: 093849-D)

Supplemental 00108099

EG & G, Bionomics (1975) Chronic Toxicity of Glyphosate to

the Fathead Minnow (Pimephales promelas, Rafmesque).

(Unpublished study received Dec 27, 1978 under 524-308;

submitted by Monsanto Co., Washington, DC; CDL:097759-B)

Data Supporting Guideline

Requirements for the

Reregistration of Glyphosate—

Reregistration Eligibility

Decision (RED)

00108171

Fink R and J Beavers. (1978) Final Report: One-generation

Reproduction study—Bobwhite Quail: Glyphosate Technical:

Project No. 139-141. (Unpublished study received Nov 13, 1978

under 524-308; prepared by Wildlife International, Ltd.,

submitted by Monsanto Co., Washington, DC; CDL:235924-B)

Data Supporting Guideline

Requirements for the

Reregistration of Glyphosate

Reregistration Eligibility

Decision (RED)

00108207

Fink R and J Beavers. (1978) Final Report: One-generation

Reproduction Study—Mallard Duck: Glyphosate Technical:

Project No. 139-143. (Unpublished study received Nov 13, 1978

under 524-308; prepared by Wildlife International Ltd., submitted

by Monsanto Co., Washington, DC; CDL:235924-A).

Data Supporting Guideline

Requirements for the

Reregistration of Glyphosate

Reregistration Eligibility

Decision (RED)

00111953

Ridley, W, Dietrich, M.; Folk, R.; et al. (1983) A Study of the

Plasma and Bone Marrow Levels of Glyphosate following

Intraperitoneal Administration in the Rat: Study No. 830109.

(Unpublished study received Nov 15, 1983 under 524-308;

submitted by Monsanto Co., Washington, DC; CDL:251737-F)

Data Supporting Guideline

Requirements for the

Reregistration of Glyphosate—

Reregistration Eligibility

Decision (RED)

00132685

Stout, L.; Johnson, C. (1987) 90-day Study of Glyphosate

Administered in Feed to Sprague/Dawley Rats: Proj. ID ML-86-

351/EFIL 86128. Unpublished study prepared by Monsanto

Agricultural Co. 267 p.

Data Supporting Guideline

Requirements for the

Reregistration of Glyphosate

Reregistration Eligibility

Decision (RED)

40559401

81
Study EPA Classification MRID#

Ridley, W.; Mirly, K. (1988) The Metabolism of Glyphosate in

Sprague Dawley Rats—Part I. Excretion and Tissue Distribution

of Glyphosate and Its Metablites following Intravenous and Oral

Administration: Laboratory Project No. 86139 (MSL-7215): R.D.

No. 877. Unpublished study prepared by Monsanto Co. 587 p.

Data Supporting Guideline

Requirements for the

Reregistration of Glyphosate

Reregistration Eligibility

Decision (RED)

40767101

Howe, R.; Chott, R.; McClanahan, R. (1988) Metabolism of

Glyphosate in Sprague-Dawley Rats. Part II. Identification,

Characterization, and Quantitation of Glyphosate and Its

Metabolites after Intravenous and Oral Administration:

Laboratory Project No. MSL-7206: R.D. No. 877. Unpublished

study prepared by Monsanto Co. 155 p.

Data Supporting Guideline

Requirements for the

Reregistration of Glyphosate

Reregistration Eligibility

Decision (RED)

40767102

Forbis A. (1989). Uptake, Depuration and Bioconcentration of

14C-Glyphosate to Bluegill Sunfish (Lepomis macrochints):

Project ID MSL-9304. Unpublished study prepared by Analytical

Biochemistry Laboratories, Inc. 425 p.

Data Supporting Guideline

Requirements for the

Reregistration of Glyphosate—

Reregistration Eligibility

Decision (RED)

41228301

Reyna, M. (1990) Two Generation Reproduction Feeding Study

with Glysophate in Sprague-Dawley Rats: Lab Project No: MSL10387. Unpublished study prepared by Monsanto Agricultural

Co. 1158

Acceptable, core-guideline data

(D176984,-85)

41621501

Stout, L.; Ruecker, F. (1990) Chronic Study of Glyphosate

Adminitered in Feed to Albino Rats: Lab Project Number: MSL10495: R.D. 1014. Unpublished study prepared by Monsanto

Agricultural Co. 2175 p.

Acceptable, core-guideline data

(EPA Letter dated August 25,

1992)

41643801

Moxon, M. E. (2000) Glyphosate Acid: Multi-generation

Reproduction Toxicity Study in Rats; Report No. CTL/P/6332.

Unpublished study prepared by Syngenta, to be submitted.

Not Reviewed—Study to be

Submitted

to be

assigned

Dhinsa, N. K., Watson, P. and Brooks, P. N. (2007) Glyphosate

Technical: Dietary Two Generation Reproduction Study in the

Rat. Unpublished study prepared by Nufarm Americas, to be

submitted.

