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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

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

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

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

.

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

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

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

CHEMICAL: ATRAZINE

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYATRAZINE FINAL

ii TABLE OF CONTENTS

Abbreviations...................................................................................................iii
Executive Summary..........................................................................................1

I. Introduction.......................................................................................4

II. Sources of Scientific Data and Technical Information......................................6

A. EDSP Tier 1 Screening Assays............................................................6

B. Other Scientifically Rele vant Information (OSRI)......................................6

III. Weight of Evidence (WoE) Evaluation.......................................................7

A. EDSP Tier 1 Screening Assays...........................................................10

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis............................11
1. Effects on Estrogen Pathway...................................................11
2. Effects on Androgen Pathway..................................................26

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................38

IV. Committee’s Assessment of Weight of Evidence..........................................41

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................41

1. Tier 1 in vivo Assays.............................................................42

2. OSRI...............................................................................42

B. Estrogen Pathway...........................................................................51

C. Androgen Pathway.........................................................................52

D. Thyroid Pathway...........................................................................53

E. Conclusions.................................................................................53

V. EDSP Tier 2 Testing Recommendations....................................................53

VI. References.......................................................................................54

LIST OF TABLES: Table 1. Tier 1 Assays for Atrazine...................................................................10
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Atrazine.....................................17
Table 3. Androgenic/Anti-Andr ogenic Pathway for Atrazine....................................30
Table 4. Thyroid Pathway for Atrazine.............................................................39

APPENDIX 1. EDSP Tier 1 Screening Assays.........................................................64
APPENDIX 2. Other Scientifically Relevant Information (OSRI)................................66

APPENDIX 3. References Not Utilized in the Atrazine WoE Analysis.........................155ATRAZINE FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ACTH Adrenocorticotropic hormone

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

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

CG Cowper’s Gland

ChE Cholinesterase
ChEI Cholinesterase inhibition
CMC Carboxymethyl cellulose
CMG Common Mechanism Grouping
CTA Comparative Thyroid Assay
CV Coefficient of Variation
CYP Cytochrome 450
DACT Diamino chlorotriazine

DEA Desethyl- s -atrazine

DE R Data Evaluation Recor d DIA Desiso p ro py l- s -atrazine DMSO Dimeth y l Sulfoxide DNA Deox y ribonucleic Aci d DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
E R Estro g en Rece p to rATRAZINE FINAL

iv Abbreviation Terminology

EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase ) ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g eneration
F2 Second filial g eneration
Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS High Pressure Liquid Chroma tography/Mass Spectroscopy

HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Interagency Coordinating Committee on the Validation of Alternative M hd K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

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

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

M Male
MDL Minimum Detection Level MEOGRT Medaka Extended One Generation Re p roduction Test
MH Medium hi g h
ML Medium lowATRAZINE FINAL

v Abbreviation Terminology

MoA Mode of Action MOE Mar g in of Ex p osure MRID Master Record Identifier

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

OPP Office of Pesticide Programs

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

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th SV Seminal VesiclesATRAZINE FINAL

vi Abbreviation Terminology

T Th y roid ( hormonal p athwa y) T1WoERC EDSP Tier 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)

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

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Va g inal O p enin g VP Ventral Prostate VTG Vitello g eni n WoE Wei g ht-of-EvidenceATRAZINE FINAL

Page 1 of 159 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide the necessary empirical data to evaluate the poten tial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) hormone pathways. This interaction includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as perturbations of the hypothalamic-pituitary-gon adal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. For atrazine, test order requirements for all eleven Tier 1 assays were satisfied by
submissions of other scientifica lly relevant information (OSRI), comprised primarily of studies that were generally consistent w ith, but conducted prior to issuance of , the Tier 1 test guidelines.
In addition to the available Tier 1-like data , OSRI including general toxicity data and open literature studies of sufficient quality were c onsidered in this wei ght of evidence (WoE)
evaluation. Also considered in the determina tion were the FIFRA Scientific Advisory Panel (SAP) reports on atrazine and the common mechanism grouping (CMG ) of atrazine with other
similar triazines.

In determining whether atrazine interacts with E, A, or T hormone pathways, the number and type of effects induced, the magnitude of respons es, and the pattern of responses observed across studies, taxa, and sexes and levels of biological organization were considered. Additionally, the
conditions under which effects o ccur were considered, in partic ular, whether or not endocrinerelated responses occurred at dose(s) that also resulted in general system ic toxicity or overt toxicity.

On December 17, 2014, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-of-evidence (WoE) analysis of the potential interaction of atrazine with the E, A or T signaling pathways. The T1Wo ERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

With respect to the estrogen pathway, although the Tier 1 guideline-like OSRI submissions
showed few effects indicative of a potential interaction with the estrogen pathway, the rich dataset of supporting OSRI pr ovided additional lines of ev idence to suggest potential downstream, estrogen pathway effects that ma y be consistent with the known, upstream
neuroendocrine mechanism of action for atrazine and other triazines in the common mechanism grouping (CMG). In the in vitro Tier 1-like assays, atrazine was negative in numerous estrogen receptor (ER) binding and ER transactivation assays (ERTAs). Mixed results were obtained in
the aromatase studies. Sanderson et al. (2001 and 2002) demonstrated induction of aromatase activity in H295R and JEG-3 cells, but no induc tion occurred in MCF- 7 cells. Increased aromatase activity in human gra nulosa-lutein cells, but not in human endometrial stromal cells, was noted by Holloway et al. , (2008). Hinfray et al. (2006) demonstrated that atrazine had noATRAZINE FINAL

Page 2 of 159 effect on aromatase activity in rainbow trout brai n and ovarian microsomes. However, atrazine
acted as an inducer of 17 β -estradiol in the steriodogenesis assay.

In the in vivo mammalian Tier 1-like assays, atrazine was negative for estrogenic activity in several uterotrophic assays. In the female pubert al assay, delays in estrus and vaginal opening, decreases pituitary and uterine weights were obs erved at the mid-dose in the absence of overt toxicity. There were no estrogen-related effects se en in the Part 158 mammalian toxicity studies in the absence of overt toxicity.

Effects seen in OSRI with nonmammalian wildlife species were similarly sporadic across the
data set and sometimes co-occurr ed with non-specific effects such as reductions in growth parameters ( i.e. , offspring weight or size) . Reproduction was lower in response to atrazine exposure in two studies with fish, in one study with the Northern wa ter snake, and in two of three studies with different species of birds exposed through the diet. Adverse effects on parental or offspring growth were also observed in these studies. There were no significant effects on offspring sex ratio in neonates of water sn akes exposed to atrazine in the diet. In ovo exposure to atrazine resulted in no statistically significant effects in aromatase act ivity or sex steroid
concentrations in chorionic fl uid or on neonate gonadal histol ogy. The percentage of female frogs was greater in atrazine-exposed treatments in one study, but othe r studies with frogs showed no effects on reproductive developmen t in the absence of overt toxicity.

Although the available data reflect varying re sponses in estrogen-related endpoints across species and studies, the potential for interaction with the estr ogen pathway is supported by the SAP conclusion that the chlorotriazines (includi ng atrazine and its degradates) function through a neuroendocrine mode of action (MoA) that supp resses the hypothalamic release of GnRH and therefore LH, which may result in downstr eam effects on estrogen and androgen signaling pathways.

With respect to the androgen pathway, the Tier 1 guideline-like OSRI submissions showed few effects indicative of a potentia l interaction with th e androgen pathway, but additional supporting OSRI provided further lines of evidence to suggest potential downstr eam, androgen pathway effects that may be consistent with the known, upstream neuroendocrine mechanism of action for
atrazine and other triazine s in the CMG. In the in vitro Tier 1-like assays, at razine was negative in the AR binding and ARTA assays. In the ster iodogenesis assay, atrazine acted as an inducer of testosterone at 100 μM at two of five studies (U SEPA, 2008c).

In the in vivo mammalian Tier 1-like assays, potential andr ogen-related effects of atrazine in two male pubertal assays (decreases in sex accesso ry sex organ weights and delayed preputial separation) were non dose-related and occurred at doses approach ing overt toxicity. Atrazine was negative in the Hershberger assay. Moreover, numerous SAP reports on atrazine and
triazine chlorinated metabolites ha ve concluded that the triazines have the potential to interact with the androgen pathway in mammals.ATRAZINE FINAL

Page 3 of 159 The data synthesized in this WoE evaluation reflect varying responses in androgen-related endpoints across species and studies. Data from OSRI beyond the Tier 1-like provide complementarity of evidence and redundancy of findings with respect to steroidogenesis ( in vitro
and in vivo ) and gonadal histology, though si milar effects were not seen in all studies. Although the available data, especially the OSRI beyond th e Tier 1 guideline-like studies, reflect varying responses in estrogen- and andr ogen-related endpoints across species and experiments, the potential for interaction with the estrogen a nd androgen pathways is supported by the SAP
conclusion that the chlorotriazine s [(including atrazine and its chlo rinated degradates 2-chloro-4- isoproylamino-6-amino-s-triazine(DEA), chlord iamino-s-triazine(DACT), and 2-chloro-21 6- ethylamino-4-amino-s-triazine (DIA)] function through an upstr eam, neuroendocrine mode of action (MOA) that suppresses the hypothalami c release of gonadotropin releasing hormone (GnRH) and therefore luteiniz ing hormone (LH), which may result in downstream effects on estrogen and androgen signaling pathways. Ther efore, based on a WoE analysis that also
considered the triazine CMG and SAP reports on atrazine ( http://www.epa.gov/pestic ides/reregistration/at razine/atrazine_update.htm ), atrazine has the potential to interact with th e estrogen and androgen pathways in mammals and other wildlife.

The available data for atrazine do not provide conv incing evidence of a potential to interact with the thyroid pathway in mammals or other wildlife.

In determining whether additional data are needed for risk assessment purposes based on the Tier 1 screen, the available data and Tier 1 conclusions are considered within the context of existing regulatory endpoints. The current point of de parture (POD) used for human health risk assessment for the triazine CMG is 2.56 mg/kg/ day and is based on a LH suppression with atrazine which is regarded as a precursor event in the neuroendocrine m ode of action (MOA) of the triazines. In a Tier 1-like male pubertal assa y, atrazine caused a delay in preputial separation at the lowest dose tested (12.5 mg/kg/day). This dose is approximately five-fold greater than ( i.e ., less sensitive) than the POD of 2.56 mg/kg/day used for huma n health risk assessment. Estrogen-related effects ( i.e ., delays in the onset of puberty a nd altered estrous cyclicity) were seen at doses greater than 50 mg/kg/da y in the female pubertal assay (Laws et al., 2000).

With respect to ecological risk assessment, the current risk assessment endpoint for chronic risk to aquatic vertebrates is based on observations of reduced fecundity in medaka ( Oryzias latipes ) exposed at 0.5 μg/L and above. For non-mammalian terrestrial vertebrates, the current chronic risk assessment endpoint is based on reduced malla rd duck hatchling weight at 75 mg/kg diet and above, with reproductive effects (e.g., reduced nu mber of hatchlings) observed only at higher
treatment levels.

Therefore, based on the WoE analysis of EDSP Ti er 1 guideline-like studies and other available data, EDSP Tier 2 testing with mammals, fish, amphibians, or birds is not recommended for atrazine at this time because it is not expected to impact current EPA-established regulatory endpoints for human health or eco logical risk assessment.ATRAZINE FINAL

Page 4 of 159 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide the necessary empirical data to evaluate the poten tial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) hormone pathways. This interaction includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as perturbations of the hypothalamic-pituitary-gon adal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In addition to the available Tier 1 data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were
considered in this assessment. Also considered in the determination were the FIFRA Scientific Advisory Panel (SAP) reports on atrazine (2009 – 2011) and the common mechanism grouping (CMG) of atrazine with other similar triazines.

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

On December 17, 2014, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-of-evidence (WoE) analysis of the potential interaction of atrazine with the E, A or T signaling pathways. The T1Wo ERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Technical atrazine is relatively insoluble in wa ter (33 mg/L at 20°C), with an octanol water partition coefficient (K ow ) of 501. The vapor pressure of atrazine is 3.0×10

− 7 mm Hg at 20°C. If released to air, atrazine will exist in both the vapor and particulate phases. Vapor-phase atrazine will be degraded in the atmosphere by reaction with photochemically-produced hydroxyl radicals (t 1⁄2 = 14 hours), and the particulate-phase will be removed by wet or dry deposition. In soil, atrazine is subject to photolys is with half-lives ranging between 12 to 45 days; photolysis degradates include 2-chloro-4-isoproylamino- 6-amino-s-triazine(DEA ), chlordiamino-striazine(DACT) and 2-chloro-21 6- ethylamino-4-amino-s-triazine (DIA ). Atrazine is expected to be mobile in soil, but volatilization from moist or dry soil surfaces, or from water surfaces, is not expected to be an important fate process. Biodegradation can occu r in soil and water; aerobic and
anaerobic soil metabolism half- lives are 146 days and 159 days, respectively. Biodegradation of atrazine in soil is influenced by the moisture co ntent, with increased biodegradation (decreased half-lives) in wetter soil s. There is no evidence that atrazine degrades via hydrolysis at pH 5, 7 or 9, and it is persistent (stable) to direct photodegration in water ( i.e. , aqueous photolysis t 1⁄2 =ATRAZINE FINAL

Page 5 of 159 335 days). A bioconcentration factor of up to 100 suggests that biocon centration in aquatic organisms is low to moderate.

The available information considered in determining the potential interaction of atrazine with the E, A, T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxicity st udies and other published studies. These data are summarized in Section III.A through III.C. An an alysis of the data submitted to the Agency, using the WoE approach outlined by the Agency (U SEPA, 2011), is presented in Section IV. The
EDSP Tier 2 studies recommendations are presented in Section V.

In the Part 158 carcinogenicity study, atrazine induced mammary tumor in Sprague-Dawley rats. Based on the mammary tumors seen in the Spra gue Dawley rats. Addi tional studies were
conducted to determine the mechanisim of ma mmary carcinogenesis. Additionally, around the time of the evalution of atrazine for the Registration Eligibility Decision (RED), several studies concerning the potential effect s of atrazine on amphibian development were published. As a result, potential human health and ecological effects of atrazine were reviewed extensively in many FIFRA Scientific Advisory Panel (SAP ) meetings held from 2003 to 2012 (USEPA 2003, 2007, 2010a, 2010b, 2011a, and 2012). Therefore, the Wo E determination also considered the SAP reports on atrazine (2009 – 2011) and the CMG of atrazine and other similar triazines. EPA
concluded in 2002 that the triazi ne-containing pesticides atrazine , simazine, and propazine and their three chlorinated degradates ( i.e ., DEA, DIA and DACT) shoul d be included in a CMG 1 (the triazine CMG) and c onsidered through a cumulative risk assessment. EPA’s triazine
cumulative risk assessment 2 considered the combined effects of atrazine, propazine and simazine, which share a common neuroendocrine m echanism of toxicity which may result in both reproductive and developmental alterations. Studies have demonstrated that the CMG triazines can cause a disruption of the HPG axis in rats by alteration of levels of luteinizing hormone (LH). Atrazine d ecreases LH levels by suppres sion of gonadotrophin-releasing hormone (GnRH) from the hypothalamus. Suppression of LH is regarded as a precursor event in
the neuroendocrine mode of ac tion (MOA) of the triazines.

The grouping of the CMG triazines based on a common MOA was presented to the FIFRA SAP
in 2000 3 . The SAP agreed with the Agency’s conclu sion that there is sufficient evidence to support the proposed grouping for the neuroendoc rine-related effects. From 2009 – 2012, EPA

1 EPA. 2002. The Grouping of a Series of Triazine Pesticides Based on a Common Mechanism of Toxicity.

http://www.epa.gov/pesticides/cum ulative/common_mech_groups.htm

2 http://www.epa.gov/pesticides/cumulative/

. 3 http://www.epa.gov/scipoly/sap/meetings/2000/062700_mtg.htm

.ATRAZINE FINAL

Page 6 of 159 presented information to several SAP meetings fo r independent scientific peer review of new
studies on atrazine 4 . The overall conclusions from these meetings provided further support to a
neuroendocrine MOA for the triazines with a decrease in LH as a precursor event to developmental and reproductive e ffects within an adverse outcom e pathway in which key events may be mediated through the E and A signaling pathways.

II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix

1.

B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Orde r recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 Guideline studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team on atrazine (USEPA, 20 10c). After reviewing the submitted OSRI, the Agency determined that the available OSRI fulfilled requirements for all the Tier 1 assays, with the ex ception of the Amphibian Metamo rphosis Assay (AMA), as the existing amphibian metamorphosis studies did no t examine the thyroid for histopathological changes. Therefore, additional data were reque sted for the AMA. In its WoE analysis, the Agency considered the previous reviews and conclusions for atrazine data submitted by the registrant and for open literatu re publications that were pres ented to the FIFRA SAP in May

2003 5 , October 2007 6 , and June 2012 7 . The Agency has also conducted a more recent search of

4 See http://www.epa.gov/pesticid es/reregistration/atrazine/atrazine_update.htm or links to specific SAP meetings.

5 US EPA. 2003. White Paper on Potential Developmental Effects of Atrazine on Amphibians in Support of an Interim Reregistration Eligibility Decision on Atrazine. Pr esented to the FIFRA Scientific Advisory Panel (SAP), June 17-20, 2003. US EPA Office of Pesticide Programs, Washington, DC. Available at http://www.epa.gov/scipoly/sap/meetings/2003/061703_mtg.htm

. 6 US EPA. 2007. Overview of Open Literature. Presented to the FIFRA Scientific Advisory Panel (SAP) on the Potential for Atrazine to Affect Amphibian Gonadal Development, October 9-11, 2007. US EPA Office of Pesticide Programs, Washington, DC. Docket ID: EPA-HQ-OPP-2007-0498.

7 US EPA. 2012. Appendix C – Open Literature Review of Amphibian Data. Presented to the FIFRA Scientific Advisory Panel (SAP) on the Problem Formulation for the Environmental Fate and Ecological Risk Assessment for Atrazine, June 12-15, 2012. 248 p. US EPA Offi ce of Pesticide Programs, Washington, DC. Docket ID: EPA-HQOPP-2012-0230.ATRAZINE FINAL

Page 7 of 159 available scientific literature fo r any additional relevant informa tion. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies c onsidered but no t utilized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and approach used in the WoE determination on the potential of a
substance to interact with endoc rine-related processes (i.e., E, A or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA SAP (SAP, 2013). As described in the 2011WoE guidance document, the weight of evidence process identifies how the indivi dual lines of evidence are assembled and integrated along two concepts ( i.e ., complementarity and redundancy) within th e conceptual framework of an adverse outcome pathway). Broadly, there are four ma in steps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the individual studies for their scientific quality and relevance in evaluating potential endocrine interaction(s). The second step is to integrate the data along different levels of biological organization while examining the extent of complementarity ( i.e ., the concordance of endpoints within an assay that measures multiple endpoints) and redundancy ( i.e ., the concordance of endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to
evaluate whether additional te sting is needed based on the evidence and conclusions described
above.

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data; or 2) OSRI.
The EDSP Tier 1 data include a ba ttery of 11 assays consisting of in vitro and mammalian and
wildlife in vivo assays. The Tier 1 assays were designed specifically to eval uate a number of key biological events including potential effects on r eceptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other e ffects on the HPG and HPT axes. The OSRI may include published literature studies as well as studies conducted under USEPA (often referred to as Part 158 data) or the Organization for Economic Cooperation and Development (OECD) guidelines submitted in support of pesticide registra tions. Each study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, concordance of the responses in the indi vidual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

Page 8 of 159 The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data, as well as characterizing the extent to which the available data suppor t a hypothesis that a chemical has the potential to interact E, A, or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, coherence, strength, and consistency of the body of evidence . The current EDSP Tier 1 screening assays are
meant to evaluate whether or not a chemical can interact with E, A and T consisting of different levels of biological organization from a molecular initiating ev ent ,such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment and fecundity at the whole organism level. The extent of expression of responses at higher levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.

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

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

For these WoE analyses, overt toxicity was gene rally defined in accordance with EPA’s current approach as used by OPP in reviewing Part 158 studies for both human and ecological risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g ., organ weights/histopathology), biochemical ( e.g ., blood chemistry), and other clinical signs ( e.g ., lethargy) were also considered when evaluating overt toxicity, es pecially if the effects were severe. In some instances, one parameter ( i.e ., death or >10% decr ease in mammalian bodyATRAZINE FINAL

Page 9 of 159 weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g ., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addressing th ese three main questions is fundamental to the WoE process and in determining whether addi tional data are need ed. In addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) discussion of ke y studies; 3) description of inc onsistent or conflicting data; 4) overall strength of evidence supporting a conclusi on; and, 5) what, if a ny, additional data are needed and why. Assessing the need for additiona l data is based on a case-by-case analysis
which took all available toxic ity data into account.

The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.ATRAZINE FINAL

Page 10 of 159 A. EDSP Tier 1 Screening Assays

The Tier 1 assays submitted in response to the agency’s test order for atrazine are shown below
in Table 1.

Table 1. Tier 1 Screening Assays for Atrazine

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement satisfied by OSRI (Tennant et al .; USEPA, 2009)

ER α Transcriptional Activation Assay (Human cel l line HeLa 9903) OSCPP 890.1300; OECD 455 Requirement satisfied by OSRI (Fujimoto and Honda, 2003)

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement satisfied by OSRI (USEPA, 2008a)

Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550; OECD 456 Requirement satisfied by OSRI (USEPA, 2008c)

Aromatase Assay (human recombinant microsomes) OSCPP 890.1200 Requirement satisfied by OSRI (Higley et al ., 2010; USEPA, 2008b)

Uterotrophic Assay (Rat) OSCPP 890.1600; OECD 440 Requirement satisfied by OSRI (Yamaski et al. , 2000)

Hershberger Assay (Rat) OSCPP 890.1400; OECD 441 Requirement satisfied by OSRI (Ashby et al ., 2004: Yamaski et al. , 2004)

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement satisfied by OSRI (Laws et al ., 2000)

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement satisfied by OSRI (Stoker et al ., 2000)

Fish Short-term Reproduction Assay OSCPP 890.1350; OECD 229 Requirement satisfied by OSRI (Battelle, 2005)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; OECD 231 Requirement satisfied by OSRI (MRID 47153501: Hosmer et al. 2007; Kloas et al ., 2009; MRID 48614801)ATRAZINE FINAL

Page 11 of 159 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for atrazine to interact with the estrogen hormone pathway is summarized in Table

2 . The various targets of the estrogen pathway acro ss the relevant Tier 1 assays are delineated so as to facilitate determination of potential for es trogenic, anti-estrogenic or HPG axis effects.

Table 2 also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reported in the table and not considered further in the WoE assessment.

All Tier 1 assays evaluating the estrogen pa thway were satisfied by OSRI. Atrazine was reported as negative/equivocal for ER binding in the Integrated Summary Report for the Validation of the ER binding assay (USEPA, 2009) and negative for ER binding up to 10

− 3 M, although pre-incubation with atrazine demonstrated a reduction of [ 3 H]-estradiol binding (Tennant et al. , 1994b). Atrazine also was negative for ER transactivation in the MtT/E-2 cell line at concentrations up to 10

− 4 M (Fujimoto and Honda, 2003). Additional OSRI that were considered also supported these negativ e conclusions for ER binding (Blair et al., 2000 and

O’Conner et al., 2000) and for ER transactivation in a variety of cell lines such as Chinese hamster ovary (CHO), human breast cancer (MCF-7 ), and human ductal br east epithelial tumor (T47-D) cells transfected w ith human estrogen receptor- alpha (hER α ) and/or human estrogen
receptor- beta (hER β ) (Kojima et al., 2004; Balaguer et al., 1999; Connor et al., 1996; Fukamachi et al., 2004; and Legler et al., 2002).

Atrazine also was negative in aromatase assays (Higley et al. , 2010 and USEPA, 2008b). Higley
et al. (2010) compared the indirect and direct effects of atrazine on aromatase activity by: 1) exposing cells to atrazine for 48 hours followed by rinsing of the cells (indirect); or 2) by exposing untreated cells to atrazine during the conduct of the tritium-release assay (direct); and 3) finally combined exposure (cells exposed to atrazine for 48 hours followed by rinsing of the cells, and then atrazine added again during the conduct of the tr itium-release assay). This study
demonstrated no direct effect on aromatase ac tivity, but induction of aromatase activity at atrazine concentrations of 10 and 100 μM af ter indirect exposure (1.9- and 2.25-fold, respectively) and combined exposure (1.5- and 1.75-fold, respectively). Additional OSRI were available to evaluate aromatase activity. Hinfray et al. (2006) reported that at razine had no effect aromatase activity in rainbow trout ( Oncorhynchus mykiss ) brain and ovarian microsomes at concentrations up to 100 μM. Sanderson et al. (2001 and 2002) demonstrated induction of human aromatase CYP19 (aromatase) mRNA levels in adrenocortical carcinoma (H295R) and plancental choriocarcinoma (JEG -3) cells, but no induction occurre d in MCF-7 cells. Similarly, atrazine exposure resulted in increased aromat ase activity in human granulosa-lutein cells, but not in human endometrial stromal cells (Holloway et al. , 2008). Finally, Tinfo et al. (2011) reported an up to 2-fold induction (at 30 μM) in aromatase activity in rat primary granulosa cells. In the OSRI that satisfied the Tier 1 testing requirements for steroidogenesis (USEPA, 2008c),ATRAZINE FINAL

Page 12 of 159 17 β -estradiol production was induced at 100 μM and 10 μM atrazine in four out of five studies (results only reporte d graphically).

Atrazine (up to 50 mg/kg/day) was negative in OSRI used to satisfy the uterotrophic assay requirement (Yamasaki et al. , 2000). No increase in uterine we ights were also reported in uterotrophic assays by Connor et al . (1996), O’Connor et al ., (2000) and Tennant et al ., (1994a).
It is noted that O’Connor et al ., (2000) reported in creases in uterine epithel ial cell height in the 150 and 200 mg/kg/day rats.

While numerous effects were observed in the OSRI that satisfied the Tier 1 testing requirements for the female pubertal rat assay (Laws et al. , 2000), it is noted that the effects that occurred at the high dose (200 mg/kg/day) were in the pres ence of overt toxicity (12% decrease in body weight). Decreases in uterine weight (without fl uid) were significantly decreased at 100 and 200 mg/kg/day and and ovarian weights were decrea sed only at 200 mg/kg/ day. Pituitary weights were significantly decreased in these two gr oups as well. Histopathological examination determined that corpus luteum development wa s decreased in the 200 mg/kg/day females, with increased atretic follicles and uterine hypopl asia. The mean age at VO was increased by approximately 3 to 7 days at 50, 100 and 200 mg /kg/day, respectively, and the weights at VO also were increased for these dose groups by appr oximately 20%, suggesting that the delay in attaining VO was not related to reductions in body weight. In addition, the mean age at first vaginal estrus was delayed by approximately se ven and ten days in the 100 and 200 mg/kg/day
animals.

Additionally, decreased uterine weights were reported on PND 30, 33 and 43 in peripubertal Wistar rats dosed at 100 mg/kg/day. Termin al body weights were d ecreased 11% in rats terminated on PND 43. In the same study, decreases in uterine weight were also reported at 100 mg/kg/day in Sprague Dawley (SD) rats, but did not reach statisti cal significance (Ashby et al

., 2002; MRID 45722401) . Delays in the attainment of vagina l opening (VO) of 3 days were noted at the high dose in Wistart rats. In SD ra ts, VO was delayed 2.5 days at the mid dose (30 mg/kg/day) and 3 days at the high dose in (Ashby et al ., 2002; MRID 45722401)

.
In a Part 158 21-day dermal toxic ity in rats, decreases in mean uterus and ovary weights in highdose (1000 mg/kg/day) females were observed in the presen ce of overt toxicity. No corresponding gross or microscopic morphology findings were reported.

In a Part 158 two-generation rat reproduc tion toxicity study (MRID 40431303), no treatmentrelated effects were reported fo r any reproductive parameters up to the atrazine high dose of 38.7 mg/kg/day. Foradori et al. (2014) investigated the effects of atrazine treat ment (bolus or diet) on estrous cyclicity and reproductive performance in SD and Long-Evans (LE ) rats. The only
treatment-related effects noted we re in the bolus-treatment gro up. The mean number of corpora lutea was significantly decreased in the 100 mg/kg/day SD females, and the number of ova shed per animal was decreased in al l 100 mg/kg/day animals and the 50 mg/kg/day SD females. InATRAZINE FINAL

Page 13 of 159 addition, the percentage of female s with irregular/abnormal estrous cycles was increased for both strains at 50 and 100 mg/kg/day. In other OSRI, Narotsky et al. (2001) examined different periods of gestation (gestation da y (GD) 6-10) to determine potenti al susceptibility to atrazineinduced resorptions in Fischer (F344), SD and LE rats. In the pilot study in F344 rats (100, 200 and 300 mg/kg/day), there was a dose-related increase in the number of full litter resporptions at all doses; however, the effect occurred in the presence of overt toxicity (>10% decrease in body weights). In the main study, F334, SD and LE ra ts were adiministered atrazine at 0 or 200 mg/kg/day from GD 6-10 or dosed at 25, 50 or 100 mg/kg/day atrazine from GD 6-10. In F344 rats, full litter resporptions were seen at mi d dose (50 mg/kg/day; non-si gnificant) and high dose (100 mg/kg/day; significant). Resporptions at thes e doses occured in the presence of significant decreases in maternal body weights. SD and LE rats did not exhibit resorptions at atrazine doses up to 100 mg/kg/day. In F344 rats, there was a d ecrease in the percenta ge of dams delivering
after GD 21 at ≥ 50 mg/kg/day, and in SD rats an d LE rats at 200 mg/kg/day.

In other OSRI, Davis et al. , (2011) examined the effects of at razine treatment in SD rats on female offspring reproductive i ndices (parental effects were no t evaluated). VO was delayed in amimals at 100 mg/kg/day (total dose) using once a day or twice a day dosing regimens. This dose also resulted in significant decrease s in offspring body weights and increased pup mortaility. The authors reported no significant effect on mammary gland development or estrous cyclicity up to 100 mg/kg/day.

In a Part 158 rat developmental/reproduc tive toxicity study (MRID 41065201), reproductive toxicity (full litter resporptions ) and decreases in pup survival and weight were observed at 125 mg/kg/day of atrazine, a dose that resulted in overt toxicity (decr eased body weight). In another Part 158 developmental toxicity study in ra ts (MRID 00143008), no treatment-related changes were observed in any reproductive parameters up to 700 mg/kg/day. Finally, in a prenatal developmental toxicity study in rabbits, no estrogen -related effects were observed in the absence of overt toxicity (>10% decrease in maternal body weight). In th is study increases in the mean number of resorptions and the percentage of pos t-implantation loss, decrease in the mean number of live fetuses were seen at 75 mg/kg/day.

In a Part 158 carcinogenicity study in SD rats (MRID 40629302) , there was a significant increase in the incidence of mammary adenocarcinomas at doses of 3.5 mg/kg/day and higher. A significant increase in fibroadenomas/adenomas was reported at 50 mg/kg/day and a significant increase in total mammary tumors was reported at 25 and 50 mg/kg/day. Overt toxicity (>10% decrease in body weight) was seen at 25 and 50 mg/kg/day. No estrogen -related effects were
observed in the other Part 158 mammalian toxicity studies.

Cooper et al . (2000) examined atrazine effects on th e HPG axis in ovariectomized SD and LE rats. Although atrazine treatment (50-300 mg/kg/day) for up to 21 days attenuated serum LH and prolactin concentrations, serum 17 β -estradiol and pituitary wei ghts were not affected. InATRAZINE FINAL

Page 14 of 159 addition, although single doses of atrazine did not affect ovulation, repeated dosing with 300 mg/kg/day prevented subseque nt proestrus and ovulation.

There were no estrogen-related effects observed in the OSRI Fish Short Term Reproduction Assay (FSTRA) study that was used to satisf y the Tier 1 testing requirements for FSTRA (Battelle, 2005) as well as an a dditional FSTRA OSRI study (Bringolf et al. , 2004).

In a chronic partial life-cycle study with the freshwater brook trout ( Salvelinus fontinalis ; MRID 00024377), statistically significan t (p<0.01) decreases in F 1 growth (weight) were noted at
concentrations ≥ 0.12 mg/L, but no effects were noted on fertility or fecundity at concentrations up to 0.72 mg/L (however, large variability in f ecundity output for both controls and treatment groups was observed). Similarly, in a chronic fu ll life-cycle study with th e freshwater fathead minnows ( Pimephales promelas ; MRID 42547103), decreases in F 0 and F 1 growth (F 0 length ↓ 5 – 10% and weight ↓ 19%; F 1 length ↓ 10 – 23% and weight ↓ 27 – 54%) were noted at
concentrations ≥ 0.24 mg/L and ≥ 0.15 mg/L, respectively, but no effects were noted on fertility or fecundity at concentrations up to 2.0 mg/L.

An early life-stage study by Le Mer et al. (2013) in stickleback ( Gasterosteus aculeatus ), and sexual development studies in freshwater zebrafish ( Danio rerio ) by Corvi et al. (2012), no effects on reproductive or sexual development we re observed. In a 38-d reproduction study (test design similar to FSTRA) by Papoulias et al. (2014) with freshwater Japanese rice fish (aka medaka; Oryzias latipes ), egg production was decreased after exposure to atrazine at ≥ 0.5 μg/L compared to solvent control ( 0.02% acetone); it is noted that the magnitude of decreased fecundity (as cumulative egg production) was similar across the exposure groups (100-fold difference) ( ↓ 40, 45, and 49% at 0.5, 5.0, and 50 μg/L, resp ectively, reviewer calculated) and embryo mortality in the control group was relativ ely high (38% mortality for 24-hr old embyros). No treatment-related effects on reproductive hormones or gonado-somatic index (GSI) were reported. In regards to gonad hi stopathology, the study author repor ted, there were significantly (p<0.01) fewer pre-vitellogenic oocytes and more vitellogenic oocytes in the 0.5 and 5.0 μg/L atrazine groups at 38 days compared to contro ls. In a short-term reproduction assay (similar to FSTRA), Tillitt et al. (20 10), reduced fecundity ( ↓ 19-39%, p<0.05) was reported and effects on ovarian stage in the fathead minnow exposed to atrazine at nominal concentrations of 0.5, 5.0
and 50 μ g/L, as compared to solvent (0.02% acetone) controls. In addition to the studies above which evaluated effects on reproductive parameters in fish (as well as survival and growth in some cases), an early life-stage studies which examined survival and growth in fish were
available for atrazine. In an early life-stage study with the estu arine/marine species sheepshead minnows ( Cyprinodon variegatus ; MRID 46648203), no effects of atrazine treatment were observed on hatching, fry survival or growth at concentrations up to 2.2 mg/L. However, at study termination, fish in the 2.2 mg/L treatment ha d mean body lengths and wet weights which were significantly (p<0.05) lower than controls by 17 and 46%, resp ectively, resulting in a NOAEC and LOAEC of 1.1 and 2.2 mg/L, respectivel y. For juvenile zebrafish (Plhalova et al . 2012), body weights and growth rates were reported to be significantly decreased (p<0.05) in fish in theATRAZINE FINAL

Page 15 of 159 high atrazine concentration group (90 μg/L; bod y weight data were not provided in study. Histopathological lesions in the liver were also reported at this concentration.

In the avian reproduction toxicity studies in mallard ducks ( Anas platyrhynchus ; MRID 42547101), dietary exposure to atrazine was associated with a statistically significant difference in hatchling body weight at > 75 mg/kg diet; fewer eggs laid per pen and mean food consumption at > 225 mg/kg diet; and fewer liv e embryos, and hatchli ngs per eggs set and weight gain in adult males at 675 mg/kg diet. Al l effects noted were sta tistically significant at p<0.05. In the bobwhite quail ( Colinus virginianus ; MRID 42547102), the numbe r of eggs laid, viable embryos, live 3-week embryos, hatchlin gs, and 14-day old survivors generally were
decreased. No treatment-related eff ects were observed for the numbe rs eggs cracked or eggshell thickness. There were no effects on female Japanese quail ( Coturnix japonica ) reproductive organs or hormones in a study by Wilhelms et al . (2006) for atrazine exposures up to 1000 mg/kg diet.

In ovo exposure to atrazine was conducted with alligator ( Alligator mississippiensis ) eggs and results were reported in two reports (Crain et al. , 1997 and Spiteri et al. , 1999) based on the same experimental group. No statistically si gnficiant effects on steroidogenesis ( i.e. , aromatase activity) were observed in hepati c or gonadal-adrenal mesonephros (GAM) complex tissues from neonatal alligators that were treated in ovo and incubated at a fema le-producing temperature. Decreases in egg chorioallantoic fluid (CAF) concentrations of 17 β -estradiol and testosterone were observed, but did not reach statistical significance. No effects were seen on hepatic aromatase activity or gonadal hi stopathology. Maternal plasma 17 β -estradiol concentration was not affected by dietary atr azine exposure in pregnant Northern water snakes ( Nerodia sipedon ; Neuman-Lee et al. 2014), but the proportion of live bi rths was decreased in a non-dose responsive manner at the medium- and high-doses of atrazine (20 and 200 ppb, respectively).

In an in vitro examination of ovulation with African clawed frogs ( Xenopus laevis ) oocytes (Orton et al. , 2009), stimulation of ovulation, with c oncomitant increases in progesterone (  63%-104%, p<0.001) and testosterone (  24%, p<0.01) concentrations in culture media, was
observed with no effect on 17 β -estradiol concentration.

Studies examining the potential effects of at razine on amphibian de velopment have been previously reviewed by the Agency and evaluations of these studies were presented to multiple SAPs (USEPA 2003, 2007 and 2012). These studies examined a variety of endpoints including: time to metamorphosis (development), growth, gona dal development, sex ratio, and other aspects of sexual development under a range of atrazine test concentrations, e xperimental designs, and test species. Further discussions of the test conditions and results of these studies are presented in the Agency’s evaluations (White Papers) in s upport of each of the SAPs . Overall, the available data reflect varying responses in sexual a nd metamorphosis development and growth across species and studies; however, the evaluations con ducted previously provide detailed analysis of study design elem ents that may have limited the util ity of the various studie s. In regards to theATRAZINE FINAL

Page 16 of 159 estrogen pathway, investigations of in vivo atrazine exposure in amphibians have primarily focused on potential effects on sexual/gonadal development ( i.e. , intersex/testicular oocytes). Atrazine exposure in X. laevis (MRID 47153501; Hosmer et al. , 2007; Kloas et al. , 2009) from Nieuwkoop-Faber (NF) stages 46 to 66 at con centrations of 0.01 to 100 μg/L did not affect amphibian gonadal development. No frogs displa ying intersex or testicular oocytes were observed, and no treatment effect was observed on sex ratios. In another investigation of sexual
differentiation in X. laevis (Oka et al. , 2008), the percentage of females was significantly increased (p<0.05) at atrazine concentrations of 10 (62.1% female) and 100 ppb (72% female) compared to controls. No treatment-related e ffects were noted on gonad histopathology or liver vitellogenin (VTG) concentration. Atrazine at concentrations up to 30 μg/L had no effect on metamorphosis, sex ratio, or gonadal development in spotted marsh frogs ( Limnodynastes

tasmaniensis ) (Spolyarich et al. , 2010).ATRAZINEFINALPage 17 of 159Table 2: Estrogenic/Anti-Estrogenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Estrogen level Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Obsereved 2 Overt Toxicity Observed 3EDSP Tier 1 AssaysER BindingRequirement satisfied by OSRI (Tennantet al., 1994b and USEPA, 2009)ERTARequirement satisfied by OSRI (Fujimoto and Honda, 2003)AromataseRequirement satisfied by OSRI (Higleyet al., 2010 and USEPA, 2008b)FSTRARequirement satisfied by OSRI (Battelle, 2005)Female Pubertal RatRequirement satisfied by OSRI (Lawset al., 2000)SteroidogenesisRequirement satisfied by OSRI (USEPA, 2008c)UterotrophicRequirement satisfied by OSRI (Yamasakiet al., 2000)OSRIER Binding (USEPA, 2009) NER Binding (MRID43598618; Tennantet al.,1994b)N4ER Binding (Blairet al.,2000)NER Binding (O’Conneret al.,2000)NATRAZINEFINALPage 18 of 159Table 2: Estrogenic/Anti-Estrogenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Estrogen level Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Obsereved 2 Overt Toxicity Observed 3ERTA (Kojimaet al.,2004)NERTA (Fujimoto and Honda,2003)NERTA (Balagueret al.,1999)NERTA (Connoret al.,1996)NERTA (Fukamachiet al.,2004)NERTA (Legleret al.,2002)NAromatase (USEPA, 2008b)NAromatase (Higleyet al.,2010)N5Aromatase (Sandersonet al.,2001)PAromatase (Sandersonet al.,2002)PAromatase (Rainbow Trout;Hinfreyet al., 2006)NAromatase (Hollowayet al.,2008)PAromatase (Tinfoet al.,2011)PATRAZINEFINALPage 19 of 159Table 2: Estrogenic/Anti-Estrogenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Estrogen level Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Obsereved 2 Overt Toxicity Observed 3FSTRA (Battelle, 2005)NN NNR N N N NFSTRA (Bringolfet al.,2004)NEN NNR N N N NModified FSTRA(Fathead Minnow; Tillittetal., 2010)NEN PFcd:↓19-39%(L, M,H)NModfied FSTRA (JapaneseMedaka; Papouliaset al.,2014)NNPFrt: NE:Fcd:↓36-42%(L, M, H)NFemale Pubertal Rat(Lawset al., 2000)NEwo/fluid:↓31%(MH)NN↓16%(L,MH)P(MH)Age:↑3.4,4.5 d(M,MH);BW:↑(M,MH)X(H)X(H)Steroidogenesis(USEPA, 2008c)PUterotrophic(Yamasakiet al., 2000)NNNNUterotrophic(Tennantet al.,1994a)↓(M)X(M,H)X(H)ATRAZINEFINALPage 20 of 159Table 2: Estrogenic/Anti-Estrogenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Estrogen level Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Obsereved 2 Overt Toxicity Observed 3Uterotrophic(Connoret al., 1996)NNENEUterotrophic (Rat; MRID45722401; Ashbyet al.,2002, Exp 1)↓41%(H)NEX(H)NUterotrophic (Rat; MRID45722401; Ashbyet al.,2002, Exp 2)↓55%(H)NEX(H)NUterotrophic (Rat; MRID45722401; Ashbyet al.,2002, Exp 3)NNX(H)X(H)Uterotrophic (Rat; MRID45722401; Ashbyet al.,2002, Exp 4)N↑2.5,3 day(M, H)X(M,H)NUterotrophic (Rat; O’Conneret al., 2000)NX(M,H)X(H)21-Day Dermal Toxicity(Rabbit; MRID 42089902)↓44%↓36%NNX(H)X(H)Two-generation reproductiontoxicity (Rat; MRID40431303, DeSessoet al.,2014)NENNNENX(H)X(H)Reproductive toxicity, Exp 1-bolus (Rat; Foradoriet al.,2014)NEPPNNATRAZINEFINALPage 21 of 159Table 2: Estrogenic/Anti-Estrogenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Estrogen level Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Obsereved 2 Overt Toxicity Observed 3Reproductive toxicity, Exp 1-diet (Rat; Foradoriet al.,2014)NENNX(H)X(H)Reproductive toxicity, Exp 2-bolus (Rat; Foradoriet al.,2014)NENNNReproductive toxicity, Exp 2-diet (Rat; Foradoriet al.,2014)NENX(H)X(H)Reproductive toxicity, Pilot(Rat; Narotskyet al., 2001)NEFrt: N;Fcd:↓63%(L)X(M)X(M, H)Reproductive toxicity, Singledose (Rat; Narotskyet al.,2001)NENXXReproductive toxicity, F344strain (Rat; Narotskyet al.,2001)NENNX(M, H)Reproductive toxicity, S-Dstrain (Rat; Narotskyet al.,2001)NENNX(M, H)Reproductive toxicity, LEstrain (Rat; Narotskyet al.,2001)NENNX(H)ATRAZINEFINALPage 22 of 159Table 2: Estrogenic/Anti-Estrogenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Estrogen level Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Obsereved 2 Overt Toxicity Observed 3Reproductive toxicity,onedose per day(Rat; Daviset al., 2011)NENAge:↑1.5day(H)NNX(H)X(H)Reproductive toxicity,twodoses per day(Rat; Daviset al., 2011)NENAge:↑1.9day(H)NNX(H)X(H)Developmental reproductivetoxicity,in uteroexposure(Rat; DeSessoet al., 2014)NENX(H)X(H)Developmental reproductivetoxicity, Post-natal exposure(Rat; DeSessoet al., 2014)NENX(H)X(H)Developmental toxicity (Rat;MRIDs 00143008,40566302; Scialliet al.,2014)NENX(H)X(H)Developmental toxicity (Rat;MRID 41065201; Sciallietal., 2014)NRNX(H)X(H)Developmental toxicity(Rabbit; MRIDs 00143006,4056630; Scialliet al.,20142)NEFrt: N;Fcd:↓33%(H)X(H)X(H)ChronicToxicity/Carcinogenicity(Rat; MRID 40629302)NE N N NEX(MH,H)X(MH,H)ATRAZINEFINALPage 23 of 159Table 2: Estrogenic/Anti-Estrogenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Estrogen level Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Obsereved 2 Overt Toxicity Observed 3Carcinogenicity (Mouse;MRID 40431302)NE NE N NEX(MH,H)X(MH,H)Chronic Toxicity (Dog;MRID 40431301, 41293801)NE N N NX(H)X(H)Avian Toxicity (MallardDuck; MRID 42547101)Frt:↓(H);Fcd:↓(H)NAvian Toxicity (BobwhiteQuail; MRID 42547102)Frt:↓(H);Fcd:↓(H)NAvian Toxicity (JapaneseQuail; Wilhelmset al., 2006)NNFish Partial Life CycleToxicity (Brook trout; MRID.00024377)NNEarly Life-Stage Toxicity(Sticklebacks; Le Meret al.,2013)NNNFish Full Life-Cycle Toxicity(Fathead minnow, MRID42547103)NNSexual development, Study 1(Zebrafish; Corviet al.,2012)NNNATRAZINEFINALPage 24 of 159Table 2: Estrogenic/Anti-Estrogenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Estrogen level Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Obsereved 2 Overt Toxicity Observed 3Sexual development, Study 2(Zebrafish; Corviet al.,2012)NNNIn ovoexposure, (Alligator;Crainet al., 1997)NNIn ovoexposure, (Alligator;Spiteriet al., 1999)NNNDevelopmental toxicity(Watersnake; Neuman-Leeetal., 2014)NFrt:NE;Fcd:↓(M, H)NIn vitro ovulation (Frog;Ortonet al., 2009)NP (L)Amphibian metamorphosisand reproductivedevelopment (Frog; Hosmeret al., 2007 and Kloaset al.,2009)NNNAmphibian reproductivedevelopment (Frog; Okaetal., 2008)N↑%♀(H)NNAmphibian reproductivedevelopment (Frog;Spolyarichet al., 2010)NNNHPG Axis (Rat; Cooperetal., 2000)NNP(H)X(H)NATRAZINEFINALPage 25 of 1591. Key to responses: Lowest treatment (L), Medium-low treatment (ML), Low-medium treatment (LM), Medium treatment (M), High-medium treatment(HM), Medium-high treatment (MH), High test treatment (H). Arrows (↓or↑) indicate the direction of the response.A shaded cell indicates parameter isnot routinely evaluated or is not applicable in this assay.Changes in weight are absolute unless otherwise indicated.2.The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an Xin this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Competitive binding against estradiol (E2) was negative (up to 10−3log M), but pre-incubation with excess atrazine inhibited E2binding (IC50= 20 μM).5. Direct aromatase activity was not different from control at any concentration tested. Atrazine significantly induced indirect and combined aromataseactivity.P Positive findingN Negative finding (in vitro)/No effect (in vivo)NE Not examinedATRAZINE FINAL

Page 26 of 159 2. Effects on Androgen Pathway

The potential for atrazine to interact w ith the androgen pathway is summarized in Table 3 . The various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential androgenic, anti-androgenic or HPG axis effects.This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reporte d in the table and not considered further in the WoE assessment.

In an evaluation of androgenic ac tivity from OSRI that satisfied the Tier 1 testing requirements,
atrazine was classified as a non-binder (negative) in in vitro androgen receptor (AR) binding (rat ventral prostate cytosol) a ssays (USEPA, 2008a) at atrazine concentrations up to 10

− 3 M. A
classification of non-binder was also determined for atrazine in a published investigation of androgen receptor (AR) bindi ng using PanVera AR (Fang et al., 2003), with no effect on binding up to 4.28×10

− 5 M. Atrazine was also negative up in an AR transcriptional activation assays using CHO-K1 cells (Kojima et al . (2004).

In OSRI that satisfied the Tier 1 testing re quirements for steroidogene sis (USEPA, 2008c),
atrazine induced test osterone production ≥ 1.5 fold in tow labs, but produced increases of <1.5- fold at the remaining three labs. Ma le steroidogenesis was also evaluated in several literature studies. Pogrmic et al. (2009) investigated eff ects on steroidogenesis in Leydig cells harvested from peripubertal male Wistar rats. After atra zine treatment (50 or 200 mg/kg/day) for four weeks, testes, adrenal, and accessory sex or gan weights were decreased. Serum androgen concentration [testosterone + di hydroetestosterone (DHT)] was d ecreased at the high dose, and

in vitro basal and hCG-stimulated androgen produc tion was decreased in Leydig cells from atrazine-treated males. Pogrmic-Majkic et al. (2010) further examined steroidogenesis in Leydig cells ( ex vivo and in vitro ) after one, three or six days of at razine treatment. Atrazine treatment
increased basal and hCG-stimulated cAMP accumulation and demonstrated concentrationdependent effects on androgen production (w ith bi-directional e ffects on progesterone concentration). The effects of atrazine (232 μM ) on Leydig cell androgenesi s were reported to be hCG-concentration dependent. The results of the ex vivo studies showed increased concentrations of cAMP and androgen afte r one, three and five days of atrazine treatment in vivo , with peak concentrations at Day 3. Effects of atrazine tr eatment on intact or castrated male rats were investigated by Riffle et al. (2014). Serum progesterone and co rticosterone concentrations generally were increased in all dose groups with increases in inta ct rats less than in castrated rats. Serum LH, androstenedione, and testosterone co ncentrations in inta ct rats produced nonmonotonic dose-response. In contrast, LH concentr ations were increased in the castrated males compared to the intact males, and androstenedi one and testosterone conc entrations were below the limit of detection. Finally, Jin et al . (2013) investigated effect s of atrazine (up to 200 mg/kg/day) on hormone synthesis and the tran scription of steroidogenic genes in male peripubertal mice. The absolute testis weights in the low- an d high-dose groups were decreased.ATRAZINE FINAL

Page 27 of 159 Serum testosterone concentrati ons were decreased, and serum 17 β -estradiol concentrations were increased, in all treated groups i ndicating that oral atrazine expo sure during puberty affected the serum hormone homeostasis in male mice. Atrazine treatment had no effect on AR, ER α , or ER β mRNA concentrations in the testes, with varied effects on hormone mRNA levels.

In the OSRI that satisfied the Tier 1 testing re quirements for the Hersberger assay, atrazine was negative for androgenic and/or anti-androgenic activity (Ashby et al. , 2004; Yamasaki et al.

, 2004). However, Ashby et al. (2004) noted non-dose-related d ecreases in levator ani-bulbo cavernosus (LABC) weights at 50 and 100 mg/kg/day, as well as an increased glans penis weight, at the high dose during th e anti-androgenic evaluations.

In the OSRI that satisfied the Tier 1 testing requirements for the male pubertal rat assay after atrazine administration (Stoker et al. , 2000), ventral prostate weight s at PND 53 were decreased
in the ≥ 50 mg/kg/day treatment groups in a dose-relate d manner, and in the pair-fed control. Other organ weight decreases ( e.g. , seminal vesicle, epididymis, and pituitary weights) occurred at 200 mg/kg/day, but were similar to control after the recovery peri od. Preputial separation (PPS) was delayed by approximately 2-3 days in all treatment groups, but the delays were not dose-related. Changes in male gonads (weight, hi stopathology, and testoste rone concentration) were generally limited to the 200 mg/kg/day group in the presence of overt toxicity, with few differences between the high-dose and pair-fed c ontrol groups. In a modified pubertal rat study (Trentacoste et al. , 2001) with a similar dosing regimen up to 200 mg/kg/day, findings included decreases in ventral prostate and seminal vesicle weights, delayed PPS, and decreased serum
testosterone concentrations were noted only in the two highest treatment groups at overtly toxic
doses.

No androgen-related effects were observed in the Part 158 two-generation rat reproduction toxicity study (MRID 40431303). Three modified reproductive toxicity studies in male rats
were considered as OSRI. Fraites et al. , 2011, in a companion study to Davis et al. (2011)
examined the effects of atrazine treatment in SD rats on male offspring reproductive indices (parental effects were not evaluated). Mean abso lute pituitary weight was decreased in male offspring in the once daily dosing regimen in the medium-low and medium-high dose groups ( ↓ 7 and 8%, respectively) and in the high-dose males ( ↓ 12%). No treatment-related effects on organ weights were noted in any dose groups in th e twice daily dosing regi men. No effects on anogenital distance (AGD), AGD index, achievement of PPS, or behavioral endpoints were observed for either dosing regimen (once daily or twice daily) at up to 100 mg/kg/day. Overt toxicity was seen at 100 mg/kg/day in pups ( ≥ 10% decrease in body weight). In a male reproductive toxicity study by Rosenberg et al. (2008), dose-dependent differences in pup survival were noted ( ↓ 25%) at 75 and 100 m g/kg/day, with de creases in AGD index at PND 21. PPS was minimally delayed at 50 and 75 mg/kg/ day, but was delayed up to three days at 100 mg/kg/day. In addition, serum testosterone c oncentrations were decreased at atrazine
concentrations ≥ 50 mg/kg/day on PND 60. Effects observed at 75 and 100 mg/kg/day occurredATRAZINE FINAL

Page 28 of 159 in the presence of overt toxicity (>10% decrease in body weight). In another male reproductive toxicity study (Song et al. , 2014), anti-androgenic e ffects were noted in the presence of overt toxicity at the atrazine high dos e (154 mg/kg/day). Decreased se rum testosterone and inhibin-B concentrations, increased FSH concentrations, di srupted testicular orga nization, and decreased sperm counts were seen in the absence of overt toxcicty at 77 mg/kg/day. Addtionally, in a developmental/reproductive toxicity study by DeSesso et al . (2014), the effects of atrazine treatment during gestation and lactation were in vestigated. The incidence of abnormal sperm was increased and pituitary weights we re decreased in pups born after in utero exposure (GD 6 to 21) at a dose (125 mg/kg/day) that was overtly toxic ( ↓ body weight).

Since simazine belongs to the triazine CMG, evidence of androgen-related effects have been
noted with atrazine and its ch lorinated metabolites (Stoker et al . 2000; 2002). There have been a number of studies demonstrating that atrazine delays male pubert y in different strains of rats following both peripubertal and perinatal exposure (Stoker et al. 2000; Friedmann, 2002;

Trentacoste et al. 2001; Rayner et al. 2006 and Rosenberg et al. 2008; cited in the SAP reports for atrazine). Stoker et al . (2002) have also demonstrated that chlorinated metabolites of atrazine possess a similar mode of action with rega rd to androgenic-related effects. In a male pubertal protocol, atrazine and the chlorinated metabolites of atrazine significantly (p<0.05) delayed preputial separation, and decreased the weights of reproduc tive tissues associated with androgenic responses. The effects are consistent with the effect of atrazine on the central nervous system ( i.e. , suppression of GnRH/decreased pituitar y secretion of LH) and subsequent alterations in the hormonal control of pubertal de velopment. Decreased LH secretion would lead to insufficient stimulation of the gonads during puberty to produce the ne cessary steroids for androgen-dependent maturation of the sex accessory tissues in the male. Therefore, the delayed growth of the reproductive tract in the male supports the reported alterati on of the hypothalamic control of pubertal progression.

There were no androgen-related effect s, except for noted differences in male testicular staging in the study that satisfied the Tier 1 testing requirements for the FSTRA (Battelle, 2005). Additionally, there were no a ndrogen-related effects observe d in an additional FSTRA considered as OSRI (Bringolf et al. , 2004). Hepatic VTG production wa s negative in male carp ( Cyprinus carpio ) hepatocytes exposed to atrazine at concentrations up to 30 μM (Sanderson et
al. , 2001). Alterations in gene e xpression associated with endocrine/reproductive development were observed in zebrafish exposed to atrazine concentrations of ≥ 0.3 μg/L ( Weber et al . 2013).

The study by Papoulias et al . (2014) with medaka fish exposed to atrazine at up to 50 μg/L reported no treatment-related effects on ma le steroid hormone concentrations, E/T ratios, or GSI values; however, treatment-related delayed testes staging (stage III instead of IV) and increased aberrant spermatogonia and testicul ar lesions were reported. Tillitt et al . (2010) showed no clearly treatment-realted effect s on androgen-relevant parameters in a short-term reproductions study with fathead minnow. A published study by Kroon et al . (2014) with juvenile barramundiATRAZINE FINAL

Page 29 of 159 failed to demonstrate consistent atrazine treatment-related effects (up to 100 μg/L) on gill ventilation rate, growth, CYP19B and VTG expression, or concentr ations of testosterone and

17 β -estradiol in the catadromous fish. In freshwat er zebrafish exposed to atrazine up to 10 μM (Corvi et al ., 2012), decreased growth was observed at 1 and/or 10 μM concentrations. Sex ratios were not affected by treatment, and minimal incide nces of testicular oocy tes were observed, with no dose dependence. In Le Mer (2013), no e ffects on sex ratio or VTG was observed.

In ovo exposure to atrazine was conducted with a lligator eggs and reported in two published reports (Crain et al. , 1997 and Spiteri et al. , 1999), with Spiteri et al . (1999) conducting subsequent hepatic aromatase and gonadal histopat hology analyses on a subset of the eggs used
in the Crain et al . (1997) experiment. No statistically significant effects were observed on sex
steroid concentrations in c horionic fluid, on aromatase act ivity or on gonadal histology.

An in vitro examination of ovulation with X. laevis oocytes (Orton et al. , 2009) demonstrated increases in progesterone and te stosterone concentrations w ith stimulation of ovulation.

As discussed in the estrogen pathway, many studi es with amphibians have been previously reviewed and presented at multiple SAPs (USEPA 2003, 2007 and 2012). In regards to the androgen pathway, investigations of in vivo at razine exposure in amphibians have primarily focused on potential effects on metamorphosis (discu ssed separately in the section on the thyroid pathway) and sexual/gonadal development. Se xual differentiation and development generally were not affected across these studies. Although Oka et al. (2008) noted a shift in sex ratio (female>male) at atrazine concentrations ≥ 10 ppb, normal testes and ovaries were observed in all froglets, with no sex revers al. Atrazine exposure in X. laevis [MRID 47153501; Hosmer et al.

, 2007; Kloas et al. , 2009] from NF stage 46 to 66 at con centrations of 0.01 to 100 μg/L did not affect amphibian gonadal development. No frogs displaying intersex or testicular oocytes were observed, and no treatment effect was observed on sex ratios. Spolyarich et al. (2010) also observed no effect of atrazine up to 30 μg/L on sex ratio in the spotted marsh frog, with minimal effects on male gonadal histology an d only a single incidence of te sticular ovarian follicles.

Wilhelms et al . (2005) exposed different groups of male Japanese quail to atrazi ne in a series of seven experiments using either dietary ( 0, 10, 100 or 1000 mg/kg diet) or 10 mg/kg/day subcutaneous ( s.c. ) exposures. When data from different age classes were combined, atrazine exposure did not elicit any dose-dependent effects on reproductive organs. Ciruclating
testosterone concentration was three-fold greater at 1,000 m g/kg diet (p<0.01), based on pooled data for four-week old and six-week old quail exposed through the diet in two separate experiments, with no other consis tent effects on testosterone, 17 β -estradiol or LH throughout the experiments.ATRAZINEFINALPage 30 of 159Table 3: Androgenic/Anti-Androgenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1Study Type/LiteratureCitation AR Binding AR Activation Steroidogenesis Testosterone level Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AsssaysAR BindingRequirement satisfied by OSRI (USEPA, 2008a)FSTRARequirement satisfied by OSRI (Battelle, 2005)HershbergerRequirement satisfied by OSRI (Ashbyet al., 2004 and Yamasakiet al., 2004)Male Pubertal RatRequirement satisfied by OSRI (Stokeret al., 2000)SteroidogenesisRequirement satisfied by OSRI (USEPA, 2008c)OSRIAR Binding(USEPA, 2008a)NAR Binding(Fanget al.,2003)NReceptorTransactivation(Kojimaet al.,2004)NFSTRA (Battelle,2005)NNPNENNNFSTRA (Bringolfet al., 2004)NENNNNNNModified FSTRA(Fathead minnow;Tillittet al., 2010)NENNNNENENATRAZINEFINALPage 31 of 159Table 3: Androgenic/Anti-Androgenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1Study Type/LiteratureCitation AR Binding AR Activation Steroidogenesis Testosterone level Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Modified FSTRA(JMedaka;Papouliaset al.,2014)NNP(L, M,H)Frt: NENVitellogenin(Sandersonet al.,2001)NHershberger(Ashbyet al.,2004)LABC:,↓16-18%(MH, H)GP:↑10%(H)NNEHershberger(Yamasakiet al.,2004)NX(H)NEMale Pubertal Rat(Stokeret al.,2000)NNNN↓VP:↓(M, HM,MH)NX(H)X(H)Modified MalePubertal (Rat;Trentacosteet al.,2001)↓34(H)NRNENENENESV:↓32 (H);VP:↓26(H)Age:↑3 day(H); BW:NRX(H)X(H)Steroidogenesis(USEPA, 2008c)P21-Day DermalToxicity (Rabbit;MRID 42089902)NNNNNNH(H)X(H)ATRAZINEFINALPage 32 of 159Table 3: Androgenic/Anti-Androgenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1Study Type/LiteratureCitation AR Binding AR Activation Steroidogenesis Testosterone level Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Two-generationreproductiontoxicity (Rat;MRID 40431303,DeSessoet al.,2014)NNNENNENNX(H)X(H)Reproductivetoxicity,oncedaily dosing(Rat; Fraitesetal., 2011)NNNENNE↓7, 8,12%(ML,MH,H)NNX(H)X(H)Reproductivetoxicity,twicedaily dosing(Rat; Fraitesetal., 2011)NNNENNENNNX(H)X(H)Reproductivetoxicity (in uteroexposure in Rat;Rosenberget al.,2008)↓(M)NNENENENENNX(MH,H)X(MH,H)Reproductivetoxicity (Rat;Songet al., 2014)↓10%(M)NP(M)NNENEN↓Sperm(M)X(H)X(H)ATRAZINEFINALPage 33 of 159Table 3: Androgenic/Anti-Androgenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1Study Type/LiteratureCitation AR Binding AR Activation Steroidogenesis Testosterone level Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Developmentalreproductivetoxicity,in uteroexposure (Rat;DeSessoet al.,2014)NNP(MH)NNENPR: N;SV: NX(H)X(H)Developmentalreproductivetoxicity, Postnatal exposure(Rat; Desessoetal., 2014)N↓7, 6%(H)P(NS;H)↓12%(H)NENPR: N;SV: NX(H)NChronic Toxicity/Carcinogenicity(Rat; MRID40629302)NNNNNEPR: NX(MH,H)X(MH,H)Carcinogenicity(Mouse; MRID40431302)NNNENNENEX(MH,H)X(MH,H)Chronic Toxicity(Dog; MRID40431301,41293801)NNNNNNEX(H)X(H)Reproductivedevelopment inmales (JapaneseQuail (Wilhelmset al., 2005)↑200%(H)↑46%(L)↑49%(L)NNATRAZINEFINALPage 34 of 159Table 3: Androgenic/Anti-Androgenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1Study Type/LiteratureCitation AR Binding AR Activation Steroidogenesis Testosterone level Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Steroidogenesis,in vivo(JuvenileBarramundi;Kroonet al.,2014)↑(L, M)NN NEarly Life-StageToxicity(Sticklebacks; LeMeret al., 2013)NNNReproductivedevelopment(Zebrafish; Corviet al., 2012)NNNReproductivedevelopment (inovoexposure inAlligator; Crainetal., 1997)NNNNEReproductivedevelopment (inovoexposure inAlligator; Spiteriet al., 1999)N NNNNEIn vitroovulationassay (Frog;Ortonet al.,2009)↑24%(M)ATRAZINEFINALPage 35 of 159Table 3: Androgenic/Anti-Androgenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1Study Type/LiteratureCitation AR Binding AR Activation Steroidogenesis Testosterone level Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Amphibianmetamorphosisand reproductivedevelopment(Frog; Hosmeretal., 2007 andKloaset al.,2009)NNNAmphibianreproductivedevelopment(Frog; Spolyarichet al., 2010)NNAmphibianreproductivedevelopment(Frog; Okaet al.,2008)N↑%♀(H)NNTumorDevelopment(Rat; Fukamachiet al., 2004)NN NMaleSteroidogenesis(Rat; Pogrmicetal., 2009)N↓8%(L)SV:↓30% (L)VP:↓22% (L)X(L,H)X(H)ATRAZINEFINALPage 36 of 159Table 3: Androgenic/Anti-Androgenic Pathway for AtrazineLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1Study Type/LiteratureCitation AR Binding AR Activation Steroidogenesis Testosterone level Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3MaleSteroidogenesis,In vitro(Rat;Pogrmic-Majkicet al., 2010)↑31, 69,98%(LM,HM, H)MaleSteroidogenesis,In vivo(Rat;Pogrmic-Majkicet al., 2010)↑MaleSteroidogenesis(Rat; Riffleet al.,2014)NX(M,H)NMaleSteroidogenesis(Mouse; Jinet al.,2013)↓25-30%(L, M)↓10%(L)X(H)X(H)1. Key to responses: Lowest treatment (L), Medium-low treatment (ML), Low-Medium (LM), Medium treatment (M), High-Medium (HM),Medium-hightreatment (MH), Highest treatment (H). Arrows (↓or↑) indicate the direction of the response. A shadedcell indicates that is parameter is not routinelyevaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues:Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’sgland (CG), glans penis (GP).2.The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an Xin this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. AP Positive findingATRAZINEFINALPage 37 of 159N Negative finding (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedATRAZINE FINAL

Page 38 of 159 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

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

The potential for atrazine to interact with t hyroid regulation is summa rized in Table 4. The various targets of the th yroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. This table also includes HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the ta ble and not considered further in the WOE assessment.

In the OSRI that satisfied the requiremen ts for the female pubertal rat assay (Laws et al. , 2000), thyroid weights were not determined, but there were no effects on thyroi d histopathology, serum total thyroxine (T 4 ) or thyroid stimulating hormone (TSH) concentrations (up to 200 mg/kg/day). Pituitary weights were decrease d at atrazine concentrations ≥ 50 mg/kg/day. In the OSRI that satisfied the requirements for the male pubertal rat assay (Stoker et al. , 2000), triiodothyronine (T 3 ) concentrations were increase d in all treatment groups, with significance achieved only in the presence of overt toxicity (200 mg/kg/day). There were no signifi cant change in T4 or TSH. Thyroid weights were not determined, but there were no effects on thyroi d histopathology. In another literature study examining the HPG axis in the rat (Cooper et al. , 2000), no treatment
effects were noted on TSH concentr ation or pituitary we ight in animals treated with atrazine up to 300 mg/kg/day.

There were no thyroid-relate d effects observed in the ma mmalian Part 158 studies

In the amphibian study [which is Tier 2-like in that frogs complete metamorphosis and gonadal development is assessed; MRID 48614801; Hosmer et al. , 2007; Kloas et al. , 200]) that satisfied the Tier 1 Amphibian Metamorphosis Assay (AMA), there were no treatmen t-related effects of atrazine in the follow-up thyroid tissue analysis from atrazine-treated frogs that had completed metamorphosis. There were also no treatment-re lated effects on time to metamphosis in this study. In this study, although decreases (p<0.05 ) were noted in mean body weight ( ↓ 7%) and SVL ( ↓ 2-3%) in females, these changes were not dos e dependent (also were observed in only the data from one of the two performing laboratorie s). Besides this Tier 2-like study, several other published studies investigated amphibian metamorphosis ( e.g. , Brodeur et al. , 2013; Oka et al.

, 2008; and Spolyarich et al. , 2010). None of these studies ex amined gross or microscopic pathology of the thyroid. Brodeur et al. (2013) reported a non-dose-dependent decrease in the time to complete metamorphosis in the South American toad ( Rhinella arenarum ), but the other studies reported no effect on metamorphosis (e.g., Choung et al . 2011).ATRAZINE FINAL

Page 39 of 159 Table 4. Thyroid Pathway for Atrazine.

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

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth(BW, SVL)Systemic Toxicity2Overt ToxicityObserved3

EDSP Tier 1 Assays

Female Pubertal Rat Requirement satisfied by OSRI (Laws et al. , 2000)

Male Pubertal Rat Requirement satisfied by OSRI (Stoker et al. , 2000)

OSRI

Female Pubertal Rat (Laws et al. , 2000) NE N N N ↓ 16, 16 (L, MH) X (H) X (H)
Male Pubertal Rat (Stoker et al. , 2000) NE N N N N X (H) X (H) 21-Day Dermal Toxicity (Rabbit; MRID 42089902) N N N X (H) X (H)

Chronic Toxicity/Carcinogenicity (Rat; MRID 40629302) N N NE X (MH, H) X (MH, H)

Carcinogenicity (Mouse; MRID 40431302) NE N NE X (MH, H) X (MH, H) Chronic Toxicity (Dog; MRID 40431301, 41293801) N N N N X (H)

HPG Axis (Rat; Cooper et al. , 2000) N N X (H) N

Amphibian Metamorphosis (Toad; Brodeur et al. , 2013) NE P (ML, M,

MH) BW: ↑ 12% SVL: ↑ 4-5% (ML,M) X (H)

Amphibian Metamorphosis (Frog;
Hosmer et al. , 2007; Kloas et al. , 2009) NE N BW: ↓ 7% (L, M, H ) SVL: ↓ 2-3% (L, M, H ) N

Amphibian Metamorphosis (Frog; Oka et al. , 2008) NE N N

Amphibian Metamorphosis (Frog; Spolyarich et al. , 2010) NE N N NATRAZINE FINAL

Page 40 of 159 Table 4. Thyroid Pathway for Atrazine.

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

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth(BW, SVL)Systemic Toxicity2Overt ToxicityObserved3

HLL and Thyroid Histopathology of Archived Specimens from Hosmer et al

. (2007) and Kloas et al

. (2009) (MRID 48614801) 4 N 5 N 5

1. Key to responses: Lowest treatment (L), Medium-low treatment (ML), Low-Medium (LM), Medium treatment (M), High-Medium (HM), Medium-high treatment (MH), Highest treatment (H). Arrows ( ↓ or ↑ ) indicate the direction of the respon se. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are
absolute unless otherwise indicated.

2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 3. The overt toxicity in the Tier 1 assays are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 4. Tissues derived from archived specimens from the Hosmer et al. , 2007 and Kloas et al. , 2009 studies. 5. Archived specimens were from NF stage 66 metamorphs. P Positive finding N Negative finding
NE Not examinedATRAZINE FINAL

Page 41 of 159 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of atrazine to interact with endocrine related processes ( i.e. , E, A, or T signaling pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with the OSRI ( e.g. , 40 CFR Part 158 test guidelines and published or publicly available peer-reviewed studies). WoE an alysis in the context of the EDSP follows the Agency’s guidance (USEPA, 2011b) and is conducted on a case-by-ca se basis by first assessing the different lines of evidence ( i.e. , specific Tier 1 assays and OSRI), then performing an integrated analysis of those lines of evidence.

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

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses are mediated through an endocrine pathway, therefore nonendocrine toxic responses (incl uding but not limited to mortalit y or body weight changes) are
also considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence evaluation of the results of the Tier 1 assays stated that “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All effects in endocrine se nsitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects related to toxicity in non-endocrine organs or overall disruptions in homeostasis ” (USEPA, 2013).

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. In addition to the endocrine -related effects described above
for the in vivo Tier 1 assays and OSRI, other eff ects observed which may be considered overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. However, in other instances, mo re than one parameter was needed to determine overt toxicity. Effects which were considered to be signs of system ic toxicity were generally less severe forms of toxicity (e.g., changes in organ weights or blood chemistry).ATRAZINE FINAL

Page 42 of 159 1. Tier 1 in vivo Assays

The requirements for the in vivo EDSP Tier 1 assays were satisfied by OSRI.

2. OSRI

In mammals, overt and/or systemic toxicity we re observed at higher dos es of atrazine (100 mg/kg/day and above), and presented primarily as decreased body weights and body weights gains.

The requirements for the Tier 1 FSTRA were satisfied by Battelle (2005), a multi-chemical evaluation (including atrazine) of the short-term reproduction assay with fathead minnow. In this study, overall survival was 100% in all treated groups, and body we ights of male or female minnows were unaffected by treatment. Similarl y, in a published study on adult fathead minnows by Bringolf et al . (2004), there were no effect s of atrazine treatment on mortality, body length or weight, or larval survival.

The requirements for a Tier 1 He rshberger assay were satisfied by Ashby et al. (2004) and
Yamasaki et al. (2004) . In Ashby et al . (2004), body weights were d ecreased (NS) by 5-6% at 100 mg/kg/day, while in Yamasaki et al. (2004), body weights were decreased by 7% at 80 mg/kg/day.

The requirements for a Tier 1 female ra t pubertal assay were satisfied by Laws et al . (2000). In this study, terminal body weights (PND 41) were decreased (p<0.05) by 12% at 200 mg/kg/day; body weight gains during treatment were decreased (p<0.05) by 16% at this dose. Similarly, in the rats used for estrus cyclicity observations , body weights were decreased (p<0.05) by 11% and body weight gains also were decreased (p< 0.05). By ANOVA analysis, kidney and adrenal weights were decreased ( ↓ 15%) at 200 mg/kg/day, and pituit ary weights were decreased ( ↓ 16- 31%) 100 and 200 mg/kg/day. By ANCOVA anal ysis, liver weights were increased ( ↑ 6%) and kidney ( ↓ 8%) and adrenal ( ↓ 3%) weights were decreased at 200 mg/kg/day, and pituitary weights were decreased ( ↓ 13-16%) at 100 and 200 mg/kg/day.

The requirements for a male rat pubert al assay were satisfied by Stoker et al. , 2000. In this study, body weights were decreased (p<0.05) on PND 43 ( ↓ 14%) and PND 53 ( ↓ 17%) at 200 mg/kg/day. Similarly, in a modifi ed pubertal rat study by Trentacoste et al. (2001), mean body weights were decreased over the study period at 100 mg/kg/day, and terminal body weights were decreased (p<0.05) at 100 ( ↓ 9%) and 200 ( ↓ 21%) mg/kg/day. In another section of the study (feed deprivation examination), rats admi nistered 100 mg/kg/day at e an average of 10% less food per day with concomitant decreases in daily m ean body weight and decreased (p<0.05) terminal mean body weight ( ↓ 10%). Terminal mean body weight was decreased (p<0.05) to a similar extent ( ↓ 10%) in the feed- restricted vehicle group.ATRAZINE FINAL

Page 43 of 159 The requirements for a rat uterotrophic study were satisfied by Yamasaki et al . (2000). In this study, there were no clinical signs of toxicity and body weights we re unaffected by treatment at up to 50 mg/kg/day. In a published, multicomponent , modified uterotrophic study by Tennant et
al . (1994a), body weights were decreased (p<0.0 5) by 11% at 300 mg/kg/day. In rats coadministered 17 β -estradiol, body weights were decr eased (p<0.05) by 7% at 100 and 300 mg/kg/day. In a published, multicomponent, modified uterotrophic study by Connor et al

. (1996), systemic toxicity parameters (body weight s, body weight gains, or gan weights) were not reported. In a published, multicomponent, modifi ed uterotrophic study by Ashby and Tinwell (2002) [also see Ashby et al . (2002)], terminal body weights we re decreased (p<0.01) by 7% and body weight gains were decreased (NS) by 14% at 100 mg/kg/day in the first experiment with AP female rats. In the second experiment with AP female rats, terminal body weights were decreased (p<0.01) by 7% and body weight gains were decreased (NS) by 13% at 100 mg/kg/day. In the third experiment with AP female rats, terminal body weights were decreased (p<0.01) by 11%, and body weight gains were decreased (NS) by 13% at 100 mg/kg/day. In the fourth expe riment (with SD female rats), terminal body weights were decreased (p<0.05) at 30 and 100 mg/kg/day ( ↓ 4%, respectively), with decreases (NS) of 6 and 7% in body-weight gain, respectively. Finally, in a published modified uterotrophic study by

O’Connor et al. (2000), after four days of treatment, fi nal body weights were decreased (p<0.05) by 9 and 14% at 150 and 200 mg/kg/day.

In a 21-day dermal study in rabbits, animals dosed with atrazine at 1000 mg/kg/day displayed sporadic instances of mucoid and/or few feces on Days 4-21. Decreased (p<0.05) mean body weights ( ↓ 10-12% males; ↓ 19-22% females) were generall y observed at 1000 mg/kg/day on Days 7, 14, 21, and 25, and total percent body weight gain on Day 21 was decreased for males ( ↓ 4.4% vs ↑ 9.5) and females ( ↓ 11% vs ↑ 8%). These decreases in body weights and body weight gains were consistent with decrea ses (p<0.05) in food consumption ( ↓ 28-67% males; ↓ 37-85% females) during Days 7-21. Relativ e (to body) and absolute spleen weights were increased in males and females, and may have been relate d to changes in hemato crit and red blood cell counts. Dermal findings were limited-to-slight eryt hema and/or scaling for both sexes in all dose groups with the exception of minimal-to-moderate acanthosis and focal sub-acute inflammation in three 1000 mg/kg/day female s (compared to 0 controls).

In a two-generation reproduction toxicity study in rats, mean premating body weights in the P
and F 1 males and females were decreased (p<0.05) at all measurement times ( ↓ 9-16%) at 500 ppm (equivalent to 25 mg/kg/da y, calculated by reviewers). In addition, mean body weights in
the P and F 1 females were decreased (p<0.05) on GD or LD 0 by ≥ 10% ( ↓ 10-14%), with decreases (p<0.05) lessening throughout each period ( ↓ 6-10%) at 500 ppm. Additionally at 500 ppm, food consumption was decreased (p<0.05) fo r the males and females in both generations, with recovery in females during gestation. P up body weight decreases (p<0.05) on PND 21 at 50 (F 2 , ↓ 8%) and 500 (F 1 and F 2 , ↓ 7 and 10%, respectively) ppm were considered to be related to

treatment.ATRAZINE FINAL

Page 44 of 159 In a published female rat reprodu ctive toxicity study by Foradori et al. , (2014), two experiments were performed. In the first experiment, body wei ghts were decreased (p< 0.05) at 100 mg/kg/day in both the SD (Day 2; ↓ 5%) and LE (Days 2, 3, and 4; ↓ 9-12%) rats. Food consumption was decreased (p<0.05) at 100 mg /kg/day on Days 1-3 or 4 ( ↓ 21-41%) and at 50 mg/kg/day on Days 1 or 2 ( ↓ 19-23%) in both strains. In the second experiment, administ ration of atrazine by oral gavage did not affect body weight during treatment or gestation in SD or LE rats. In the LE rats administered atrazine in the diet, mean body we ights were decreased (p<0.05) on Days 2, 3 and 4 at 1460 ppm ( ↓ 9-11%), and on GD 0 (p<0.05; ↓ 8%). Food consumption was decreased (p<0.05) during treatment in SD female s at 50 mg/kg/day on Day 2 ( ↓ 14%), and on all days at 100 mg/kg/day ( ↓ 20-27%); food consumption also was d ecreased at 100 mg/kg/day in the LE animals on all treatment days ( ↓ 20-27%).

In another published female reproductiv e toxicity study in rats (Narotsky et al. , 2001), at 200 mg/kg/day, maternal body weight losses were noted during the respective treatment periods (

− 9.1 g treated vs. +1.5 g control during GD 8-10;

− 11.7 g treated vs. +2.4 g control during GD 11-15). Mortality occurred in 2/10 and 1/10 dams during the early- and late-treatment periods, respectively. For dams dos ed on GD 8-10, full litter resorption was seen in 3/8 dams and was considered treatment-related. Pup weights in the late-treatment group were decreased (p<0.05) on PND 6 ( ↓ 7%). Concurrent exposure of F344, SD, and LE rats to 200 mg/kg on GD 6-10 yielded maternal body weight losses (p <0.05) in all three strains on GD 6-7 (F344:

− 8.6 g treated vs. +3.6 g control; SD:

− 14.3 g treated vs. +10.4 g control; LE:

− 9.1 g treated vs. +12.4 g control), with overall body wei ght decreases of 97% (p<0.05), 48% (NS), and 40% (p<0.01), respectively. Each strain also was administered atrazine on GD 6-10 at 0, 25, 50 and 100 mg/kg. F344 rats had maternal body weight losses of

− 6.2 g and

− 11.2 g at 50 and 100 mg/kg/day, respectively, compared to cont rol (+0.6 g) during GD 6-7, with overall body weight decreases (NS) of 24 and 101%, respectively. The SD dams had maternal body weight losses of

− 6.8 g,

− 6.7 g, and

− 15.5 g at 25, 50, and 100 mg/kg/day, respec tively, compared to control (+1.6 g) during GD 6-7, with overall body weight decreas es. LE dams had maternal body weight losses
after the first dose at 100 mg/kg/day (

− 8.2 g treated vs. +3.6 g control).

In a published fe male offspring reproduc tive toxicity study in rats (Davis et al. , 2011), conducted in conjunction with the i nvestigation of effects of atrazine on male offspring reproductive indices (Fraites et al. , 2011), two dosing regimens [a single full dose in the morning ( s.i.d .; Block 1) or a half-dose twice per day ( b.i.d .; Block 2) were used to admi nister equal amounts of atrazine. Postnatal mortality (birth to PND 4) was signi ficantly increased (p<0.05) for both sexes at 100 mg/kg/day in Blocks 1 and 2. Body weight s at birth were decreased (p<0.01; ↓ 10 and 17%, respectively) in the 50 and 100 mg/kg/day pups in Block 1. PND 4 body weights at 50 mg/kg/day recovered; PND 4 body weights at 100 mg/kg/day remained decreased (p<0.05; ↓ 15%), but recovered by PND 21. In Block 2 ( b.i.d .), body weights of the 100 mg/kg/day offspring were significantly decreased at birth and PND 4 (p<0.01; ↓ 15 and 12%, respectively), with recovery by PND 21.ATRAZINE FINAL

Page 45 of 159 In a published male offspring reproduc tive toxicity study in rats (Fraites et al. , 2011) utilizing the

same s.i.d . and b.i.d . dosing regimens as in Davis et al , dams administered 100 mg/kg/day in both portions of the study showed decreases in mean body weight beginning on GD 16 in Blocks 1 and 2. Maternal weights at th is dose level were further de creased (p<0.001) by GD 19-21. In contrast, the 100 mg/kg/day male offspring were significantly decrease d (p<0.01) at birth ( ↓ 13 and 11%) and PND 4 ( ↓ 10 and 9%) with both dosing regi mens, respectively. In Block 1, significantly decreased (p<0.01; ↓ 6-7%) body weights in male o ffspring continued through PND 59. In Block 2, however, offspring weights in the high-dose group recovered after PND 4 through PND 59. Mean absolute pituitary weights were significantly decreased in Block 1 at 5 mg/kg/day and above (p<0.05; ↓ 7-12%).

In a published male offspring reproductiv e toxicity study in rats (Rosenberg et al. , 2008), decreases in maternal body weight were obser ved from GD 18-21 at 75 and 100 mg/kg/day. Decreased mean daily food consumption dur ing gestation was observed at 50, 75, and 100 mg/kg/day (decreased by approximately 33%, 38%, and 47%, respectively). With increasing atrazine dose, the percentage of dead pups increased from <1% in the control and 1 mg/kg/day groups to approximately 10% in the 10 and 50 mg/kg/day groups and approximately 25% (p<0.05) in the 75 and 100 mg/kg/day groups. No di fferences were noted in body weights in the PND 60 males, although there was a downward trend at 100 mg/kg/day.

In a published male reproductive toxicity study in rats (Song et al. , 2014), mean terminal body weights were decreased (p <0.05) by 18% at 154 mg/kg/da y after 30 days dosing.

In the first of two special studies examining male reproductive development in rats (DeSesso et
al. , 2014), decreases (p<0.05) were observed in maternal food consumption and body weights and/or body weight gains during ge station and dosing in females at 125 mg/kg/day, with lesser decreases noted at 25 mg/kg/day. Only 13/21 pregnant, 125 mg/kg/day dams delivered live litters, with total in utero litter loss in eight dams. Additiona lly, embryo-fetal mortality occurred in live-born litters demonstrated by an increase (NS) in post-implantation loss ( ↑ 455%) and a decrease (p<0.05) in the mean number of pups per litter at birth at 125 mg/kg/day ( ↓ 39%). Postnatal pup survival also was affected in th e high-dose group with su rvival decreased by 75% on PND 4, and decreased by 85% on PND 21. Body weights were decreased (p<0.05) on PND 1 for male and female pups through PND 7 for male s and PND 14 for females at 125 mg/kg/day.

In the second of two special st udies examining male reproductive development in rats (DeSesso
et al. , 2014), significant decr eases (p<0.05) were observed in maternal food consumption and body weights and/or body weight gains during lact ation and dosing in females at 125 mg/kg/day. Body weights were decreased (p<0.05) on PND 4 for m ale and female pups through PND 21 at 125 mg/kg/day. After weaning, body we ights of the 125 mg/kg/day F 1 males remained decreased through PND 63, but food consumption was comparable to controls.ATRAZINE FINAL

Page 46 of 159 In a prenatal developmental toxicity study in rats (Accession No. 00143008; MRID 40566302;
Scialli et al. , 2014), a mortality rate of approximate ly 78% was observed at 700 mg/kg/day (21/27). Clinical signs at this dose included salivati on (13/27), oral/nasal discharge (12/27), ptosis (11/27), swollen abdomens (8/27), and blood on the vulva (7/27). Decreases (p<0.05) in body weight were observed on GD 14, 18 and 20 ( ↓ 8%, ↓ 22% and ↓ 30%, respectively) at 700 mg/kg/day. Overall maternal body weight gains were decreased by 264%, and food consumption was decreased (p<0.05) by 39%. At 700 mg/kg/day, fetal weights we re decreased (p<0.05) in the male ( ↓ 44%) and female ( ↓ 45%) pups.

In a second prenatal developmental toxicity st udy in rats, salivation was observed in 18/26 females at 100 mg/kg/day. Mean body weights were decreased on GD 8, 12, and 16, with overall maternal body weight gains decreased by 20% at 100 mg/kg/day. Food consumption in the highdose dams was decreased (p<0.05) by 13%.

In a prenatal developmental toxi city study in rabbits, stool change s (little, none, and/or soft) were increased (p<0.01; 19/19) at 75 mg/kg/day. Blood on the vulva or in the cage also was increased (<0.05) at this dose (4/19 vs. 0/19 control). Decreases (p<0.01) in body weight were observed on GD 14, 19, 21, 25 and 29 ( ↓ 12%, ↓ 19%, ↓ 18%, ↓ 10% and ↓ 8%, respectively) at 75 mg/kg/day. Additionally at this dose, body weight losses were observed (p<0.01) during GD 7-14 (

− 522 g treated vs. +113 g control), GD 14-19 (

− 204 g treated vs. +120 g cont rol), and overall (GD 0-29 adjusted for gravid uterus;

− 393 g treated vs.

− 73 g control). An intermittent decrease (p<0.05) in body weight gain was observed during GD 14-19 ( ↓ 63%), with no significant effect on body weight at 5 mg/kg/day. Treatme nt-related changes in food consumption were observed at 75 mg/kg/day only, with decreases (p<0.01) from GD 7-20. Three abortions occurred during the study; one 5 mg/kg/day female on GD 21 a nd two 75 mg/kg/day females on GD 20 and 25, respectively.

In a combined two-year feeding/carcinogenic ity study in rat , female survival at study
termination decreased to 37% (NS) and 26% (p<0.01) in the 500 and 1000 ppm groups (equivalent to 25 and 50 mg/kg/day, respectivel y) compared to 50% in the controls. Body weights were significantly d ecreased (p<0.01) at 500 ppm ( ↓ 8% males; ↓ 19% females) at Week 104. Body weights also were significantly decrea sed (p<0.01) for males and females at 1000 ppm through Week 104 ( ↓ 11-21 and 10-27%, respectively). Decr eases in food consumption were noted only during the first year of the study. Significant decreases (p<0.05 or 0.01) in hematology parameters (RBC, hematocrit and he moglobin) were noted in fe males during the study, but were not significantly different at study terminatio n. Absolute liver and kidney weights were decreased in the 1000 ppm males at the interim sacr ifice. Gross pathology findings were minimal, but increased incidences of masses ( e.g. , abdominal, axillary and thoracic) were noted in females in all treatment groups ex cept the low-concentration group. Histopathology findings in the 1000 ppm females included incr eased incidences of retinal degeneration, centrilobular necrosis in the liver, transitiona l epithelial hyperplasia in the bladder and the kidney, splenic extramedullary hematopoiesis and myeloid hyperplasia in both the femur andATRAZINE FINAL

Page 47 of 159 sternum bone marrow. Histopathology findings in the 1000 ppm males consisted of increased incidences of prostate epithelial hyperplasia, calculi in the kidney pelv is and acinar hyperplasia of the mammary gland.

There was an increased incidence of mammary adenocarcinoma in the medium-low, mediumhigh and high-concentration females, as well as an increase in fibroadenoma in the highconcentration females, in the carcinogenicity subgroup. Some females developed multiple mammary tumors. There also was an increase (N S) in adenocarcinomas in high-dose females at
the 12- and 13-month sacrifices. Incidences of carcinomas were increased (p<0.05) in the medium-low, medium-high and high-concentration females, and incidences of adenomas and
fibroadenomas were increas ed (p<0.05) in the high -concentration females.

In a mouse carcinogenicity study, there was a signif icant increase in mortal ity (p<0.05) in the 3000 ppm females, with only 25% of the females surviving vs. 39-45% of females surviving in the other dose groups. Mean body weight gain decreases were reported at Weeks 12 and 91 for the 1500 and 3000 ppm animals (equivalent to 194/247 and 386/483mg/kg/da y, respectively). At Week 91, body weight gain was significantly de creased (p<0.05) by 25% and 24%, respectively, in males and decreased by 10% (NS) and 52% (p<0.01) in females at 1500 ppm and above. Treatment-related decreases in food consump tion were observed in the 1500 ppm males and 3000 ppm animals. Organ weight ch anges were minimal, with decr eased kidney weights noted in the 3000 ppm females. Gross pathology findings incl uded enlarged atria/atri um of the heart and
tan lesions of the heart in th e 1500 and 3000 ppm animals. The in cidence of cardiac thrombi in animals that died prior to study termination was increased in the 3000 ppm males and 1500 and 3000 ppm females. The 1500 ppm male s and 3000 ppm males and females also had decreases in several hematological parameters ( e.g. , RBC, hematocrit and hemoglobin).