Not Reviewed—Study to be

Submitted

to be

assigned

82
MRID 00046362

International Research

and Development Corporation

MATTAWAN, MICHIGAN, U.S.A. 49071 TELEPHONE (616! 668-3336

SPONSOR: The Monsanto Company, IH.-79-016

TEST ARTICLE: Technical Glyphosate

SUBJECT: Teratology Study in Rats

DATE OF SUBMISSION: March 21, 1980

CONTAINS TRADE SECRET OR

OTHERWISE CONFIDENTIAL

INFORMATION OF MONSANTO

COMPANY

A 0 0 7 4 2 3

(c) Monsanto Company 1980

401-054

"credence through research"

83
MRID 00046363

international Research

and Development Corporation

MATTAWAN, MICHIGAN, U.S.A. 49071 TELEPHONE (616) 668-3336

SPONSOR: Monsanto Company , IR-79-018

TEST ARTICLE: Technical Glyphosate

SUBJECT: Teratology fitudy in Rabbits

DATE OF SUBMISSION: February 29, 1980

COMPANY °N °F

(c) Monsanto Company 1980

A A 0 0 7 4 2 1

401-056

'credence through research'

84
MRID 00081674

Bicyaynamics Inc.

Division of Biology and Safety Evaluation

PROJECT NO. 77-2063

BDN 77-417

A THREE GENERATION REPRODUCTION STUDY IN RATS

WITH GLYPHOSATE

Final Report

Submitted to: Monsanto Company

St. Louis, Missouri 63166

' Date: March 31, 1981

AA00743 7

MOHSKNTO COMPANY 1981

\JN IAINS I KAUt StCKtl Ok

;. fHERWISE CONFIDENTIAL

85
MRID 00105995

ADDITIONAL INFORMATION SUBMITTED IN

SUPPORT OF THE REGISTRATION OF

ROUNDUP® HERBICIDE

EPA REG. NO. 524-308

A THREE-GENERATION REPRODUCTION STUDY

IN RATS WITH GLYPHOSATE

R. D. #374

SPECIAL REPORT MSL #1724

JULY 31, 1981

COMPILED BY

R. W. STREET

MONSANTO AGRICULTURAL PRODUCTS COMPANY

800 NORTH LINDBERGH BOULEVARD ST. LOUIS, MISSOURI 63166

CONTAINS TRADE SECRET OR

OTHERWISE CONFIDENTIAL

INFORMATION OF MONSANTO

86
MRID 00108099

MONSANTO COMMERCIAL PRODUCTS CO.

AGRICULTURAL DIVISION

RESEARCH DEPARTMENT

Agricultural Research Report No. 298

Final Report

on

CP 67573 RESIDUE AND METABOLISM

Part 9: The Gross Distribution of N-Phosphonomethylglycine-14C

(CP 67573-14C) in the Rabbit

Job No. 9-23-760.06-7863

Date Written: June 20, 1973

Written By

and

Work Done By: L. B. Colvin

J. A. Miller

Group Leader: J. T- Marvel

AA016678

This report contains confidential information which is the property

of Monsanto Company and which shall be disclosed only to duly authorized persons. The recipient is held accountable for the filing

and safe custody of this report, which must be returned on demand.

87
MRID 00108171

CHRONIC TOXICITY OF GLYPHO5ATE TO THE

FATHEAD MINNOW (Pimephales promelas,

Rafinesque).

SUBMITTED TO: .

MONSANTO COMPANY

ST. LOUIS, MISSOURI

E G & G, Bionomics

Aquatic Toxicology Laboratory

790 Main Street

Wareham, Massachusetts

October, 1975

PM013542

88
I

'fitlt

MRID 00108207

I

I

I

I

I

I

I

ONE-GENERATION REPRODUCTION STUDY - BOBWHITE QUAIL

GLYPHOSATE TECHNICAL

FINAL REPORT

I

I

I

1

I

I

I

»

I

Submitted to

Monsanto Company

St. Louis, Missouri 63166

WILDLIFE INTERNATIONAL LTD.

^031368

November 1, 1978

CONTAINS TRADE SECRET OR

OTHERWISE CONFIDENTIAL

INFORMATION OF MONSANTO

COMPANY

89
MRID 00111953

ONE-GENERATION REPRODUCTION STUDY - MALLARD DUCK

6LYPHOSATE TECHNICAL

FINAL REPORT

Submitted to

Monsanto Company

St. Louis, Missouri 63166

WILDLIFE INTERNATIONAL LTD.