In a chronic toxicity study in dogs, one 1000 ppm female was sacrificed in a moribund condition on Days 113. At 1000 ppm (equivalent to 33.7/33.8 mg/kg/day), treatment-related clinical
observations noted at low incidenc es attributed to myocardial de generation included ascites (one male and female), cachexia (one female) and labored/shallow breathing (one male). These animals, and three others, also had EKG and/or morphologic evidence of heart disease (irregular heartbeat, tachycardia, and increased heart rates) that were correlated with pathological findings. Mean terminal body weights we re decreased at 1000 ppm ( ↓ 20% males; ↓ 16% females). Mean body weight gain was decreased by 21% (NS) and 36% (p<0.05) in the 150 (equivalent to 5.0/5.0 mg/kg/day) and 1000 ppm males and decreased (NS) by 40% in the 1000 ppm females. Males and females in the 1000 ppm group also had reduced food consumption throughout the course of the study, with decr eases of 22% in the males a nd 20% in the females at study termination. Absolute heart weights were decr eased in the 1000 ppm females. Gross pathology findings were also seen in the heart in the 1000 ppm survivors and consisted mainly of moderate to severe dilatation of right and/or left atria. Fluid-filled pericardium and an enlarged soft heart also were noted in 1000 ppm males. Cardiac myol ysis and focal atrophy of the heart were noted at 1000 ppm.ATRAZINE FINAL

Page 48 of 159 In a special study by Fukamachi et al. (2004), the potential modifyi ng effects of atrazine on 7,12- dimethylbenz[ a ]anthracene (DMBA)-induced mammary and skin carcinogenesis was examined. This investigation used a rat line carrying copies of the human c-Ha- ras proto-oncogene with its own promoter region, which is highly susceptibl e to DMBA-induced carcinogenesis. Final mean body weight in female rats fed 500 pp m atrazine was decreased (p<0.01; ↓ 14%) compared to control, and food intake was si gnificantly decreased (p<0.05; ↓ 10%).

In a mechanistic study in peripubert al male Wistar rats by Pogrmic et al. (2009) examining steroidogenesis gene expression in Leydig cells, mean body weights were decreased (p<0.05) in the 200 mg/kg/day group beginning on PND 27-29, with further decreases on PND 31-35 and 37-49. Mean terminal body weight in this gr oup remained significantly decreased (p<0.01)
24 hours after the final dose.

The relative roles of adrenal and testis hormones af ter intact or castrated male rats were exposed to atrazine with and without androgen maintenance were investigated by Riffle et al. (2014). The mean body weights were similar among all treatmen t groups within each intact or castrated category. However, the overall mean body weights were decreased (signifi cance not reported) in the castrated groups (339.0 g) compared to th e intact groups (370.4 g). After three days of treatment in both groups, weight losses (significa nce not reported) were observed in the intact and castrate 75 mg/kg (

− 5.1 g and

− 8.4 g, respectively) and 200 mg/kg (

− 16.7 g and

− 17.2 g, respectively) groups.

In a published study by Jin et al . (2013), the effects of atrazi ne on hormone synthesis and the transcription of steroidogenic genes was investigated in male peripubertal mice. Body weights were decreased (p<0.05) in the 200 mg/kg/day group at all of the examined time points. Absolute liver weights decreased (p<0.05) in a dose-de pendent manner at 50, 100 and 200 mg/kg/day. However, relative (to body) liver weights were similar to control.

In a published study by Cooper et al . (2000), body weight was unaffected in female rats administered atrazine for 1 or 3 days. Similarl y, atrazine did not affect body weights in females dosed for 21 days at 75 or 150 mg/kg. However, the rate of body weight gain in those females administered 300 mg/kg was signi ficantly decreased (p<0.05).

In a avian reproduction study in mallard duck, adult male body weight gain was reduced ( ↓ 102%), although adult female body gain was not a ffected. Mean food consumption by adults
was measured and determined to be 8-12% lower at 225 and 675 mg/kg diet than in controls. In another avian reproduction study in bobwhite quail, there were no differences in adult body weight throughout the study at dietar y concentrations up to 675 ppm.

In a published study by Wilhelms et al . (2005), a series of seven e xperiments investigating the
effects of atrazine on sexual maturation in male Japanese quail were conducted. In Experiments 1 and 2, 1000 ppm atrazine decreased (p<0.05) body weight gains and feed intake by 32% and 15%, respectively. In Experim ent 3, 1000 ppm atr azine significantly de creased (p<0.05) bodyATRAZINE FINAL

Page 49 of 159 weight gain by 64% with no effect on liver we ight or liver-somatic index. In contrast, body weight gain and liver weight, but not liver-somatic index, were reduced by 91 and 26% in pairfed birds. In Experiment 4, atrazine administered at 1 and 10 mg/kg/day by s.c . injection had no effect on indices of growth or reproductive develo pment. In Experiment 5, atrazine administered by silastic implant had no effect on indices of growth and repr oductive development. Atrazine significantly decreased (p<0.05) body weight gain s and feed intake 32% and 14%, respectively, in Experiment 6. In Experiment 7, addition of 1 000 ppm atrazine to the diet did not influence growth, feed intake, or liver weights versus control. In a second published study by Wilhelms et
al . (2006), a series of five experiments investig ating the effects of atrazine (>98% purity) on sexual maturation in female Japanese quail were conducted. Dietary admi nistration of atrazine up to 1000 ppm had no effect on mortality, averag e body weight gain (ADG) or feed intake. Subcutaneous or implanted atrazine had no e ffect on mortality, ADG, feed intake, or ovary, oviduct and liver weights. Atrazine did not influence circulating plasma concentrations of LH irrespective of the route of administration. Systemic at razine delivered either by s.c . injection or implants had no effect on plasma concentra tions of corticoster one or aldosterone.

In a published study by Hermanutz et al . (1973; Accession No. 00057846) on fathead minnow embryos exposed to atrazine for 45 weeks, surviv al of parental fish wa s decreased (p<0.05) in the mean-measured 63.5 μg/L treatment group ( ↓ 98%). The parental fish length and the F 1 fish survival and length were also decreased (p< 0.05) in the mean-measured 39.5 μg/L treatment group. Percent hatchability of embryos from unexpos ed parents incubated at 63.5 μg/L atrazine was similar to that of control embryos, but al l larvae died 5-8 days after hatching. There was 100% mortality of 1-day-old larvae from unexpo sed parents incubated at 63.5 μg/L atrazine within 24 hours. Survival of 3-day-old hatchl ings in the medium-high concentration group was decreased (p=0.05) approximately 50%.

In a early life-stage toxicity study in sheepsh ead minnow embryos exposed to atrazine for 33 days, the total length and wet weights were ad versely affected at the 2.2 mg/L concentration, with significant decreases (p<0.05) of 17% in mean body length and 46% in wet weight compared to the dilution water control at study termination (28-days post-hatch).

In a fish chronic partial life-cycle toxicity study conducted by the EPA Office of Research and Development with yearling brook trout ( Salvelinus fontinalis ) exposed to atrazine for 44 weeks, exposure in the P generation at concentrations ≥ 0.24 mg/L resulted in d ecreases in body length ( ↓ 17-25%) and weight ( ↓ 56-63%) compared to the negative (w ater) control. Growth of the F 1 fry was decreased (NS) at 90 days at 0.12 mg/L; length was decreased by 7%, and wet weight was decreased by 16%, compared to control. Furthe r decreases of 39% to 45% (p<0.01) were noted in length and weight, respectively, at atrazine concentrations ≥ 0.24 mg/L.ATRAZINE FINAL

Page 50 of 159 In a published non-Guideline study by Le Mer et al . (2013) on larval threespine sticklebacks, mortality was <11% for the fish exposed to at razine over a 42-day exposure period. There were no differences in wet weight, body length, or condition of juvenile fish between atrazine-exposed fish and controls (acetone or seawater, respectively).

In a fish life-cycle toxicity study on fathead minnow embryos (<24-hour-old), a significant effect was noted on larval growth. For the P genera tion, total length was decreased by 7-10% at 0.99 mg/L and 2.0 mg/L at Day 30, and by 5% at ≥ 0.46 mg/L at Day 60. Body weights were also significantly decreased by 19% at 0.99 and 2.0 mg /L at Day 60. Total length at 30 days posthatch in the F 1 generation was decreased at 0.15, 0.25, 0.46, 0.99 and 2.0 mg/L by 7, 10, 10, 17 and 23%, respectively, and wet weights were d ecreased by 17, 27, 34, 44, and 54%, respectively.

In a published study by Kroon et al . (2014) on juvenile barramundi , no treatment-related effects were observed on gill ventilati on rate, total le ngth or weight due to atrazine exposure.

In a published study by Papoulias et al . (2014) on medaka, no adult mortality was observed during the study.

In a published study by Corvi et al . (2012) on zebrafish exposed in two four-month chronic exposure studies, mortality was low in both of the chronic toxicity studies, with no differences in percent survival in ei ther study. In Study I, significantly de creased (p<0.05) body weights were observed in the 1 and 10 μM males ( ↓ 22 and 13%, respectively), and in the 10 μM females ( ↓ 21%) relative to controls. In Study II, signif icantly decreased (p<0.05) body weights were observed in the 1 and 10 μM males ( ↓ 11 and 15%, respectively), and in the 10 μM females ( ↓ 18%). Body lengths in the 10 μM males and fe males were significantly decreased (p<0.01) compared to control ( ↓ 5% for both sexes).

In another published study by Plhalova et al . (2012) on zebrafish, body we ights and growth rates were significantly decreased (p<0.05) at st udy termination in fish in the 90 μg/L group. Hepatotoxicity (based on histolog y) and clinical signs of toxicity (behavior) were also noted, but
the duration of behavioral effects was not reported.

In lieu of conducting a Tier 1 AMA, the regi strant cited a previous study of amphibian metamorphosis and gonadal development in X. laevis (Hosmer et al. 2007; see also Kloas et al.

, 2009) and submitted additional data obtained from archived tissue samples from that study (MRID 48614801). It was reported that there was no effect on mortality or dry weight; these were the only two parameters a ssessing overt/systemic toxicity.

In an investigation of reproductive toxicity/metamorphosis in frogs by Oka et al. (2008), survival rates in two experiments were determine d. Survival rates in Experiment I were ≥ 90% in control tadpoles and in the 1 and 10 ppb atrazine-treatment groups, while low survival rates were noted in one tank each for the 0.1 ppb (73.3%) and 100 ppb (26.7%) atrazine-treatment groups. In Experiment II, ≥ 96% survival rates were observed in all groups.ATRAZINE FINAL

Page 51 of 159 Brodeur et al. (2013) examined the effects of exposure to atrazine at environmentally relevant concentrations on the timing of metamorphosis and body size at metamorphosis in the South American toad ( R. arenarum ). Tadpole survival was decreased ( ↓ 30%) at 1000 μg/L compared to water and positive controls, with most mortalities occurring during the first 20 days of exposure. This group was not discussed further. There were no other sign s of overt toxicity.

In another developmental frog study by Spolyarich et al. (2010) in develo ping spotted marsh frogs, mortality was limited (2.3%; 18/800 animals) across all experiments, and occurred mainly during the first week of exposure and was attr ibuted to handling st ress. Atrazine had no observable effect on the development of the spotte d marsh frog larvae at concentrations up to 30 μg/L.

B. Estrogen Pathway

Although the Tier 1 guideline-like OSRI submissions showed few effects indicative of a potential interaction with the estrogen pathwa y, the rich dataset of supporting OSRI provided
additional lines of evidence to suggest potential downs tream, estrogen pathway effects that may be consistent with the known, upstream neuroendocrine mechanism of action for atrazine and
other triazines in th e common mechanism grouping (CMG). In the in vitro Tier 1-like assays, atrazine was negative in numer ous ER binding and ERTA studies. Mixed results were obtained
in the aromatase studies. Sanderson et al. (2001 and 2002) demonstrated induction of aromatase activity in H295R and JEG-3 cells, but no induc tion occurred in MCF- 7 cells. Increased aromatase activity in human gra nulosa-lutein cells, but not in human endometrial stromal cells, was noted by Holloway et al. , (2008). Hinfray et al. (2006) demonstrated that atrazine had no effect on aromatase activity in rainbow trout brai n and ovarian microsomes. However, atrazine
acted as an inducer of 17 β -estradiol in the steriodogenesis assay.

In the in vivo mammalian Tier 1-like assays, atrazine was negative for estrogenic activity in several uterotrophic assays. In the female pubert al assay, delays in estrus and vaginal opening, decreases pituitary and uterine weights were obs erved at the mid-dose in the absence of overt toxicity. There were no estrogen-related effects se en in the Part 158 mammalian toxicity studies in the absence of overt toxicity.

Effects seen in OSRI with nonmammalian wildlife species were similarly sporadic across the
data set and sometimes co-occurr ed with non-specific effects such as reductions in growth parameters ( i.e. , offspring weight or size) . Reproduction was lower in response to atrazine exposure in two studies with fish, in one study with the Northern wa ter snake, and in two of three studies with different species of birds exposed through the diet. Adverse effects on parental or offspring growth were also observed in these studies. There were no significant effects on offspring sex ratio in neonates of water sn akes exposed to atrazine in the diet. In ovo exposure to atrazine resulted in no statistically significant effects in aromatase act ivity or sex steroid
concentrations in chorionic fl uid or on neonate gonadal histol ogy. The percentage of fem aleATRAZINE FINAL

Page 52 of 159 frogs was greater in atrazine-exposed treatments in one study, but othe r studies with frogs showed no effects on reproductive developmen t in the absence of overt toxicity.

Although the available data reflect varying re sponses in estrogen-related endpoints across species and studies, the potential for interaction with the estr ogen pathway is supported by the SAP conclusion that the chlorotriazines (inc luding atrazine and its DEA, DIA and DACT degradates) function through a ne uroendocrine MOA that suppresse s the hypothalamic release of GnRH and therefore LH, which may result in downstream effects on estrogen and androgen signaling pathways.

C. Androgen Pathway

As with the estrogen pathway, the Tier 1 guidelin e-like OSRI submissions showed few effects indicative of a potential interaction with the androgen pathway, but additional supporting OSRI provided further lines of evidence to suggest potential downstream, androgen pathway effects that may be consistent with the known, upstream neuroendocrine mechanism of action for
atrazine and other triazine s in the CMG. In the in vitro Tier 1-like assays, at razine was negative in the AR binding and ARTA assays. In the ster iodogenesis assay, atrazine acted as an inducer of testosterone at 100 μM at two of five studies (U SEPA, 2008c).

In the in vivo mammalian Tier 1-like assays, potential andr ogen-related effects of atrazine in two male pubertal assays (decreases in sex accesso ry sex organ weights and delayed preputial separation) were non dose-related and occurred at doses approach ing overt toxicity. Atrazine was negative in the Hershberger assay. Moreover, numerous SAP reports on atrazine and
triazine chlorinated metabolites ha ve concluded that the triazines have the potential to interact with the androgen pathway in mammals.

The data synthesized in this WoE evaluation reflect varying responses in androgen-related endpoints across species and studies. Data from OSRI beyond the Tier 1-like provide complementarity and redundancy of findi ngs with respect to steroidogenesis ( in vitro and in

vivo ) and gonadal histology, though similar effects were not seen in all studies. Overall, the potential for interaction with the androgen path way is supported by the SAP conclusion that the chlorotriazines (including atra zine and its DEA, DIA and DACT degradates) function through a neuroendocrine MOA that suppresses the hypotha lamic release of GnRH and therefore LH, which may result in downstream effects on estrogen and androgen signaling pathways.ATRAZINE FINAL

Page 53 of 159 D. Thyroid Pathway

The available data for atrazine do not provide conv incing evidence of a potential to interact with the thyroid pathway in mammals or other wildlife.

E. Conclusions

Based on the weight-of-evidence anal ysis, atrazine has the potential to interact with the estrogen and androgen pathways in mammals and other wi ldlife. There was no convincing evidence of potential interaction of atrazine with the thyroid pathway in mammals or other wildlife.

V. EDSP Tier 2 Testing Recommendations

In determining whether additional data are needed for risk assessment purposes based on the Tier 1 screen, the available data and Tier 1 conclusions are considered within the context of existing regulatory endpoints. The current point of de parture (POD) of 2.56 mg/kg/day is based on a luteinizing hormone suppression with atrazine wh ich is regarded as a precursor event in the
neuroendocrine mode of action of the triazines. In a Tier 1-li ke male pubertal assay, atrazine caused a delay in preputial separation at the lo west dose tested (12.5 mg /kg/day). This dose is approximately five-fold greater ( i.e ., less sensitive) than the P OD of 2.56 mg/kg/day used for human health risk assessment. Estrogen-relate d effects (delays in the onset of puberty and altered estrous cyclicity) discussed in this WoE were seen at doses greater than 50 mg/kg/day in the female pubertal assay (Laws et al., 2000).

With respect to ecological risk assessment, the current risk assessment endpoint for chronic risk to aquatic vertebrates is based on observations of reduced fecundity in medaka ( Oryzias latipes ) exposed at 0.5 μg/L and above. For non-mammalian terrestrial vertebrates, the current chronic risk assessment endpoint is based on reduced malla rd duck hatchling weight at 75 mg/kg diet and above, with reproductive effects (e.g., reduced nu mber of hatchlings) observed only at higher
treatment levels.

Therefore, based on the WoE analysis of EDSP Ti er 1 guideline-like studies and other available data, EDSP Tier 2 testing with mammals, fis h, amphibians or birds is not recommended for atrazine at this time because it is not expected to impact current EPA-established regulatory endpoints for human health or eco logical risk assessment.ATRAZINE FINAL

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60.ATRAZINE FINAL

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Wistar rats: an evaluation in the prot ocol for the assessment of pubertal development and thyroid
function. Toxicol. Sci. 58:50-59.

Stoker, T.E., Guidici, D.L., Laws, S.C., et al . (2002) The effect of atrazine metabolites on puberty and thyroid function in the male Wistar Rat. Toxicol. Sci. 67:198-206.ATRAZINE FINAL

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chloro- S -triazines in rat uterus. Toxicol. Environ. Health 43(2):183-196.

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USEPA (2002) The Grouping of a Series of Tria zine Pesticides Based on a Common Mechanism of Toxicity. Health Effect s Division, Office of Pesticid e Programs, Washington, D.C. 20460March 2002. ID : EPA-HQ-OPP-2005-0481-0011

USEPA (2003) Transmittal of Meeting Minutes of the FIFRA SAP Meeting held June 17 - 20, 2003 on Potential Developmental Effects of Atrazine on Amphibians. Office of Chemical Safety and Pollution Prevention, U.S. Environmental Protection Agency, Washington, DC.

USEPA (2007) Transmittal of Meeting Minutes of the FIFRA SAP Meeting held October 9 - 12,
2007 on The Potential for Atrazine to Affect Amphibian Gonadal Development. Office of Chemical Safety and Pollution Prevention, U.S. Environmental Protection Agency, Washington,

DC.

USEPA. (2008a) Integrated summary report for the validation of an androgen receptor binding assay as a potential screen in the Endocrine Di sruptor Screening Program . Revision of February 18, 2008.ATRAZINE FINAL

Page 62 of 159 USEPA. (2008b) Peer review results for the aromatase assay. January 22, 2008.

USEPA. (2008c); Multi-laboratory validation of the H295R steroidogenesis assay to identify
modulators of testosterone and estradiol production. February, 2008.

USEPA. (2009) integrated summary report for vali dation of an estrogen re ceptor assay using rat uterine cytosol as source of r eceptor as a potential screen in th e Endocrine Disruptor Screening Program Tier 1 Battery. March, 2009.

USEPA (2010a) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting held April 26-29, 2010 on the Re-evaluati on of the Human Health Effects of Atrazine: Review of Experimental Animal and In Vitro Studies and Drinking Water Monitoring Frequency. Office of Chemical Sa fety and Pollution Prevention, U.S. Environmental Protection Agency, Washington, DC.

USEPA (2010b) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting held September 14-17, 2010 on the Re-eva luation of the Human Health Effects of Atrazine: Review of Non-cancer Effects and Drinking Water Monitori ng Frequency. Office of Chemical Safety and Pollution Prevention, U.S. Environmental Protection Agency, Washington,

DC.

USEPA (2010c) ATRAZINE - Report of the Endocrine Disruptor Review Team- Test Order#: EDSP-080803-1; -2; -3; a nd -4. December 16, 2010. Office of Chemical Safety and Pollution
Prevention. U.S. Environmental Pr otection Agency, Washington, D.C.

USEPA (2011a) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting held July 26-28, 2011 on the Re-evaluati on of the Human Health Effects of Atrazine: Review of Non-cancer Effects, Drinking Water Monitoring Frequency and Cancer Epidemiology. Office of Chemical Safety and Pollution Prevention, U.S. Environmental Protection Agency, Washington, DC.

USEPA (2011b) Endocrine Disrup tor Screening Program – Wei ght of Evidence: evaluating results of EDSP Tier 1 screening to identify th e need for Tier 2 tes ting. Office of Chemical Safety and Pollution Prevention, Office of Scien ce Coordination and Policy. U.S. Environmental Protection Agency, Washington, DC.

USEPA (2012) Transmittal of Meeting Minutes of the FIFRA SAP Meeting held June 12-14,
2012 on the Scientific Issues Asso ciated with “Problem Formula tion for the Reassessment of Ecological Effects from the Us e of Atrazine”. Office of Chemical Safety and Pollution Prevention, U.S. Environmental Pr otection Agency, Washington, DC.ATRAZINE FINAL

Page 63 of 159 USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
held July 30 - August 1, 2013 on Weight-of

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

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Wilhelms, K.W., Cutler, S.A., Proudman, J.A., et al . (2006) Lack of effects of atrazine on estrogen-responsive organs and circulating hormone concentrations in sexually immature female Japanese quail ( Coturnix coturnix japonica ). Chemosphere 65:674-681.

Williams, B.K. and Semlitsch, R.D. (2010) Larval responses of three midwestern anurans to chronic, low-dose exposures of four herbicides. Arch. Environ. Contam. Toxicol. 58(3):819-827.

Wolf, J.C. (2012) Atrazine: study to sa tisfy the EPA Test Order # EDSP-080803-1,2,3,4 Guideline Study 890.1100 Amphibian Metamorphosis Assay (AMA) data requirement for atrazine using existing frog ti ssues available from Kloas et al. studies. Experimental Pathology Laboratories, Inc., Sterling, VA. Labora tory Report No. 140-121, February 10, 2012. Unpublished. (MRID 48614801)

Yamasaki, K., Sawaki, M., Noda, S., et al . (2000) Immature rat uterotrophic assay of diethylstilbestrol, ethynyl estradiol and atrazine. J. Toxicol. Pathol. 13:145-149.

Yamasaki, K., Sawaki, M., Noda, S., et al . (2004) Comparison of th e Hershberger assay and androgen receptor binding assay of twelve chemicals. Toxicology 195(2-3):177-186.

Zaya, R.M., Amini, Z., Whitaker, A.S., et al . (2011) Atrazine expos ure affects growth, body
condition and liver health in Xenopus laevis tadpoles. Aquat. Toxicol. 104:243–253.ATRAZINE FINAL

Page 64 of 159 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100)

A Tier 1 AMA was not submitted. The requirement for the AMA was addressed by citing a Tier 2-like study on amphibain metamorphosis and gonadal development (Hosmer et al. 2007; see
also Kloas et al. , 2009) and by submitting additional data on thyroid-relevant endpoints from archived tissues from that study (MRID 48614801). A summary of these data is presented in Appendix 2.

Although these submisisons do not, by themselves, pr ovide data equivalent to a Tier 1 AMA,
when reviewed in context with Part 158 data and other OSRI for mammals and other wildlife,
there is sufficient information to make a Tier 1 screening determina tion on the potential of atrazine to interact with the thyroid and HPT axis . Therefore, the AMA te st order requirement is satisfied by the available data.

Androgen Receptor (AR) Bi nding Assay (OCSPP 890.1150)

The requirement for a Tier 1 AR binding assay is satisfied based on the results of USEPA (2008a) . A summary of this study is presented in Appendix 2

.

Aromatase (OCSPP 890.1200)

The requirement for a Tier 1 AR binding assay is satisfied based on the results of Higley et al. (2010) and USEPA (2008) . Summaries of these studies are presented in Appendix 2

.

Estrogen Receptor (ER) Binding (OCSPP 890.1250)

The requirement for a Tier 1 AR binding assay is satisfied based on the results of Tennant et al. (1994b) and USEPA (2009) . Summaries of these studies are presented in Appendix 2

.

ER α Transcriptional Activatio n Assay (ERTA, OCSPP 890.1300)

The requirement for a Tier 1 AR binding assay is satisfied based on the results of Fujimoto et al. (2003) . A summary of this study is presented in Appendix 2

.

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350)

The requirement for a Tier 1 AR binding assay is satisfied based on the results of Battelle (2005)

. A summary of this study is presented in Appendix 2

.ATRAZINE FINAL

Page 65 of 159 Hershberger (OCSPP 890.1400)

The requirement for a Tier 1 AR binding assay is satisfied based on the results of Ashby et al. (2004) and Yamasaki et al. (2004) . Summaries of these studies are presented in Appendix 2.

Female Pubertal (OCSPP 890.1450)

The requirement for a Tier 1 AR binding assay is satisfied based on the results of Laws et al. (2000) . A summary of this study is presented in Appendix 2

.

Male Pubertal (OCSPP 890.1500)

The requirement for a Tier 1 AR binding assay is satisfied based on the results of Stoker et al. (2000) . A summary of this study is presented in Appendix 2

.

Steroidogenesis (OCSPP 890.1550)

The requirement for a Tier 1 AR binding assay is satisfied based on the results of USEPA (2008c) . A summary of this study is presented in Appendix 2

.

Uterotrophic (OCSPP 890.1600)

The requirement for a Tier 1 AR binding assay is satisfied based on the results of Yamasaki et al. (2000) . A summary of this study is presented in Appendix 2

.ATRAZINE FINAL

Page 66 of 159 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Amphibian Metamorphosis Assay

In an assessment of metamorphosis and gonadal morphology ( MRID 47153501; Hosmer et al.
, 2007 and Kloas et al. , 2009 ), African clawed frogs ( X. laevis ) at NF stage 46-48 were exposed to atrazine (97.1% purity) under flow-through c onditions at nominal concentrations of 0 (negative control), 0.01, 0.1, 10, 25 and 100 μg/L. A positive control, 17 β -estradiol (0.2 μg/L), also was included. Separate studies were conducted at two laboratories [ i.e. , Wildlife International, Inc. (WLI), and the Leibniz Inst itute of Freshwater Ecol ogy and Inland Fisheries (IGB)]. Mean-measured concentrations were <0.01, 0.011, 0.092, 0.957, 23.4, and 86.7 μg/L at WLI and <0.01, 0.0069, 0.055, 0.716, 22.0, and 87.9 μg/L at IGB. The study duration was 75 days. The number of replicates per treatment was sixteen for the negative control group and eight each for the positive contro l and atrazine treatment groups, with 25 larvae per replicate. Exposures continued through completion of metamor phosis (NF stage 66) or test termination at Day 75, if NF stage 66 had not been attained. Animals were sacrificed at the end of the exposure. Time to complete metamorphosis and size (length and weight) at metamorphosis were recorded; gross morphology and histology were conducted on animals that completed metamorphosis. Gonadal assessments were condu cted with a dissecting stereomicroscope, and extra-gonadal anomalies were determined. Phenot ypic sex was identified as male, female, or mixed; final sex determinations were made based on histopathology.

Mortality and time to metamorphosis were not aff ected by atrazine treatment. Survival rates in both experiments were ≥ 94%, and the average time to metamorphosis was 50.9-52.9 days. Time to metamorphosis in the 17 β -estradiol positive control gr oup males was delayed (p<0.05) approximately 3-4 days (WLI and IGB) and delayed (p<0.05) 1.7 days in females (IGB). Gonadal area in the positive control group was increased (dat a and significance not reported) compared to the negative control. In addition, atra zine exposure concentrations did not result in any consistent effect on size at metamorphosis, shif t in sex ratio, or on any of the indices used to gauge amphibian gonadal development. Although decreases (p<0.05) were noted in mean body weight and SVL in females (IGB only), these changes were not dose dependent.

The study examined a number of gross morphologi cal and histological e ndpoints, with effects occasionally noted in the positive control and at razine treatment groups. No frogs displaying intersex or testicular oocytes were observed in these experiments. The frequencies of morphological and histological ch anges tended to be low, and none of the measurements exhibited consistent responses across laboratories or dose-depende nt responses due to atrazine exposure. The positive control group responses also were inconsistent between the laboratories. Finally, the relevance of the gross morphological and histological endpoints examined in this study, other than mixed-sex tissue type, is uncertain.ATRAZINE FINAL

Page 67 of 159 This study is consistent with recommendations made by the Agency regarding study design
elements needed to conduct studies to determ ine whether atrazine affects amphibian gonadal development. Atrazine exposure in X. laevis from NF stage 46-66 (0.01 to 100 μg/L) did not affect amphibian gonadal development, and did not affect time to or size at metamorphosis.

The objective of the s ubsequent submission ( MRID 48614801 ) was to evaluate preserved X.
laevis tissues from Hosmer et al. (2007; see also Kloas et al. , 2009) for potential effects of atrazine on thyroid-dependent endpoints similar to those identified in OCSPP 890.1100, Amphibian Metamorphosis Assay. Tissue samples from the lowest two treatment levels (0.01 and 0.1 μg/L) were not included in these secondary analyses. Preserved tissu es were analyzed for thyroid pathology and growth pa rameters (hind-limb length, s nout-vent length, and wet weight) were measured by image analysis. Tissue sample s from amphibian larvae (100 frogs from the control and each treatment group) were evaluate d from the original study. These archived samples were generally all of NF stage 66 fr ogs which had completed metamorphosis; no data were available for endpoints specific to the Day 7 (prior to endogeneous thyroid activity) and Day 21 (peak thyroid activity) sampling poi nts in the OCSPP Guideline 890.1100 AMA.

Normalized hind-limb length (HLL) was 3, 3, and 5% greater in the low-, medium-, and highconcentration groups at NF stage 66 than in ne gative controls (p<0.05). There were no effects detected on snout-to-vent length or dry weight endpoints. Since al l frogs were at NF 66 at test termination, no effects on NF stage were repor ted. According to the data in Hosmer et al. (2007)
and Kloas et al. , (2009), there were no effects on time-to -metamorphosis, as well as no observed abnormal development. There were observations of thyroid follicular cell hypertrophy, follicular cell hyperplasia, and decreased colloid in the archived NF stage 66 thyroid samples from control and all treatment groups, with no indication of a treatment-related eff ect. Other histological endpoints such as changes in thyroid follicular lumen, cell height, or shape were not reported. Overall, atrazine exposure in X. laevis from NF stage 46-66 (1 to 100 μg/L) did not affect amphibian thyroid-dependent endpoints.

Androgen Receptor (AR) Binding Assay

In the Integrated Summary Report for the validat ion of an androgen receptor (AR) binding assay ( USEPA, 2008a ), an in vitro rat ventral prostate cytosol AR binding assay was optimized and validated for use as an EDSP Tier 1 testing requi rement to identify chemicals capable of binding to the AR. Atrazine was selected to be a negati ve chemical in the vali dation of an AR binding assay in the EDSP and, as such, provides supporting information on the performance characteristics of a negative compound in the assay. An inter-laboratory validation of the AR binding assay was conducted with five testing facilit ies; the performance of atrazine in the AR binding assay was determined in four of the five te sting facilities.ATRAZINE FINAL

Page 68 of 159 Ventral prostate cytosol from SD rats was used as the source of AR to conduct Saturation and Competitive Binding Experiments. The Competit ive Binding Experiment measured the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (logarithmic increase from 10

− 11 to 10

− 3 M) of atrazine (98% purity). A total of three runs were conducted, and each run included dexamethasone as a weak positive control, and R1881 as the ligand reference standard.

In the inter-laboratory Satura tion Binding Experiments, the mean dissociation constant (K d ) for [ 3 H]-R1881 was 0.613±0.041 nM and the mean estimated B max was 0.817±0.049 fmol/100 μ g protein for a standard batch of prostate cyto sol that was tested. The overall ranges for K d and

B max values were 0.685 to 1.57 nM and 6.67 to 15.6 fmol/100 μ g protein, respectively.

In the inter-laboratory Competitive Bi nding Experiments, the mean log IC 50 values for R1881 ranged from

− 8.9 to

− 8.6 log M) and for the weak positive control, dexamethasone, ranged from

− 4.6 to

− 4.0 log M. The mean relative binding affi nity (RBA) for the weak positive control ranged from 0.0017 to 0.0097%.

Precipitation and/or cytotoxicity with atrazine was not reported up to a concentration of 10

− 3 M.
Atrazine had no effect on specific binding of the [ 3 H]-ligand over the con centration range of

10

− 11 to 10

− 3 M; therefore, a log IC 50 and RBA could not be calculat ed for atrazine as it did not
result in 50% reduction in bindi ng at any concentration. Consiste nt with its use as a negative chemical, atrazine either did not have model convergence or had poorly fitted curves for all
laboratories and runs. Atrazine was classified as a “non-binde r” in the AR binding assay.

In a published study by Fang et al . (2003) , over 200 natural, synthetic, and environmental chemicals that encompass a broad range of structural classes, including steroids, diethylstilbestrol and related chemicals, antie strogens, flutamide deri vatives, bisphenol A derivatives, alkylphenols, para bens, alkyloxyphenols, phthalates, siloxanes, phytoestrogens, DDTs, PCBs, pesticides, and organophosphate insect icides were screened for their potential to
bind with the AR.

PanVera AR, a recombinant rat protein expressed in Eshcerichia coli , was used as the source of AR to conduct Saturation and Competitive Binding Experiments. The Competitive Binding Experiment measured the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (log arithmic increase from 4.28×10

− 9 to 4.28×10

− 4 M) of atrazine (purity not reported). A R1881 standard curve (10

− 11 to 10

− 7 ) also was included with each run.

In the Saturation Binding Experiment , the mean dissociation constant (K d ) for [ 3 H]-R1881 was
2.03±0.15 nM. The mean estimated B max was not reported. In the Competitive Binding Experiment, the mean log IC 50 value for R1881 was 3.07×10

− 9 ± 9.12×10

− 10 M.ATRAZINE FINAL

Page 69 of 159 Precipitation and/or cytotoxicity with atrazine were not reported. At razine had no effect on specific binding of the [ 3 H]-ligand over the conc entration range of 4.28×10

− 9 to 4.28×10

− 5 M; therefore, a log IC 50 = >4.28×10

− 5 M was determined for atr azine. The RBA could not be
calculated for atrazine. Atrazi ne was classified as a “ non-binder” with the AR. The nonchlorinated triazine pesticid es (including atrazine) were a ll non-binders; these results are
considered to be consistent with an endocri ne disruption mechanism of action for triazine pesticides on the central nervous system.

Aromatase Assay

In the Integrated Summary Report for the validation of an in vitro aromatase (CYP 19) aromatase assay ( USEPA, 2008a ), a microsomal aromatase assay was optimized and validated for use as an EDSP Tier 1 testing requiremen t to identify chemicals capable of inhibiting aromatase activity. The U. S. EPA selected two assay systems for validation, the human placental microsomal assay and the human recombinant microsomal assay. The placental microsomal and recombinant assays are similar excep t that the protein activity is higher in the latter system. Atrazine was one of two com pounds selected as a negative (non-inhibitor)
chemical in the validation of an aromatase a ssay in the EDSP, and as such provides supportinginformation on the performance characteristics of a negative compound in the assay. An interlaboratory validation of the aromatase assay wa s conducted with four te sting facilities; the performance of atrazine in the AR binding assay was determined in four of the five testing
facilities.

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation period for each c oncentration of chemical. Tritiated water was quantified with liquid scintillation counting (LSC). Three independent runs were conducted, and each run included a full activity control, a bac kground activity control, a positive control series (10

− 9 to 10

− 6 M) with a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), and the test chemical series. Atrazine (98% purity) in DMSO was incubated with human recombinant or placental aromatase and tri tiated androstenedione ([1 β

3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]- ASDN) at concentrations from 10

− 10 to 10

− 3 M for 15 minutes at 37°C to assess the effect on aromatase activity. After Run 1, tw o of the four testing laborat ories modified the atrazine
concentrations selected for tes ting in Runs 2 and 3, and two la boratories used the same eight concentrations for all three runs. The modifications (addition of mid-level concentrations) were made in an attempt to characteri ze the shape of the curve between 10

− 3 and 10

− 5 M.

Aromatase activity in the full activity controls determined by the four test laboratories ranged from 0.312±0.016 to 1.258±0.400 nmol·mg-protein

− 1 ·min

− 1 . Activity in the background controls was reported to be negligible. Results for the positive control (4-OH ASDN, 5×10

− 8 ) yielded aromatase activity ranging from 0.166±0.019 to 0.642±0.226 nmol·mg-protein

− 1 ·min

− 1

.ATRAZINE FINAL

Page 70 of 159 For atrazine, the percentage of control aromatas e activity in the recombinant microsomal assay averaged 96.46±3.51% at 10

− 10 M and 88.06±2.02% at 10

− 3 M. The mean percentage of control aromatase activity in the placental microsom al assay was similar with 93.62±1.57% at 10

− 10 M
and 81.31±1.46% at 10

− 3 M. Based on the data from the average response curve, atrazine was classified as negative for the aromatase assay.

In a published study by Higley et al . (2010) , H295R cells were exposed to atrazine (97.1% purity) at concentratio ns ranging from 0.01-100 μM for 48 hours. The culture medium was removed and analyzed for 17 β -estradiol and testosterone con centrations, after which the live cells were subjected to a 3 H 2 O -release assay for determinatio n of aromatase activity. Based on these results, three concentrati ons (low, medium, and high) were selected, and atrazine exposures
were conducted in three different ways: i) indirect aromatase activity with cells exposed to atrazine for 48 hours, the cell rins ed with PBS, and aromatase act ivity determined; ii) combined aromatase activity with cells exposed as describe d in the indirect assay, and the same atrazine concentrations were added again during the conduc t of the tritium-release assay to evaluate the combined effects of pre-exposure and direct interaction with catal ytic enzyme activity; and iii) direct aromatase activity with atrazine added to untreated cells during the conduct of the tritiumrelease assay at the same concentrations as desc ribed in the combined aromatase assay to assess direct interaction with the enzyme.

Atrazine induced indirect (p <0.001) and combined (p<0.05) ar omatase activity, but direct aromatase activity was not different from control at any concentration tested. Aromatase activity
measured in the indirect and combined aromatas e assays was approximately 2.0-fold greater than control levels. However, the increase in the combined assay was less pronounced than that observed in the indirect assay, and was increas ed (p<0.05) compared to the solvent control starting at 10 μM. Atrazine increased (p<0.05 or 0.01) 17 β -estradiol production in a dosedependent manner at concentrations ≥ 1 μM. 17 β -estradiol concentrations were approximately 7.2-fold greater at the high dose. Testosterone was increased (p<0.01) at the two highest concentrations (10 and 100 μM), but the magnit ude of the change was less than observed for

17 β -estradiol (maximum fold cha nge of testosterone = 1.4).

In a published, multicomponent study, Sanderson et al . (2001) investigated the effects of atrazine, and the metabolites DIA and DEA, on human aromatase (CYP19) activity and mRNA levels in H295R, JEG-3 (placental choriocarcin oma), and MCF-7 (breast cancer) cells. The cell lines were exposed to atrazine or the meta bolites at concentrations of 0, 0.3, 1.0, 3.0, 10.0, or 30 μM.

Atrazine and the metabolites were able to i nduce the catalyti c activity of aromatase in a concentration-dependent manner to an apparent maximum of just over 2-fold (data presented graphically) in H295R cells. Addi tionally, in H295R cells trea ted with 30 μM atrazine or metabolites, tritiated water release and estrone production were increased in a 1:1 ratio. The
mechanism of induction of arom atase activity appeared to in volve the induction of CYP19ATRAZINE FINAL

Page 71 of 159 mRNA, because atrazine and the metabolites were able to increase mRNA concentrations for
CYP19 relative to control.

In JEG-3 cells, atrazine induced aromatase ac tivity in a concentra tion-dependent manner, producing increases (p<0.05) in ac tivity above control at concentr ations above 1 μM. The most
noticeable difference between JEG-3 and H295R cells is the basal activity of aromatase, which was at least an order of magnitude greater in JEG-3 cells (about 15 pmol androstenedione/hour/mg cellular protein) than in H295R cells (about 1–1.5 pmol androstenedione/hour/mg cellular protein). In MCF-7 cells, basal ar omatase activity was undetectable, and atrazine was not able to induce the activity to de tectable levels; the same was
true for mRNA concentrations.

In another published study by Sanderson et al . (2002) , atrazine and other pesticides were
screened in H295R cells for eff ects on the catalytic activity and mRNA expression on aromatase. To investigate the mechanism of aromatase inducti on in H295R cells, the ab ility of atrazine to
increase intracellular cAMP concentrations was examined. As previously reported ( Sanderson et al., 2000 ), atrazine increased CYP19 mRNA levels in H295R cells. In the pr esent study, atrazine increased (NS) cAMP concentrations in a time -dependent manner in H295R cells, producing a time-response course with an appare nt post-maximal decline after 4 hours.

In a published study by Hinfray et al . (2006) , brain and ovarian microsomes prepared from rainbow trout ( O. mykiss ) were exposed to atrazine at conc entrations from 10 nM to 100 μM, and aromatase activity measured by the 3 H 2 O -release assay. Atrazine had no effect on aromatase activity at the concentrations tested.

In a published study by Holloway et al . (2008) , human granulosa-lutein cells and human endometrial stromal cells were exposed to atrazine at 1 nM to 100 μM for 24 hours. Prostaglandin E 2 (PGE 2 ) and vomitoxin were included as pos itive control groups. The medium was removed and aromatase activity was measured by the 3 H 2 O-release assay. Additionally, a cell-free human recombinant aromatase assay was c onducted with atrazine at concentrations of 1 pM to 100 μM; aromatase activity was determined with a fluorescent substrate (dibenzyfluorescein).

Atrazine induced a dose-dependent increase (p<0.05) in aromatase activity in granulosa-lutein cell cultures at both 0.1 and 1.0 μM, causing a greater than 2-fold increase in aromatase activity
over baseline levels. Atrazine had no effect on cell number as demonstrated by a lack of change in protein concentrations acro ss the treatment groups, suggesting that the increase in aromatase activity was not due to an increase in the number of cells present. In contrast to granulosa-lutein cells, atrazine had no effect on aromatase activity at any dose tested in endometrial stromal cells, whereas the positive controls PGE 2 (1.0 μM) and vomitoxin (250 ng/ml) induced an increase in aromatase activity (132% and 149% of control, respectively; p<0.05). There was no effect of atrazine on endometrial stromal cell protein concentrations across the treatment groups,ATRAZINE FINAL

Page 72 of 159 suggesting that treatments had no e ffect on cell number and that at razine, at the concentrations studied, was not cytotoxic to ei ther endometrial stromal cells or granulosa-lutein cells.

To determine if the increase in granulosa-lutein cell aromatase activity was due to an increase in the proportion of cells expre ssing aromatase, slides were prepared and stained immunohistochemically with a monoclonal anti -human aromatase antibody. Treatments had no effect on the percentage of cells that stained positively for aromatase. Atrazine also had no effect on aromatase protein expression in immunoblots. The direct eff ect of atrazine on aromatase activity was tested with a cell-fr ee aromatase assay. Atrazine at 1 μ M had no effect on human recombinant microsomal aromatase activity. By comparison, testosterone, used as a positive control in this study, competitively inhibited di benzyfluorescein (10.7% of control activity).

Finally, in a published study by Tinfo et al . (2011) , rat primary granulosa cells were exposed to atrazine (97.1% purity) at 0, 1, 10, or 30 μM for either 24 or 48 hours. Production of 17 β

estradiol and progesterone was determined in th e media, and aromatase activity was measured by
the 3 H 2 O-release assay. Positive (8-Br-cAMP; 100 μM) and negative (4- hydroxyandrostenedione; 10 μM) controls were included. H295R cells were also cultured and
treated with atrazine as above for 48 hours.

The 8-BrcAMP and 4-hydroxyandrostenedione were successfully used to demonstrate
stimulation and inhibition of 17 β -estradiol production as compared with control. Atrazine did not
alter 17 β -estradiol production over the 24-hour exposure period. However, these experiments demonstrated the ability of atrazine to alter the production of progesteron e. Cells treated with atrazine (10 and 30 μM) increase d (p<0.05) progesterone producti on compared to control during the 24-hour exposure. Two additional treatment regimens (immediate exposure 24-48 hours) also
were used to evaluate the effects of atrazine on steroid production. In these experiments, the cells were exposed to the test chem icals immediately after plating. Again, no effects were observed on

17 β -estradiol production determined after 24 or 48 hours of atrazine; however, atrazine increased progesterone production after 24- hour (significance not report ed; 30 μM) and 48-hour (p<0.05; 10 and 30 μM) exposure in granulosa cells.

The production of 17 β -estradiol and progesterone, as well as aromatase activity, were determined in granulosa cells during a 1-hour period after 24-hour exposure. Concentrations of 17 β -estradiol were significantly increased for atrazine (p<0.05; 10 and 30 μM) when evaluated over the 1-hour period. To investigate th e possible mechanism behind the increase in 17 β -estradiol observed after treatment with atrazine, aromatase activ ity was evaluated during the same time period. Positive and negative controls, 8-Br-cAMP ( 100-200 μM) and 4- hydroxyandrostenedione (10 μM), respectively, were successfully used to de monstrate stimulation and inhibition of aromatase activity. Atrazine (10 and 30 μM ) increased (p<0.05) aromatase activity by up to two-fold. The magnitude of change in 17 β -estradiol production was similar to that observed for the increase in aromatase activity after atrazine treatment. In addition, pr ogesterone production was increased (p<0.05) during the 1-hour post-exposure period for atrazine (1, 10, and 30 μM).ATRAZINE FINAL

Page 73 of 159 Finally, H295R cells were exposed to 1, 10, or 30 μM atrazine prior to determining the concentrations of estrone, 17 β -estradiol, and progesterone in the culture medium. Atrazine (10 and 30 μM) increased (p<0.05) the concentrations of estrone, 17 β -estradiol, and progesterone. These studies confirm previous reports that atrazine treatment stimulates estrone and

17 β -estradiol production, and extend these findings to demonstrate the ability of atrazine to alter progesterone production in H295R cells.

Estrogen Receptor (ER) Binding Assay

In the Integrated Summary Report for the validat ion of an estrogen receptor (ER) binding assay ( USEPA, 2009 ), an in vitro rat uterine cytosol ER bi nding assay was optimized and validated for use as an EDSP Tier 1 testing requirement to identify chemicals capable of binding to the ER.
After an initial validation of the ER binding assay with five laboratories, a second interlaboratory validation of the ER binding assay wa s conducted with three te sting facilities; the performance of atrazine in the ER binding assay was determined in all three testing facilities.
Atrazine was selected to be a negative chemical (non-binder, al beit estrogen active) in the validation of an ER binding assay in the EDSP and as such provides supporting information on the performance characteristics of a negative compound in the assay.

Uterine cytosol from SD rats was used as the source of ER to conduct Saturation and Competitive Binding Experiments. The Competit ive Binding Experiment measured the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in th e presence of increasing concentrations (testing range was not reported) of atrazine (purity not reported). A total of three
or four runs were conducted at each testing facility, and each run included a weak positive control, norethynodrel; a negative cont rol, octyltriethoxysilane); and 17 β -estradiol as the ligand
reference standard.

In the second set of inter-laboratory Satura tion Binding Experiments, the mean dissociation constant (K d ) for [ 3 H]-17 β -estradiol ranged from 0.1137 to 0.6588 nM and the mean estimated

B max ranged from 25.85 to 89.97 fmol/100 μ g protein for the site-speci fic batches of prostate cytosol that were prepared and tested.

In the second set of inter-laboratory Competitive Binding Experiments, the mean log IC 50 values
for 17 β -estradiol ranged from

− 9.1 to

− 8.9 log M (with an outlier of

− 9.7) and for the weak positive control, norethynodrel, ranged from

− 6.1 to

− 5.9 log M. The mean relative binding affinity (RBA) for the weak positive control ranged from approximately 0.0009 to 0.0011 or 0.09
to 0.11%.

Precipitation and/or cytotoxicity with atrazine we re not reported. Atrazine binding data were not reported for the three test laboratories. Although two of the three laboratories concluded that atrazine was equivocal in the ER binding assa y, the reasons for these conclusions were not reported. This category covers compounds which app ear to be interacting with the receptor atATRAZINE FINAL

Page 74 of 159 high concentrations but are so weak that they displace only 25 to 50% of the radioactive estradiol. Additionally, equivocal compounds may have a precipitous slope that may be an artifact of the fitting algorithm rather than a reflection of the behavior of the chemical. Although the third laboratory reported atrazine as a non-binder, overall , atrazine was classified as “equivocal” in the ER binding assay.

In a published study by Tennant et al . (1994b) , the potential for atrazi ne (as well as other
chloro- s -triazines and metabolites) to bind to rat ut erine ER in vitro was examined. Suspensions of atrazine (97.7% purity) were prepared in 0.5% carboxymethylcellulose in tap water. The in vitro binding studies were conducted with lyoph ilized pools of uterine cy tosol prepared from
intact adult female SD rats. Determination of ER binding in uteri of tr iazine-dosed rats was conducted with freshly prepared cytosols from individual ovariectomized female SD rats.

Binding was determined by incubating cytosol, 5 nM [ 3 H]-estradiol, and competitor ( e.g. , atrazine, unlabeled estradiol, or blank) at room te mperature (25°C), refrigerated at 4°C, or in an ice-water bath. All incubations were complete w ith the tubes at 4°C for the charcoal separation step (addition of a dextran- charcoal suspension for a 10-minute incubation). Tubes were centrifuged at 2000×g for 15 minutes at 4°C, and receptor-bound radiolabelled estradiol was determined in the supernatant samples by LSC.

Ovariectomized rats were given atrazine suspen sions of 50 or 300 mg/kg by oral gavage for two days. The uteri were dissected at necropsy, slic ed, and incubated in modified Eagle’s medium with 10 nM [ 3 H]-estradiol for 30 minutes at 37°C in 95% O 2 /5% CO 2 . After incubation, the tissue fragments were homogenized and cytosol was prepared as described for the in vitro binding experiment with the addition of ice- cold TEG hypotonic buffer system [Tris (10 mM ), EDTA (1 mM), sodium molybdate (10 mM), dith iothreitol (1 mM), and glycerol (10%) at pH 7.40 with 0.4 M KCl] prior to ultracentrifugation. Portions of cytosol were counted by LSC, and binding data were normalized for protein content.

Atrazine, at concentrations of 10

− 8 to 10

− 3 M, did not displace [ 3 H]-estradiol from the ER in the standard in vitro competitive binding assay. Due to these findings, disequilibrium binding experiments were conducted next. Dose-related competition of atrazine against [ 3 H]-estradiol-ER binding was assessed by pre-incubati ng cytosol at 25°C with various concentrations of atrazine (10

− 7 to 10

− 3 M) or unlabeled estradiol (10

− 11 to 10

− 7 M) prior to addition of [ 3 H]-estradiol. Under conditions that strongly favored the competitor, binding of [ 3 H]-estradiol was reduced by atrazine, but to a much lesser extent than th e reduction caused by estradiol. Reduction of radioligand binding to a 50% exte nt required approximately 20 μM atrazine, which was four to five orders of magnitude gr eater than the approximate IC 50 of estradiol.

Finally, a determination of the degree of competition after atrazine administration in vivo was conducted. Binding of [ 3 H]-estradiol in incubated uterine s lices was decreased (p<0.05) by 33%
in ovariectomized rats that were administered atrazine (300 mg/kg) by gavage for two days.ATRAZINE FINAL

Page 75 of 159 Administration of atrazine at 50 mg/kg/day redu ced estrogen binding by 18% (NS). These results demonstrated that, in the presence of a high dos e of atrazine, rat uterine ER had a diminished ability to bind estrogen.

In a published study by Blair et al . (2000) , the potential for 188 various natural ligands and xenochemicals, including atrazine (98% purity), to bind to ra t uterine ER in vitro in a competitive binding assay was examined. The in vitro binding studies were conducted with ERenriched uterine cytosol prepared fr om intact adult female SD rats.

The [ 3 H]-estradiol (1×10 –9 M final assay concentration) was incubated with increasing concentrations of radio-inert competitor, uterin e cytosol preparation, and 50 mM Tris buffer (pH 7.4) in duplicate tubes. Reaction-mixture tubes were placed incubated at 4°C for 20 hours. After the incubation period, a cold 60% hydroxylapat ite (HAP) slurry was added to each tube, followed by incubation in an icewater bath fo r 20 minutes. After centrifugation, the resulting HAP pellets were resuspended in cold 50 mM Tris buffer, washed three times, and 2.0 ml of cold (4°C) 100% ethanol was added to e ach tube to extract the radiolabeled estradiol from the HAP. The resulting extracted supernat ants were counted by liquid sc intillation counter (LSC).

Atrazine (and other test chemi cals) was dissolved in 100% ethanol at the highest concentration possible. Stock solutions were subsequently diluted in ethanol for analysis in the ER competitive-binding assay. Due to the large numbe r of chemicals tested, the ER competitivebinding assays were conducted in a tiered design. Unless known to bind to the ER, test chemicals were initially run at only two high concentrations spanning 3 log concentrations (Tie r 1). If a test compound exhibited binding to the ER, then a s econd assay (Tier 2) was conducted with a wider range of concentrations, ranging generally from 1×10 –4 to 1×10 –9 M in 10-fold increments. A Tier 3 assay (consisting of one-half log molar concentra tions which bracketed the IC 50 observed in the Tier 2 assay) was conduc ted if necessary. Chemicals whic h failed to bind the ER were designated as “non-binders.” All assays were replicated a minimum of two times and IC 50 values of positive chemicals are the means of the replicates.

Atrazine was negative in the ER competitive binding assay (Tier 1), with an estimated IC 50 of

>1×10

− 4 (the highest concentration tested).

In a published study by O’Connor et al . (2000) , the potential for atrazi ne (as well as other
chloro- s -triazines and metabolites) to bind to rat uterine ER in vitro also was examined. Atrazine (purity not reported) , and other chloro- s -triazines and metabolites, were evaluated for their ability to compete for binding to the ER in competition binding assays. The competitive binding assays were conducted at Novascreen (Hanover, MD); assay details were not reported. Three concentrations (10

− 7 , 10

− 5 , and 10

− 3 M) of atrazine were incubate d with ER homogenates and the appropriate radioligand (0.3 nM [ 125 I]-estradiol), and the per cent binding inhibition was calculated. Atrazine was negative in the ER competition binding assay.ATRAZINE FINAL

Page 76 of 159 ER/AR Transactivation Assays

In a published study by Kojima et al . (2004) , two hundred pesticides covering nine chemical classes were screened for their potential to interact with two human ER subtypes (hER α and
hER β ) and one human AR (hAR) in transactivation assays in Chinese hamster ovary cells (CHOK1 cells). Cells were grown in DMEM/F-12 (Dulbecco’s modified Eagle medium plus Ham’s F-12 nutrient mixture) supplemented with 10% fe tal bovine serum (FBS) and antibiotics at a temperature of 37°C in an atmosphere of 5% CO 2 under saturating humidity. Atrazine was
dissolved in DMSO to a final c oncentration of 10 mM and diluted to the desired concentration(s)
in DMEM/F-12. The final solvent concentration in the culture medium did not exceed 0.1% and did not affect cell yields.

The hER α and hAR expression vectors (pcDNAER α and pZeoSV2AR, respectively) were constructed as described previously ( Kojima et al. , 2003 ). The hER β expression vector was constructed by cloning the hER β cDNA with reverse transcript ase-polymerase chain reaction from human placental RNA. The sequence was verified and inserted into the mammalian expression vector pcDNA 3.1Zeo(-) creating pcDNAER β . The construction of the estrogen or androgen responsive elements containing re porter plasmid (pGL3-tkERE and pIND-ARE, respectively) was described previously ( Kojima et al. , 2003 ). pRL-SV40 containing the Renilla luciferase gene was used as an intern al control for transfection efficiency.

Host CHO cells were plated into 96-well microt iter plates at a dens ity of 8,400 cells/well in phenol red-free DMEM/F-12 cont aining 5% CD-FBS (complete medium) one day before
transfection. For detection of hER α or hER β activity, cells were transfected with 5 ng pcDNAER α or pcDNAER β , 50 ng pGL3-tkERE, and 5 ng pRL-SV40. For detection of hAR activity, cells were transfected with 2.5 ng pZeoSV2AR, 50 ng pIND-ARE and 5 ng pRL-SV40. After a 3-hour transfection period, cells were dosed with various con centrations of test compounds (95-100% purity) or with 0.1% DMSO (v ehicle control) in complete medium. For measurement of antagonistic activity to hER α , hER β and hAR, either 10

− 11 or 10

− 10 M E 2 (17 β

estradiol, > 97% purity) or 10

− 10 M DHT (5 α -dihydrotestosterone, 95% purity) was added to the cell cultures along with the test compound, resp ectively. Cells were incubated for 24 hours, rinsed with phosphate buffered saline (pH 7.4), and lysed with passive lysis buffer (50 μL/well). Luciferase activity was measured with a Mini Lumat LB 9506 luminometer and was normalized based on the activity of co-transfected pRL-SV4 0. At least three independent experiments were conducted for each compound.

Agonistic activities were evaluated by relative ac tivity expressed as REC 20 (20% relative effective concentration). The REC 20 is the concentration of the compound showing 20% of the activity of 10

− 10 M E 2 , 10

− 9 M E 2 , or 10

− 9 M DHT for ER α , ER β and AR, respectively. Antagonistic activities were evaluated by relative activity expressed as RIC 20 (20% relative inhibitory concentration). The RIC 20 is the concentration of the compound showing 20% of theATRAZINE FINAL

Page 77 of 159 inhibition of the activity of 10

− 10 M E 2 , 10

− 9 M E 2 or 10

− 9 M DHT for ER α , ER β and AR, respectively. The range of concentrations tested was approximately 10

− 8 to 10

− 5 M; all pesticides
were tested at concentrations ≤ 10

− 5 M to avoid cytotoxicity. Wh en the activity of the test compound was higher than the REC 20 the pesticide was judged to be positive for activity. When the activity of the test co mpound was higher than the RIC 20 the pesticide was judged to be positive for inhibitory activity. The statistical si gnificance of differences was evaluated with a
two-tailed Student’s t test with a nominal level of significance of p<0.05.

Atrazine exhibited no activity against hER α , hER β , or hAR in these screening studies.

In a published study by Fujimoto and Honda (2003) , a pituitary tumor cell line, MtT/E-2, which is characterized by high sensitivity to estrogen regarding pro liferation, was used to investigate the ability of atrazine to activate the ER. Int act MtT/E-2 cells were exposed to
atrazine at concentrations of 10

− 7 to 10

− 4 M to examine if atrazine could stimulate cell proliferation. MtT/E-2 cells were transiently tran sfected with an ERE-luci ferase reporter gene and exposed to atrazine at the sa me concentrations to see if luci ferase activity was stimulated. In a third experiment, six week-old surgically ova riectomized F344 rats were inoculated with
MtT/E-2 cells at two sites within the subcutaneous fat pads and admi nistered atrazine in the diet at dose levels of 0, 5, 50, or 500 mg/kg. The resulti ng tumors were allowed to grow to 1-2 cm diameter, then the rats were euthanized and th e pituitary glands and uteri were collected and weighed. Blood samples were collected for se rum prolactin concentr ation determination.

Atrazine had no effect on cell proliferation, i nduction of luciferase act ivity, tumor promotion, pituitary or uterus weig hts, or serum prolactin.

In a published study by Balaguer et al . (1999) , MCF-7 cells were stably transfected with an ERE-luciferase reporter gene, a nd HeLa cells were stably transfected with an ERE-luciferase reporter gene and then ER α or ER β constructs. These cell lines were exposed to atrazine (>98% purity) to examine activation of the ER. Atrazine had no effects in any of the cell lines tested.

In a published, multicomponent study by Connor et al . (1996) , MCF-7 cells and the recombinant estrogen-dependent yeast strain PL3 were exposed to atrazi ne (>97% purity) to
determine if atrazine could activate the ER. MCF-7 cells were treate d with atrazine at concentrations of 10 nM to 10 μM in the presence and absence of 17 β -estradiol to investigate ER-mediated cell growth; MCF-7 cells were transi ently transfected with Gal4-ER chimera and a Gal4-luciferase reporter construc t to measure atrazine effects on reporter gene activity in the presence or absence of 17 β -estradiol; and the recombinant es trogen-dependent yeast strain PL3 was exposed to 10 μM atrazine to measure ER-mediated growth in the absence of 17 β -estradiol. Additionally, a gel electrophoretic mobility shift assay for progesterone receptor (PR) - progesterone responsive element (PRE) comple x formation was conducted on nuclear extracts
from 17 β -estradiol, atrazine, and 17 β -estradiol + atrazine treated MCF-7 cells.ATRAZINE FINAL

Page 78 of 159 Atrazine did not alter MCF-7 cell proliferation with or without 17 β -estradiol at any concentration tested. Similarly, atrazine alone did not induce reporter gene activ ity in transiently transfected MCF-7 cells, and atrazine di d not inhibit or enhance 17 β -estradiol -induced reporter gene activity
in these cells. 17 β -estradiol caused a 3.5-fo ld increase in PR-PRE band intensity, whereas
atrazine did not induce the form ation of the PR-PRE complex. In cells co-treated with 17 β

estradiol + atrazine, neither com pound affected the intensity of 17 β -estradiol -induced PR-PRE complex bands relative to E 2 alone. Finally, atrazine di d not stimulate growth of 17 β -estradiol - dependent PL3 yeast cells.

In a published, multicomponent study by Fukamachi et al . (2004) , MCF-7 cells were exposed to atrazine (purity not reported) at unspecified concentrations for th ree days, and cell proliferation
was measured. Atrazine had no effect on MCF- 7 cell growth at any concentration tested.

In a published study by Legler et al . (2002) , the effects of atrazine (purity and concentrations used not reported) in an ER-mediated lucife rase reporter gene (ER-CALUX) assay were examined. The ER-CALUX assay uses T47-D hum an breast adenocarcinoma cells expressing endogenous ER α and ER β , which are stably transfected with an estrogen-responsive luciferase reporter gene. Exposure of stably-transfected T47-D cells to (xeno-) estrogens results in
transactivation of the ER and consequent induction of the luci ferase gene. Atrazine had no estrogenic activity in the ER-CALUX assay at the concentrations tested (not reported).

Fish Short Term Reproduction Assay (FSTRA)

In a multi-chemical evaluation of the short-te rm reproduction assay with the Fathead minnow ( Battelle, 2005 ), the FSTRA was optimized and validated for use as an EDSP Tier 1 testing requirement to identify chemicals capable of affecting the short-te rm reproductive fitness in fish.

In a 21-day FSTRA study c onducted under flow-through conditions with Fathead minnows ( P. promelas) , 12 spawning groups of 17-week-old fish (2 males and 4 females in each group; 4 replicates per treatment) were exposed to atrazine (purity not reported) at nominal concentrations of 0 [negative or solvent c ontrol (acetone) not specified], 25, and 250 μg/L. Mean-measured concentrations were <2.5 (<MDL; control) , 24.5±18.9, and 223.7±156.6 μg/L. The test system was nominally maintained at 24-26°C; pH was not reported.

One female in the control group died during the a ssay; overall survival was 100% in all atrazine treatment groups. No treatment-related effects on s econdary sex characterist ics or clinical signs were reported, although secondary sex characterist ics were reportedly assessed as part of the daily observations to determine overall health. B ody weights of male or female minnows did not demonstrate any treatment-related differe nces compared to control weights.

Spawning frequency in the control group was not reported for the 9-day pre-exposure period or for the 21-day assay period. No differences in to tal fecundity were noted during the pre-exposure or assay periods between the study groups, althou gh total fecundity was decreased (NS) by 17%ATRAZINE FINAL

Page 79 of 159 in the treated groups. Similarly, mean fecundity per female reproductive day was 53.6 eggs in the control group and 42.1 and 42.2 eggs, resp ectively in the treated groups (NS; ↓ 21%). Fertilization success was 100% in all replicat es, except for a single replicate at the low concentration. In addition, no trea tment-related difference was noted in hatchability or larval development.

Plasma vitellogenin (VTG) concentrations were variable, but no treatment effect on plasma VTG
concentrations in male or female fish were obs erved at any treatment c oncentration. Sex-steroid concentrations (estradiol and testosterone) were measured in male and female fish; no treatmentrelated effects were noted in either sex for sex-steroid concentrations.

Treatment-related effects on gonadal histopathology were not observed in female fish. In male fish, a difference in testicular staging was observed with both treatment groups having a greater proportion of cells in Stage 2A, and the low-c oncentration group having a lower proportion of cells in Stage 2B, compared to control. Finall y, there were no changes in gonado-somatic index (GSI) for either sex.

In a published study by Bringolf et al . (2004) , ten spawning groups of 22-month-old adult fathead minnows ( P. promelas ; 2 males and 4 females in each group; 2 replicates per treatment) were exposed to atrazine (>98% purity) at concentrations of 0 (water and solvent [10 μL/L 50:50 methanol:acetone] controls), 5, or 50 μg/L fo r 21 days under static conditions. An additional group exposed to 0.5 μg/L 17 β -estradiol was treated as above.

Fish mortality, number of spawns, and number of eggs spawned (fecundity) were monitored daily. Fecundity was monitored by counting all eggs adhered to the inside surface of the spawning substrate. Incubation of eggs was used for assessment of fertilization success (number of eyed eggs by 48-hours post-spawn), hatching success (% of 50 eyed eggs that hatched by 120 hours post-spawn), and larval fish survival (% of hatched fish that survive to 48 hours posthatch). On Day 21, coloration of male fish was evaluated in situ based on the presence or absence of light and dark vertical bands on the body surface, and the presence or absence of an ovipositor was noted for female fish. After observa tion of secondary sexual characteristics, all fish were anesthetized, weighed, measured, and bled. Fish sex was confirmed by gross examination of the gonads. The gonads were removed, weighed for determination of GSI, and fixed. Nuptial tubercles were counted unde r a dissecting microscope. Plasma VTG concentrations were quantifie d with a commercially-availab le ELISA method. Fixed gonads were processed and stained with hematoxylin and eosin.

Fish survival was 100% during the exposure peri od. No differences were observed in length or weights of males or females at the end of the study. Cumulative fecundity (total eggs spawned/treatment) of fish exposed to atrazi ne was slightly decreased (NS) compared to controls. A rapid divergence in fecundity was evident in the 17 β -estradiol treatment group; noATRAZINE FINAL

Page 80 of 159 eggs were produced in the 17 β -estradiol treatment group after the seventh day of the exposure period.

Fertilization success was not si gnificantly reduced in the atrazine treatment groups. No significant or obvious effects from atrazine exposure of adult fish were observed on hatching
success or larval fish survival to 48 hour s post-hatch. Male and female GSI and gonad maturation were reduced by exposure to 17 β -estradiol but no statistica lly signficiant differences
from controls were observed for atrazine-treat ed animals. No mixed gonads (testis-ova) were identified in fish in any of the treatment groups.

Atrazine did not have an appare nt effect on the secondary sexual characteristics of male fish, although the effects of 17 β -estradiol exposure were evident. Exposure to atrazine did not reduce the number of breeding tubercles compared with control fish; however, fish exposed to 17 β

estradiol did not have breeding tubercles. It was not known if the fish exposed to 17 β -estradiol had breeding tubercles that re gressed in response to the 17 β -estradiol exposure or if the tubercles failed to develop. No obvious effects of atrazi ne on coloration of male fish were observed, although all males exposed to 17 β -estradiol lacked vertical ba nding patterns on their lateral body
surfaces. Plasma VTG concentrations were incr eased (p<0.0001 and 0.05, respectively) in male and female fish exposed to 17 β -estradiol compared to all other treatments. Plasma VTG concentrations were greater in male fish exposed to both atrazine treatme nt concentrationsand in solvent control group males, as compared to ne gative controls. Although a pparently greater than in negative controls (statistic al significance unreported), the plasma VTG concentrations in
atrazine treatments and solvent controls remained three to four orders of magnitude lower than those in male fish exposed to 17 β -estradiol. In female fish, neither atrazine nor solvent affected plasma VTG concentrations.

In a portion of a published, multicomponent study, Sanderson et al . (2001) investigated the
effects of atrazine and atr azine metabolites on ER-mediated induction of VTG in primary hepatocyte cultures of adult male carp ( Cyprinus carpio ) was examined. Isolated male carp hepatocytes were exposed to atrazine or th e metabolites at 0, 0.3, 1.0, 3.0, 10.0, or 30 μM in the presence and absence of 17 β -estradiol.

VTG concentrations in unexposed or dimethylsulfoxide (DMSO )-exposed male carp hepatocytes
were undetectable. A lowest observe d- effect concentration of 17 β -estradiol of about 2 nM produced a detectable amount of VTG of about 100–400 ng/mg cellular protein; the EC 50 of 17 β

estradiol-induced VTG concentrations to 4,000–6,000 ng/mg protein. Exposure of male carp hepatocytes to various concentrations (0–30 μ M) of the atrazine di d not induce VTG production. None of the compounds could produ ce a concentration-dependent an ti-estrogenic re sponse in the presence of 100 nM 17 β -estradiol.ATRAZINE FINAL

Page 81 of 159 Hershberger Assay

In a Hershberger antagonist assay ( Ashby, 2004 ), six-week old male rats [Alpk:APfSD (AP)] were castrated, acclimated for 24 hours, and rand omized into treatment groups (6/group) based on body weight. The rats were dosed by oral ga vage at dose volumes of 5 mL/kg for ten consecutive days in three separate studies with the following test substances: atrazine (purity 97.6%), flutamide (FLU; positive control AR blocki ng agent), or finasterid e (FIN; inhibitor of the enzyme testosterone-5 α -reductase). The vehicle for all treatments was 0.5% (w/v) carboxymethylcellulose (CMC) sodium salt in dei onized water. Each treatment group included a

s.c . injection of 0.4 mg/kg/day testos terone propionate (TP) in toc opherol-stripped corn oil at a dose volume of 1 mL/kg. The first study include d a vehicle control gr oup, four atrazine dose groups (10, 25, 50, or 100 mg/kg/day), and the pos itive control groups FLU (3 mg/kg/day) and FIN (25 mg/kg/day). The second study include d a vehicle control gr oup, two atrazine dose groups (50 or 100 mg/kg/day), and FLU (3 mg /kg/day). The third study included a vehicle control group, atrazine (100 mg/kg/day), and FLU (3 mg/kg/da y). Animal weights were recorded on each day of treatment and at termination.

The rats were terminated 24 hours after the last dose in each study and the following tissues were removed and weighed: liver, kidneys, adrenal gl ands, and accessory sex tissues (AST; Cowper’s glands, LABC, seminal vesicles [with coagulatin g gland], ventral prosta te, and glans penis).

Body weights in the high-dose atrazine group sh owed small decreases (NS) compared to the
controls in all three studies. A similar decrea se was also observed in the medium-high dose group in the first study. Based on this, the study au thor concluded that higher doses would not
have been tolerated. In the fi rst study, the LABC weights were decreased (p<0.05) in the highdose group. In the second study, the LABC wei ghts were decreased in the 50 mg/kg/day (p<0.01) and 100 mg/kg/day (p<0.0 5) atrazine dose groups. LABC weights also were decreased (p<0.01) in the high-dose group, and the glans peni s weight was increased (p<0.01). The positive controls FLU and FIN performed as expected in the three studies.

Inconsistent non-AST organ weight changes were noted in the th ree portions of this study. Adrenal weights were increased (p<0.05; ↑ 10%) in the high-dose group in Study 1. Liver and kidney weights were increased (p<0.05; ↑ 7%, respectively) in the medium-high dose group in Study 2. Kidney weights also were increased (p<0.01; ↑ 8%) in the high-dose group in Study 3. The study does not fully comply with the O PPTS 890.1400 Guideline, as it was conducted prior to Guideline publication. In addition, the st udy was not conducted according to Good Laboratory
Practices.

In another Hershberger antagonist assay ( Yamasaki et al. , 2004 ), six-week old male rats [Brl Han: WIST Jcl (GALAS)] were cas trated and acclimated for 14 days. Rats were randomized into treatment groups (6/group) based on body weight. Th e rats were dosed by oral gavage for ten consecutive days with vehicle control (olive oil) or atrazi ne (purity 99.9%) at 3.2, 16, orATRAZINE FINAL

Page 82 of 159 80 mg/kg/day in the agonist portion of the study. In the antagonist por tion of the study, the
vehicle and atrazine doses were ad ministered in conjunction with a s.c. injection of 0.2 mg/kg/day TP, as well as a combined TP +FLU (10 mg/kg/day) group. Animal weights were recorded on each day of treatment and at termination. Tissues were collected at study termination as described previously.

Mean body weight was decreased (p<0.05) by 7% in the high-dose group in the agonist portion only. No changes in AST weights were observed. The LABC muscle complex is not considered to be the primary or most sensitive AST in the Hershberger assay. The results of Yamasaki et al. (2004) were consistent with the findings of Ashby (2004) . Based on the results of these two studies, the study authors concluded that atrazine was without activ ity in the Hershberger assay.

Female Pubertal Assay

In a published study by Laws et al . (2000) , female Wistar rats (15/dose group) were administered atrazine (97.1% purit y) in aqueous 1% methyl cellulo se by oral gavage (5 mL/kg) at doses of 0, 12.5, 25, 50, 100, or 200 mg/kg/day during post natal days (PND) 22-41. The study was conducted in two blocks, with eight treatme nt groups included in the first block. Seven females administered 0, 50, 100, or 200 mg/kg/day were terminated on PND 41, the remaining females were evaluated for changes in estrus cyclicity from PND 42-149. All females in the 12.5 and 25 mg/kg/day groups were terminated on PND 41. An additional pair-fed control group (n=15) was given the amount of food consumed by the 200 mg/kg/da y rats on the previous day; half were terminated on PND 41, and the remaining rats were evaluated for estrus cyclicity as above. Body weights were recorded daily, and the dosing volume adjusted for changes in body weights. For females killed on PND 41, liver, ki dney, adrenal, ovary, uterus, and pituitary weights were recorded, and serum T 3 , T 4 , TSH, and prolactin concentrations were measured. The uterus, ovaries, and thyroid were fixed and processed, and tissues from the pair-fed control, 0, and 200 mg/kg/day were examined microscopicall y. All rats were evaluated daily for VO; the day of complete VO, and body weight on that day, were recorded. Beginning on the day of complete VO, vaginal smears were collected and evaluated in the 0, 50, 100, 200 mg/kg/day and pair-fed control groups to determine the age of the first complete es trus cycle after VO.

Terminal body weight (PND 41) was decreased (p<0.05) by 12% in the 200 mg/kg/day rats; body weight gain during treatmen t was decreased (p<0.05) by 16% in this group. Similarly, in the rats used for estrus cyclicity observations , body weight was decreased (p<0.05) by 11% and body weight gain also was decreased (p< 0.05). VO was delayed (p<0.05) by 3.4, 4.5, and >6.8 days in the 50, 100, and 200 mg/kg/day grou ps, respectively, and VO did not occur during treatment in 4/15 high-dose rats. Body weights at VO were increased (p <0.05; data presented graphically) in the 50, 100, and 200 mg/kg/day groups . In the females used for estrus cyclicity observations, age at VO was delayed by 1.5, 3.5, a nd 8.0 days in the 50, 100, and 200 mg/kg/day groups, respectively, and VO did not occur in 8/15 high-dose rats. However, once treatment wasATRAZINE FINAL

Page 83 of 159 stopped on PND 41, VO occurred within 3-4 days in these rats. Body weights at VO were increased (p<0.05) by 7%, 11%, and 21% in the 50, 100, and 200 mg/kg/day groups.

In the pair-fed control group, body weights and body weight gains we re similar between the pairfed control and the 200 mg/kg/day rats, and were d ecreased compared to vehicle control. A slight delay (NS) in VO was observed in the pair-fed rats (1.6 days).

Organ weights were analyzed both by ANOVA and Bonferroni’s t-test for multiple comparison and by ANCOVA with terminal body weight as the covariate. By ANOVA, kidney weights were decreased by 15% and adrenal weights were d ecreased by 15% at in the high-dose group, and pituitary weights were decreased by 16% and 31% in the medium-high and high-dose groups. By ANCOVA, liver weights were increased by 6% , kidney weights were decreased by 8%, and adrenal weights were decreased by 3% in the high-dose grou p, and pituitary weights were decreased by 13% and 16% in the medium-high and high-dose groups.

In the high-dose group, seven female s were not cycling, and ovary ( ↓ 38%), uterus with fluid ( ↓ 56%) and uterus without fluid ( ↓ 42%) weights were decreased (p <0.05). Uterus without fluid weight also was decreased (p<0.05) by 31% in the medium-high dose group. In the pair-fed control group, kidney and pituitary weights were decreased compared to vehicle control, but were greater than the respective organ weight s in the 200 mg/kg/day group. Ovary and uterine weights were not affected in the pair-fed group.

There were no effects of treatment on serum or pituitary prolactin concentrations, or on T 3 , T 4 , or TSH concentrations.

Microscopically, there were no effects of treatment on the thyroid. A decrease in corpus luteum development was noted in the high -dose group that was associated with underdevelopment of the reproductive tract on PND 41. Uterine hypoplasia, characterized by decreased uterine horn diameter, decreased myometrial development, d ecreased or absent endometrial glands, and/or
immature endometrial stroma was observed in 60% of the high-dose females, and was restricted
to those females that demonstrated a marked decrease in corpus luteum development.

The day of the first complete 4-5 day estrus cy cle was delayed (p<0.05) by 6.9 days and 9.6 days in the 100 and 200 mg/kg/day group s, respectively, and the number of 4-5 day cycles observed for 15 days after VO was reduced (1.5 cycles at 100 mg/kg/day and 1.7 cy cles at 200 mg/kg/day vs. 2.7 cycles for control). A similar response was not ed in the pair-fed cont rol (1.6 cycles). This effect was no longer apparent in any group by PND 57-71. Females with regular cycles were decreased by 53% in the 100 and 200 mg/kg/day groups, respectively, but with a decrease of only 29% in the pai r-fed control group.ATRAZINE FINAL

Page 84 of 159 Male Pubertal Rat Assay

In a pubertal rat assay ( Stoker et al. , 2000 ), male Wistar rats were dosed daily by oral gavage with atrazine (97.1% purity) from PND 23 to 53 (or 54). The treatment groups received 0 (vehicle control), 12.5, 25, 50, 100, 150, or 200 mg/kg/ day of atrazine in 1% methyl cellulose suspension. The total number of ma le rats in each treatment gr oup was: 38 rats for the vehicle control; 14 rats each for the 12.5, 25, and 50 mg/kg/day groups; 28 rats each for the 100 and 150 mg/kg/day groups; and 35 rats for the 200 mg/ kg/day group. The study also included a pairfed control group (n=18) in which the rat’s diet wa s restricted to match th e dietary intake of the rats in the 200 mg/kg/day dose group. Pups were initially assi gned to the various treatment groups such that the groups had similar body weight means and variance on PND 22. Six rats each from the vehicle control and high-dose gr oups were sacrificed on PND 45 to evaluate leutinizing hormone (LH) receptor number in the testes, and intratesticular and serum testosterone concentrations, and eight rats from each group were sacrificed at PND 120 (67 days after dosing) to evaluate the re covery of reproductive tract weights and horm one concentrations.

Animals were weighed daily from PND 23 to 53, and PPS was monitored daily beginning on PND 33 until all males showed separation. On PND 53 or 54, 6-24 males per group were sacrificed, blood was collected for hormone assays , and weights of the pi tuitary, testes, ventral and lateral prostates, epididymides, and seminal vesicles with coagula ting gland (with fluid)
were determined. Serum and the ante rior pituitary we re stored at

− 80°C for hormone analysis. The epididymides, left testis, and thyroid were collected for histologic al evaluation. The right
testis was stored at

− 80°C for subsequent LH receptor assa ys. LABC weights were not evaluated in this study. Hormone assays at PNDs 45 a nd 53 included serum and pituitary LH, prolactin (PRL), and TSH concentrations. Serum test osterone, estradiol, estrone, and total T 3 and T 4 concentrations were also determined.

Body weights for the 200 mg/kg/da y (high-dose) group were decrea sed (p<0.05) compared to the vehicle control on PND 43 ( ↓ 14%) and PND 53 ( ↓ 17%). The pair-fed control body weights were also decreased (p<0.05) compared to control on PND 43 ( ↓ 20%) and PND 53 ( ↓ 15%), and the decreases were comparable to the decreases observed in the 200 mg/kg/day group. By PND 120, the mean body weights for all atrazine-dosed r ecovery groups were comparable to control.

Ventral prostate weights at PND 53 were decreased (p<0.05) in the ≥ 50 mg/kg/day treatment groups in a dose-related manner, and in the pair-fed control. Seminal vesicle and epididymal weights at PND 53 also were decreased (p<0.05) in the high-dose and the pair-fed control groups. No differences were observed between the high-dose group and th e pair-fed control group in the weights of these two tissues. No effects of treatment were observed on lateral prostate or testicular weights. After recovery, on PND 120, the ventral prostate weights remained lower (p<0.05) in the high-dose group, but the ot her reproductive organ weights were similar to
control.ATRAZINE FINAL

Page 85 of 159 PPS was delayed (p<0.05) compared to control by 2.3, 1.8, 1.7, 1.6, and 3 days in the 12.5, 50, 100, 150, and 200 mg/kg/day treatment groups, respectively. PPS for the 25 mg/kg/day treatment group was also delayed, but the delay was not statistically significan t (p=0.07), and not doserelated. PPS for the pair-fed control was delayed (p <0.05) compared to cont rol (2 days) and this was shorter (p<0.05) compared to the high-dose group (3 days).

Serum testosterone concentrations were not di fferent from control in any treatment group at PND 53, or in the high-dose group at PND 45, a lthough concentrations were decreased (NS) by
16-49%. Intratesticular testoste rone was decreased (p<0.05; ↓ 34%) in the high-dose group on PND 45 and decreased (NS) by 30% on PND 53. Se rum estradiol and estrone concentrations were increased in the high-dose group at PND 53 relative to the vehicle and pair-fed controls;
these values were similar between the vehicle and pair-fed controls.

Pituitary weights were decreased in the high-dose group on PND 53, but recovered by PND 120. On PND 53, serum LH concentrations were decrea sed (NS) in a dose-dependent manner in all treatment groups, and serum PRL concentratio ns were decreased (NS) in the 150 and 200 mg/kg/day dose groups. Serum LH and PRL c oncentrations were similar for the vehicle control and pair-fed control. No differences in mean pituitary LH or PR L concentrations were
observed.

T 4 and TSH serum concentrations in the treated gr oups were not different from vehicle control. However, serum T 3 concentrations were increased in a generally dose-dependent manner in all treated groups, but only the 200 mg/kg/day dos e group was significantly increased (p<0.05).
Serum concentrations of T 3, T 4 and TSH were similar for the ve hicle and pair-fed controls.

No effect on the number of testicular LH recepto rs was observed between the vehicle control and high-dose group on PNDs 45 or 53. However, mild hypospermia was observed in 33% of the rats from the high-dose group. The thyroid gland sect ions for the vehicle control and high-dose groups showed no treatment-related differences.

The results of this study demonstr ate that atrazine delays puberty in male rats dosed from PNDs 23 to 53, with a LOAEL of 12.5 mg/kg/day, the lo west dose group tested. The pubertal effect was based on the time of PPS. Although a NOAEL was not observed in this study, the authors noted that data from another recent study (no data provided) showed that there was no effect on
PPS in rats dosed with at razine at 6.25 mg/kg/day.

In a modified pubertal rat study ( Trentacoste et al. , 2001 ), weaned 22-day-old male SD rats (9- 12/group) were dosed with atrazine (96.1% purity ) by oral gavage at doses of 0 (0.5% CMC; n=11), 1, 2.5, 5, 10, 25, 50, 100, or 200 mg/kg/day th rough PND 47 (26 days). Animal weights were recorded on every other da y of treatment (PNDs 22-46; cont rol and 100 mg/kg/day) and at termination (PND 48; all treatments). The day of PPS was determined for the control, and the 100 and 200 mg/kg/day, animals. After sacrifi ce, trunk blood was collected and serum wasATRAZINE FINAL

Page 86 of 159 harvested at stored at

− 80°C for analysis of testosterone and LH concentrations by RIA methods.
Testicular interstitial fluid also was collected and stored at

− 80°C for analysis of testosterone. Organ weights determined at sacrifice included the testes, epididymides, ventral prostate, and seminal vesicles (dry we ight after removal of the coagulating gland).

In a second portion of the study designed to examin e the effects of food in take/deprivation on the above-measured parameters, a group of 22 day-old male SD rats (n=13; Group 1) was dosed with atrazine by oral gavage at 100 mg/kg/day. The amount of food consumed per day was determined. A second group of 22 day-old male SD rats (n=14) was dosed with vehicle by oral gavage, and on each day of the study the rats were fed the mean da ily intake of feed consumed by the Group 1 rats on the previous day. A second group of 22 day-old male SD rats (n=16) was dosed with vehicle by oral gavage and fed ad libitum . Animals were treated through PND 47, sacrificed on PND 48, and evaluated as described for the main study.

In the main study, treatment-related effects genera lly were noted in the two highest atrazine treatment groups (100 and 200 mg/kg/day). Mean body weight over the study period was decreased in the 100 mg/kg/day group compared to control, and terminal body weights were decreased (p<0.05) in the 100 and 200 mg/kg/day animals ( ↓ 9 and 21%, respectively). Serum
testosterone concentrations we re decreased (p<0.05) in the 100 and 200 mg/kg/day dose groups by 34 and 32%, respectively, and inte rstitial testosterone concentr ations were decreased (p<0.05) 41 and 45%, respectively, in these groups. Serum LH concentrations also were decreased in the 100 and 200 mg/kg/day dose groups with decr eases of 17% (NS) and 20% (p<0.05), respectively. Effects on androgen-dependent organs were observed as decreases (p<0.05) in the weights of ventral prostate ( ↓ 26 and 48%, respectively) and seminal vesicles ( ↓ 32 and 55%, respectively) in the two highest dose groups. Finally, PPS was delayed in the 100 mg/kg/day (PND 44 vs. PND 41 control) and 200 mg/kg/ day animals (PND 45 vs. PND 41 control).

Over the course of the second portion of th e study (feed deprivati on examination), rats administered atrazine at 100 mg/ kg/day ate an average of 10% le ss food per day than the vehiclegavage control group (420 g vs. 463 g control) with concomitant decreases in daily mean body weight and decreased (p<0.05) terminal mean body weight (~ ↓ 10%). Terminal mean body weight was decreased (p<0.05) to a similar extent (~ ↓ 10%) in the food- restricted vehicle group.
Decreases in serum testosterone and LH concentr ations, and ventral prostate and seminal vesicle weights, were observed to genera lly similar extents as well in the treated and food-restricted dose groups. One noted difference was an approxima tely two-fold increase in control serum
testosterone concentration in th e second portion of the study compared to the first with a similar response in the 100 mg/kg/day dose group betwee n the two portions of the study. The serum
testosterone concentration in th e food-restricted vehicle group also was decreased (p<0.05), but
was similar to the control concentration of testos terone determined in the main study. The results indicate that treatment-related differences observed in this study may be due wholly, or in part, to overt toxicity ( ≥ 10% decreases in m ean body weight).ATRAZINE FINAL

Page 87 of 159 Steroidogenesis Assay

In the multi-laboratory validation of the H295R steroidogenesis assay ( USEPA, 2008 ), an in

vitro H295R human adrenocarcinoma cel l line assay was optimized and validated for use as an EDSP Tier 1 testing requirement to identify chemicals capable of affecting steroidogenesis. Seven laboratories were chosen to participate in the validation study; five of the seven validation study laboratories tested atrazine. Atrazine was selected for inclusion in the study because it was
considered to be a weak inducer of aromatase in vitro . NCI-H295R cells were obtained from American Type Culture Collection (ATCC CRL -2128, Manassas, VA). The cells were cultured
for ≥ 5 passages, and 24-well plates were seeded at a density of 300,000 cells/mL and incubated
at 37°C and 5% CO 2 for 24 hours. Medium was replaced, and the cells were incubated with atrazine (purity not reported) at serial log concentrations of 0.0001 to 100 μM and incubated at
37°C and 5% CO 2 for 48 hours. The medium was removed from the cells and extracted with ether for measurement of hormone concentration, and the cells were immediately examined for viability. A Quality Control (QC) plate was run concurrently with each independent run of a test chemical plate to demonstrate that the assay resp onded properly to positive control agents at two concentrations. The positive co ntrols included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol a nd testosterone production.

Atrazine did not demonstrate cytotoxicity. One laboratory re ported a 2.4-fold increase and another laboratory reported a 1.5- fold increase in testosterone production relative to solvent control (SC) treatment with at razine at the high concentratio n (100 μM). The remaining three laboratories reported a less than 1.5-fold increas e. A less than 1.5-fold increase is generally considered to be a negative response. Atrazine act ed as an inducer of estradiol at 10 and 100 μM (approximately 5- and 10- to 20-fold, respectively) re lative to SC in four of the laboratories; the magnitude of the increase was expressed graphica lly, but not numerically, in the report. Atrazine produced interference with the testosterone assay th at was considered likely to have an impact on
the final result in one of the laboratories. No laboratories reported in terference with hormone
measurements in the estrad iol assay with atrazine.

Uterotrophic

In a published study by Yamasaki et al . (2000) , groups of sexually immature (PND 21) female SD rats (6/dose group) were admi nistered atrazine (98.7% purity) in olive oil by oral gavage at doses of 0, 0.5, 5.0, or 50 mg/kg/day for three or seve n days. A second set of rats were treated as
above and co-administered 17 β -estradiol by s.c. injection at a dose of 0.3 μg/rat/day. After three or seven days, the rats were terminated by exsanguination under anes thesia approximately
6 hours after the last dose administration. Th e uterus (without oviducts) was removed and trimmed of fat and facia, the vagina removed at the level of the cervix, and the uterus was weighed with luminal contents.ATRAZINE FINAL

Page 88 of 159 There were no clinical signs of toxicity and body weights were unaffected by treatment in all groups. Premature VO was observed in all of the rats administered 17 β -estradiol after six days, and uterine fluid was present in all rats administered 17 β -estradiol for three or seven days. In rats administered atrazine only, uter ine weights were decreased (NS) by 15% at 5 mg/kg/day and by 11% at 50 mg/kg/day after seven days of dosing. In rats co-administered 17 β -estradiol and 50 mg/kg/day atrazine, uterine weights were decreased by 15% (p<0.05) after three days of dosing and by 24% (NS) after seven days of dosing.