September 25, 1978

D

90
MRID 00132685

MONSANTO COMPANY

ENVIRONMENTAL HEALTH LABORATORY

645 S. NEWSTEAD

ST. LOUIS, MISSOURI 63110

A Study Of The Plasma And Bone Marrow Levels

Of Glyphosate Following Intraperitoneal

Administration In The Rat

Study Number:830109

DMEH Project Number: ML-83-218

Submitted to: Monsanto Agricultural Products Company (Sponsor)

Through: Richard C. Dirks, Product Toxicologist

Author and Study Director: William P. Ridley

24,* •

William P. Ridley, StudjU Director

Martin W. Dietrich, Gr/up Leader / '

/<y^y/£

Roger M. Polk, Director EHL / /

© MONaANTO COMPANY 1983

ALL RIGHTS RESERVED

AA00456 2

I EHL 830109

i PAGE 1

91
MRID 40559401

STUDY TITLE

90-Day Study of Glyphosate Administered In

Feed To Sprague/Dawley Rats

DATA REQUIREMENTS

Guideline 82-1, 90-Day Feeding Study - Rodent

AUTHORS

L.D. Stout and C.W. Johnson

STUDY COMPLETED

November 30, 1987

PERFORMING LABORATORY

Monsanto Agricultural Company

Environmental Health Laboratory

645 S. Newstead

St. Louis, Missouri 63110

LABORATORY PROJECT I.D.

ML-86-351/EHL 86128

R.D. No. 860

'A'""A 0 0 038 8

Page 1 of 267

92
MRID 40767101

STUDY TITLE

Volume 1: The Metabolism of Glyphosate in Sprague Dawley Rats

- Part I. Excretion and Tissue Distribution of

Glyphosate and Its Metabolites following Intravenous

and Oral Administration.

DATA REQUIREMENTS

Guideline 85-1: General Metabolism

AUTHORS

W.P. Ridley and K. Mirly

STUDY COMPLETED

March 23, 1988

PERFORMING LABORATORY

Monsanto Company

Environmental Health Laboratory

645 S. Newstead

St. Louis, Missouri 63110

LABORATORY PROJECT HO

86139 (MSL-7215)

R.D. No. 877

Page I of VII

Followed By

Pages 1 thru 587

86139 (MSL-7215)

93
MRID 40767102

STUDY TITLE

Volume 2: Metabolism of Glyphosate in Sprague-Dawley Rats.

Part II. Identification, Characteriziation, and

Quantitation of Glyphosate and Its Metabolites

After Intravenous and Oral Administration

DATA REQUIREMENTS

Guideline 85-1: General Metabolism

AUTHORS

R.K. Howe, R.C. Chott, and R.H. McClanahan

STUDY COMPLETED

February, 1988

PERFORMING LABORATORY

Monsanto Company

800 N. Lindbergh Blvd.

St. Louis, Missouri 63167

LABORATORY PROJECT NO

MSL-7206

R.D. No. 877

Page I of IX

Followed By

Pages 1 thru 155

MSL-7206

AA00776 3 I 9 4
MRID 41228301

STUDY TITLE

Uptake, Depuration and Bioconcentration of

14C Glyphoaate to Bluegill Sunfish (Lapomis macrochirue)

Part I: MSL-9304

PATA REQUIREMENT

Guideline 72-6

AUTHOR

A. D. Forbis

STUDY COMPLETED

August, 1989

PERFORMING LABORATORY

Analytical Bio-Chemistry Laboratories, Inc.

Aquatic Toxicology Division

7200 East ABC Lane

P.O. Box 1097

Columbia, MO 65205

LABORATORY PROJECT NO.

MSL-9304

R.D. No. 955

Volume 1

Page 1 of()OOOf<M25

AA00783 4

95

MSL-9304
MRID 41621501

STUDY TITLE

Two Generation Reproduction Feeding Study with

Glyphosate in Sprague-Dawley Rats

DATA REQUIREMENT

Guideline 83-4

AUTHOR

| ' M.S. Reyna

STUDY COMPLETED

August 27, 1990

PERFORMING LABORATORY

Monsanto Agricultural Company

800 N. Lindbergh Blvd.

St. Louis, Missouri 63167

LABORATORY PROJECT NO.

MSL- 10387

VOLUME 1 of 1

Page 1 of 1158

•

A A 0 0 2 (f b~
MRID 41643801

STUDY TITLE

Chronic Study of Glyphosate Administered

in Feed to Albino Rats

DATA REQUIREMENT

Guideline 83-5

AUTHORS

L.D. Stout and F.A. Ruecker

STUDY COMPLETED

September 26, 1990

PERFORMING LABORATORY

Monsanto Agricultural Company ; 800 N. Lindbergh Blvd.

St. Louis, Missouri 63167

LABORATORY PROJECT NO.

MSL-10495

R.D. NO. 1014

VOLUME 1 of 1

Page I of IX

followed by pages 1 to 2175

AA00202 0

97