In a published, multicomponent study by Tennant et al . (1994a) , groups of young adult, ovariectomized, SD female rats were admini stered atrazine (97.7% purity) in aqueous 0.5% CMC by oral gavage at dos es of 0, 20, 200, or 300 mg/kg/day for three consecutive days.
A second set of rats were treated as above and co-administered 2 μg 17 β -estradiol by s.c. injection on Days 2 and 3 of treatment. Rats we re euthanized 24 hours after the final dose, and the uteri dissected, trimmed, and weighed.

Body weights were decreased (p<0.05) by 12% in the 300 mg/kg/day atrazine group. In rats coadministered 17 β -estradiol, body weights were decrea sed (p<0.05) by 7% in the 100 and 300 mg/kg/day atrazine group. Uterin e weights of ovariectomized female rats were not increased by atrazine at doses up to 300 mg/kg/day. In ovariectomized rats co-administered 17 β -estradiol, atrazine decreased (p<0.05) the 17 β -estradiol -induced uterine weights at 100 and 300 mg/kg/day (data presented graphically).

In a published, multicomponent study by Connor et al . (1996) , groups of four immature (PND 21) female SD rats (n=5 in each of tw o vehicle control groups and n=4 per treatment group) were administered atrazi ne (>97% purity) in aqueous hydroxypropyl cellulose by oral gavage at doses of 0, 50, 150, or 300 mg/kg/day for three days. A second set of rats were treated as above and co-administered 10 μg/kg/day 17 β -estradiol by i.p. injection on the same treatment days; control rats were administered corn oil only. The rats were euthanized 20 hours after the last dose, and the uteri were removed, weighed, bl otted, and cut into halves such that each half
contained a uterine horn. The ut erine bisections were pooled by treatment group, homogenized, and centrifuged, and the resulting cytosols used in competitive binding assays to measure PR concentrations. The pellets from the centrif ugation step were re-suspended, clarified by centrifugation, and uter ine peroxidase activity of th e supernatant was determined.

Atrazine alone did not induce ut erine wet weights, cytosolic PR binding, or uterine peroxidase activity compared to control. Rats treated with 10 μg/kg/day 17 β -estradiol alone displayed increases in these responses as expected [increased (p<0.05) by 6.2-, 8.9-, and 9.7-fold, respectively]. Decreases (p<0.05) were obs erved in uterine PR binding activity ( ↓ 43 and 44 for
the m edium- and high-dose groups ) and peroxidase activity ( ↓ 40, 58, and 95 for the low-, medium- and high-dose groups) after atrazine tr eatment. Co-treatment of rats with 17 β -estradiol plus atrazine did not affect 17 β -estradiol -induced uterine wet weight increases, PR binding, orATRAZINE FINAL

Page 89 of 159 uterine peroxidase activities after co-administrati on of atrazine relative to animals treated with

17 β -estradiol alone; however, these we re not dose-dependent effects.

In a published, multicomponent study by Ashby and Tinwell (2002) [also see Ashby et al

. (2002) ], groups of prepubescent (PND 20-21) female AP (Alpk:ApfSD, Wistar-derived) or SD
rats were administered atrazine (98.2% purity) in aqueous 0.5% CMC by oral gavage (10 mL/kg) at dose levels of 0, 10, 30, or 100 mg/kg/day in a series of four experiments. Groups of rats administered the gonatotropin-re leasing hormone (GnRH) antagoni st Antarelix (2.5 mg/kg/day)
were included to examine atrazine interfer ence with GnRH signaling. Interference with the GnRH signaling pathway results in inhibition of LH release from the pituitary. The studies
evaluated the effects of atrazi ne treatment on the timing of ut erine growth and VO. Uterine weights (blotted and dry) were r ecorded for AP rats 24 hours afte r 8, 11, or 21 daily exposures to vehicle, atrazine, or Antarelix, or for SD rats after 24 daily exposures to vehicle or atrazine. Females exposed for either 21 or 25 days were mo nitored on a daily basis from the start of each study for VO. The age at the start of VO as well as the body weight on that day were recorded for each female. Four separate experime nts were conducted. The first two involved determination of uterine weight on either P ND 30 (start of puberty) or on PND 33 (uterine growth assumed to have been completed) for AP rats. The third experi ment involved dosing AP rats until completion of VO in the vehicle control group, at which time the animals were terminated and uterine and body weights determin ed (PND 43). The fourth experiment involved dosing SD rats until completion of VO in the ve hicle control group, at which time the animals were terminated (PND 46) and ut erine and body weights recorded.

In the first experiment with AP female rats, th ere was a dose-related decr ease in uterine weight (both blotted and dry) after atrazine exposure on PND 22-29 (t ermination PND 30). Decreases (NS) at the low- ( ↓ 16/11%, blotted/dry) or medium-doses ( ↓ 22/16%) were observed, with a decrease (p<0.01) of 41/34% in the high-dose animals. Decreased (p<0.01) uterine weights also were observed after Antarelix exposure ( ↓ 61/47%). Terminal body weight was decreased (p<0.01) in the high-dose group ( ↓ 7%), with a decrease (NS) of 14% in body-weight gain. Body weight and body-weight gain of the Antarelix-treated rats we re comparable to control.

In the second experiment with AP female rats, there also was a dose-related decrease in uterine weight (both blotted and dry) after atrazi ne exposure on PND 22-32 (termination PND 33). Decreases (NS) at the medium-dose ( ↓ 11/8%) were observed, with a decrease (p<0.01) of 55/50% in the high-dose animals. Decreased (p< 0.01) uterine weights also were observed after Antarelix exposure ( ↓ 79/71%). A comparison of uterine we ight (blotted 20.2 mg/dry 4.0 mg) of
naive control animals at PND 22 with uterine we ights (blotted 23.1 mg/dry 5.2 mg) in Antarelixtreated rats on PND 30 shows a l ack of uterine growth. Terminal body weight also was decreased (p<0.01) in the high-dose group ( ↓ 7%), with a decrease (NS) of 13% in body-weight gain. Body weight and body-weight gain of the Antarelix-treated rats w e re comparable to control.ATRAZINE FINAL

Page 90 of 159 In the third experiment with AP female rats, th ere also was a dose-relate d decrease in uterine weight (both blotted and dry) after atrazi ne exposure on PND 22-42 (termination PND 43). Decreases (NS) similar to Expe riment 1 at the medium-dose ( ↓ 18/19%) were observed, with a further decrease of 21/23% in the high-dose anim als (only the decrease in dry weight was significant at p<0.05). The decreases (p<0.01) in uterine weights after Antarelix exposure ( ↓ 87/85%) were similar to the decreases noted in Experiment 2. Terminal body weight also was decreased (p<0.01) in the high-dose group ( ↓ 11%), with a decrease (N S) of 13% in body-weight gain. Body weight and body-weight ga in in the Antarelix-treated ra ts were again comparable to
control.

Finally, in the fourth experiment (with SD female rats), there was a slight decrease in uterine weight (both blotted and dry) after atrazi ne exposure on PND 22-45 (termination PND 46). Decreases (NS) were only observe d in the high-dose animals ( ↓ 13/11%). Terminal body weights were decreased (p<0.05) in the medium- and high-dose groups ( ↓ 4%, respectively), with decreases (NS) of 6 and 7% in body-weight gai n, respectively. Antarelix was not administered to

SD rats.

Assessments of VO also were conducted duri ng Experiments 3 and 4. There was a delay (p<0.01) in the age at VO in AP females dos ed from PND 22 to PND 42. Although not different than control, the standard devi ation of the age at VO in the medium-dose females was increased compared to the other dose groups and the numbe r of females greater than 40 days old at VO was increased compared to the low-dose and control groups (3 vs. 0). Although there was a decrease in terminal body weight [ ↓ 11%; p<0.01] in this group, body weight on the day of VO
was not different from control in the medium- or high-dose groups. None of the Antarelix-treated rats attained VO by study termination (PND 43), and body weights were not affected in this

group.

Finally, there were delays (p<0.05 and 0.01, respectiv ely) in the age at VO in SD females in the medium- and high-dose groups dosed from PND 22 to PND 45. Although terminal body weight at these doses were decreased ( ↓ 4%; p<0.05), body weights on the day of VO were not different from control in the medium- or high-dose groups.

In another modified uterotrophic study, O’Connor et al. (2000 ) examined the role of prolactin in
chloro- S -triazine-related mammary tumorigenesis. Ov ariectomized, six-week-old, female Crl:CD BR rats were acclimated for one week prior to st udy initiation. Atrazine (purity not reported) in 0.25% methylcellulose vehicle was administer ed at doses of 50, 150, and 200 mg/kg/day. Atrazine doses were administered by i.p . injection three times daily ( 1⁄3 of the total daily dose
was administered at each of th e three dosing intervals) at ap proximately 8-hour intervals (0800, 1600, and 2300 hours) for four days in order to faci litate detection of we ak endocrine activity.
The i.p . route of administration was selected in orde r to enhance the sensitivity of the assay and to facilitate potency comp arisons. Between 0600 and 0900 hours on the m orning of test dayATRAZINE FINAL

Page 91 of 159 (Day 5), rats were anesthetized with CO 2 and euthanized by exsanguination. The presence of
fluid in the uterine horns was recorded. Processe d tissues were embedded in paraffin, sectioned nominally at 5 μm, and fixed to slides for staini ng. Uterine stumps from all rats were saved in 10% neutral-buffered formalin and processed by the pathologist to confirm the absence of
ovarian tissue.

Rats assigned for hormonal analysis also were evaluated for vaginal cytology. Vaginal washes were collected once daily, and slides were air dried, stained by the Wright-Geimsa method, and evaluated for the presence of cornifed cells indicative of estrous convers ion. Blood was collected at the time of sacrifice (under CO 2 anesthesia) from all animals. Serum was harvested and stored
between

− 60 to

− 80°C until analyzed for serum prolactin concentration with a commercially
available RIA kit.

After four days of atrazine treatment, fina l body weights were decrea sed (p<0.05) by 9 and 15% in the medium- and high-dose females. There were no treatment-related effects on absolute uterine weight, uterine fluid imbibition, and uter ine stromal cell proliferation due to atrazine exposure. Additionally, estrus conversion was not detected in any dose group. In contrast, uterine epithelial cell height was significantly increased (p<0.05) in rats treated with atrazine in a similar manner in the medium- and high-dose groups ( ↑ 32 and 33%, respectively). Finally, mean serum prolactin concentrations were increased in th e medium- and high-dose atrazine-treated female rats 229 and 216%, respectively.

Dermal Toxicity (Rabbit, OCSPP 870.3200)

In a 21-day dermal study in rabbits ( MRID 42089902 ), New Zealand White rabbits (5 rabbits/sex/dose) were treated with atrazi ne (97.6% purity) for six hours per day for 25-26 days at concentrations of 0 (sterile water), 10, 100, or 1000 mg/kg/day. Atrazine, in sterile water, was applied to a shaved portion (approximately 5-10% of the total body surface) and covered with a dressing.

No mortality occurred during th e study. Treatment-related clinical observations in the high-dose males and females on Test Days 4-21 included spor adic instances of mucoid (2/2, respectively) and/or few feces (4/5, respectively). Significant mean body weight decreases (p<0.05) generally were observed in the high-dose animals at all determinations (Days 7, 14, 21, and 25) of 10-12% in males and 19-22% in females. Total percent body weight gain on Day 21 was decreased for males ( ↓ 4.4 vs ↑ 9.5%; ↓ 146%) and females ( ↓ 11 vs ↑ 8%; ↓ 244%). These decreases in body weight and weight gain were c onsistent with significant decreas es (p<0.01) in food consumption ( ↓ 28-67% for males and ↓ 37-85% for females) from Days 7- 21 in the high-dose animals.

Relative (to body weight) and abso lute spleen weights were in creased (p<0.05 for absolute weight only) in males ( ↑ 81 and 105%, respectively) and females ( ↑ 86 and 126%, respectively). These changes may be related to changes in hematocrit and red blood cell counts that wereATRAZINE FINAL

Page 92 of 159 observed. Significant decreases also we re noted in mean uterus (p<0.01; ↓ 44%) and ovaries (p<0.05; ↓ 36%) weights in high-dose females, wit hout corresponding gross or microscopic findings. Dermal findings were limited-to-slight erythema and/or scaling for both sexes in all dose groups with the exception of minimal- to-moderate acanthosis and focal sub-acute inflammation in three high-dose females (c ompared to 0 in control animals).

The LOEL is 1000 mg/kg/day based on decrease d food consumption, mean body weight, and percent body weight gain, and several organ weight changes. The NOEL is 100 mg/kg/day.

Multi-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a 2-generation reproducti on study (TXR 0050644; MRID 40431303 ; DeSesso et al. , 2014)), atrazine (purity not specified but said to be technical grade) was administered to 240 Charles River (CRCD, VAF/PLUS) rats 30/sex/dose in the diet at dose levels of 0, 10, 50, and 500 ppm. There was very little variation in test ar ticle consumption betw een generations; the F 0 and F 1 males had similar test article consumption dur ing the 70-day premating period as did the F 0 and

F 1 females. The average values for the two generations are 0, 0.75, 3.78, 39.0 mg/kg/day for males and 0, 0.86, 3.70, 42.8 mg/kg/day for females. Test article consumption for the F 0 and F 1 generation females during their ge station period did not vary great ly between generations. Mean compound consumption for both generati ons were 0, 0.66, 3.33 and 35.43 mg/kg/day

Parental body weights, body wei ght gain, and food consumption we re statistically significantly reduced at the 500 ppm dose (highest dose tested ) in both sexes and bot h generations throughout the study. Compared to controls, body weights for F 0 high-dose males and females at 70 days into the study were decreased by 12% and 15%, respectively while F 1 body weight for the same time period was decreased by 15% and 13% for males and females, respectively. The only other parental effect that may have been treatment related was a slight, but statistically significant, increase in relative testes weight, which occu rred in both generations at the high dose. There did not appear to be any reproductive effects from compound expos ure. Measured reproductive parameters from both generations did not appear to be altered in a dose-related manner.

The LOAEL is 500 ppm (39 mg/kg/day in males, 42.8 mg/kg/day in females) based on decreased body weights, body weight gains and food consum ption. The NOAEL is 50 ppm (3.78 mg/kg/day in males, 3.7 mg/kg/day in females)

The developmental toxicity LOAEL is 39 mg/kg/ day in males, 42.8 mg/kg/day in females, based on decreased body weights in both generations of males at postnatal day 21. The developmental toxicity NOAEL is 3.78 mg/kg/day in ma les and 3.7 mg/kg/day in females.ATRAZINE FINAL

Page 93 of 159 Reproductive Toxicity in Females

In a female reproductive toxicity study ( Foradori et al. , 2014 ), the effect of orally administered or dietary atrazine on estrous cyclicity in rats was examined. Young adult female (60- to 90-dayold) Sprague Dawley (SD) and Long-Evans (LE) ra ts were used in the study and fed Certified
Rodent Lab Diet 5002 with ad libitum access to water. Animals were housed individually in suspended wire-mesh cages and maintained on a 14-hour light:10-hour dark photoperiod under controlled temperature (22±3°C) and humidity (50±20%). Vaginal lavages were conducted daily immediately after lights on to de termine the stage of the estrou s cycle beginning at least two weeks prior to the first day of treatment. Only females displaying regular, 4-day estrous cycles for at least two consecutive cycles during the pretest period were used.

Atrazine (97.5% purity) was admini stered continually in the diet (distributed dose) or daily for four days by gavage (bolus dose) to intact fema le rats. Beginning a minimum of 7 days before the first dose, all animals were acclimated to treatment by administering daily doses of the gavage vehicle (l% methylcellulose in deioni zed water) at a volume of 5 mL/kg. Animals assigned to the gavage treatment groups continue d to receive the vehicle daily by gavage, and animals assigned to the dietary groups did not re ceive further gavage admi nistrations; all animals
were fed the control diet.

In the first experiment, atrazine treatments were initiated on the day of estrus for all animals, and continued for four consecutive days. Animals were administered daily oral gavage doses of atrazine of 0 (vehicle), 0.75, 1.5, 3, 6, 10, 12, 50, or 100 mg/kg/day for SD females, or 0, 1.5, 3, 6, 12, 50, or 100 mg/kg/ day for LE females at lights on (05:00 hours). The second cohort of
animals received atrazine-fortified diet on a continuous basis beginning at 19:00 hours on the fourth day of the previous estrous cycle (estrus) and continuing over the next 4-day estrous cycle.
Nominal atrazine concentrati ons in the diet were 30, 100, or 500 ppm for SD females and 160, 660, or 1460 for LE females. The mean-measured atrazi ne doses in the dietary-fed SD rats were 2.7±0.06, 8.6 ± 0.14, and 41.8±1.07 mg/kg/day in the 30, 100, or 500 ppm groups, respectively. The corresponding mean-measured atrazine doses in dietary LE females were 10.2±0.27, 30±11, and 45.6±1.97 mg/kg/day, in the 160, 660, or 1460 ppm groups, respectively. Control animals were given ad libitum access to control diet; 11 to 43 an imals per treatment group were used. Vaginal lavages were conducted daily just after lights on during th e treatment period. On the last day of treatment, 250 μ L of blood was collected in pre-chilled heparinized tubes from each animal by jugular vein puncture at 6-, 11- , 13-, and 18-hours post-l ights on. The 18:00-hour sample (4-hours post-lights out) was collected un der red light. Plasma samples were harvested, frozen, and stored at

− 70°C until analysis by RIA to determine the plasma LH concentrations. Because each female was expected to begin a ne w estrous cycle the morning after the LH blood sample collection, a vaginal smear was collected to confirm that the animal was in estrus. If a female was not in estrus the next morning, the previously collected blood samples were discarded, treatment continued, and a second set of blood samples was collected. Vaginal smearsATRAZINE FINAL

Page 94 of 159 were again obtained the next morning, and the fe male was continued in th e study if it displayed an estrus smear. If the animal was not in estrus, the stage of estrous was recorded and the animal was excluded from subsequent analysis. If estrus was confirmed, the animal was euthanized, the abdominal cavity was opened, and the uterus was carefully dissected and trimmed to retain the
luminal fluid. Each uterus with fluid was weighed intact, after which it was opened longitudinally and blotted with filter paper to remove the luminal fluid. The ampulla of each oviduct was removed and placed on a clean glass s lide. The ampulla was opened to allow exit of the eggs within into saline for counting. Ovar ies were grossly examined, and the number of corpora lutea was recorded. The number of corpora lutea and ova were not recorded for SD
females administered atrazine in the diet; these atrazine dietar y groups in the first experiment were a subset of animals from another expe riment where only the LH surge was assessed.

Atrazine administration by gavage or in the diet did not affect behavior, clinical signs, or
survival in either SD or LE rats in the firs t experiment. No appare nt effect on body weight occurred due to gavage administration, but body weights were decrease d (p<0.05) in high-dose SD (Day 2; ↓ 5%) and LE (Days 2, 3, and 4; ↓ 9-12%) rats. Food consumption also was affected by treatment with decreases (p <0.05) regardless of strain or route of administration. Food consumption was decreased after oral gavage administration in th e 100-mg dose groups on Days 1-3 or 4 ( ↓ 21-41%) and in the 50-mg dose groups on Days 1 or 2 ( ↓ 19-23). In diet-treated SD and LE animals, all groups administered ≥ 500 ppm of atrazine had decreased food consumption on all days of treatment (Days 1-4; ↓ 20-65%).

Administration of atrazine by oral gavage during estrus affected estrous cyclicity in SD and LE rats, but dietary administration of atrazine had no effect. The number of SD females with an irregular/abnormal cycle (5-day estrous cycle or displaying no estrus) was increased by 20 and 16% in the 50 and 100 mg/kg/day dose groups, re spectively. The number of LE females with an irregular/abnormal cycle was increased by 19 and 46% in the 50 and 100 mg/kg/day, in respectively, at the same doses.

Mean LH concentrations were significantly d ecreased (p<0.0001) in SD females at 11 and 13 hours post-lights on in the 50 and 100 mg/kg/day dose groups. Mean LH concentrations also were decreased (p<0.0001) in LE females at 11-hours post-lights on in the 100 mg/kg/day dose group only. There was no effect of dietary atrazine on LH concentrations at any sampling time point in SD or LE animals. Peak LH concentrat ions were decreased (p<0 .0001) in bolus-treated, SD females in the 50 and 100 mg/kg/day dose gr oup; decreases in the bolus-dosed LE groups were not significant. Administration of atr azine in the diet had no effect on peak LH concentrations at any dose for SD or LE females.

The mean numbers of corpora lut ea and ova per animal also were determined for each treatment group. In the SD females administered atrazine by oral gavage, the number of corpora lutea were decreased in the 50 (NS; ↓ 27%) and 100 mg/kg/day (p<0.05; ↓ 31%) groups, with concomitantATRAZINE FINAL

Page 95 of 159 decreases (p<0.05) in the num ber of ova per animal ( ↓ 40 and 43%, respectively). Non-dosedependent decreases (p<0.05) were noted in the LE females ad ministered atrazine by oral gavage. The number of corpor a lutea were decreased in th e 3 and 100 mg/kg/day groups ( ↓ 46 and 56%, respectively), with concomitant decrea ses (p<0.05) in the number of ova per animal ( ↓ 50 and 66%, respectively). No treatment e ffects were noted for the LE dietary exposure

groups.

In the same female reproductive toxicity study ( Foradori et al. , 2014 ), the effect of orally administered or dietary atrazine on female repr oductive performance also was evaluated. Animal husbandry and pretreatment preparations were de scribed previously for the first experiment.

In the second experiment, intact female rats were administered four daily gavage doses of atrazine at doses of 0, 12, 50, or 100 mg/kg/ day (SD) or 0, 15, 3, 6, 12, 50, or 100 mg/kg/day (LE) to determine if an atrazine-induced re duction in the pre-ovulatory LH surge affected fertility. A subgroup of LE females were admini stered atrazine in the feed at dietary concentrations of 160, 660, or 1460 ppm. Mean-measured daily atrazine doses in the dietary groups of LE rats were 11.2±0.3, 35±0.8, and 51±2.0 mg/kg/day. Control animals were provided
access to control diet ad libitum or gavaged with vehicle; 16 to 21 animals per treatment group
were used. All animals received atrazine or the vehicle over one complete 4-day estrous cycle beginning at 05:00 hours on the day of estrus. The start of dosing for each female was based on the stage of estrus. On the last day of the treatment period, females were paired with untreated, intact young adult males of the same strain by placement into the male's home cage in the evening. If evidence of mating ( i.e. , the presence of a vaginal pl ug) was noted the next morning, the female was returned to an individual cage and the day was designated as GD 0. If there was no evidence of mating, a vaginal la vage was taken and the stage of estrous was recorded. If the female was in estrus, the female was separate d from the male without further opportunity for mating. Finally, if the female was not in estrus on the morning after the in itial pairing, treatment continued for an additional day and the female wa s paired with a second male. In the absence of mating on the second day, the stage of the estr ous cycle was recorded and the female was separated from the male without further oppor tunity for mating. Pregnant females were euthanized on GD 20, and the gravid uterine we ights and dam body weight were recorded. The number of fetuses was counted, and individual fetuses were weighed and sexed. The uterus, placenta, and ovaries were examined, and the total number of implantation sites, corpora lutea, and the number of late resorptions were counted.

Atrazine administration by gavage or in the diet did not affect behavior, clinical signs, or
survival in either SD or LE rats in the sec ond experiment. Administratio n of atrazine by oral gavage in SD animals apparently did not affect body weight during the treatment period, but did significantly decrease mean body weight (p <0.0001) on GD 0. There was no effect on body weights during treatment or gestation in LE rats after oral gavage administration. Finally, in the
LE animals administered atrazine in the diet , mean body weights were decreased (p<0.05) onATRAZINE FINAL

Page 96 of 159 treatment Days 2, 3, and 4 in the high-dose group ( ↓ 9-11%). In addition, body weight in the high-dose group also was decreased (p<0.05; ↓ 8%) on GD 0. Food consumption was decreased (p<0.05) during treatment in SD females in the 50 mg/kg/day oral gavage group on Day 2 ( ↓ 14%), and on all days in the 100 mg/kg/day treated animals ( ↓ 20-27%); food consumption also was decreased in the 100 mg/kg/day oral gavage LE animals on all treatment days ( ↓ 20-27%). During gestation, food consumption was increased (p<0.05) in SD rats dosed by oral gavage on GD 0. There also were increases in food consumption in the high-dose oral gavage LE animals (NS). There was no treatment eff ect on food intake in the LE dietary administration group at any dose or day.

There were no effects of atrazine treatment on mating, fertility, or conception indices in SD or LE rats, regardless of dose or route of administ ration. Likewise, there was no effect of atrazine
treatment on the mean number of fetuses or fertil ity per litter at the term ination of pregnancy. An increase (p<0.05) in post-implantation loss ( ↑ 175%) was observed in th e 100 mg/kg/day SD group that received atrazine by gavage. There were no effects of treatment on fetal body weight or sex ratio. Although decreases in the numbers of corpora lute a and ova per animal in the high-dose SD and LE bolus dose groups, but no dose-dependent effects on mating, fertility, and conception indices were observed.

In another female reproductive toxicity study ( Narotsky et al. , 2001 ), different periods of gestation (during and after the LH -dependent period) were evalua ted to determine susceptibility to atrazine-induced resorption. In addition, thre e rat strains (F344, SD, and LE) were compared to identify relative differences in sensitivity to atrazine during the LH-dependent period of

pregnancy.

Timed-pregnant, 60–90 day old F344, SD, and LE ra ts were used for the study. The day that evidence of mating was detected (copulatory pl ug or vaginal sperm) was designated GD 0. All animals were individually housed in polycarbonate cages; animal s were provided feed and tap
water ad libitum and maintained on a 12 hour:12 hour light:dark cycle. Room temperature and relative humidity were maintained at 22.2±1.1°C and 50±10%, respectively.

Except for the experiment evaluating alternate dosing periods (see below), all animals were administered atrazine (purity 97.1%) by gavage on GD 6-10, and maternal body weights were determined on GD 5-16 and 20. Beginning on GD 20, th e dams were observed periodically to determine the approximate time of parturition. Pups were individually examined and weighed on PNDs 1 and 6. PND 1 was defined as GD 22, indepe ndent of the actual time of parturition; therefore, pups were examined at the same time post-coitus. After PND 6 examinations, the dams and pups were sacrificed, and the number of uterine implantation sites was recorded. The
uteri of females that did not de liver were stained with 10% (v/v) ammonium sulfide to enhance detection of resorption sites.ATRAZINE FINAL

Page 97 of 159 In a pilot study, atrazine was administered to F344 rats on GD 6-10 at 0, 100, 200, and 300 mg/kg. Maternal toxicity was evidenced by weight losses duri ng the treatment period at all three doses, and mortality in 7/12 dams in th e 300 mg/kg/day group. Dose-related incidences of full litter resorption were seen at all three doses; 63, 80, and 100% of the surviving dams were affected at 100, 200, and 300 mg/kg/day, resp ectively. The 200 mg/kg/day dams that successfully maintained their litte rs had increased prenatal loss, and decreased litter sizes and pup weights.

Initially, F344 rats were dosed with atrazine at 200 mg/ kg on GD 8-10 or 11-15 (control group received vehicle on GD 8-15). This experiment attempted to esta blish an experimental model with a shorter dosing period that would cause a high rate of pregna ncy loss, as well as to assess the effect of exposure after the LH-dependent period of pregnancy. Mate rnal body weights were determined on GD 7-16 and 20. In addition, groups of F344, SD, and LE rats were dosed concurrently on GD 6-10 at 0 or 200 mg/kg/day. Separate dosing studi es in each strain included
atrazine administration at 0, 25, 50, and 100 mg/kg/day.

Maternal toxicity was observed in both atrazi ne-treated groups, with maternal weight loss evident during the respective treatment periods ( ↓ 9.1 g vs. ↑ 1.5 g control during GD 8-10 and ↓ 11.7 g vs. ↑ 2.4 g control during GD 11-15). Mortality occurred in 2/10 and 1/10 dams during the early- and late-treat ment periods, respectively; no clear signs of gavage trauma were found. For dams dosed on GD 8-10, full litter resorption wa s seen in three of eight (38%) dams. This incidence (NS) was attributed to at razine due to the rarity of full resorption in historical controls. Gestation length, and pup survival and weights were comparable to control in dams with surviving litters. For dams dosed on GD 11-15, after the LH-dependent period, full resorption was not observed. Delayed parturition was margin ally significant (p=0.051) in this group, with two dams that delivered on GD 23 and 24 (vs GD 21- 22 in control). Neither of these two late litters survived. An additional litter (delivered on GD 22) failed to survive to PD 6. Litter sizes and pup weights in the late-treatment group were decreased (p<0.05) on PND 6 ( ↓ 46 and 7%, respectively).

Concurrent exposure of F344, SD, and LE rats to 200 mg/kg on GD 6-10 yielded similar rates of litter resorption in the three strains (53, 67, and 67%, respectiv ely). Among the surviving litters, parturition was delayed (p<0.05) for the F344 da ms but not statistically significantly delayed (p=0.077) for the SD dams. Also, prenatal loss was significantly increased (p<0.001; ↑ 657%) in the surviving F344 litters, resulting in decreased (p<0.01) litter sizes ( ↓ 40%). Maternal toxicity, evidenced by maternal body weight loss, was obser ved in all three strain s. Significant decreases (p<0.001) of 8.6-14.3 g compared to weight increases of 3.6-12.4 g in the respective control groups were noted on GD 6-7, with overall body weight decreases of 97 (p<0.05), 48 (NS), and 40% (p<0.01), respectively.ATRAZINE FINAL

Page 98 of 159 Each strain also was administered atrazine on GD 6-10 at 0, 25, 50, and 100 mg/kg. F344 rats
had dose-related incidences of litter resorption in the medium- and high-dose groups of 36 (NS; p=0.055) and 64% (p<0.01), respectively. Due to the dose-related occurre nce and low incidence in historical controls, 50 mg/ kg was considered the LEL for this endpoint. For surviving litters, parturition was delayed at 100 mg/kg (p<0.05). There were no treatment-related effects on pup weight or postnatal viability. Decreases in mate rnal weight of 6.2 and 11.2 g in the medium- and high-dose dams, respectively, compared to control ( ↑ 0.6 g) were noted during GD 6-7, with overall body weight decreases (N S) of 24 and 101%, respectively.

All SD dams exposed to atrazine at ≤ 100 mg/kg maintained their litte rs. Parturition was delayed (p<0.01) in the high-dose group, but there were no effects on pre- or postnatal survival, or pup weights. Decreases in maternal body weights (p<0.05 or 0.001) of 6.8, 6.7, and 15.5 g compared to a weight increase of 1.6 g in the control group were noted during GD 6-7, with overall nonsignificant body weight decrease s. With one exception in the low-dose group (atypical late resorption), all LE dams exposed to atrazine at ≤ 100 mg/kg also maintained their litters. This atypical resorption was not considered to be due to treatment. Gestation lengths, and pup survival and weights, were comparable to control. Matern al weight loss after th e first dose was observed in the high-dose group only ( ↓ 8.2 g vs. ↑ 3.6 g control). The results of this study indicate that atrazine caused pregnancy loss in F344 rats due to administration during the LH-dependent period of pregnancy, but not if ad ministered after this period.

In a published study ( Davis et al. , 2011 ), the effects of atrazine on female offspring reproductive
indices in SD rats were examined. This study was conducted in conjunction with the investigation of eff ects of atrazine on male o ffspring reproductive indices ( Fraites et al. , 2011). Timed-pregnant SD rats (70-90 days old) were individually hous ed and provided with commercial laboratory feed and water ad libitum . Two different dosing regimens (Blocks 1 and 2) were used in the study to administer equal amounts of atrazine, and each regimen was
conducted in two blocks. Atrazi ne (97.1% purity) dosing soluti ons were prepared in 1% methylcellulose. Timed-pregnant female rats (12/ dose group/block) were administered atrazine as a single full dose in the morning ( s.i.d. ; Block 1) or a half-dose twice per day ( b.i.d. at 0900 and 1900 hours; Block 2) at 0 (vehicle), 1, 5, 20, or 100 mg/kg/day by or al gavage from GD 14
to 21. The b.i.d . dosing was included due to the rapid me tabolism of atrazine, and was expected to produce a more consistent exposure of atrazi ne relative to the single daily dose. Additional timed-pregnant dams (5/dosing regimen) that di d not receive treatment were included in the second block; these females were weighed daily and functioned as cage controls for additional
behavior studies. Females were allowed to deliver naturally.

Pups in each litter were grossly examined, sexe d, counted, and weighed co llectively by sex at birth. Postnatal days (PND) were defined such that PND 0 coincided with GD 22 to allow litter examinations at the same time post-coitus. Pups were weighed on PND 4, and the litters were culled to eight pups. Pups were weighed and anogenital distance (AGD) was determined onATRAZINE FINAL

Page 99 of 159 PND 7. At weaning (PND 21 for Block 1 or PND 22 for Bl ock 2), pups were weighed and housed two per cage with a same-sex sibling or same-sex animal from the same treatment group (as possible). Dams were euthanized post-weanin g and uterine implantation sites were counted. Female offspring were examined daily begi nning on PND 28 to determine the day VO was completed. Female offspring were weighed dail y during VO evaluations and weighed weekly
afterwards.

One female in each litter was sacrificed by decapitation on PND 45. The fourth abdominal mammary gland was excised from each animal, fixed, and stained with carmine alum solution. Photographs of each mammary gland mount were made with commercial software linked to a camera mounted on a microscope. Representative im ages of ductal branching were evaluated and the total number of terminal end buds having a diameter of ≥ 100 μm at the edge of the ductal field was counted. Additionally, th e distance between th e lymph node and the edge of the ductal field was determined with a stage micrometer. A subjective assessment of the mammary glands was also conducted. Each mammary gland was as signed a score on a scale of 1-4, based on the degree of development (a score of 1 represented poor developmen t and a score of 4 represented normal mammary development at PND 45 in rats). Scoring was based on a holistic appraisal of the tissue including the extent of branching growth relative to the lymph node and the presence
of terminal end buds.

To determine whether gestational atrazine e xposure resulted in premature emergence of reproductive senescence, estrous cy clicity of offspring was examined through PND 272. After all female rats achieved VO, estrous cycles were ex amined at 14-day intervals. Vaginal smears were conducted and evaluated; females were categorized as cycling including regular and irregular cycles (<4 days of estrus and <6 days of dies trus) or non-cycling indica ting persistent estrus (>4 consecutive days of estrus). The number of animals in ea ch cyclicity category from the
combined s.i.d . and b.i.d . blocks was determined.

All dams in Block 1 ( s.i.d .) gave birth to live litters [GD 21 (69%), GD 22 (23%), or GD 23 (8%)]. Litters born on GD 23 occurred only in th e second block, and pren atal and neonatal mortality in these litters were unremarkable, indicating that there was no association with treatment. No differences among groups were f ound for the number of live pups at birth or sex distribution. Postnatal mortality (birth to PND 4) was increased (p<0.05) for both sexes in the high-dose group; no differences in postnatal mortal ity were seen from PND 4 to 21. AGD on
PND 7 was similar to control fo r all atrazine treatment groups.

All dams in Block 2 ( b.i.d .) in the vehicle control, and lo w and high-dose groups gave birth to
live litters. One dam had a dead litter in the medium-low dose group and one dam had dystocia
and did not deliver her litter (full term fetuses were found in utero after euthanasia of the dam on GD 22) in the medium-high dose group. Gestatio n lengths (21-22 days) were not different between groups, but postnatal mortality was signi ficantly increased (p<0.05) at PND 4 in theATRAZINE FINAL

Page 100 of 159 high-dose group; no differences in postnatal mortality were seen from PND 4 to 21. Litter size, sex distribution, and AGD were similar to c ontrol for all atrazi ne treatment groups.

Body weights at birth of offspring in the medium-low and high-dose s.i.d . groups were decreased (p<0.01; ↓ 10 and 17%, respectively). Body weights at PND 4 of offspring in the medium-low dose group had recovered and were similar to cont rol. Body weights of offspring from the highdose group remained decreased (p<0.5; ↓ 15%) at PND 4, but recove red by PND 21. At PND 35, body weights of offspring from the low-dose gr oup were increased (p<0.05) compared to control. In Block 2 ( b.i.d .), body weights of offspring from the high-dose group were decreased at birth and PND 4 (p<0.01; ↓ 15 and 12%, respectively), with recovery by PNDs 21 and 35. Offspring body weights in the other treatment groups in the b.i .d. study were similar to vehicle
control.

In Block 1, VO was significantly de layed (p<0.01) in female offs pring from the high-dose group (PND 34.9±0.4) compared with vehicle contro l (PND 33.3±0.4). In Bl ock 2, VO also was significantly delayed (p<0.05) in the high-dose female of fspring (PND 34.9±0.3 vs. PND 33.0±0.3 control). Body weights on the day of VO we re not different between groups in either
block.

In both studies, there were no treatment-related differences observed in the number of branch points, the number of terminal end buds, or the distances from the lymph node to the tip of the most distal ducts in mammary gland whole moun ts. Additionally, it was noted that subjective scoring of the mammary glands did not show any differences among the treatment groups; data were not provided.

Finally, there were no differences in cyclicity between the studie s or dose groups. Estrous cycles continued in all female offspring through PNDs 104 and 132 in Studies 1 and 2, respectively. In Block 1 ( s.i.d .), several females from the low- and medium-low dose groups stopped cycling and entered persistent estrus between PNDs 119 and 132; a single female from the high-dose group was in persistent estrus from PNDs 119 to 160, but began cy cling again from PNDs 175 to 216. There were fewer non-cycling females in Block 2 ( b.i.d .) at later time points than in Study 1.

There were few effects of gestational atrazine trea tment on female offspring at doses <100 mg/kg/day and few differences in the measured parameters between the two dosing regimens. Treatment-related effects o ccurred only in the high-dose group ( i.e. , offspring body weights decreased at birth, mo rtality increased shortly afte r birth, and decreased VO). No differences in mammary gland development at PND 45 or in estrous cyclicity through PND 272
were observed.ATRAZINE FINAL

Page 101 of 159 Reproductive Toxicity in Males

In a reproductive toxicity study ( Fraites et al. , 2011 ), the effects of atrazine on male offspring reproductive indices in SD rats were examined. This study was conducted in conjunction with the investigation of effects of atrazine on female offs pring reproductive indices ( Davis et al.
, 2011). Husbandry and dosing details were discussed previously for Davis et al. (2011). Maternal body weights were monitored prior to (GD 8 and 11) and during atrazine treatment (GD 14-21)
in both the s.i.d . and b.i.d . dosing regimens. Litter details th rough weaning also were discussed previously. Male offspring were examined daily beginning on PND 38 to determine the day PPS was completed. Male offspring were weighed da ily during PPS evaluations and weighed weekly
afterwards.

At PND 59, two male offspring fr om each litter were randomly selected for necropsy to assess AGD, serum testosterone concentration, ex vivo testosterone producti on by the testis, and androgen-sensitive organ weights. To examine the ex vivo testicular testoste rone production at birth, two males from each litter were decapitated and testes were removed and weighed. The
testes from the first male and one testis from the second male of each litter were added to separate microcentrifuge tubes with 0.5 mL pre-warmed media [Medium-199 with reduced (10%) methionine], buffered with sodium bi carbonate, and supplemented with bovine serum albumin (0.2%) insulin-transferri n-selenium mix sodium pyruvat e and non-essential amino acids. Five microliters of 100 ng/mL ovine LH was added to one testis incubation from each male prior to 3-hour incubation at 34°C; the second testis incubation from the first male was not stimulated with ovine LH. After incubation, the media from the three tubes per litter were transferred to new microcentrifuge tubes and stored at

− 80°C until assayed for testosterone concentrations. The second testis from the second male from each litter was placed in Bouin's fixative for subsequent histological analysis.

On PND 30-33, male pairs were assessed for ro ugh-and-tumble play behavior. Cage-mates (littermates; one pair per litter) were separa ted for approximately 24 hours prior to testing, reunited in their home cage at the onset of the dark phase of th e light cycle, and observed under red light for a 10-minute period between 1 and 4 hour s after the onset of the dark cycle. Both rough-and-tumble play beha vior and anogenital sni ffing, as a measure of social interaction, were scored. Play bouts were scored fo r both duration (number of seconds the pair spent engaged in a play bout during the 10-minute scor ing period) and frequency (the total number of bouts that occurred during the scoring period) . Social interactions were sc ored for frequency only; female vehicle control animals were scored (one pair pe r litter) to provide a comparison to the males for this sexually dimorphic behavior.

At study termination, two males per litter were sacr ificed by decapitation and trunk blood was collected in serum-separation tubes. The pituitar y, testes, ventral prostate , left epididymis, and seminal vesicles with coagulat ing gland (with fluid) were removed and weighed. Serum wasATRAZINE FINAL

Page 102 of 159 harvested and divided into tw o aliquots, and stored at

− 80°C. The whole brain and pituitary were frozen separately on dry ice and stored at

− 80°C for subsequent analys is. The right testis and epididymis from one male from each litter wa s fixed in Bouin's fixative for subsequent histological analysis. The righ t testis from the second male of the litter was immediately processed for interstitial fluid extraction. Th e testis was weighed decapsulated, and the parenchyma was slowly extruded through a syringe into a conical tube and kept on ice. The parenchyma was then centrifuged and the re sultant interstitial fluid was stored at

− 80°C. Serum and media testosterone, and LH, anal yses were conducted by RIA methods.

Dams administered atrazine at the high dose in both portions of the study showed decreases in mean body weight after the firs t day of dosing (GD 14). Matern al body weights in this group were significantly decreased relative to cont rol (p<0.05) beginning on GD 16 in Blocks 1 and 2, respectively. Maternal weights in the high- dose group were also significantly decreased (p<0.001) by GD 19-21. All dams gave birth to live litters and gestation lengths were comparable across groups in both dosing regimens . The numbers of implantation sites and live pups per litter also were comparable across group s in both dosing regimens. Pre- and postnatal mortalities were discussed previously ( Davis et al. , 2011)

.

There was no effect of atrazine treatment on male offspring body weight with either dosing regimen for dams treated with 1- 20 mg/kg/day. Cage control pup wei ghts were also similar to the vehicle control group. In contrast, high-dose ma le offspring were significantly decreased (p<0.01) at birth ( ↓ 13 and 11%) and PND 4 ( ↓ 10 and 9%) with both dosing regimens, respectively. In the s.i.d . regimen, decreased (p<0.01; ↓ 6-7%) body weights in male offspring continued through PND 59. In the b.i.d . regimen, however, offspri ng weights in the high-dose group recovered after PND 4 through PND 59.

There were no treatment-related effects on AGD, AGD index, or achievement of PPS observed at any dose of atrazine for eith er dosing regimen. Additionally, there were no treatment-related effects observed on any behavioral endpoint ( e.g. , rough-and-tumble play or social interactions) at any dose.

Unstimulated and stimulated testosterone producti on per milligram testis was not different from control for any dose group in males treated with atrazine s.i.d . Unstimulated testosterone production was decreased (p <0.05) in the high-dose b.i.d. regimen, but no differences were observed with stimulated production. Serum and in terstitial fluid testos terone and serum LH
concentrations were not different between th e control and any dose gr oup in either dosing regimen. Testosterone production in stimulated or unstimulated te stis incubations also was not different between dose groups in either regimen, and there were no treatment-related effects on androgen-dependent organ weights. Mean absolute pituitary we ight was decreased in male offspring in the s.i.d . regim en in the medium-low and medium-high dose groups (p<0.05; ↓ 7 and 8%, respectively) and in th e high-dose males (p<0.01; ↓ 12%). No treatment-related effects onATRAZINE FINAL

Page 103 of 159 organ weights were noted in any dose groups in the b.i.d . regimen. There were generally no
effects on endocrine-related endpoints in this study.

In a reproductive toxicity study ( Rosenberg et al. , 2008 ), pregnant SD rats (14-16/group) were dosed with atrazine (98.5% purity) by oral gavage at doses of 0 (0.5% CMC), 1, 10, 50, 10, 75, or 100 mg/kg/day from GD 14 through parturition. Dam body weights were recorded daily from GD 14-21, and daily food consumption by the pregnant dams was recorded.

The male pups from seven to eight dams per dos age group (half of the dams) were euthanized by decapitation on PND 0. The testes from all male pups in each given litter (40–50 pups) were snap-frozen in liquid nitrogen and stored at

− 80°C. For steroid extraction, the testes were thawed, and all testes from a gi ven litter were pooled. Intra-tes ticular testosterone was assayed by RIA. With the remaining seven to eight da ms per dosage group, the number of pups per dam was culled to eight (four male s and four females) on PND 2. The body weights and AGD were measured for each pup on PND 21. The AGD inde x was calculated as AGD/cube root of body weight. An average AGD index was determined per litter; means per treatment group were determined by averaging litter averages. Af ter AGD measurements on PND 21, the male pups were weaned, and the four male pups from a given litter were housed together. PPS was monitored daily beginning on PND 37, and an average day of PPS was determined for each litter.

The same four male pups per litter from wh ich the AGD and PPS data were obtained were euthanized by decapitation on PND 60. Trunk blood was collected and kept on ice. Serum was
collected and stored at

− 20°C. Testes were removed and wei ghed individually. Intra-testicular fluid, which included fluid from both the semini ferous tubules and interstitial space, was collected and frozen in liquid nitrogen. For test osterone determinations , steroid was extracted from 10-mL portions of intra-testicular fluid or 100-mL portions of serum from each animal. The ventral prostate and semina l vesicles were weighed.

Maternal body weights were comparable in al l groups at the start of dosing (GD 14). No differences in body weights were observed at doses ≤ 50 mg/kg/day. Decreases (significance not reported) in body weight in the medium-hi gh and high-dose groups we re observed from GD 18-21. Decreased mean daily food consumption dur ing gestation was observed in the medium, medium-high, and high-dose groups; consumpti on was decreased by approximately 33, 38, and 47%, respectively.

The average number of pups born to individual dams in the c ontrol and atrazine groups ranged from 11 to 14, with no treatment-related differe nces. However, there were dose-dependent differences in pup survival. With increasing atrazi ne dose, the percentage of dead pups increased from <1% in the control and low-dose groups to approximately 10% in the medium-low and medium-dose groups and approximately 25% (p <0.05) in the medium-high and high-dose groups. Pup weights on PND 2 showed a downward trend, but there were no treatment-related
differences in means.ATRAZINE FINAL

Page 104 of 159 No differences were seen in mean testosterone concentrations of testicular fluid in the PND 0 pups due to treatment. No differences were seen in the AGD index in female offspring at any
atrazine dose. Decreases in AGD index in male offspring were noted in the medium-high (NS) and high-dose groups (p<0.05). PPS occurred at approximately PND 40.5-41 in the control, low-, and medium-low dose groups. PPS was delaye d by approximately 1 day in the medium and medium-high dose groups (p<0.05 and 0.01, respec tively) and by 2.5-3 days in the high-dose group (p<0.004).

No differences were noted in body or testis we ights in the PND 60 males, although there was a downward trend for both measures in the high -dose group. In addition, no differences were observed among dosage groups with respect to semi nal vesicle or ventral prostate weights.
Serum testosterone concentrations were decreased compared to control in the medium, mediumhigh and high-dose groups, and intr a-testicular testosterone concentrations were decreased in the medium-high and high-dose groups.

In another reproductive toxicity study ( Song et al. , 2014 ), 5-week-old male SD rats (10/group) were dosed with atrazine ( ≥ 98% purity) by oral gavage at doses of 0 (corn oil), 38.5, 77, or 154 mg/kg/day for 30 days. Animal body weight s were recorded at study initiation and termination. Organ weights determined at sacrific e included the testes, epididymides, prostate, and seminal vesicles. Testes were fixed, secti ons, and stained with hematoxylin and eosin. One epididymis from each rat was isolated, minced, and incubated in 10-mL portions of Ham’s F-12 medium at 37°C for one hour. After incubation, 1-mL portions were further diluted with an additional 9-mL portion of Ham’s F-12 medium , and spermatozoa were counted with a hemocytometer. Microscopic examinations of seminal smears stained with eosin also were conducted. A portion of the testis from each rat also was minced and homogenized in 0.9% saline; the supernatants harvested after centrifugation were analyzed for acid phosphatase (ACP), alkaline phosphatase (ALK), lactic de hydrogenase (LDH), and succinate dehydrogenase (SDH). The effects of atrazine on testicular antioxidant capacity were measured by determination of the total antioxidant capacity, as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). Finally, serum samples we re collected and analyzed to determined concentrations of testosterone, LH , FSH, and inhibin-B by ELISA methods.

Mean terminal body weight was decreased (p<0 .05) in the high-dose group by 18%,and testes weight was decreased (p<0.05) by 11% in this group. No other androgen-dependent tissue weights were affected by atrazi ne treatment. Rats in the cont rol and low-dose group had compact and regular arrangement of cells in the semi niferous tubules, and nearly all stages of spermatogenesis were present. In the high-dose group, normal sp ermatogenic cell organization was disrupted with visible holes among the cells in the tubules; cel l disorganization was demonstrated by a decrease in the total number of germ cells inside the tubules and connection of spermatocytes to the lumen. Damage to the semi niferous tubules in the medium-dose group was present, but less severe.ATRAZINE FINAL

Page 105 of 159 Spermatozoa counts were decreased (p<0.01) in the epididymis of male rats in the medium- and high-dose groups ( ↓ 28 and 31%, respectively, and the per centages of the total number of spermatozoa abnormalities were increased in a dose-dependent manner. The percentage of double-headed and hookless spermatozoa were in creased (p<0.05) by 44 and 41%, respectively, in the medium-dose males, and all abnorma lities (including amorphous and coiled-tailed spermatozoa) were increased by ≥ 100% in the high-dose males.

The activity of the four testicular marker en zymes examined decreased in a generally dosedependent manner. Activities for ACP and LDH we re decreased (p<0.01) only in the high-dose group ( ↓ 13% each). Activities for AKP and SDH were decreased (p<0.01 or 0.05) in a slightly dose-dependent manner ( ↓ 26 and 28% and ↓ 18 and 19%, respectively) in the medium- and highdose groups. The total antioxidant capacity in th e testes was decreased (p<0.05 or 0.01) in all atrazine treatment groups in a dose-dependent manner. Mean test icular GSH concentration was decreased (p<0.05) and MDA concentration wa s increased (p<0.05) in the high-dose males ( ↓ 40% and ↑ 27%, respectively).

Finally, serum testosterone and inhibin-B concentrations were decreased in medium- (p<0.05) and high-dose (p<0.01) animals. In contrast, serum FSH concentra tions were increased in male rats in the medium- and high-dose groups (p<0.0 5 and 0.01, respectively), and LH concentration was increased (p<0.05) in the high-dose group only.

Developmental Reproductive Toxicity in Males

In the first of two special studies ex amining male reproductive development ( DeSesso et al.
, 2014 ), groups of 25 time-mated female Wistar rats were administered atrazine (99.5% purity) by gavage at doses of 0 (vehicle ; 2% corn starch in water), 1, 5, 25, and 125 mg/kg/day from GD 6- 21, inclusive. Dams were allowed to litter a nd rear their offspring to weaning; pregnancy, parturition, and litter parameters were evaluated. The parental animals, female offspring, and, where possible, one male pup per litter were euthanized at weani ng and necropsied. After weaning, the remaining F 1 male offspring were group-housed . At PND 70, 25 males per dose group were randomly selected for sperm evaluation. Thirty-five of the remaining F 1 males were randomly selected to remain on study through P ND 170, with an additional 25 males per group randomly selected for sperm evaluation at PND 170. Plasma testosterone concentrations also
were determined on PNDs 70 and 170.

Males selected for sperm analysis were necropsied, and the adrenal glands, epididymides, pituitary, prostate, seminal vesicl es, and testes were weighed. The left testis and left cauda epididymis were separately minced and ho mogenized in 0.9% NaCl with 0.5% Triton X-100. Homogenization-resistant spermatids or sper matozoa in the testes and epididymides, respectively, were counted in a hemocytometer. Spermatozoa obtained from the ductus deferens
were stained with 2% eosin and assessed mor phologically with a light microscope at 40×ATRAZINE FINAL

Page 106 of 159 magnification. Plasma testosterone concentrations were determined in duplicate with an ELISA
method.

There were no treatment-related e ffects on clinical signs or mortal ity in pregnant female rats administered atrazine up to 125 mg/kg/day during GD 6-21. Decreases (p<0.05) were observed in food consumption and body weight and/ or body weight gain during gestation and dosing in females in the high-dose group, with lesser decreases noted in the medium-high dose group. After the treatment period, a compensatory incr ease in body weight gain occurred in the highdose group; overall mean body wei ght was not different from control during lactation. The maternal LOAEL is 25 mg/kg/day, based on decr eased body weight, body weight gain, and food consumption, and the maternal NOAEL for the study is 5 mg/kg/day.

Evidence of reproductive toxicity was observed in the high-dose dams. Only 13/21 pregnant, high-dose dams delivered live litters, with total in utero litter loss in eight dams. Additionally, embryo-fetal mortality occurred in live-born litt ers demonstrated by an increase (NS) in postimplantation loss ( ↑ 455%) and a decrease (p<0.05) in the mean number of pups per litter at birth in the high-dose group ( ↓ 39%). Postnatal pup survival also wa s affected in the high-dose group with survival decreased by 75% on PND 4, and decreased by 85% on PND 21. Neither pre- nor postnatal survival was affected in the othe r treatment groups. Body we ights were decreased (p<0.05) on PND 1 for male and female pups in the high-dose group through PND 7 for males
and PND 14 for females. There wa s no effect on pup weight at doses ≤ 25 mg/kg/day. There were
no treatment-related effects on surv ival or body weight in the subs et of male pups selected for developmental studies post-weaning.

Due to excessive pre- and postnat al mortality in the high-dose pups , there were too few males to evaluate male reproductive endpoints on PND 170; therefore, males in this group were only
evaluated on PND 70. Atrazine treatment in ut ero resulted in decr eased (p<0.05) relative pituitary weights on PND 70 in the high-dose group ( ↓ 41%), but other organs were unaffected.
Prenatal atrazine treatment did not alter spermatid counts in test es, spermatozoa counts in the epididymides, or plasma testosterone concen trations on PND 70 or PND 170 at any dose. Increases (p<0.05) in the percen tage of abnormal sperm were observed on PND 70 in the highdose group ( ↑ 80%) and on PND 170 in the medium-high dose group ( ↑ 32%). Therefore, the NOEL for male developmental toxicity was 5 mg/kg/day.

In the second of two special studies examining male reproductive development ( DeSesso et al.
, 2014 ), groups of 27-31 time-mated female Wistar rats were assigned to five experimental groups. After delivery, atrazine (97.2% purity) was administer ed by gavage at doses of 0 (vehicle; 2% corn starch in water), 1, 5, 25, and 125 mg/kg/day from PNDs 2-21, inclusive.
Dams were allowed to litter and rear their offspring to weaning; pregnancy, parturition, and litter parameters were evaluated. The parental animal s, female offspring, and, where possible, one male pup per litter were euthanized at weaning and necropsied. After weaning, the remaining F 1ATRAZINE FINAL

Page 107 of 159 male offspring were group-hous ed. At PND 70, 25 males per dose group were randomly selected for sperm evaluation. Thirty-five of the remaining F 1 males were randomly selected to remain on study through PND 170, with an additional 25 ma les per group randomly selected for sperm
evaluation at PND 170. Plasma test osterone concentrations also were determined on PNDs 70
and 170.

There were no treatment-related e ffects on clinical signs or mortal ity in pregnant female rats administered atrazine up to 125 mg/kg/da y during PNDs 2-21. Decreases (p<0.05) were observed in food consumption and body weight and/ or body weight gain during lactation and dosing in females in the high-dose group only. Th e maternal LOAEL is 125 mg/kg/day, based on decreased body weight, body weight gain, and food consumption, and the maternal NOAEL for the study is 25 mg/kg/day.

Body weights were decreased (p<0.05) on PND 4 for male and female pups in the high-dose group through PND 21.

After weaning of the high-dose group, body weights of F 1 males remained decreased through PND 63, but food consumption and clinical observations for F 1 males were comparable to control offspring. There was no effect on pup weight at doses ≤ 25 mg/kg/day. On PND 70, decreases in absolute testis ( ↓ 7%) and epididymis ( ↓ 12%) weights were observed in high-dose F 1 males. By PND 170, there were no differences in ep ididymis weight, but te stis weight remained decreased in the high-dose males ( ↓ 6%). No changes in prostate, seminal vesicle, or pituitary weights were seen at any dose, and lactational exposure to atrazine had no effect on plasma
testosterone concentrations or spermatid and sperm counts in F 1 males on PNDs 70 or 170. An increase (p<0.05) in the per centage of abnormal sperm was noted in the high-dose males on PNDs 70 and 170 ( ↑ 46 and 43%, respectively). Therefore, the NOEL for male developmental toxicity after lactat ional exposure to atra zine was 25 mg/kg/day.

Developmental Toxicity (Rat, OCSPP 870.3700)

In a prenatal developmenta l toxicity study in rats ( Accession No. 00143008; MRID 40566302;
Scialli et al. , 2014 ), pregnant COBS CD (SD) BR rats (27/dose group) were administered atrazine (96.7% purity) in 3% aqueous corn starch containi ng 0.5% Tween 80 by oral gavage at doses of 0, 10, 70, or 700 mg/kg/day during GD 6-15, inclusive. All dams were sacrificed on GD 20, and the number of corpora lut ea, implantations, and resorptions were recorded. Fetuses were examined for external abnormalities, sexed, and weighed. Visceral examination was performed on approximately 1/3 of the fetuses in each litter . The remaining fetuses (2/3) were prepared for
examination of skeletal abnormalities.

No mortalities occurred during the study in th e control and low- or medium-dose groups; a mortality rate of approximately 78% was observed in the high-dose dams (21/27). Clinical signs in the high-dose dams included sa livation (13/27), oral /nasal discharge (12/ 27), ptosis (11/27),ATRAZINE FINAL

Page 108 of 159 swollen abdomens (8/27), and blood on the vulva (7/27). Clinical signs in the low-dose group were limited to rales in a single dam; alopeci a occurred in the medi um- and high-dose groups, but was not considered biologi cally significant. Decreases (p <0.05) in body weight were observed in the high-dose dams on GD 14, 18, and 20 ( ↓ 8, 22, and 30%, respectively). Overall maternal body weight gain in the high-dos e dams was decreased by 264%, and food consumption was decreased (p<0.05) by 39%.

The maternal LOAEL is 70 mg/kg/day, based on decreased body weight, body weight gain, and food consumption, and the maternal NOAEL is 10 mg/kg/day.

No treatment-related changes were observed in pregnancy rate, number of corpora lutea, mean number of implantations/litter, pre-or post-implantation loss, early or late resorptions, number of live fetuses/litter, fetal sex ratio, or soft tissue abnormalities at any dose. At the high dose, fetal weights were decreased (p<0.05) by 44 and 45% in males and fema le pups, respectively. Skeletal findings were not assessed in the high-dose pups due to fetal size and weight reductions. In the medium-dose pups, there were increases of both feta l and litter incidence in skeletal variations indicating delayed ossification. These included delayed ossification; vertebral centra or sternebrae bipartite, misaligned, or fused; and ribs rudimentary, wavy, bi furcated, or cervical.

The developmental LOAEL is 70 mg/kg/day, base d on decreased fetal weight and skeletal variations, and the developm ental NOAEL is 10 mg/kg/day.

In a second prenatal developmen tal toxicity study in rats ( MRID 41065201; Scialli et al. , 2014 ), pregnant COBS CD (SD) BR rats (26/dose gro up) were administered atrazine (purity not reported) in 3% aqueous corn st arch containing 0.5% Tween 80 by oral gavage at doses of 0, 5, 25, or 100 mg/kg/day during GD 6-15, inclusive. All dams were sacrificed on GD 20, and the number of corpora lutea, implan tations, and resorptions were r ecorded. Fetuses were examined for external abnormalities, sexed, and weighe d. Visceral examination was performed on approximately one half of the fetuses in each lit ter. The remaining fetuses were prepared for
examination of skeletal abnormalities.

No mortalities occurred during the study, except for a single death of a high-dose dam on GD 20.
This death was not considered to be related to treatment because there were no clinical signs or abnormal necropsy findings in this animal. Saliva tion was observed in 18/26 high-dose females, with no other signs in any dos e group except for alopecia. Mean body weight in the high-dose dams on GD 8, 12, and 16, with overall maternal body weight gain in the high-dose dams decreased by 20%. Food consumption in the hi gh-dose dams was decreased (p<0.05) by 13%.

The maternal LOAEL is 100 mg/kg/day, based on decreased body weight, body weight gain, and food consumption, and the maternal NOAEL is 25 mg/kg/day.ATRAZINE FINAL

Page 109 of 159 No deleterious treatment-related changes were observed in pregnancy rate, number of corpora lutea, mean number of implan tations/litter, pre-or post-implantation loss, early or late resorptions, number of live fetuses/litter, fetal se x ratio, fetal weights, or soft tissue abnormalities at any dose. Although the pregnancy rate was decreased (NS) at the high dose ( ↓ 15%), the number of implantations per dam was increased (p<0.05) by 14% and the number of live fetuses per dam was increased (p<0.05) by 15% in the hi gh-dose group. No skeletal malformations were observed. Increases (p<0.05) in th e fetal and litter inci dence of incomplete ossification of the hyoid, occipitals, and parietals we re observed in the high-dose pups, and increases (p<0.05) in the fetal and litter incidences of incomplete ossifi cation of the interparietals was reported in all
treatment groups.

The developmental LOAEL is 100 mg/kg/day, based on skeletal variations, and the developmental NOAEL is 25 mg/kg/day.

Developmental Toxicity (Rabbit, OCSPP 870.3700)

In a prenatal developmental toxicity study in rabbits ( Accession No. 00143006; Scialli et al.
, 2014 ), inseminated New Zealand White rabbits ( 19/dose group) were admi nistered atrazine (96.3% purity) in 3% aqueous corn starch cont aining 0.5% Tween 80 by oral gavage at doses of 0, 1, 5, or 75 mg/kg/day during GD 7-19, inclusive. All does were sacrificed on GD 29, and the number of corpora lutea, implan tations, and resorptions were r ecorded. Fetuses were examined for external abnormalities, sexed, and weighed. Each fetus underwent a visceral examination, followed by an examination of skeletal a bnormalities by the alizarin red S method.

All animals survived, with the exception of th ree deaths in the low-dose group that were attributed to dosing error. Stool changes (little, none, and/or soft ) were noted in the control group and the low- and medium-dose groups to a simila r extent, but were incr eased (p<0.01) in the high-dose does ( ↑ 111%; 19/19). Blood on the vulva or in th e cage also was increased (<0.05) in the high-dose group (4/1 9 vs. 0/19 control).

Decreases (p<0.01) in body we ight were observed in the high-dose dams on GD 14, 19, 21, 25, and 29 ( ↓ 12, 19, 18, 10, and 8%, respectively). In addi tion, body weight gains in the high-dose group were significantly decrea sed (p<0.01) at GD 7-14 ( ↓ 562%) and 14-19 ( ↓ 270%), with significantly increased (p<0.01 or 0.0 5) body weight gains at GD 21-25 ( ↑ 1348%) and 25-29 ( ↑ 319%). The overall body weight gain for the hi gh-dose group for the en tire gestational period was decreased (p<0.01; ↓ 638%). An intermittent decrease (p< 0.05) in body weight gain in the medium-dose group was observed at GD 14-19 ( ↓ 63%), with no significant effect on body weight. Treatment-related cha nges in food consumption were observed in the high-dose group only, with decreases ((p<0.01) from GD 7-20, a nd increases (p<0.05) from GD 24-28. Three abortions occurred during the study; a medi um-dose female on GD 21 and two high-dose females on GD 20 and 25, respectively.ATRAZINE FINAL

Page 110 of 159 The maternal LOAEL is 75 mg/kg/day, based on clinical signs and d ecreased body weight, body weight gain, and food consumption, and the maternal NOAEL is 5 mg/kg/day.

No treatment-related changes were observed in pregnancy rate, number of corpora lutea, and mean number of implantations/litter. The mean number of resorp tions was increased (p<0.05) in the high-dose group by 269%, and the percent post-implantati on loss was 42.6% compared to 12.0% for control. In addition, the mean number of live fetuses/litter was decreased (p<0.05) by 33% in the high-dose group. At the high dose, fe tal weights were decreased (p<0.05) by 23 and 19% in males and female pups, respectively. Skelet al findings indicated a slight increase in
skeletal variations concer ning delayed ossification.

The developmental LOAEL is 75 mg/kg/day ba sed on increased resorptions, decreased live fetuses, decreased fetal weight, and skelet al variations. The developmental NOAEL is 5 mg/kg/day.

Chronic Toxicity/Carcinoge nicity (Rat, OCSPP 870.4300)

In a combined two-year f eeding/carcinogenicity study ( MRID 40629302 ) atrazine (98.9% purity) was administered to SD ra ts (20/sex/treatment in the ch ronic toxicity subgroup and 50- 70/sex/treatment in the carcinogenicity subgroup) in the diet at nominal concentrations of 0, 10, 70, 500, and 1000 ppm, (calculated doses of 0, 0.5, 3.5, 25, and 50 mg/kg/day) for two years. The animals were observed daily, and body wei ght and food consumption were determined pretreatment, weekly through Week 13 of treatme nt, and monthly for the remainder of the study. Ophthalmoscopic examinations were conducted pr etreatment, at the 12- or 13-month interim sacrifices, and prior to study term ination. Organs that were weighe d included the liver, kidneys, adrenals, thyroids, epididymid es, prostate, testes, and ova ries. Gross and microscopic pathological examinations were conducted on th ese tissues as well as the seminal vesicle, uterus/cervix, urinary bladder, vagina, p ituitary, parathyroids, and mammary gland.

Female survival was 50% at study termination in the control group, and decreased to 37 (NS) and 26% (p<0.01) in the medium-high and high-c oncentration animals, respectively. Survival was increased (p<0.01) in the high-concentratio n males (67%) compared to control (44%) at study termination. Body weights were decreased (p <0.01) for males and females in the mediumhigh concentration group through Week 78 ( ↓ 7-14 and 6-21%, respectively) with decreases (NS or p<0.05) of 8 and 19%, respectively, at Week 104. Body weights also were decreased (p<0.01) for males and females in the high co ncentration group through Week 104 ( ↓ 11-21 and 10-27%, respectively). Decreases in food consumption were noted only during the first year of the study. Decreases (p<0.01) were noted through Week s 26 and 52 in the medium-high and highconcentration males, respectivel y. Decreases (p<0.01) were noted through Weeks 1 and 26 in the medium-high and high-concentration females (p <0.05 at Week 26), respectively. No treatmentrelated ophthalmic changes were observed dur ing the study. Decreases (p<0.05 or 0.01) in hematology parameters ( e.g. , ↓ RBC, hematocrit, and hemoglobin) were noted in females duringATRAZINE FINAL

Page 111 of 159 the study, but were not significan tly different at study termina tion. No hematological effects
were noted in males.

Absolute liver and kidney weights were decreas ed (p<0.05) in the high-concentration males at the interim sacrifice (Month 12) with decreases of 25 and 16%, respectively. Decreases in these organ weights at study termination were not signi ficantly different from control. Gross pathology findings were minimal, but increased incide nces of masses (e.g., a bdominal, axillary, and thoracic) were noted in females in all trea tment groups except the low-concentration group. Histopathology findings in high-concentration female s included increased in cidences of retinal degeneration (p<0.05; ↑ 83%); centrolobular necros is in the liver (p<0.01; ↑ 300%); degeneration
of the rectus-femor is muscle (p<0.05; ↑ 160%); transitional epithelial hyperplasia in the bladder (p<0.05; ↑ 150%) and the kidney (p<0.01; ↑ 82%); splenic extramedullary hematopoiesis (p<0.05; ↑ l33%); and myeloid hyperplasi a in both the femur (p<0.01; ↑ 108%) and sternum bone marrow (p<0.01; ↑ 119%). Histopathology findings in high-con centration males consisted of increased incidences of degeneration rectus femoris muscle (p<0.01; ↑ 366%); prostate epithelial hyperplasia (p<0.01; ↑ l41%); calculi in the kidney pelvis (p<0.01; ↑ l06%); and acinar hyperplasia of the mammary gland (p<0.01; ↑ 200%). No changes in e ndocrine-related endpoints were observed in this study, with the exception of epithelial hyperplasia in the prostate in the presence of overt toxicity.

There was an increased incidence of mammary adenocarcinoma in the medium-low, mediumhigh, and high-concentration females, as well as an increase in fibroadenoma in the highconcentration females, in the carcinogenicity subgroup. Some females developed multiple mammary tumors. There also was an increase (N S) in adenocarcinomas in high-dose females at
the 12- and 13-month sacrifices. Incidences of carcinomas were increased (p<0.05) in the medium-low, medium-high, and high-concentration females, and incidences of adenomas and fibroadenomas were increased (p<0 .05) in the high-concentration females. No neoplastic lesions were observed in males except for an increase (N S) in interstitial cell tumors at the high dose.

The LOEL is 500 ppm (25 mg/kg/day) base d on decreases in body weight and food consumption; the NOEL is 70 ppm (3.5 mg/kg/day).

Carcinogenicity (Mouse, OCSPP 870.4200)

In an carcinogenicity study ( MRID 40431302 ), atrazine (purity not re ported) was administered to CD-1 mice (58-60/sex/treatment ) in the diet at nominal con centrations of 0, 10, 300, 1500, and 3000 ppm (male/female mean-measured daily doses of 1.4/1.6, 38.4/47.9, 194.0/246.9, and 385.7/482.7 mg/kg/day) for 91 weeks. The animal s were observed daily, and body weight and food consumption were determined pretreatme nt, weekly through Week 12 of treatment, biweekly during Weeks 14-25, and every four weeks for the remainder of the study.ATRAZINE FINAL

Page 112 of 159 Ophthalmoscopic examinations were conducte d pretreatment and at Weeks 26, 52, 78, and 90 during treatment. Organs that were weighed included the liver, kidneys, adrenals, and testes. Gross and microscopic pathological examinations were conducted on these tissues as well as the epididymides, prostate, seminal vesicle, ovaries , uterus, urinary bladder, vagina, pituitary, thyroids/parathyroids, and mammary gland.

Female mice in the 300, 1500, and 3000 ppm groups (medium-low, medium-high, and highconcentration) received a daily atrazine dose of approximately 25% higher compared to males in these treatment groups. There was an increase in mortality (p<0.05) in high-concentration females, but not males, with onl y 25% of the females surviving vs 39-45% of females surviving in the other dose groups. Male survival rates ranged from 55-65%. There were no treatmentrelated clinical signs observed during the study. Mean body weight gain decreases were reported at Weeks 12 and 91 for the medium-high and high-concentration animals. At Week 91, body weight gain was decreased (p<0.05) by 25 and 24%, respectively, in males and decreased by 10% (NS) and 52% (p<0.01) in females in the tw o highest treatment groups. Treatment-related decreases (significance not repor ted) in food consumption we re observed in medium-high concentration males and high-dose animals, w ith sporadic changes noted in medium-high
concentration females and medium-low concentra tion animals; no changes were noted in the low treatment group. No ophthalmic change s were observed during the study.

Organ weight changes were minimal, w ith decreased (p<0.0 1) kidney weights ( ↓ 16%) weights noted in the high-concentration fe males. Gross pathology findings in cluded enlarged atria/atrium of the heart (1-13/58-60 dose group) and tan lesions of the heart (2-3/58-60 dose group) in the medium-high and high-concentration animals. The incidence of cardiac thrombi in animals that died prior to study termination was increased in high-concentr ation males (p<0.05; 9/23) and medium-high (p<0.01; 11/33) and high-concentra tion (p<0.05; 24/45) females, respectively. The incidence of cardiac thrombi in surviving animal s was minimal. The high-concentration animals, and medium-high concentration males, also had decreases (p<0.05 or 0.01) in several hematological parameters ( e.g. , RBC, hematocrit, and hemoglobin). No dose-related increases in neoplasms were observed. No changes in endocri ne-related endpoints we re observed in this study.

The LOEL is 1500 ppm (194.0 and 246.9 mg/kg/day in males and females, respectively), based on decreased body weight gain in both sexes an d increased cardiac thrombi in females. The NOEL is 300 ppm (38.4 and 47.9 mg/ kg/day in males and females, respectively). At the doses tested, there was not a treatment-related increas e in tumor incidence compared to control.

Chronic Toxicity (Dog, OCSPP 870.4300)

In a chronic toxicity study ( MRIDs 40431301 and 41293801 ), Beagle dogs (6/sex/dose group for the control and high concentrati on and 4/sex/dose group for the low and medium concentration) were administered atrazine (9%7 purity) in the feed at nominal concentrations of 0, 15, 150, orATRAZINE FINAL

Page 113 of 159 1000 ppm for 52 weeks. The mean-measured daily dos es were identical for the low and mediumconcentration groups (0.48 and 4.97 mg/kg/day, re spectively) and were similar for the highconcentration group (33.65 and 33.80 mg/kg/day for males and fe males, respectively). The animals were observed daily, and body weight and food consumption were determined pretreatment, weekly through Week 13 of treatme nt, and monthly for the remainder of the study. Auditory and ophthalmoscopic examinations were conducted pretreatment and quarterly during treatment, as were electrocardiographic (EKG) tracings (10 leads). Orga ns that were weighed included the liver, kidney, pituitar y, thyroid/parathyroid, ovary, and testis with epididymis. Gross and microscopic pathological examinations we re conducted on these ti ssues as well as the adrenals, uterus, urinary bladder, pr ostate, vagina, and mammary gland.

Three animals were sacrif iced during the study in a moribund condition: one mediumconcentration male on Day 75, and one high-concentration female and male on Days 113 and 250, respectively. The death in the medium-concen tration dose group was not considered to be related to treatment; the deaths in the high-c oncentration group were either treatment-related (female) or treatment may have been a contributing factor (m ale). Treatment-related clinical
observations were noted at low in cidences in the high-dose group that were attributed to atrazineinduced myocardial degeneration. These include d ascites (one high-dose male and female), cachexia (one female), and labored/shallow breath ing (one male). These high-dose animals, and three others, also had EKG and/ or morphologic evidence of heart disease (characterized as irregular heartbeat, tachycardia, and increased he art rates). These treatment-related clinical signs were correlated with pathologic findings attributed to atrazi ne administration.

Mean body weights at Week 52 were decreased (NS) in the high-c oncentration males ( ↓ 20%) and females ( ↓ 16%), but baseline mean body weight in the high-dose males was decreased (NS) by 10% relative to control. Mean body weight gain was decreased by 21 (NS) and 36% (p<0.05) in the medium- and high-concentration male s and decreased (NS) by 40% in the highconcentration females. Males and females in th e high-concentration group also had reduced food consumption throughout the course of the study (decr eases were NS after the first quarter). At study termination, mean food consumption was decr eased by 22% in males and 20% in females. No compound-related ocular ch anges were observed during ophthalmoscopic examination.

The major treatment-related findings in this study were cardiac findings. EKG alterations such as tachycardia and increased P wave amplitude (P -II) were observed at many time points during the study in the high-concentration gr oup animals. Mean heart rate was increased (p<0.05) by 59% and P-II was decreased (p<0.01) by 78% on Day 361 in high-concentration males; female heart rates at the same time point were increased (NS) 46% and P-II was decreased (p<0.01) by 71%. Absolute heart weight was decreased (p<0.05) by in females by 14%. Gross pathology findings were also seen in the heart in the high-dose su rvivors (4/5 males and 5/5 females) and consisted mainly of moderate to severe di latation of right and/or left atri a. Fluid-filled pericardium (1/5) and an enlarged soft heart 2/5) also were not ed in high-concentration males. Minimal atrialATRAZINE FINAL

Page 114 of 159 dilation, seen in 3/6 males and 2/6 females, was the only cardiac finding observed at necropsy in the control animals. Cardiac myolysis was noted in the high-concentration animals (3/6 males and 6/6 females vs. 0 control). Focal atrophy of th e heart also was observed in 2/6 males and 5/6 females in the high-concentration group, with no si milar findings in the control group. Two highand one medium-treatment animals were sacrifi ced in moribund condition during the course of the study. Moribundity in the high-concentratio n animals may have been due to compoundrelated cardiac dysfunction. No changes in endoc rine-related endpoints we re observed in this study.

The LOEL for both sexes is 1000 ppm (33.65 mg/kg/ day for males and 33.80 for females), based on cardiac effects (electrocardiography alterati ons as well as microscopic and macroscopic cardiac abnormalities). The NOEL is 150 pp m (4.97 mg/kg/day) for both sexes.

Avian Reproduction (OCSPP 850.2300)

In avian reproduction study ( MRID 42547101 ), 23-week old mallard ducks ( A.platyrhynchos ) were fed a diet containing at razine (97.1% purity) at nominal concentrations of 75, 225 and 675 mg/kg diet for up to 20 weeks, with a three-week recovery period; actual concentrations were 101.1, 116.6 and 105.1% of nominal, respectively. E ach treatment group cons isted of 16 males and 16 females, with a single male and female per pen (16 pens/group). The photoperiod was seven hours of light per day duri ng the first 8 weeks (pre-egg-lay ing), and was increased to 17 hours of light per day for the remainder of the study. All adult birds were observed daily for mortality, signs of toxicity, and abnormal be havior. Body weights were recorded at study initiation and at Weeks 2, 4, and 6, and at study termination, but not during egg-laying. Feed consumption was measured bi-weekly throughout the study. Eggs were collected daily during egg-laying and stored at 57-65°F. Once a week th e collected eggs were removed from storage and eggs that were uncracked or not used for shell thickness measurements were placed in an incubator. All eggs were candled to detect cr acks and determine embryo viability on Days 14 and 21, and eggs were placed in a hatcher on Day 23. On Day 27, all hatchlings, unhatched eggs, and eggshells were removed. Hatchlings were hous ed at 77-98°F at 38-90% relative humidity, and weighed at hatching and at 14 days post-hatch. Eggshell thickness was determined in eggs collected on eight separate occasions.

Decreased hatchling weight was significant at a ll concentrations teste d, with decreases ranging from 5.3 to 12.3% at 75 to 675 mg a.i./kg-di et, respectively. At a concentration of ≥ 225 mg a.i./kg-diet, there were effect s on egg production and mean food consumption while live embryos and hatchlings per eggs set and ma le weight gain were affected at 675 mg a.i./kg-diet No clinical signs of toxicity were noted. Two females, one low- and one medium-concentration, died during the study; the deaths were not treatment-related, but it is unclear whether the loss of birds from the test may have affected mean food consump tion values. Examination of one-half of the adultATRAZINE FINAL

Page 115 of 159 birds at study termination found no abnormal findings. Unless otherwise specified, all
differences were considered stat istically significant at p<0.05.

In another avian reproduction study ( MRID 42547102 ), 18-week old bobwhite quail ( C. virginianus ) were fed a diet containing atrazine (97.1% purity) at nom inal concentrations of 75, 225 and 675 ppm for up to 20 weeks, with a three-w eek recovery period; actual concentrations were 101.1, 116.6 and 105.1% of nominal, respectively. Each treatment group consisted of 12 males and 24 females, with a single male and two females per pen (12 pens/group). The photoperiod was seven hours of light per day duri ng the first 8 weeks (pre-egg-laying), and was increased to 17 hours of light per day for the remainder of the study. All adult birds were observed daily for mortality, signs of toxicity, and abnormal behavi or. Body weights were recorded at study initiation and at Weeks 2, 4, 6, 8, 10, and 20, and at study termination (Week 23), but not during egg-laying. F eed consumption was measured biweekly throughout the study. Eggs were collected daily during egg-laying and stored at 58-66°F. Once a week the collected eggs were removed from storage and eggs that were uncracked or not used for shell thickness measurements were placed in an incubator. All eggs were candled to detect cracks and determine embryo viability on Days 11 and 18, and eggs were placed in a hatcher on Day 21. On Day 24, all hatchlings, unhatched eggs, and eggshells were removed. Hatchlings were housed at 95-115°F at 20-46% relative humidity, and wei ghed at hatching and at Day 14 post-hatch. Eggshell thickness was determined in eggs collected on seven separate occasions.

One male in the medium-concentr ation group and eleven females (1 control, 4 low-, 4 medium-, and 2 high-concentration) died during the st udy. Post-mortem examinations showed abnormal findings, but none of the deaths were attributed to the test substa nce. Examination of one-half the surviving birds in each group at study termination also showed no treatment-related effects. There were no differences in adult body wei ght throughout the study. Feed consumption was higher during Week 12 in the me dium-concentration group (p<0.05; ↑ 13%), and during Week 16 in the medium- and high-concentration groups (p<0.01: ↑ 16 and 10%, respectively), but the
differences were not cons idered treatment-related

Although not statistically significant (p=0.08), egg production ( both total and number per hen) was decreased by 29% at the high concentration. In addition, the number of viable embryos (per eggs set) on Day 18 and at hatch (Day 24) wa s decreased by 6 and 10%, respectively, in the 675 ppm group, compared to the control. There we re no` differences in egg shell thickness. Although changes were seen in body weights of ha tchlings and 14-day survivors, these changes
were minor and not considered to be treatment related. There was no treatment-related effect on hatchling survival.

HPG Axis Effects in Japanese Quail

In a published study by Wilhelms et al . (2005) , a series of seven expe riments investigating the effects of atrazine (99.9% purit y) on sexual maturation in male Japanese quail were conducted.ATRAZINE FINAL

Page 116 of 159 In the first experiment, groups of 12 male quail (s ix-week-old) were administered atrazine at 0, 10, 100, or 1000 ppm in the diet. A positive control of 17 β -estradiol was administered at 100 ppm (n=12). In the second experiment, groups of four-week-old quail were administered 0 or 100 ppm atrazine (n=11) or 1000 ppm atrazine (n=13) in th e diet. The third experiment investigated the effect of 0 or 1000 ppm atrazine on growth and sexual maturation in six-weekold birds with an additional group of pair-fed birds (n=7). Pair-fed birds were used to
differentiate the effects of redu ced feed consumption and those due to exposure to atrazine. In experiment 4, atrazine in pr opylene glycol was administer ed daily by subcutaneous ( s.c .) injections at dose levels of 0, 1, or 10 mg/kg (n=8). For comparison, vehicle and estradiol -3- benzoate (1 mg/kg/day) contro ls were run concurrently. In experiment 5, atrazine was administered by silastic implants (n=8) at approximately 1.42 mg/kg/day. For comparison, implant controls of cholesterol (negative control) and estradiol- 3-benzoate (positive control) were run concurrently. In experiment 6, treatments of 100 ppm tamoxifen, 100 ppm E 2 , 1000 ppm atrazine, and combinations of 100 ppm 17 β -estradiol plus 100 ppm tamoxifen and 100 ppm

17 β -estradiol plus 1000 ppm atrazine were administered in th e diet for two weeks (n=8). In experiment 7, silastic implants of testosterone (p ositive control) or cholesterol (negative control) were implanted, and quail were assigned to dietary treatments containing 0 or 1000 ppm atrazine (n=9).

Concentrations of atrazine up to 1000 ppm in the diet had no effect on mortality in sexually-maturing male Japanese quail. 1000 ppm atrazine decreased (p<0.05) body weight gains and feed intake by 32% and 15%, respectively. Howe ver, atrazine had no effect on liver weights
or the liver-somatic inde x. In contrast, 100 ppm 17 β -estradiol reduced (p < 0.05) body weight gains and feed intake by 21% each, respectively, and increased (p<0.05) liver weights and liversomatic index by 50% an d 58%, respectively.

Atrazine, up to 1000 ppm, had no effect on testes we ight or the ratio of th e left-to-right testis weight. In contrast, 100 ppm 17 β -estradiol decreased (p<0.05) te stes weight and GSI by 92% and 91%, respectively, and increased (p <0.05) the left-to-right testis weight ratio by 879% versus
control.

Atrazine, up to 1000 ppm, had no effect on circulat ing concentrations of LH. However, atrazine at 1000 ppm increased (p<0.05) circulating concentra tions of testosterone by 3-fold compared to the negative control. In a ddition, atrazine at 10 ppm in creased (p<0.05) circulating
concentrations of 17 β -estradiol by 3.6-fold compared to control. There were no changes in circulating 17 β -estradiol at the higher concen trations of atrazine tested.

In the pair-fed examination, the high concentr ation of atrazine (1000 ppm) decreased (p<0.05) body weight gain by 64% with no effect on liver weight or liver-somatic index. Although there
were no effects of atrazine on total feed intake , daily feed intake was statistically lower in atrazine treatments on days 2, 3, 6, and 11 of th e study (p<0.05).. Neither atrazine nor feed restriction in the pai r-fed birds had any effect on testes weights, GSI, or circulatingATRAZINE FINAL

Page 117 of 159 concentrations of 17 β -estradiol and testosterone. Similarl y, there were no differences in the measured indices between atrazine-treated and pair-fed birds.

Atrazine administered at 1 and 10 mg/kg/day by s.c . injection had no effect on indices of growth or reproductive development. In contrast, s.c. injections of estradio l-3-benzoate decreased (p<0.05) both testes weights and GSI by 47% and 43%, respectively. Ho wever, the estrogen control had no effect on the other parameters anal yzed. Atrazine administered by silastic implant had no effect on indices of growth and reproduc tive development. In contrast, the estrogen control delivered by silastic implant increased (p<0.05) liver we ights and liver-somatic index 44% and 50%, respectively. Furthermore, estr ogen delivered by impl ant decreased (p<0.05) testes weights and GSI by 77% and 83%, respectively.

Atrazine and pair-feeding had no effect on the stag e of development of the testes, lumen size, or seminiferous tubule diameter. However, atrazine at 10 ppm decreased (p<0.05) the tubule diameter-to-testis weight ratio by 33%. No othe r concentration of atrazi ne (or pair feeding) altered this parameter.

Atrazine decreased (p<0.05) body weight gains and feed intake 32% and 14%, respectively, in Experiment 6. Addition of 17 β -estradiol to the diet decreased (p<0.05) feed intake by 16% and tended to decrease body weight gains. Furthermore, 17 β -estradiol increased (p<0.05) the liversomatic index by 27%. In contrast, c oncurrent administration of 100 ppm 17 β -estradiol and 1000 ppm atrazine in the diet de creased (p<0.05) body weight gains and feed intake by 63% and 29%, respectively. Addition of tamoxifen (an anti-es trogen) to the diet ma rginally inhibited the
deleterious effects of 17 β -estradiol on growth, feed in take, and liver-somatic index.

Atrazine at 1000 ppm in the diet (Experiment 6) did not influence testes weight versus control.
Administration of 17 β -estradiol in the diet decreased (p<0.05) testes weight by 84%. Concurrent
administration of atrazine and 17 β -estradiol in the di et decreased (p<0.05) testes weight by 97%.
This decrease was observed to be 79% more than the effect of 17 β -estradiol alone (p<0.05). In contrast, addition of tamoxifen to a 17 β -estradiol-containing diet marg inally inhibited the effect
of 17 β -estradiol on testes weight.

In Experiment 7, administration of 1000 ppm atrazi ne in the diet or syst emic administration of testosterone via an implant did not influence growth, feed intake, or liver weights versus control. However, concurrent administra tion of 1000 ppm atrazine and te stosterone decreased (p<0.05) growth by 42%. Atrazine at 1000 ppm in the diet had no effect on testes weight but reduced (p<0.05) circulating concentrations of LH by 36 %. Systemic administration of testosterone by subdermal implants decreased (p<0.05) testes we ight and circulating co ncentrations of LH by 92% and 69%, respectively. Administ ration of atrazine in the diet in the presence of testosterone decreased (p<0.05) testes weights by 96%. This d ecrease was observed to be 49% m ore than the effect of testosterone alone (p <0.05). However, the combination of atrazine and testosterone had
no additive effect on circulat ing concentrations of LH.ATRAZINE FINAL

Page 118 of 159 In a second study by Wilhelms et al . (2006), a series of five experi ments investigating the effects of atrazine (>98% purity) on sexual maturation in female Japanese quail were conducted. All treatments were administered for 14 days. In these studies, atrazine and estrogen (17 β -estradiol or estradiol -3-benzoate) were dissolved in ch loroform, mixed in 10 g of feed, and the solvent evaporated. These premixes were used to produce the required dietary treatments. Experiments 1 and 2 used low doses of atrazine or estrogen in the feed (atrazine: 0.001-10 ppm; estrogen: 0.1 and 1 ppm). In Experiment 1, there were ten bi rds/treatment and in E xperiment 2, there were eight birds/treatment. In E xperiment 3, atrazine and 17 β -estradiol were weighed and directly mixed into feed to yield doses of 0, 10, 100, or 1000 ppm atrazine and 100 ppm 17 β -estradiol (n=8). The alternative feed wa s used due to limited availa bility of the starter diet.

Experiments 4 and 5 compared the effects of system ically administered atra zine to the effects of dietary atrazine (n=8). In Experiment 4, vehicle (p ropylene glycol), atrazine (1 and 10 mg/kg) or

17 β -estradiol (1 mg/kg) was administered by daily s.c. injection. In Experiment 5, quail received atrazine, 17 β -estradiol or a negative contro l (cholesterol) in silastic implants. Atrazine implants were determined to release approximately 1.42 mg/kg per day.

Dietary administration of atrazine up to 1000 ppm had no effect on mortality, average body weight gain (ADG), or feed intake. The 17 β -estradiol positive contro l also had no effect on ADG
or feed intake. Atrazine did not exert any consistent effect on liver, ovary, or oviduct weights. Oviduct weight was increased (p<0.01) by 38% by atrazine treatment in Experiment 1 at 10 ppm, and increased (p<0.05) by 50% in Experiment 2 at 0.1 ppm. This effect was not observed when data from Experiments 1-3 were combined. In contrast, dietary 17 β -estradiol (100 ppm) increased (p<0.001) liver weight by 53%. Additiona lly, estradiol-3-benzoate increased (p<0.05) oviduct weight by 44% at 1 ppm and 17 β -estradiol increased (p <0.05) oviduct weight by 100-fold at 100 ppm.

Subcutaneous or implanted atrazine had no e ffect on mortality, ADG, feed intake, or ovary, oviduct, and liver weights. However, in quail re ceiving estradiol, liver weights were increased (p<0.001) by 40%, and oviduct weights were in creased (p<0.001) by 37- and 133-fold in quail
administered 17 β -estradiol and estradiol-3-benzoate by injections and implants, respectively.

Atrazine did not influence circul ating plasma concentrations of LH irrespective of the route of administration. Systemic atrazine delivered either by s.c. injection or implants had no effect on plasma concentrations of co rticosterone or aldosterone.

Fish Early Life-stage Toxi city (Sheepshead minnow)

In an unpublished, early lif e-stage toxicity study ( MRID 46648203 ), groups of fertilized sheepshead minnow ( C. variegates ) embryos (<26-hours-old) were exposed to atrazine (97.1% purity) at nominal concentrati ons of 0 (dilution water cont rol), 0.20, 0.40, 0.80, 1.6, or 3.2 m g/L, under flow-through conditions for 33 days. Each tr eatment level included two replicate aquaria with 60 embryos per tank (120 embryos/treatment group). Mean-measured concentrations wereATRAZINE FINAL

Page 119 of 159 <0.028 (<LOQ, control), 0.15, 0.30, 0.57, 1.1, and 2.2 mg/L , respectively. The test system was maintained at 24-27°C and a pH of 7.8-8.2.

No treatment-related effect was observed on hatching (71-79%) or fry survival ( ≥ 97%), and no abnormal behavior or other clinical signs of toxicity were reporte d. At study termination (28-days post-hatch), the total length and wet weights were adve rsely affected at the 2.2 mg/L concentration, with decreases (p<0.05) of 17% in mean body length and 46% in wet weight compared to the dilution water control. The LO AEC is 2.2 mg/L based on decreased growth, and the NOAEC is 1.1 mg/L.

Fish Chronic Toxicity (Trout, OCSPP 850.1500)

In a fish chronic partial life-cycle toxicity study ( Accession No. 00024377 ), yearling brook trout ( S. frontinales ) were continuously exposed to atrazine (purity not reported) at concentrations of 0 (dilution water), 0.065, 0.12, 0.24, 0.45 and 0.72 mg/L , under flow-through conditions for 44 weeks. Each treatment consisted of duplicate aquaria each containing 15 fish. The length and weight of adult fish were determined at study initiation and after 90 days of exposure. Other parameters that were assessed included number of eggs spawned (total and number per female), percent fertilization, per cent hatchability, and growth (length and wet weight) and survival of the

F 1 generation at 90 days.

Atrazine exposure in the F 0 generation at concentrations ≥ 0.24 mg/L resulted in decreases (significance not reported) in body length ( ↓ 17-25%) and weight ( ↓ 56-63%) compared to the negative (water) control. No treatment -related effect was observed in the F 0 generation on number of eggs spawned, fertilization or hatchab ility at atrazine concen trations up to 0.72 mg/L. However, growth of the F 1 fry was decreased (NS) at 90 days at 0.12 mg/L; length was decreased by 7%, and wet weight was decreased by 16%, compared to control. Further decreases (p<0.01) were noted in length and weight ( ↓ 39% to ↓ 45% , respectively) at atrazine
concentrations ≥ 0.24 mg/L.

In a published non-Guideline study, Le Mer et al . (2013), examined the effect of atrazine ( ≥ 97.5% purity) exposure on larval estuarine/marine threespine sticklebacks ( G. aculeatus ), with growth, survival, sexual differentiation, and th e reproductive biomarke rs VTG and spiggin 8 (SPG) as endpoints in exposed larvae. The tested concentrations were 0.1, 1, 10, and 100 μ g/L

8 Spiggin is a glycoprotein secreted by male sticklebacks in response to androgens; the protein is used by males to stick together their nests (Sloman, K. A. 2003. Spiggin: A sticky toxicity indicator. Journal of Experimental Biology 206. 1102)ATRAZINE FINAL

Page 120 of 159 for atrazine, and the tests included a seawater control, an acetone (5 μ g/L) control, a positive
control of 17 α -ethinylestradiol (0.05 μ g/L), and a positive control of DHT (3 μ g/L). The three
lowest atrazine concentrations were consid ered to be representa tive of environmental
concentrations found in estuarin e and coastal waters of eastern North America. Atrazine was initially dissolved in acetone and then dilute d with deionized water such that the final concentration of acetone in exposure containe rs was 0.0005%. Measured concentrations of
atrazine were ≥ 78% of nominal.

The larvae utilized for testing were obtained fr om laboratory matings of wild caught adults. The newly-hatched larvae (seven days post fertilization) were exposed to atrazine in 1-L glass
beakers with 500-mL aerated seawater at 18°C with a 16:8 hour light:dark photoperiod. Each
treatment consisted of three re plicates, with 11-13 fish per replicate. Fish were exposed throughout the embryo-larvae and juvenile life st ages (from hatching to Day 42 post-hatching) until fish had a mean length of ~20 mm. Larvae were fed once per day with Artemia nauplii; water was changed every day (>80% replacement) before feeding. Dissolved oxygen, temperature, and fish mortality were monitored daily.

At the end of the 42-day exposure period, survivi ng fish were sacrificed and total body lengths (L) and wet weights (W) were determined, and a condition factor (CF) wa s calculated as CF = (W/L 3 )100. Half of the fish from each replicate, se lected randomly, were frozen at –80°C for VTG and SPG determinations, and the remaining fi sh were preserved for intersex evaluations; a subset of these fish were fixed in phospha te-buffered formalin, dehydrated, embedded in paraffin, sectioned, and stained (hematoxylin and eosin) for hi stological evaluation. VTG was isolated from the whole-body homogenates of ju venile sticklebacks and analyzed using ELISA.
Stickleback SPG was determined with a competitive ELISA method. For histological evaluations, the gonads were examined by light microscopy; all sections were examined for phenotypic sex determination. A gon ad was determined to be an ovary when the entire gonad was packed with oocytes, and was determined to be a testis when it contained spermatogonialike germ cells packed in lobul es and no oocytes. Gonads with an appearance intermediate between an ovary and testis, containing at least one oocyte but showing a te stis-like structure or spermatogonia-like germ cells, was determined as ovotestis.

The tests were conducted over tw o years (2007 and 2008), with data for the seawater control groups being pooled for both years. Mortality was <11% for the fish exposed to atrazine over the 42-day exposure period. There were no differen ces in wet weight, body length, or condition of juvenile fish between at razine-exposed fish and controls (ace tone or seawater, respectively) in either test year.

Treatment with 17 α -ethinylestradiol resulted in an increase (p<0.05) of whole-body VTG compared to the other treatments, but VTG induc tion was not observed in fish treated with DHTATRAZINE FINAL

Page 121 of 159 or atrazine, or fish in the ne gative control groups. No induction of SPG was observed in any of the treatment groups, including the positive DHT control.

Atrazine treatment had no effect on the proportion of mixed-sex individuals. A single mixed-sex individual was found in the group exposed to atrazine at 1 μ g/L. The proportion of mixed-sex individuals was higher in the positive controls (17 α -ethinylestradiol, DHT) compared to the negative controls (seawater, acet one). The proportion of female individuals did not differ among treatments, but tended to be hi gher in the fish exposed to 17 α -ethinylestradiol in the experiment
run concurrent with atrazine treatments. The stud y authors concluded that atrazine treatment did not demonstrate estrogenic or an drogenic effects to early life stages of sticklebacks at environmentally realistic exposure concentrations.

Fish Life Cycle Toxicity (Minnow, OCSPP 850.1500)

In an unpublished fish life-cycle toxicity study ( MRID 42547103 ), fathead minnow ( P. promelas ) embryos (<24-hour-old) were continuously exposed to at razine (97.1% purity) at nominal concentrations of 0 (dilution water and solvent control [0.002% dimethylformamide (DMF)]), 0.13, 0.25, 0.50, 1.0 or 2.0 mg/L; mean-measured concentrations were <0.013, 0.15, 0.25, 0.46, 0.99 and 2.0 mg/L, respectively. Duplicate a quaria were used for each treatment, and two groups of thirty-five eggs were incubated in each aquarium. Dead eggs were removed and counted each day until hatching was completed (Day 4). Percent hatch was based on the number of live fry from 140 eggs (35 per cup; 70 per a quaria). Twenty five larvae from each embryo incubation group were randomly selected and distri buted to growth chambers in each aquarium (four groups per treatment level). Two growth cham bers were used to facilitate handling of fry for 30- and 60-day measurements by a photogr aphic method. Percent survival, based on cumulative mortality, and length were determined at these intervals. At Day 60, the two larval groups in each aquarium were combined and thinned to 25 larvae per aquarium, and were assessed for percent survival, to tal length, and wet weight afte r 274 days. At approximately 140 days, two spawning groups, consis ting of one male and two fema les per group, were established in each aquarium. For each spawning group, the number of eggs spawned and the number of eggs incubated were recorded. Fifty embryos from the first ten spawns consisting of >50 eggs in each aquarium were incubated to determine the percentage hatch. Subsequently, every third spawn of ≥ 50 eggs in each aquarium was incubated fo r determination of percentage hatch. As F 1 embryo groups hatched, two groups of 25 newly-hatched fry were established in each aquarium, one from each spawning group. After 30-days post-h atch, each larval group was terminated, and percentage survival and individual body length an d wet weights were determined. Parental fish (F 0 ) were terminated when no spawning had occu rred in any aquarium for 14 days (Day 274), and body length, wet weight and sex were determined for each fish.

Compared to the solvent control, there was no si gnificant effect on hatching success, survival or reproduction (number of eggs/spawn, total number of eggs, number of spawns/female, or numberATRAZINE FINAL

Page 122 of 159 of eggs/female) at concentra tions up to 2.0 mg/L. However, a significant effect was noted on larval growth. For the parental generation (F 0 ), total length was decreased (p<0.05) by 7-10% at 0.99 mg/L at Day 30 and 2.0 mg/L, and by 5% at ≥ 0.46 mg/L at Day 60. Body weights were significantly decreased (p<0.05) by 19% at 0.99 and 2.0 mg/L at Day 60. Although apparent solvent effects were noted on growth in the F 1 generation, the differences between solvent control and negative control were not statistically significant; ther efore, the reviewer compared treatment results to the negative (dilution water) control. Total length in atrazine-treated F 1 fish at 30 days post-hatch was lower at 0. 15, 0.25, 0.46, 0.99 and 2.0 mg/L by 7, 10, 10, 17 and 23%, respectively (p<0.05), and wet weights were lower by 17, 27, 34, 44, and 54%, respectively (p<0.05). Based on decreases in larval growt h, the LOAEC was the lo west level tested 0.15 mg/L, and a NOEAC was not observed.

Fish Endocrine (Steroidogenesi s) development (Barramundi)

In a published study by Kroon et al . (2014 ), the effects of short-term (2-day) exposure to atrazine on CYP19B brain transcript abunda nce, plasma hormone (testosterone and 17 β

estradiol) concentrations, and an increase in hepatic VTG transcript abundance were investigated. Juvenile barramundi [ Lates calcarifer (Bloch); approximately five-months old; n=40] were obtained from the Betta Barra Tropical Fish Hatchery at Lake Eacham on 29 January 2013 and were established individually in gla ss aquaria. Tanks were filled with 45 L of quadruple-filtered rainwater one week before introduction of the catadromous fish, and water was not renewed during the experiment. Each ta nk was continuously aerated (150 L/minute), and kept at an ambient temperature of 25±0.5°C. Determination of no prior known chemical exposure in the fish preceding th e experiment was conducted. Fish were acclimated for seven days prior to the two-day exposure period. Afte r acclimation, fish (5 /group) were randomly assigned to the following groups: negative control, solvent control (ethanol ; SC), positive control (17 α -ethynylestradiol), and atrazi ne (analytical standard, purit y not reported; 0.1, 0.5, 5, 50, and 100 μg/L). The SC and atrazine-treatme nt aquaria containe d 10 μL/L ethanol.

All fish were observed for gill ventilation ra te (gill beats/minute) on Days -2, 1, and 2 of exposure. After 48 hours of exposure to atrazine, individual fish were removed, anesthetized, measured and weighed, and bled with a heparini zed 23-gauge needle. Fish were subsequently euthanized by gill slit and cranial dislocation, and brain and liver tissues were collected for quantitative polymerase chain reaction (qPCR) analyses. Plasma was harvested from blood samples immediately after collection for plasma steroid determinations. Plasma, liver, and brain samples were frozen at

− 80°C. Water samples were collected from each tank in 1-L amber glass bottles (containing 80 mg sodium thiosulfate as preservative) for pesticide/chemical analyses immediately after the addition of treatment solu tions on Day 0, and immediately after removal of fish on Day 2. Samples were stor ed at 4°C prior to analysis.ATRAZINE FINAL

Page 123 of 159 Species-specific gene sequences were isolated (VTG) or obtained from the National Center for Biotechnology Information database ( i.e. , CYP19B, AY684259; β -actin, and GU188683). RNA was extracted from liver and brain specimens of sexually-mature Barramundi, and RNA was reverse transcribed into complementary DNA (cDNA) with a high-capacity RNA to cDNA synthesis kit. Complementary DNA was subsequen tly used as a template for all downstream PCR reactions after PCR amplification. RNA from laboratory tissue samples was extracted with a modification of the above procedure. Quantita tive PCR analyses on brain and liver samples were conducted by investigators under blinded co nditions. Expression levels of CYP19B and
VTG were normalized to β -actin concentration. Plasma concen trations of testosterone and 17 β

estradiol were determined with EIA kits, and validated for Barramundi.

No treatment-related effects were observed on gill ventilation rate, total length, or weight due to atrazine exposure. The concentrations of atrazine in all treatment groups were close to nominal values and remained constant during the exposure period. No other herbicid es were detected in the water samples.

Expression of CYP19B and VTG in juvenile barramundi did not exhi bit solvent effects. Relative expression levels for VTG were significantly increased (p=0.007) in the positive control, but no effect was observed for CYP19B (p = 0.14). Atrazi ne treatment did not a ffect CYP19B or VTG expression compared to the ne gative (water) control.

Concentrations of testosterone and 17 β -estradiol were detectable in all fish. Mean plasma
testosterone concentrations we re not affected by the solvent or positive control treatments.
Plasma testosterone concentrations were increa sed (p<0.05) in the atrazine low- and mediumconcentration groups compared to the water cont rol, but this increase was not dose-dependent (testosterone concentrations in the low-, medium-low, and me dium-concentration groups were similar, with lower mean testosterone concentra tions at the higher atrazine treatments). Mean plasma 17 β -estradiol concentrations were not affected in the SC, but were increased (p<0.05) in the positive control group. Expos ure to atrazine in the medi um-concentration group yielded increased (p<0.05) plasma 17 β -estradiol concentrations compared to the water control, but there was no dose-dependent effect noted . The ratio of testosterone/17 β -estradiol was not affected by any treatment. Finally, no treatment-related e ffect on gill ventilation rate (biomarker for oxidative stress) was obs erved in this study.

Developmental Reproductive Toxicity in Fish

In a published study by Papoulias et al . (2014 ), the effects of atrazine exposure on egg production and additional reproductive effects along the HPG axis of male and female Japanese medaka ( O. latipes ) were evaluated. Mature virgin medaka were exposed to atrazine at 0.5, 5.0, and 50 μg/L (equivalent to 2.3, 23.2, and 231 nM, respec tively) in a static renewal system with a
0.02% acetone control. Atrazine (98% purity; Fluka Chemical, Dorset, UK) stock solutions were prepared in 60:40% acetone:wat er solution. The fish were obtained from the ColumbiaATRAZINE FINAL

Page 124 of 159 Environmental Research Center stock with ini tial mean weights of 0.57 g for females and 0.64 g
for males. The fish were acclimated for one week prior to testing, at whic h point all females had produced some eggs. One male and four female fish were randomly assigned to each 6.5-L exposure tank, and each treatment group include d six replicate tanks for each of the three observation time points at Days 0, 14, and 38. The test water was renewed every 24 hours. The
fish were maintained at 25±1°C with a 14- hour light:10-hour dark photoperiod. Tanks and plumbing were made of glass or Teflon to mi nimize chemical inferences or adsorption of
atrazine. The sides of each tank were covered with opaque contact paper to prevent interactions
of fish tank to tank. The atrazine test concentrations were m easured weekly with an ELISA method, and confirmatory analysis of atrazine stock preparations and selected tank water samples were conducted using gas chromatogr aphy with a nitrogen-phosphorous detector (GC/NPD).

Adult mortality and egg production were determin ed daily. Eggs were collected from each tank (by stripping eggs from females or by examin ing siphoned waste water), placed in petri dishes with exposure water, incubated at 25±1°C for 24 hours, and inspected for viability and counted. On Days 0, 14, and 38, fish were euthanized with tricaine methanesulfonate and weighed prior to histological and biochemical assa ys; for all tanks, fish were collected for euthanasia during the same time period (19:00 to 24:00) to minimize th e diurnal effects of re productive hormones. The brain, liver and gonads were harvested, and th e liver and gonads were weighed. Aromatase protein content was determined from the fresh brain and ovary samples of two female fish from each replicate of each treatment group with a tritiated-water assay. The 17 β -estradiol and
testosterone concentrations in eviscerated carcasses were measur ed by RIA, and the estradiol to testosterone ( E/T ) ratio was calculated; GSI also was calculated.

Histological examinations of gonads were c onducted to evaluate reproductive stage and pathological lesions. For each post-exposure co llection time point (Days 14 and 38), one whole ovary of one female from each replicate of each treatment and the testis of one male from three replicates of each treatment were preserved in Davidson’s solution and prepared with standard techniques. Sections of the ovari es were stained with hematoxylin and eosin, and testes sections were stained with Giemsa or hematoxylin/eos in, for histological analysis with a compound microscope. Two ovary sections from each fi sh were evaluated for oogenic stage: previtellogenic (stage II), early vi tellogenic [stage III; central germ inal vesicle (GV)], mid-late vitellogenic (stage IV; GV moving toward animal pole), and mature (stage V; GV at the animal pole). The oocytes in each section were coun ted, and the percentages and average were
determined for each fish based on the two sections. Th e testis sections (seria l sections of at least one-half of each testis) were evaluated for deve lopmental stage and pathological lesions. The testis reproductive stages also were evaluated: tubules containing sper matogonia singly or in clusters predominate (stage II); spermatocytes prev alent (stage III); mostly spermatids with some spermatozoa in duct (stage IV); and duct greatly expanded and filled with spermatozoa (stage V). Clastogenic abnormalities ( e.g. , bridges, chromosome fragments, micronuclei) were counted forATRAZINE FINAL

Page 125 of 159 all stages of mitotic spermatogonia but primarily at anaphase and telophase. The percent of cells
with chromosomal abnormalities was calculated fo r 1000 spermatogonial cells in two testicular lobes of each fish and averaged per fish. Abnormalities were observed visually with oil immersion and a compound microscope at 1000× magnification.

Mean atrazine concentrations in tank wa ter over the 38-day exposure were <0.05 (below detection limit), 0.56, 5.63, and 51.5 μg/L for the nominal 0, 0.5, 5.0, and 50 μg/L atrazine dose groups, respectively. No adult mortal ity was observed during the study.

Egg production was decreased in all atrazine-exposed groups co mpared to control. The study authors reported significant de creases in total egg production, weekly egg production, but not for daily egg production. According to the reviewer s’ analysis, cumulativ e total egg production (using the tanks exposed for 6 weeks (n=6)), was reduced 40, 45, and 49% at 0.5, 5.0, and 50 μg/L, respectively (reviewer calculated). There were no significant differences between the control and treatment groups for mean per cent mortality (±SD) of embryos 24 hours after collection (38±18 in contro l vs. 44±23, 41±19, and 45±20 for the 0.5, 5.0 and 50 μg/L, respectively. It is noted th at the magnitude of the decrea sed fecundity (as cumulative egg production) was similar across the 100X fold in exposure (0.5-50 μg/L) and embryo mortality in the control group was relatively high (38% mortality for 24-hr old embyros).

No treatment-related effects were observed for the male endpoints of steroid hormone concentrations, E/T ratios, or GSI values. For testes stag ing, all fish at all time periods were found to be at stage III or IV. All control fish sampled at Days 14 and 38 were at stage IV. However, for the atrazine treatment groups, a ma ximum of one out of three sampled fish at Day 14 were at stage IV, and two of three sample d fish at Day 38 were at stage IV. The percent of abnormal mitotic events based on aberrant sp ermatogonia was significan tly increased (p<0.01) compared to control for all atrazine treatment groups (sampling points combined). Minimal
testicular lesions were observed for fish in each of the control and 0.5 μg/L groups (2/10), and minimal or moderate lesions were observed for fish in each of the 5.0 and 50 μg/L groups (10/12). Observed lesions consisted of ge rm cell syncytia and tissue degeneration.

Similarly, no effects were observed for the female end points of steroid hormone concentrations,

E/T ratios, GSI values, or the gonad and brain arom atase protein content at 14 or 38 days. There were significantly fewer (p<0.01) pre-vitellogenic oocytes and more vitell ogenic oocytes in the
low- and medium-concentration atra zine groups at Day 38 compared to control. No pathological
lesions were observed in the ovaries.

Fish Sexual development (Zebrafish)

In a published study by Corvi et al . (2012 ), the potential for chronic atrazine exposure to affect
zebrafish (D. rerio) sexual development and gonad differentiation was evaluated. Two fourATRAZINE FINAL

Page 126 of 159 month chronic exposure studies were conducte d in 2009 (Study I) and 2010 (Study II) which targeted sensitive gonadal developmental windows during sexual maturity.

Adult wild-type zebrafish were housed in poly carbonate tanks filled with fish water (reverseosmosis water containing 0.6% Instant Ocean) an d equipped with a recirc ulating system. Fish were group-spawned, and embryos were collected at 2 to 3 hours post-fertilization. Embryos were bleached, rinsed with water, and incubate d in petri dishes with 0.1% methylene blue until Day 5 post-fertilization (dpf). Next, embryos were raised in the recirculating fish water system until 17 dpf. At 17 dpf, a total of 400 fish were randomly transferred to either forty black polycarbonate tanks (Study I) or forty stainless steel tanks (Study II). Each tank was equipped with an oxygen bubbler to maintain optim um dissolved oxygen concentrations.

A 20 mg/L stock solution of atrazi ne (97.6% purity) in fish wate r was prepared every third day, and a 1 mg/mL 17 β -estradiol ethanol stock solution was d iluted in fish water to produce the working stock solution. A solvent (ethanol) cont rol was not used in this study as ethanol comprised only ≤ 0.00005% of the final 17 β -estradiol expo sure solution.

For chronic toxicity studies, ten zebrafish (17 dpf) were randomly distributed to each of forty tanks containing approximately 3 L (Study I) or 3.75 L (Study II) of water. Fish were continuously exposed for 113 days to atrazine at nominal concentrations of 0, 0.1, 1, and 10 μM (0, 21.6, 216, and 2160 μg/L) or 17 β -estradiol at 1 nM (0.27 μg/L ). Each treatment included eight replicate tanks, and solutio ns were freshly prepared and re newed every third day; control tanks contained untreated water only. Fish we re observed for mortality and any abnormalities daily over the 113-day exposure period. Dead fish were removed, and discarded or fixed for histological analysis. Fish were fed newly-hatched Artemia one to four times daily, and commercial flake adult zebrafish diet was suppl emented one to three times daily. In Study II, feedings were reduced mid-study to twice a day, and typically consisted of one Artemia feeding and one flake feeding. For Study II, from Day 90 to 108, viable embryos (6-78 hours postfertilization) were collected every third da y from all treatment tanks, examined with a microscope, and euthanized. The age, number, and quality of offspring were estimated and
recorded for each tank.

At study termination, all fish were euthaniz ed, and wet weight and total body length were
determined. Fish were fixed in Dietrich's fi xative for 60 hours under continuous agitation. Fish were transferred to buckets containing Dietrich's fixative with 5% trichlor oacetic acid and further agitated for 18 hours to decalcify bones. Finally, fi sh were rinsed and stored in 70% ethanol until histologic preparation. Concen trations of atrazine and 17 β -estradiol were determined every third day, during solution renewals, during the chronic t oxicity studies. Atrazine concentrations were determined with a commercial enzyme immuno assay (EIA) kit during Study I and with a liquid chromatography tandem mass spectroscopy (LC/ MS/MS) method during Study II. For both studies, 17 β -estradiol concentrations were determined with a commercial EIA kit.ATRAZINE FINAL

Page 127 of 159 After fixation, 130-dpf whole fish were bisected sagittally, processed overnight, embedded in paraffin, and cut into sections containing representative gonadal planes. Tissue sections were stained with hematoxylin and eosin for evaluatio n. Gonads were categorized as: female (100% ovarian tissue); male (100% testicular tissue); te sticular oocyte(s) (TO; phenotype: presence of one or two primary oocytes in a male fish, or presence of mixed sex gonad); undefined gonad (gonadal tissue could not be defi ned); or abnormal male [male fish with immature testicular development ( e.g. , the presence of spermato cytes but no spermatids), testicular degeneration,
reduced amount of testicular tissu e, or ectopic oocyte in a male fish]. Gonadal developmental abnormalities were defined as any deviation fr om mature male or female gonadal tissue.

In Study I, mean measured atrazine tr eatment concentrations were 0.088, 0.88, and 8.9 μ M (19, 190 and 1,920 μ g/L) in the nominal 0.1, 1, and 10 μ M (0, 21.6, 216, and 2160 μg/L) treatment groups, respectively, and in Study II, mean m easured concentrations were 0.096, 0.92, and 9.4

μ M (20.7, 198, and 2,027 μ g/L, respectively. For the 1 nM 17 β -estradiol treatment, meanmeasured concentrations were 0.72 nM for Study I and 0.32 nM for Study II; no explanation was provided for the decreased concentration in Stud y II. Atrazine was detected in some Study I control samples. It was detected at 0.00028-0.022 μ M (0.06 – 4.7 μ g/L) on seven sampling days, and at concentrations above the upper range of the calibration curve (>0.023 μΜ ; >5.0 μ g/L) on three sampling days. The detectable atrazine co ntamination was attributed to contaminated glassware, but the experiment was repeated in Study II. Atrazine was not detected above the lower limit of quantitation (LOQ=0.00023 μ M) in any control sample from Study II.

Mortality was low in both of the chronic toxicity studies, with no differences in percent survival in either study. In Study I, ove rall (across treatments and cont rols) weights and lengths were decreased compared to Study II wei ghts and lengths; the study aut hors attributed the differences to changes in feeding protocols and tank desi gn in Study II. Untreated female weights were higher compared to untreated male weights in both studies.

In Study I, statistically significant decreases (p<0.05) in body weights were observed in the medium- and high-concentration males ( ↓ 22 and 13%, respectively), and in high-concentration females ( ↓ 21%) relative to controls. Mean body weight s for males and females exposed to 17 β

estradiol or the low concentrati on of atrazine, and females exposed to the medium concentration of atrazine, were not different comp ared to control. Atrazine or 17 β -estradiol treatments did not affect body lengths of males or females in Study I.

In Study II, the mean body weight of males exposed to 17 β -estradiol ( ↑ 26%) was significantly increased (p<0.01); however, mean body weight of females exposed to 17 β -estradiol was similar
to control. S ignificantly decr eased (p<0.05) body weights were observed in the medium- and high-concentration males ( ↓ 11 and 15%, respectively), and in high-concentration females (p<0.05; ↓ 18%). Mean body weights for males and female s exposed to the low concentration of atrazine, and females exposed to the medium co ncentration of atrazine, were not differentATRAZINE FINAL

Page 128 of 159 compared to control. Body lengths if males and females in the high atrazine concentration were significantly decreased (p<0.01) compared to control ( ↓ 5% for both sexes). Body lengths of 17 β

estradiol-exposed males were increased (p<0.01) by 5%, and body lengths of females exposed to

17 β -estradiol were decrea sed (p<0.01) by 5%.

The percentage of females was significantly in creased (p<0.01; 77%) and the percentage of males was significantly decreased (p<0.01; 23%) co mpared to untreated female and male counts (46 and 54%, respectively) in fish exposed to 17 β -estradiol in Study I. Similarly, the percentage of females was significantly increased (p< 0.01; 86%) and the percen tage of males was significantly decreased (p<0.01; 14 %) compared to untreated female and male counts (59 and 41%, respectively) in fish exposed to 17 β -estradiol in Study II. Th e number (proportion) of males or females in the atrazine exposure gr oups was not different from their respective counterparts in unexposed fish tanks for eith er study at any treatment concentration.

In Study I, three fish in the 17 β -estradiol-exposed group and one fish in the 10 μM (2160 μg/L) atrazine exposed group could not be identified as male or female because gonadal tissue was not observed within any of the fifteen histologic secti ons prepared. The percentage of fish, in either treatment, with unidentifiable gonads was not differe nt from control in Study I. In Study II, fish exposed to 17 β -estradiol had a significantly increase d (p<0.01) number of abnormal males (12 fish) and fish with indistinguishable gonads (6 fi sh) compared to the control group (1 and 0 fish, respectively). Abnormal males were observed at a low frequency in atrazine treatment groups (1- 2 fish) in Study II, and no fish with undifferentia ted gonads were present in the Study 2 atrazine
treatment groups.

In Study I, examination of gonadal tissues across all treatments revealed five male fish with the testicular oocyte(s) (TO) phenot ype; all were primordial oocytes present in mature testes. The TO(s) were identified in the atrazine treatment groups (one, three, and one fish in the low-, medium-, and high-dose groups, respectively). Examination of gonadal tissues from Study II
across all treatments also found five fish with the TO(s) pheno type. Pre-vitellogenic oocytes
were observed in fish in the low- an d medium-dose atrazine groups and the 17 β -estradiol group. Two zebrafish in the low-dose group and one fish in the medium-dose group were identified with a single primary oocyte. Two TO fish were obser ved with mixed gonad feat ures including testesova and ova-testes in the 17 β -estradiol treatment group. There were no differences observed in
the incidence of TO for Study I or II fish when co mpared to untreated fish.

Limited spawning data were obtained from St udy II. Embryos (6-78 hours post-fertilization) were collected from treatment tanks from exposure Day 90 to 108. No viable embryos were observed in any of the 17 β -estradiol tanks. The age, number, and quality of offspring were estimated, and no statistical analyses were conduc ted on these data. The re sults only show that spawning occurred for atrazine-exposed and unex posed zebrafish in a similar manner. TheATRAZINE FINAL

Page 129 of 159 results of these investigations demonstrate that chronic atrazi ne exposure does not affect sex percentages or gonadal development in zebrafish.

In another published study by Plhalova et al . (2012 ), the potential for subchronic atrazine exposure to affect zebrafish (D. rerio) growth and development was examined. Juvenile (30-dayold) zebrafish were randomly distributed (40/tank ) into 30-L glass aquari ums; atrazine treatment groups were conducted in duplicate. Fish were exposed to atrazine (98.8% purity) at 0.3, 3.0, 30.0, or 90.0 μ g/L dissolved in water with DMSO for 28 days in a flow-through system. A negative control group (dilution water) and a solvent control group (DMSO, 0.01%) were included. The test solution volume was replaced twice daily and fish were fed dried Artemia
salina at 8% of body weight each day. Food amount s were based on initial fish weights and recalculated on Day 14 of treatment. During the experiment, tank conditions (mean water temperature 25±1°C, oxygen saturation >60%, an d pH 7.95-8.28) were checked every 24-hours,
and the number of dead fish was recorded for each treatment. Atrazine concentrations were determined by gas chromatography with ion trap tandem mass spectrometry. Fish were weighed initially and at study completion; food was withheld for 24 hours prior to weighing. After Day 28 of treatment, fish were euthanized with car bon dioxide, weighed, and the tank average growth rate was calculated (the slope of the line determin ed by the change in the averaged log of fish weights over time). Ten fish from each treatm ent group were used for histopathological examination of the gills, kidneys, and liver. Tissu es were fixed in buffered 10% neutral formalin and stained with haematoxylin and eosin; five tissue sections from each fish were examined microscopically.

Mortalities were reported to be ≤ 5% in the control and treatment groups over the course of the study (actual numbers were not provided). No ch anges in behavior were noted except for the

90.0 μ g/L treatment group, in which decreased food intake was observed compared to control; the fish reportedly only swam in the middle of the tank and showed no interest in food. There were no differences between the negative and solv ent control groups, but it was not stated which group was used in comparison to the treated groups.

Initial body weights were reported to be similar to control(s) in all treatment groups, but at study termination, body weights were significantly decreas ed (p<0.05) in fish in the high atrazine concentration group; body weight data were not provided. Growth rates al so were significantly decreased (p<0.05) in fish in the high-concentration group.

Histopathological examinations re vealed no pathological lesions in atrazine-exposed fish, except at the highest test concentra tion. In the high-concentration gr oup, moderate dystrophic lesions of the hepatocytes were observed, and there were signs of initial ce ll injury as indicated by the hydropic to vacuolar degeneration of the hepatocytes, the dila tation of the capillaries, and hyperaemia, compared to the negative control. All fish examined from the 90 μg/L group,ATRAZINE FINAL

Page 130 of 159 exhibited the same intensity of morphological changes in the liver throughout the organ. The study authors reported a NOEC of 30.0 μ g/L and a LOEC of 90.0 μ g/L.

Developmental Reproductive Toxicity in Reptiles (Alligator)

In an examination of steroidogenesis after in ovo treatment ( Crain et al. , 1997 ), the authors describe that alligator ( Alligator mississippiensis ) eggs were collected from seven nests on a
control lake at Lake Woodruff National Wildlife Re fuge, Florida. Eggs were transported to the laboratory and placed in an incubator at 30°C, an d one egg from each clutch was opened to stage the embryos. Five days after collection (priorto sex determination during the temperaturesensitive period), eggs were separated into two groups; half of the eggs from one clutch were incubated at 30°C (female-producing temper ature) and half at 33°C (male-producing temperature). Eggs were maintained at appr oximately 90% humidity with sphagnum moss as incubation material. Within each incubation gr oup, eggs from each clutch were distributed among various treatment groups, including atrazine (purity not reported) at doses of 0.14, 1.4, and 14 ppm. There were separate controls with ei ther water or 95% ethanol, used as a test substance solvent. A positive control group, 17 β -estradiol, also was in cluded with the same
treatment concentrations in addition to a lo w dose of 0.014 ppm. Treatments were applied topically to the eggshell in 50 μ L of 95% ethanol. The treatments were applied at stage 21 of embryonic development, which is the beginning of the critical period of gonadal differentiation. Upon pipping, egg chorioallantoic fluid (CAF) was collected and frozen at

− 72°C prior to analyses for steroid hormones. To tal protein content in the CAF samples was determined with a commercially available kit, and CAF steroid hormone concentrations were presented per microgram protein.

Hatchlings were individually hous ed for ten days prior to tissue collection, and s acrificed after the collection of blood from the dorsal postcranial sinus. The right gonadal-adrenal mesonephros (GAM) complex was immediately removed for th e aromatase bioassay. Aromatase activity was measured indirectly based on the release of tritium ( 3 H) from 1 β

3 H-androstenedione during aromatization of the substrate into estroge n. GAMs from three additional control female alligators were used to determine th e specificity of the aromatase assay.

Histology was conducted to determine histological sex in order to document induction of sex reversal. A complete histopatholog ical examination of the GAMs wa s not included in this report, but was conducted and reported separately ( Spiteri et al. , 1999 ).

There were no differences in results among cont rols with and without the solvent. The 17 β

estradiol and testosterone concen trations were determined in pl asma and CAF of all hatchlings that provided sufficient fluid volume. Radio-immuno assays (RIAs) for 17 β -estradiol and
testosterone were conducted. Cross- reactivities of th e testosterone antisera to other ligands were as follows: DHT, 44%; -1-testosterone, 41% ; -1-DHT, 18%; 5 -androstan-3ß,17ß-diol, 3%; 4- androsten-3ß,17 β -diol, 2.5%; -4-andro stenedione, 2%; 5 β -androstan-3 β ,17 β -diol, 1.5%;ATRAZINE FINAL

Page 131 of 159 estradiol, 0.5%; all other ligands <0.2%. Cross-reactivities of the 17 β -estradiol antisera to other ligands were as follows: estrone, 1.3%; estriol, 0.6%; 16-keto-estriol, 0.2%; all other ligands
<0.2%.

Results of the aromatase enzyme assay were e xpressed both as fmol/hr and pmol/g/hr. Four response variables were measured for the alligators treated in ovo : sex reversal, CAF hormone concentrations, hatchling plasma hormone con centrations, and GAM aromatase activity. Total (100%) sex reversal (male to fema le) was noted for all doses of 17 β -estradiol. Atrazine treatment
did not result in sex reversal. Atrazine treatment had no statis tically significant effects on any
parameter.

Spiteri et al. (1999) published histology and hepatic aromatase findings for a subset of the alligator eggs and treatment groups from the experiment reported in Crain et al. (1997).

Hatchlings were individually hous ed for ten days prior to tissue collection, and s acrificed after the collection of blood from the dorsal postc ranial sinus. The ri ght GAM complex was immediately removed for the aromatase bioassay ( Crain et al. , 1997 ). Histology was conducted to determine histological sex in order to doc ument induction of sex reversal. The left GAM complex was preserved in Bouin's fixative, serial sectioned at 7 μ m after paraffin embedding, and stained. Gonads were inspecte d and scored as testis or ovar y by two independent researchers. In brief, criteria for testis included reduced cortex and medullary sex cord diameter and proliferation, and criteria for ovaries include d hypertrophied cortex, medullary reduction, the presence of lacunae in the medulla, and germ ce lls in the cortex. In a ddition, both Mullerian duct epithelial cell height in the ovari es and the degree of degenerati on in the ovarian medulla were
measured.

There was no difference between control male and control female animals in hepatic aromatase activity. Atrazine treatment did not affect hepatic aromatase activity in males or females. In addition, none of the histological measurements were different in atrazine-treated animals compared to control. All animals incubated at 30°C had ovaries, and all alligators incubated at 33°C had testes. All eggs treated with 17 β -estradiol produced female hatchlings at the maleproducing temperature. Epithelial cell height of the Mullerian duct was significantly increased (p<0.001) in the high-concentration 17 β -estradiol treatment groups in both male and female temperature regimes. The regression of the ovari an medulla was not different. The study authors reported slight reductions (NS) in sex-cord diameter in the atrazine-treated males.

Developmental Reproductive Toxici ty in Reptiles (Water snake)

In an investigation of reproductive/developmental toxicity ( Neuman-Lee et al. , 2014 ), the effects of atrazine ingestion on w ild-caught Northern watersnakes ( N.sipedon ) and their offspring were examined with multiple endpoints.ATRAZINE FINAL

Page 132 of 159 A total of 25 female and 12 male watersnakes were collected from two lakes in Illinois (previous exposure history unknown). Each snake was hous ed in a 30×30×60 cm plastic cage under standard laboratory conditions. After sex verification, males were paired with females; males
were rotated to a new female after one week to increase the likelihood of insemination. Females were mated for 2-3 weeks prior to treatment.

Pregnant females were administered one of the following randomly select ed dietary atrazine (98.9% purity) treatments: 0 (deionized water with 95% ethanol), 2, 20, or 200 ppb. The diet consisted of live minnows ( Notropis sp.) that received intramus cular injections of 0.1 mL of atrazine solution per gram of fi sh. The amount of fish with each solution was mass-corrected for each snake on a weekly basis such that each snak e received the identical atrazine concentration. These concentrations were meant to represent potential environmental le vels of atrazine, not necessarily within the fi sh. After injection, the fish were im mediately offered to the respective snake for a period of not more than 1 hour to av oid any degradation of atrazine. In rare cases when the fish were not consumed, the mothers were force fed by coaxing the snake to swallow
the fish to ensure that each female was c ontinuously exposed to th e appropriate atrazine concentration throughout gestation. Food intake was standardized, w ith pregnant females fed 5% of their body mass twice weekly.

At approximately Week 12, mothers were observed for signs of parturitio n. Stillborn offspring were measured (weight and SVL), probed for sex determination, marked uniquely, and frozen at

− 20°C. Live born offspring were measured (wei ght and SVL), probed for sex determination, and photographed for identification by pattern. All neonates, regard less of their condition (born live/stillborn/removed from oviduct), were analyzed for effects on weight, SVL, and sex ratio. The proportion of live to dead offspring for each snake was recorded. Offspring from females that died before parturition were not incl uded in the analyses of stillborn neonates.

Blood samples (approximately 1 mL) were obtained from each pregnant female from the caudal vein on a weekly basis. Plasma was harvested and stored frozen at

− 20°C until further analysis. Plasma samples were prepared for enzyme li nked immunosorbent assay (ELISA) evaluation of

17 β -estradiol concentration after solid-phase extraction. A known concentration of 17 β -estradiol was extracted to determine the percent recovery.

Neonates were measured for asymmetry to asse ss possible aberrations in development. All offspring that had full scalation we re used in the analyses of thes e characteristics. The left and right dorsal scales exte nding from the mid-dorsal scales were counted at 25, 50, and 75% of the SVL for each snake using a dissecting microscope. After all offspring were measured, the count was repeated for each individual to ensure accuracy. Additionally, clefts in ventral scales were
counted for each individual because clefts in snakes are considered abnormal. Digital radiographs of the dorsal view of each offspri ng were produced to analyze left and right jawATRAZINE FINAL

Page 133 of 159 length. The distance from the anterior margin of the premaxilla to the posterior margin of the quadrate bone was recorded.

There were no treatment-related effects on weight or SVL for o ffspring. In addition, there was no
interaction between the number of stillborn neonates, sex, and treatm ent, and no treatmentrelated effect on mortality was observed in any treatment group. No effects were seen on maternal estradiol. However, atrazine-treated females displayed an increased incidence of
infection which led to death (2-3 treated vs. 1 control).

The proportions of live born offspring were decr eased in all atrazine treatment groups in a nondose-dependent manner compared to control. Atrazine treatment was also associated with effects on scale row symmetry at the mid-body, w ith more asymmetries occurring in offspring born to mothers in the medium-dose (p = 0.002). Neither of these effects showed a linear pattern of dose-response.

In Vitro Ovulation (Frog)

An in vitro investigation by Orton et al. (2009) examined the endocrine disrupting potential of
atrazine in a cultured X. laevis oocytes assay used to measure ovulatory response and ovarian steroidogenesis. Ovaries from sexually mature X. laevis were removed, cultured in Modified Barth’s Medium, and cut into ten oocyte fragme nts. The oocytes were incubated with 6.25, 12.5, 25, or 50 IU of human chorionic gonadotropin (h CG) or control media for 20 hours. The media was sampled and samples were stored frozen a

− 80°C. After the initial incubation, a submaximal concentration of hCG (to yield approximately 60% of maximum ovulatory response) was chosen for the co-incubation with atrazine and other pe sticides. Atrazine (>97% purity) was initially tested at concentrations of 6.25 and 62.5 μM (13,500 μg/L); due to effects at these concentrations, additional tests were conducted at 0.00625, 0.0625, and 0.625 μM (1.35, 13.5 and 135 μg/L). In addition, the 3 β -HSD inhibitor (epostane) wa s tested at 0.001, 0.01, 0.1, and 1 μM to verify inhibition of ovula tion by inhibition of steroid hormone synthesis. Incubations were conducted for 20 hours, and media sa mples were stored frozen at

− 80°C. Hormone analyses in the media samples ( i.e. , progesterone, testosterone, and 17 β -estradiol) were conducted by RIA
methods.

In the ovulation assay, nine pesticides, includ ing atrazine, had an effect on the ovulatory response or hormone production. At razine exposure was associated with apparent stimulatory (( ↑ 50% at 6.25 μM; p<0.05) and inhibitory ( ↓ 68% at 0.0625 μM; p<0.01) e ffects on ovulation at different exposure levels. In response to atrazi ne, progesterone levels were higher at 6.25 5 μM ( ↑ 63%) and at 62.5 μM ( ↑ 104%) (p<0.001). Testosterone leve ls were higher) at 6.25 μM ( ↑ 24%, p<0.01) but not statistically diffe rent at 62.5 μM. No effects on es trogen levels were detected.ATRAZINE FINAL

Page 134 of 159 In Vivo (Frog)

In an investigation of reproduc tive/metamorphosis in frogs by Oka et al . (2008) , the potential for
atrazine to affect sex differentiat ion were examined. Adult wild-type X. laevis were purchased for this study. All-ZZ (male) cohorts of X. laevis tadpoles were obtained by mating sex-reversed males (phenotypic female, genetic male) and wild -type males to produces all-male offspring. Frog colonies were kept in de-c hlorinated tap water that has b een left to stand for two days; residual chlorine analyses confirmed that no chlorine was present. Frogs were maintained at 20– 22°C on natural light and fed diet for X. laevis (XL-No. 3 pellets), which showed slight estrogenic activity (possible phytoestrogen contamination) in the yeast screening assay with human estrogen receptor.

Sex-reversed male and wild-typ e male were injected with 50 0 i.u. per 100 g body weight of gonadotropin. They were allowed to mate semi-natur ally in de-chlorinated tap water. Fertilized eggs were collected and maintained in 10-L portions of test medium consisting of 0.25 g/L commercial salt mixture (Tropic Marin Meersalz, pH 7.5) in de ionized water. The water was aerated and the temperature was adjusted to 22–23° C. This solution was also used as the test medium during treatment exposures. Tadpoles we re fed with Seramicron daily. The estrogenic activity of the Seramicron food had been tested with a yeast two-hybrid system and found to be free of estrogenic activity.

Wild-type X. laevis tadpoles were used in Experiment I and an all-ZZ male cohort of X. laevis tadpoles were used in Experiment II. Thirty ta dpoles were assigned to each glass tank, and tanks were filled with 10-L portions of aerated test medium at 20–22°C. The test medium was changed three times per week (semi-static condition), and a 12-hour light/dark cycle was maintained. In Experiment I, each test treatment was comp rised of duplicate (for atrazine exposure concentrations) or triplicate (cont rol group) test tanks. In Experi ment II, a single tank per test
treatment was used. Atrazi ne (97.4% purity) and 17 β -estradiol were dissol ved in 99.5% ethanol
and diluted to a final vehicle concentration of 0.01% in test medium. In both experiments, NF stage 49 tadpoles were prepared and used beca use previous studies have demonstrated that NF stages 49-56 are critical st ages for sex determination in X. laevis

.
In Experiment I, NF stage 49 wild-type X. laevis tadpoles were exposed to 0.1, 1, 10, and 100 ppb atrazine until all tadpoles completed metamor phosis (NF stage 66). In Experiment II, all
ZZ male X. laevis tadpoles were exposed to 0.1 and 1 ppb atrazine and 0.27 ppb (1 nM)

17 β -estradiol for a positive control was exposed fr om NF stages 49 to 66. In both experiments, control tadpoles were maintain ed in test medium containi ng the vehicle (0.01% ethanol). Developing larvae were staged ev ery week in Experiment I. In both experiments, froglets were collected upon completion of me tamorphosis (NF stage 66), and total length and time to metamorphosis of the tadpoles were recorde d. Any dead tadpoles found during exposure wereATRAZINE FINAL

Page 135 of 159 removed and recorded. Chemical analysis of at razine in the test medium was conducted with GC-MS according to the standard guidelines.

Froglets were collected at NF stage 66, an aesthetized, and sex was determined by gross morphology of the gonad with a dissecting microscope . All froglets were recorded as being male, female or hermaphrodite (gonad having characte ristics of both testes and ovaries). After recording of sex categories, the left side of gonad with kidney was removed from 16 (control), 20 (0.1 and 1 ppb atrazine), 22 (10 ppb atrazine), and 19 (100 ppb atra zine) froglets to examine the expression levels of P450 aromatase. The right gonad was used for histological analysis. In Experiment II, five froglets in each group we re examined for P450 aromatase induction by atrazine in the gonad with kidney.

Liver samples were taken for VTG analysis from 14 (control), 20 (0.1 and 100 ppb atrazine), 21 (1 ppb atrazine), and 22 (10 ppb atrazine) frogle ts in Experiment I and from 30 (control and atrazine groups) and 28 (17 β -estradiol group) frogle ts in Experiment II. The body of each froglet, including gonads, was fixed in Boui n’s fixative (after removal of the intestine), and preserved in 70% ethanol until histological analysis. Gonad samples and the kidney were removed from the body under the dissecting microscope and embedded in paraffin. Cross-se ctions were cut and stained with hematoxylin and eosin.

In Experiment I, gonads of male or female froglets in each group were analyzed for expression levels of P450 aromatase. Total RNA was isolated, followed by DNase I treatment. The cDNA was synthesized from purified total RNA, and PC R amplifications were conducted in triplicate. In Experiment II, five gonads of male or fe minized froglets from each treatment were used to examine the expression levels of aromatas e mRNA. Total RNA was isolated, followed by DNase I treatment. The cDNA was synthesized fro m purified total RNA, but PCR amplifications were conducted in a single assay.

The livers from control frogl ets and atrazine- or 17 β -estradiol-exposed froglets were removed and weighed. For VTG determinations, livers we re individually homoge nized, centrifuged, and the supernatants were collected and frozen at

− 70°C until analyzed with an ELISA assay. For the

in vitro VTG investigation, hepatocytes we re isolated from adult male X. laevis . Cells were
inoculated at 4×10 4 cells/well into 96-well tissue culture plates and incubated in air at 22°C. After incubation for 48 hours, the cells were expo sed to a test compound by addition of the test compound-containing medium. Atrazine and 17 β -estradiol were dissolved in DMSO at concentrations of 10,000 and 27.24 ppm, respectively, as stock solutions. Each solution was used after dilution with the medium at the following final concentrations for atrazine: 1000, 333, 111, 37, and 12 ppb and for 17 β -estradiol: 2.724 (10 nM), 0.681 (2.5 nM), 0.172 (0.63 nM) 0.044 (0.16 nM), 0.011 (0.04 nM), and 0.003 ppb (0.01 nM). Control cultures were exposed to DMSO at concentrations up to 0.01% (v /v) in the test medium. The culture media were renewed after three days by replacement of two-thirds of th e volume. Three days after medium renewal, theATRAZINE FINAL

Page 136 of 159 culture media were harvested into 96-well microtiter plates and concentrations of VTG in the culture media were determined by a sandwich ELISA method.

The survival rates in Experiment I were ≥ 90% in control tadpoles and in the 1 and 10 ppb atrazine-treatment groups, while low survival rates were noted in one tank each for the 0.1 ppb (73.3%) and 100 ppb (26.7%) atrazine-treatment groups. Since these groups showed high variability in survival between tanks of the same group, statistical analysis of sex ratio excluded these tanks. In Experiment II, ≥ 96% survival rates were obser ved in all groups. All tadpoles completed their metamorphosis between Days 35 and 56 after exposure, and time to completion of metamorphosis was similar among treatment groups. Neither acceleration nor inhibition of development related to atrazine treatment was observed up to 28 days of exposure ( i.e. , initiation of larval metamorphic climax, NF stage 58, characterized by emergence of forelimbs).

All froglets were classified as male or female by gross morphology at NF stage 66 in Experiment I; hermaphroditic gonads were not observed in any larvae exposed to atrazine at the
concentrations and conditions used in this st udy. The sex ratios (male:female) in the 0.1 and 1ppb atrazine treatment groups were not different from control; 10 and 100 ppb atrazine groups were 37.9:62.1%, and 28.0:72.0%, respectively, and differed (p<0.05) compared to control. Normal testis and ovaries were observed in frogl ets in each group, and sex ratios ascertained on the basis of gross morphology were co rroborated by histological analysis.

The all-(ZZ) male cohort of froglets exposed to 0.1 and 1 ppb atrazine, and control froglets, were classified as male, which had gonads displaying typical testis with seminiferous tubules and spermatogonia. There was no evidence of sex-revers al in any of the -male cohorts of larvae exposed to atrazine; in contrast, sex-reversal ( i.e. , female phenotype in genetically ZZ male froglets) was observed in almost all froglets exposed to 0.27 ppb 17 β -estradiol as positive control. Twenty-one feminized (female) frogs an d seven hermaphroditic frogs were confirmed in
the 17 β -estradiol group.

The expression level of P450 aromatase in gonad- kidney complexes was si gnificantly increased (p<0.05) in females compared to males in the control group and the 1-100 ppb atrazine treatment groups, with no difference in the 0.1 ppb group. The P450 aromatase mRNA was significantly induced (p<0.05) in the all-male ZZ froglets exposed to 0.27 ppb 17 β -estradiol.

There were no differences in VTG concentratio ns of NF stage 66 froglets between males and females in any experimental group including the control gr oup; therefore, VTG concentrations in both sexes were combined. In addition, there were no increases in hepatic VTG concentrations in any atrazine-exposed groups for wild-type (Exper iment I) and all-male cohorts (Experiment II). On the other hand, hepatic VTG was induced in the all-male ZZ frogl ets exposed to 0.27 ppb

17 β -estradiol. A concentration-dependent increase of VTG in culture me dia was observed after primary-cultured hepatocytes were exposed to 17 β -estradiol. However, no VTG was observed in hepatocyte cultures exposed to atrazine concentrations up to 1000 ppb, indicating no estrogenicATRAZINE FINAL

Page 137 of 159 activity of atrazine under the study conditions. Atrazine did not inhibit 17 β -estradiol-induced hepatocyte VTG synthesis after co-exposure with 17 β -estradiol in vitro

.
On Day 0, actual concentrations in the 0.1, 1, 10, and 100 ppb atrazi ne groups were 200% (0.2 ppb), 170% (1.7 ppb), 140% (14 ppb), and 120% (120 ppb) of the nominal concentrations, respectively. The concentrations after 2 days exposure were 100% (0.1 ppb), 170% (1.7 ppb), 170% (17 ppb), and 150% (150 ppb) of the nominal concentrations, respectively. Analyzed 17 β

estradiol concentratio ns were 84% of the nominal concentration.

Brodeur et al. (2013) examined the effects of exposure to atrazine at environmentally-relevant concentrations on the timing of metamorphosis and body size at metamorphosis in the common South American toad, Rhinella arenarum . This study is a follow-up of an earlier study ( Brodeur et al., 2009 ) which demonstrated a non-monotonic accel eration of the time to metamorphic climax (Gosner stage 42) and delayed tail resorption. Exposures examined in the current study were longer (beginning at stage 25 instead of stage 38) and the test concentrations were lowered
to be similar to concentrations detected in the environment.

Oocytes for this test were collected from w ild-caught adult toads (previous exposure history unknown), and fertilized in vitro with fresh sperm suspended in AMPHITOX solution (AS). The resulting embryos were maintained in AS at 20±2°C until reaching Gosner stage 25. Tadpoles were pooled from four different clutches, and twenty replicates were conducted for each concentration tested. In each rep licate, ten tadpoles (stage 25) were placed in AS with or without atrazine (98% purity). Tadpoles were exposed to atrazine at 0.1, 1, 10, 100 or 1000 μg/L. The atrazine was prepared in AS with acetone as a so lvent; the final acetone c oncentration in AS was 0.02% (v/v). The study also incl uded a control group exposed to AS only; a solvent control group exposed to AS containing 0.02% (v/v) ace tone; and a positive control group exposed to 100 μg/L of 17 β -estradiol. Test solutions were replaced entirely every 48 hours, and the temperature was maintained at 20±2°C. Dead tadpoles were removed and the Gosner stage and survival were evaluated every other day during solution renewal. Frogs completing metamorphosis were examined for malfor mations, and their body mass and SVL were measured.

Tadpole survival was decreased (significance not reported; ↓ 30%) in the high-concentration group (52.0±10.0%) compared to control (74.2±6.5%) and the pos itive control, with most mortalities occurring during the first 20 days of exposure. Due to overt toxicity at the high concentration, results from this group are not further discussed. At the lowest atrazine concentration tested (0.1 μg/L), there were no effects (NOEC) on timing of metamorphosis, body weight, or SVL. However, concentrations from 1-100 μg/L decreased the time to each stage of metamorphosis in at non-monotonic U-shaped con centration-response, with the greatest effect occurring at the 10 μg/L concentration. Compared to control, the m edian time required to reach stages 39 and 42 and to complete metamorphosis was decreased by 4.2, 4.3 and 4.7 days, respectively, at 10 μg/L, by 5.8, 8.0 and 8.3 days at 10 μg/L, and by 3.3, 4.0 and 4.0 days at 100ATRAZINE FINAL

Page 138 of 159 μg/L. The effect of 17 β -estradiol at 100 μg/L was simila r to the 1 and 100 μg/L atrazine concentrations, decreasing the median times to each stage by 2.9, 4.3 a nd 4.7 days, respectively. Atrazine at 1-100 μg/L and 17 β -estradiol at 100 μg/L had no eff ect on the intervals between each stage of development, suggesting that the a cceleration in development occurred primarily between stages 25 and 39.

A similar U-shaped response was noted for e ffects on body weight, SVL, and the ratio of BW/SVL. Compared to control, terminal body weight was significantly increased (p<0.05) by ~14% at 1 and 10 μg/L of atrazine, and SVL was increased by ~4-5%, with the 17 β -estradiol group having similar increases in body weight (18%) and SVL (5%). In addition, the body mass of the metamorphs at specific SVLs was signifi cantly increased by appr oximately 10% at at 1, and 10 μg/L atrazine and at 100 μg/L 17 β -estradiol, compared to control (p<0.05).

In another developmental frog study, Spolyarich et al. (2010) examined the effects of exposure to atrazine (98% purity) alone and in combination with othe r pesticides on the growth, development, and sex ratio of developing spotted marsh frogs ( Limnodynastes tasmaniensis ) in a series of seven experiments based on the Xenopus Metamorphosis Assay. Only the results from the three tests (Tests 1, 6 and 7) with atrazine alone are discussed further.

For all experiments, fertilized eggs were colle cted from the wild (previous exposure history unreported) and the resulting larv ae were reared in the laborator y in aquaria. Exposures were started at early limb bud emergence (Gosner stage 28) and continuing until th e majority of larvae underwent tail resorption (G osner stage 42). In Test 1, larvae were exposure to atrazine at concentrations of 0, 0.1, 1 or 3 μg/L for three weeks, with each treatment consisting of two replicates (10 tadpoles/replicate, n=20). For Tests 6 and 7, larvae were exposed to atrazine at concentrations of 0, 0.1, 3 or 30 μg/L for four we eks, with each treatment consisting of three replicates (10 tadpoles/replicate, n=30). Each replicate was cont ained in 10 L of water within 14.5-L glass aquaria fitted with a glass lid to lim it evaporation. Each aquarium was aerated and was on a 12 hour:12 hour light:dark cycle. Spiking took place afte r every water renewal after every third or fourth day of e xposure. Atrazine concentrations were analyzed weekly on the fourth day after water renewal. No atrazine was detected in the control aquaria, and the average recovery of atrazine from the treated aquaria was 121.5%. Sex ratios were initially based on gross gonadal morphology, and male gonads from Test s 1, 6, 7 (atrazine only) were examined histologically for the presence of oocytes. Body le ngths were measured from the snout to the tip
of the tail.

Mortality was limited (2.3%; 18/800 animals) ac ross all experiments, and occurred mainly during the first week of exposure and was attr ibuted to handling st ress. Atrazine had no observable effect on the development of the spotte d marsh frog larvae at concentrations up to 30 μg/L. Atrazine treatments varied from the c ontrol by no more than one or two stages at any time period, with no differences between treated and control groups in any test. In addition,ATRAZINE FINAL

Page 139 of 159 atrazine did not have an effect on total body length at the concentrations tested. The sex ratios (male:female) were 50.3:49.7 for the combined controls. Female dominated ratios were
observed at 1 and 3 μ g/L atrazine treatments in Test 1, but the authors no ted that this trend was not strong in the 3 μ g/L atrazine treatment in Tests 6 and 7. No changes in male gonadal histology were noted after atrazine exposure, with the exception of two ma les in one experiment.
Testicular ovarian follicles were observed in one male at Gosner stage 44 after 4 weeks of exposure to atrazine at 3 μ g/L, with the testes of this animal described as "ambiguously" shaped. Another male at Gosner stage 41 had an enlarged area of the testes in th e same treatment group.

In Choung et al. 2011 , a pilot study was conducted in which the study authors stated that there was no significant difference in development rate between control and a 25 μg/L atrazine (>98%) treatment; however, the control tadpoles deve loped more quickly than the treatment with the combined pesticides. For the defi nitive test, gosner stage 26 tadpoles ( ca . 6 weeks post hatch; obtained from laboratory-reared sp awns) were exposed to a 25 μg/L atrazine concentration along with a blank (filtered, ag ed Sydney Australia ta p water) control and a mixture of 25 and 10 μg/L atrazine and te rbufos sulfone, respec tively, for 10 weeks to encompass complete metamorphosis completely. Th ere were three replicates (glass aquariums) for each treatment with 10 tadpoles in each replicat e (10L solutions); test vessels aerated and maintained at 25oC under a 12:12 light:dark peri od. Test solutions renewed twice weekly along
with measured test concentra tions (duplicate samples) and en vironmental quality (dissolved oxygen, electrical conductivity, pH, and temp erature). Each week each tadpole was photographed to examine visual inspection of physical abnormalities and measure snout-vent length (SVL) and tadpole stage. When tadpoles reached stage 42, they were transferred to another test vessel containing small amount of test solution until tail resorpbtion was complete at which time SVL and weight were measured. Th e results presented will focus on the definitive
test. There were three mortalitie s overall, one in terbufos and two in combined, with all other tadpoles completing metamorphosis. There was no si gnificant difference in survival, SVL, mass, or metamorphosis between the control and atrazine-only group.

In Weber et al. 2013 , evaluation of trascriptomic and protei n alterations in zebrafish embyros was examined after exposure to atrazine (t echnical grade) at 0.3, 3 or 30 ppb from 1-72 hours post fertilization (3 replicates per treatment; 50 embryos per replicate); dilution water control used. Culture zebrafish ( Danio rerio , wild-type AB stra in; source unspecified) were maintained at 28 °C with a pH of 7.2 and a 14:10 hour light :dark cycle. Eye diameter, head length, and total larval length were measured for 20 larvae from each treatment using light microscopy (however, it appears pseduoreplication was used in statistical analysis, so re sults not included here). Results showed alterations in gene expression at all at razine treatment groups compared to control (21, 62, and 64 genes in the 0.3, 3, and 30 ppb, respectively ). The altered genes were associated with neuroendocrine and reproductive system developmen t, function, and disease; cell cycle control; and carcinogenesis. Two of these genes (CYP17A 1 and SAMHD1) found to be altered at all three exposure concentrations.ATRAZINE FINAL

Page 140 of 159 Tumor Development (Rat)

In a special study by Fukamachi et al. (2004 ), the potential modifyi ng effects of atrazine on 7,12-dimethylbenz[ a ]anthracene (DMBA)-induced mammary and skin carcinogenesis was examined. This investigation used a rat line carrying copies of the human c-Ha- ras protooncogene with its own promoter region, whic h is highly susceptible to DMBA-induced carcinogenesis.

Human c-Ha- ras proto-oncogene transgenic (Tg) and non- Tg rats at 7 weeks of age were maintained in plastic cages at 22±2°C and 55±10% humidity. The animals were allowed access to pellet basal diet (iso flavone-free) and tap water ad libitum . A total of 276 female and male Tg and non-Tg rats (10/sex/treatment group) received a single dose of DMBA (25 mg/kg) by gavage at 50 days of age. On the following day, rats were placed on powdered basal diet containing atrazine (purity not reported) at nominal concentrations of 0, 5, 50, 500 ppm (mean-measured doses of 0.55/0.26, 6.19/2.78, and 36.4/24.2 mg/kg/day in females/males, respectively; calculated by reviewer). A non-Tg group of male and female rats were exposed (in addition to Tg rats) to 0, 10, 25, 100, or 250 ppm nonylphenol in a second experiment; it was not clear if atrazine was administered to non-Tg rats. Palp ation and observation were regularly conducted to monitor the development of mammary and skin tumors after DMBA treatment. The surviving animals were sacrificed by exsanguination under et her anesthesia at th e end of Week 8 for Tg females and Week 20 for non-Tg females a nd both groups of males. Numbers of grossly visible tumors were recorded before they were measured and sampled for histological examination. Values are expressed as average tu mor volume of total tumors per rat. Body, liver, kidney, ovary, uterus, and testis weights also were recorded. Visible mammary tumors were excised and immediately fixed in ice-cold acetone, trimmed, and embedded in paraffin for histopathological examination.

Two deaths in Tg rats (sex and dose group not id entified) occurred prior to study termination that
were considered unrelated to atrazine treatmen t. Final mean body weight in female rats fed 500 ppm atrazine was significan tly decreased (p<0.01; ↓ 14%) compared to control. There were no
consistent differences noted in th e weights of liver, kidneys, ovari es/testes, and uterus. The daily food intakes of groups fed the 5 and 50 ppm atrazi ne-treated diet were significantly increased (p<0.05) in both females and males. In contrast, food intake in the females fed the 500-ppm atrazine diet was significantly decreased (p<0.05; ↓ 10%).

In female Tg rats, palpable mammary tumors we re observed at four week s post-administration of DMBA. Incidences increased rapidly after Week 4, especially in rats fed the 5- or 50-ppm doses. In Tg males, palpable mammary tumors were firstly observed at Week 11 and the incidence increased with time, apparently independent of atrazine treatment. The appearance of a singleATRAZINE FINAL

Page 141 of 159 tumor in the mammary gland region (a sarcoma) at 20 weeks in the non-Tg male rat fed 5 ppm atrazine was reported in the te xt, but no summary tabular data was included for a atrazinetreated, non-Tg group. In female Tg rats, the ad enoma and adenocarcinoma incidences for rats fed 5 ppm atrazine were signi ficantly increased (p<0.01 and p<0.05, respectively) compared to the Tg control ( ↑ 50 and 50%, respectively). In addition, th e adenocarcinoma incidence of the 50 ppm-treated female rats also was significantly increased (p<0.05; ↑ 50%), with a tendency for increase (NS) in the 500 ppm treatment group ( ↑ 36%). No differences in mammary tumor incidences were noted in male Tg rats. In cont rast, skin tumors (squamous cell papillomas and carcinomas) were induced only in males. Initial a ppearances were at Week 10 in a control male, Week 11 in Tg rats fed 5 or 50 ppm atrazine, a nd Week 15 in Tg rats fe d 500 ppm atrazine; all incidences increased with time. Skin tumor incidence at Weeks 15/16 was significantly decreased (p<0.05) in the high-dose male Tg rats.

Mechanistic Study: Male Steroidogenesis (Rat)

In a mechanistic study by Pogrmic et al. (2009 ) examining steroidogenesis gene expression in Leydig cells, atrazine (98% purity) was administered to peripubert al male Wistar rats by oral gavage at doses of 0 (olive oil), 50, or 200 mg/kg/day from PNDs 23 to 50. Animals were
sacrificed 24 hours after the final dose was ad ministered. Body weights were determined on the first day of dosing and every other day through P ND 51 (prior to sacrifice). Organ weights were determined for the testis, seminal vesicle, ve ntral prostate, dorsal pr ostate, and the adrenal glands.

Isolation and purification of Leydig cells was conducted, and the proportion of Leydig cells present in culture was determined by staining for 3 β HSD activity (typically >95%). Purified Leydig cells obtained indi vidually from four to eight rats were pooled, and each pool/group was cultured in three to eight replicat es per experiment. The cells were allowed three hours to attach to 96-well plates (50,000 cells/0.2 mL/well), followed by stimulation for two hours with M199 medium containing different concentrations of hCG or different ster oid substrates: 22-OH cholesterol, pregnenolone, progesterone, or D4-androstenedione. Additionally, in each experiment, purified Leydig cells were plated in 50-mm Petri dishes (2.5×10 6 cells/2-mL culture medium/dish) and analyzed for androgen and progesterone concentrations in the medium, as well as the RNA transcript concentrations by real time PCR. The cells were cultured in M199-0.1% bovine serum albumin (BSA) supplemented with sa turated dose (10 ng/mL) of hCG. Purified Leydig cells were stimulated with different c oncentrations of 22-OH c holesterol (1 and 50 μM) for two hours, and the cells were plated in 50-mm Petri dishes (1×10 6 cells/2-mL culture medium/dish) and progesterone and androgen con centrations were determined in the medium. After the treatment, cell-free media were collected and stored at

− 80°C prior to measurement of cyclic-adenosine monophosphate (cAMP), andr ogen, and progesterone concentrations; cell lysates were used for real-time PCR analysis.ATRAZINE FINAL

Page 142 of 159 Serum androgen and progesterone concentrations in the incubation medium were determined by RIA. Each experiment was run in a single assay. Because the anti-testosterone serum used in the assay had a high cross-reactivity with DHT, assay values are referred to as testosterone + DHT concentrations (T + DHT). Progesterone and LH concentrations also were determined by separate RIA methods, and samples from all ex periments were run in one assay/hormone. The
amounts of cAMP secreted into the culture medium were m easured by a cAMP EIA kit.

Total RNAs from purified rat Leydig cells we re isolated with the RNAqueous 4PCR kit. Concentrations of RNA, measured by abso rbance at 260 nm, and purity estimated by the 260/280-nm absorbance ratio, were determined sp ectrophotometrically. After DNase-1 treatment to eliminate residual genomic DNA, up to 2 μg of the total RNA from each sample were reverse transcribed into cDNA in a 20-μl reaction mixt ure containing random primers and MultiScribe reverse transcriptase. Reverse transcription was estimated at 25°C for 10 minutes, 37°C for
120 minutes and after 85°C for 5 seconds in th e Veriti Thermal Cycler. RT-PCR was conducted with the 7900HT Fast RT-PCR system. The SYBR Green PCR Master Mix reagent kits were used for quantification of gene expression. Rat specific primers were designed for the relevant genes: LHR, SR-B1, StAR, TSPO, SF-1, PDE4B, 3 β HSD, CYP17A1, and 17 β HSD.

Mean body weights in atrazine-tre ated rats were decreased in the high-dose group beginning on PNDs 27-29 (p<0.05), with further decrea ses on PNDs 31-35 (p<0.01) and PNDs 37-49 (p<0.001). Mean terminal body weight in the hi gh-dose group remained significantly decreased (p<0.01) 24 hours after the final dos e. Atrazine treatment also re sulted in decreases in the androgen-dependent organ weights. Testis and ventral prostate weights were significantly decreased (p<0.05) in the low-dose group ( ↓ 8 and 22%, respectively) a nd significantly decreased (p<0.01) in the high-dose group ( ↓ 10 and 34%, respectively). Semina l vesicle and dorsal prostate weights were decreased (p<0.001 and NS, respectively) in the low-dose group ( ↓ 30 and 13%, respectively) and significantly decrea sed (p<0.001) in the high-dose group ( ↓ 60 and 37%, respectively). In addition, adrenal gland weight s were significantly increased (p<0.01 and 0.001, respectively) after atrazine treatmen t in the low- and high-dose groups ( ↑ 57 and 94%, respectively).

The serum androgen (T + DHT) and LH concentr ations were determined in samples taken
24 hours after the final administration of atrazi ne. No difference was observed in serum LH
concentrations after atrazine tr eatment, but mean serum T + DHT concentration was significantly decreased (p<0.001) in the high-dose group.

In Leydig cells obtained from atrazine-treated rats, the basal and hCG-stimulated (10 ng/mL) androgen (T + HDT) production was significantly decreased (p<0.001) in a dose-dependent manner. Furthermore, hCG-stimulated androgen (T + HDT) production in Leydig cells from the high-dose animals was shown to be significan tly decreased (p<0.001) in a dose-dependent manner across a range of hCG (0.25-20 ng/well); si milar results generally were noted for LeydigATRAZINE FINAL

Page 143 of 159 cells from the low-dose animals. Progesteron e production by Leydig cells obtained from
atrazine-treated rats was una ltered under basal co nditions, but was significantly decreased (p<0.05) in the presence of saturated dose of hCG in the high-dose group. In parallel with inhibited androgen production, at razine treatment resulted in significant dose-dependent decreases (p<0.001) in extracellu lar cAMP concentrations under both basal and hCG-stimulated
conditions.

The inhibition of T + DHT production in Leydig cells isolated from atrazine-treated animals was examined in the presence of 22-OH chol esterol, pregnenolon e, progesterone, and Δ 4

- androstenedione at substrate concentrations of 0.5-50 μM. The greatest inhibition was expressed in the presence of 22-OH cholesterol, and the least in the presence of Δ 4 -androstenedione as a
substrate.

The transcript levels of the ma jority of the genes examined ( i.e. , LHR, SR-B1, StAR, SF-1, PDE4B, CYP17A1, and 17 β HSD) were significantly decreas ed (p<0.05) in a dose-dependent manner by in vivo atrazine treatment. TSPO, a translocater protein, was decreased in the highdose group only, and the expres sion of the gene for 3 β HSD, the enzyme that catalyzes the conversion of pregnenolone to progesterone, was not affected by atrazine treatment.

In further investigations by Pogrmic et al. (2010 ) examining steroidogenesis gene expression in Leydig cells ( ex vivo and in vitro ), atrazine (98% purity) was administered to peripubertal male Wistar rats by oral gavage at a dose of 200 mg/k g/day for one, three, or six days (initial dose on PND 45, 48, or 50). Animals were sacrificed on PND 51, 24 hours after the final dose was
administered. For the in vitro investigations, untreated rats were sacrificed on PND 51. Leydig cells were isolated and purified fr om all rats as described above.

For in vitro experiments, Leydig cells were pooled, allowed three hours to attach (1×10 6 cells/2 mL culture medium/dish), and treate d for 24 hours with atrazine (0.001, 1, 10, 20, or 50 μM) in DMSO. Culture medium was collected at the end of the incuba tion period, and cells
were stimulated with different concen trations of hCG, progesterone, or Δ 4 -androstenedione for two hours. After treatment, cell-free media were co llected and stored at

− 70°C prior to determination of cAMP, androgen, and progesterone concentrations. Cell lysates were used for real-time PCR analyses. Cells also were incubate d in the presence of atrazine (232 μM) in the presence or absence of hCG (0.25 ng/mL and 10 ng/mL culture medium). The conversion of progesterone to testosterone, which refl ected the activity of CYP17A1 and/or 17 β HSD, also was measured. Additionally, the conversion of Δ 4 -androstenedione to testos terone, which reflected the activity of 17 β HSD, was measured. Leydig cells were exposed to atrazine for 24 hours and challenged with progesterone or Δ 4 -androstenedione in a concen tration-dependent manner.

For each in vivo experiment, Leydig cells obtained individu ally from four to five rats were pooled, and each pool/group was cultured in four to eight replicates per experiment. Cells (2.5×10 6 cells/2 mL culture medium/dish) were allo wed to attach for three hours and cultured inATRAZINE FINAL

Page 144 of 159 M199-0.1% BSA without or with a saturated dose of hCG (1 0 ng/mL) for 2 hours. Androgen, progesterone, and cAMP concentration analyses , and quantitative RT-PCR analyses were as
described in Pogrmic et al. (2009 ).

Atrazine treatment significantly increased (<0. 05) basal cAMP accumulation in medium during 24-hour incubation with 1 and 20 μM concentrati ons. In addition, the high concentration of atrazine (20 μM) significantly increased (p<0.05) hCG-stimul ated cAMP accumulation during a
further 2-hour incubation.

In parallel experiments, the c oncentration-dependent effects ofatrazine on basal and hCGstimulated steroidogenesis were investigated. Basal progesterone producti on was bi-directionally affected by atrazine; progestero ne concentration was significan tly increased (p<0.05) up to 41% at 1 μM atrazine, and significantly decreased (p<0.05) by 22% at 50 μM atrazine. In contrast, basal androgen production was si gnificantly increased (p<0.05) by atrazine in a concentrationdependent manner (up to 98% increase at 50 μM (11 ppm)). Atrazine treatment in the presence of hCG (0.125-10 ng/mL) demonstrated similar effects. Progesterone concentrations were decreased (p<0.05) at the highe r atrazine concentrations ( 20 and 50 μM), and significant concentration-dependent increase s (p<0.05) were observed in T + HDT concentrations at these
same atrazine doses.

The effects of atrazine (232 μM; 50 ppm) on Leydig cell androgenesis during a 3-hour incubation period demonstrated that the stim ulatory effect of atrazine on Leydig cell androgenesis is hCG-concentration dependent. In the presence of 0.25 ng/mL hCG, atrazine significantly increased (p<0.05) androgen production by 126-142%. In contrast, in the presence of 10 ng/mL hCG, atrazine-stimulated andr ogen production generally was unchanged 10-34%.

Expression of mRNA transcripts for genes invo lved in luteinizing hormone receptor (LHR) action, cholesterol transport, and steroidogenesis were affected by in vitro exposure to atrazine for 24 hours. The steriodogenic factor (SF)-1 expression increased 141 and 191% after exposure to 1 and 20 μM of atrazine, respectively. The high concentration of atrazine also increased the expression of StAR ( ↑ 162%), CYP17A1 ( ↑ 247%), and 17 β HSD genes ( ↑ 153%). In contrast, the transcript levels for LHR, SR-B1, TSPO, 3 β HSD, PDE4B, and CYP19A 1were not affected by
atrazine treatment.

Conversion of progesterone to testosterone was significantly increased (p<0.05) when Leydig cells were challenged with high er concentrations of atrazine (20 and 50 μM; 4.3 and 11 ppm) for 24 hours and incubated in the presence of progester one and atrazine for an additional two hours. The increased conversion of progesterone to testosterone in the presence of these two
concentrations of atrazine was si milar regardless of con centrations of progesterone or atrazine (average ↑ 227%). Conversion of Δ 4 -androstenedione to testosterone was also stimulated in the presence of atrazine. Androge n concentration was increase d in the presence of 0.25 μM Δ 4

- androstenedione at all concentr ations of atrazine except 0.001 μM; higher concentrations of Δ 4

-ATRAZINE FINAL

Page 145 of 159 androstenedione (0.5 and 10 μM) increased (p<0.0 5) androgen conversion only in the presence of 20 μM atrazine.

The results of the ex vivo studies demonstrated si gnificantly increased (p< 0.05) concentrations of
cAMP and T + HDT under both basal and hCG-stim ulated conditions after one, three, and five days of atrazine treatment in vivo . These experiments revealed sim ilar pattern of changes in basal
and hCG-stimulated cAMP accumulation and an drogen production: increases after single injection, peak amplitude in response after the three-day treatment, and reduced responses after the six-day treatment. These results indicate that atrazine has a transi ent stimulatory action on cAMP signaling pathway in Leydig cells, leading to facilita ted androgenesis.

The relative roles of adrenal and testis hormones af ter intact or castrated male rats were exposed to atrazine with and without androg en maintenance were investigated by Riffle et al. (2014 ). Male Wistar rats were provided water and commercial rat chow ad libitum . Rats were ranked by body weight and assigned to five treatment gr oups, and each treatment group was subdivided into castrated or intact groups (10 castrated and 10 intact per treatment group). The rats were castrated 12 days prior to initiation of treatment. Rats were ad ministered a daily oral gavage dose of atrazine (97.1% purity) at 0 (1% methyl ce llulose in water), 5, 25, 75, or 200 mg/kg/day for three days. The rats were dosed with the ve hicle or the test chem ical between 0700 and 0900 hours, and sacrificed by decap itation 30 minutes after dosi ng on Day 3. Body weights were recorded daily.

At sacrifice, trunk blood was co llected and processed to isolate serum and plasma; the portions
were stored at

− 80°C prior to analysis. Adrenal wei ghts were recorded at necropsy. The androstenedione, corticosterone, progesterone, test osterone, estrone, and es tradiol concentrations
in the serum were determined with commercial radioimmunoassay kits. The LH concentrations
were determined with a di ssociation-enhanced lanthanide fluorometric immunoassay.

At necropsy, the adrenal and testes were removed from each rat and placed into RNAlater.
Adrenal tissues were stored at

− 20°C until protein was isolated with a commercial kit. Samples
were then stored at

− 80°C until extraction. Testes extracts were prepared by homogenizing samples in Tris buffer containing EDTA, 3-[(3-cholamidopropyl)-d imethylammonio]-1- propanesulfonate, n-octyl- β -glucopyranoside, and phenylmethyl sulfonyl fluoride. The testes extracts were assayed for protein, and analyzed quantitatively with two-dimensional sodium dodecyl sulfate polyacrylamide ge l electrophoresis (2D SDS-PAGE). The proteins for which expression was significantly alte red by treatment or significantly correlated to testosterone production were isolated from the 2D gels and identified with ma trix-assisted laser desorption/ionization coupled mass spectrometry and commercial proteomics and rat species protein database software. The prot eins that were altered with at razine exposure or correlated with testosterone production also were subjected to pathway analysis. Western blot assays were
conducted for the one testicular and three adrenal proteins identified as predictive. As part of theATRAZINE FINAL

Page 146 of 159 Western blot procedure, the proteins were subj ected to electrophoresis under standard conditions
and transferred onto nitrocellulose membranes. The nitrocellulose membranes were then incubated with both primary and secondary an tibodies and washed, and the membranes were scanned with an infrared imaging system.

The mean body weights were similar among all treat ment groups within each intact or castrated category. However, the overall mean body weights we re decreased (significa nce not reported) in the castrated groups (339.0 g) compared to th e intact groups (370.4 g). After three days of treatment in both groups, weight losses (significa nce not reported) were observed in the intact and castrate 75 mg/kg (1.3% and 2.4% loss, resp ectively) and 200 mg/kg (4.3% and 5.4% loss, respectively) groups. No differences in adrenal weights were observed fo r intact or castrated
treatment groups.

Serum progesterone concentrations in intact rats were significantly increased (p<0.05) by approximately 290 and 250% in the medium-low and high-dose groups, respectively. Serum progesterone concentrations in castrated ra ts were significantly increased (p<0.05) by approximately 200, 360, and 640% in the medium-low, medium-high, and high-dose groups, respectively. Serum corticosterone concentrations in castrated rats were significantly increased (p<0.05) by approximately 370, 460, and 960% in the medium-low, medium-high, and high-dose groups, respectively, and serum corticosterone concen trations in intact rats were significantly increased (p<0.05) by approximately 370, 550, a nd 1160% in the medium-low, medium-high, and high-dose groups, respectively. Increased (NS) serum corticosterone concentrations were reported for the low-dose intact rats and castrat ed rats by 180 and 260%, respectively. The serum progesterone and corticosterone co ncentrations were significantly ( not reported) correlated with
one another in both the in tact and castrated rats.

The serum LH, androsenedione, a nd testosterone concentrations for intact rats were each observed with a similar non-monotonic dose-respons e. Significant increases (p<0.05) in LH and
testosterone concentrations we re observed in the medium-low dose group (2.9-fold increases).
The androstenedione concentrations followed the sa me pattern as LH and te stosterone in intact males, with the highest (NS) concentrations observed in the medium-low dose group. Mediumhigh and high-dose concentrations were decreas ed relative to the medi um-low dose group, with high-dose concentrations generally similar to cont rol. The LH concentrations were increased in the castrated males compared to the intact males, and were similar across all atrazine treatment groups. The androstenedione and te stosterone concentrations acro ss all atrazine treatment groups
were below the limit of detection in the castr ated rats. Concentrati on data for estrone and estradiol were not reported.

In the adrenals of intact rats, the expression s of six different protei ns were significantly decreased (p<0.05) as a result of atrazine exposure. The expr ession of one protein, PGRMC1,
was correlated in the intact ra ts with the non-monotonic increase in serum testosterone and theATRAZINE FINAL

Page 147 of 159 expression of another protein, HSPD1. In the adrena ls of castrated rats, the expression of one protein, HSPD1, was correlated with the serum pr ogesterone and corticoste rone concentrations. The expression of PGRMC1 was correlated to the HSPD1 expression levels in the castrated rats.
The correlations between HSPD1 an d PGRMC1 were inversely related in the intact compared to
castrated rats. In the intact rats, the HSPD1 expression was down-regulated, and the PGRMC1 expression was up-regulated. In the castrated ra ts, the HSPD1 expression was up-regulated and the PGRMC1 expression was down-regulated. Western blot analyses confirmed the upregulation of PGRMCl and the dow n-regulation of HSPD1 and HSPA8 in the adrenal from intact atrazine exposed male rats, and the up-regulation of the expression of HSPDl in the adrenal from castrated male rats. The expressions of six di fferent proteins were correlated with the nonmonotonic increase in serum testoste rone concentrations in male rats exposed to atrazine. The
non-monotonic increase in ATP5B in the testis of intact males was confirmed by western blotting.

Mechanistic Study: Male Steroidogenesis (Mouse)

In a published study by Jin et al . (2013) , the effects of atrazine on hormone synthesis and the transcription of steroidogenic genes was investigated in male peripubertal mice. Groups of
four-week old ICR mice were administered atrazi ne (>97% purity; vehicl e not described) at doses of 0, 50, 100, or 200 mg/kg/day for three week s. On the last day of treatment, all mice were euthanized, and serum, liver, and te stes were collected. Testosterone and 17 β -estradiol concentrations and aromatase activity were dete rmined. Total RNA was isolated from the liver and testes, and the expression of 14 steroidogenic genes was determined. All data was presented graphically in this study.

No clinical signs of toxicity were observed during treatment. Body weights were decreased (p<0.05) in the high-dose group (200 mg/kg/day) at all of the ex amined time points. Absolute liver weights were significantly decreased (p<0 .05) in a dose-dependent manner in all treated groups. However, relative (to body) liver weights we re similar to control. The absolute testis weights of the low- and high-dose groups were significantly decreased (p<0 05) compared to control; relative testis weights of the medium- and high-dose groups were significantly increased (p<0.05).

Serum testosterone concentrations were signif icantly decreased (p<0.05) in all treated groups.

Serum 17 β -estradiol concentrations in creased in all treated groups in a dose-dependent manner, and were significantly increased (p<0.05) in the medium- and high-dose groups. Similarly, the

E 2 /T ratio was increased in all treated groups in a dos e-dependent manner, and was significantly increased (p<0.05) in the medium - and high-dose groups, indicating that oral atrazine exposure during puberty affected the serum hormone homeostasis in male mice. Aromatase activity also was significantly increased (p<0.05) in the medium- and high-dose groups.ATRAZINE FINAL

Page 148 of 159 Atrazine treatment had no effect on AR, ER α , or ER β mRNA concentrations in the testes. In addition, mRNA concentrati ons of HMG-CoA synthase in both the livers and testes were not affected by atrazine; however, hepatic mRNA concentrations of HMG-CoA reductase significantly increased (p<0.05) after at razine treatment at 100 or 200 mg/kg/day. The mRNA concentrations of SR-B1, LDL-R, and StAR in the testes also were not altered by atrazine treatment. In contrast, mRNA concentrations of PBR were significantly decreased (p<0.05) in the high-dose group, and the mRNA concentrations of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) in th e testes were significantly de creased (p<0.05) by 33% and 37% after exposure in the medium- and high-dose gr oups, respectively. No differences in the
testicular mRNA concentrations of 3 β -HSD were detected in any of the treatment groups, indicating that atrazine treatment did not affect the conversion of pregnenolone to progesterone
in male mice. In the case of P450 17 α , the testicular mRNA concentrations were significantly decreased (p<0.05) in treated groups, with decrea ses of 38%, 48%, and 30% after treatment with 50, 100 and 200 mg/kg/day, respectively. Testicular 17 β -HSD mRNA concentrations were significantly decreased (p<0.05) by approxi mately 50% in all treatment groups.

Mechanistic Study: Female Reproductive Hormones (Rat)

In a mechanistic study of female reproductive hormones ( Foradori et al. , 2009 ), the effect of atrazine on the estrogen-/progester one-induced LH surge in ovariect omized rats was examined to
determine if LH inhibition was concomitant with decreased gonadotr ophin-releasing hormone (GnRH) neuronal activation.

Female adult (60-90 days old) enhanced green fluorescent protein (eG FP)-GnRH Wistar rats (expressing eGFP driven by the GnRH promoter ) were individually housed and provided food
and water ad libitum . Rats were ovariectomized, and treatments were initiated within one week after surgery. In Experiment 1, ovariectomized rats (5-8/group) were orally dosed between 1000 and 1100 hours by gavage with atrazine (purity not reported) in 1% CMC sodium salt at 0 (vehicle), 50, 100, or 200 mg/kg/day for four consecutive days. Rats were injected with 40 μg/kg 17 β -estradiol-3-benzoate (EB) in safflower oil ( s.c., between 1000 and 1100 hours) for three consecutive days to induce a LH surge be ginning on Day 2. On the final day of dosing, rats were injected with 2 mg progesterone ( s.c .) in safflower oil at 1100 hours to induce an afternoon LH surge. Blood samples were collected from fr ee-moving rats by an atrial cannula (implanted on Day 2) at 1-hour intervals for 9 hours, starti ng on the afternoon of the final day of atrazine dosing. Red blood cells (collected from the previous sam ple for that rat) in saline were transfused
back into the rat to maintain blood volume and hematocrit levels in each animal.

In Experiment 2, to evaluate the effects of atrazine on GnRH neuronal activation, ovariectomized rats (4-6/group) were treated as described for Experiment 1 with atrazine at 0, 50, 100, or 200 mg/kg/day, and injected with EB and progesterone. Close to peak LH surge (Day 4, 1700 hours), rats were anesthetized with ketamine and perfused with paraformaldehyde; the brains were removed, post fixed in paraformaldehyde, and infiltrated with sucrose for cryostat sectioning.ATRAZINE FINAL

Page 149 of 159 Concentrations of GnRH and the Finkel-Biskis-Jinkins osteosarcoma (cFOS) were determined in every fourth section. A second group of ovariectom ized rats was treated (as described) with atrazine at 0 and 50 mg/kg/day without EB and pr ogesterone injections to determine if atrazine alone has a stimulatory effect on GnRH neuronal activation. On th e afternoon of Day 4, rats were euthanized and brains collected (as above) for FOS analysis.

In Experiment 3, to evaluate the effects of at razine withdrawal on GnRH neuronal activation and LH surge, ovariectomized rats (4-13/group) we re dosed with atrazine at 0 (vehicle) or 200 mg/kg/day for four consecutive days, and the LH surge was induced during atrazine treatment, or 2 or 4 days after treatment (a s described above). For LH surge induction, rats received EB injections three c onsecutive days starting on the s econd day of atrazine dosing, the final day of atrazine dosing, or two days after atr azine dosing. On the final day of EB injections, rats were injected with proge sterone and blood samples were co llected hourly for 9 hours (as described for Experiment 1). A second group of ovariectomized rats (8-10/group) were treated, but without implantation of an intra-atrial cannula for blood co llection. Close to the LH surge peak (1700 hours), rats were perfused with pa raformaldehyde and brains were collected (as described for Experiment 2) for histological examination.

Blood and/or brain samples from th e three experiments were analyzed. Concentrations of LH and FSH in plasma were determined by RIA, and GnRH and cFOS concentrations in the brain were determined with immunohistochemistry. The lo cation and number of eGFP-GnRH-positive cells and the percent of GnRH neurons that were co-localized with FOS immunoreactivity were determined; cells were only considered doubl e labeled if eGFP-positive cytoplasm surrounded the FOS immunoreactivity nucleus through multiple fields.

The EB+ progesterone -induced LH surge was si gnificantly decreased (p<0.05) to a similar extent by atrazine at all doses. Significant decr eases (p<0.01) were noted in the peak amplitude and the area under the curve (AUC) of the LH surge. All rats from the vehicle (4/4) and 50 mg/kg (6/6) treatment groups had peak LH concentr ations >2 standard devi ations (SD) above the baseline, but fewer rats dosed at 100 mg/kg (6/8) and 200 mg/kg (2/5 ) responded to hormone priming. The follicle stimulating hormone (FSH) su rge was not affected by atrazine treatment, but the FSH AUC was significantly decreased (p <0.05) in the high-dose group (200 mg/kg/day).
All rats in the cont rol group (4/4) had peak FSH concentra tions >2 SD above the baseline, but a lower proportion of rats responded in the atrazi ne-treatment groups [50 mg/kg (5/6), 100 mg/kg (4/8), and 200 mg/kg (3/5)].

The number of activated GnRH neurons presen t during the afternoon af ter EB+ progesterone priming was decreased in atrazi ne-treated rats in Experiment 2. The percentage of GnRH neurons immunoreactive for cFOS was significantly decreased (p<0.05) by a similar extent in rats from the 100 and 200 mg/kg groups ( ↓ 67 and 64%, respectively). The mean number of
GnRH neurons immunoreactive for FOS also was si gnificantly decreased (p<0.02) with atrazineATRAZINE FINAL

Page 150 of 159 treatment at 100 and 200 mg/kg/day ( ↓ 59 and 74%, respectively). There were no atrazinetreatment effects on the number of GnRH immunoreactive cells. Fi nally, atrazine treatment alone (50 mg/kg without EB+ progester one) did not have an effect on FOS immunoreactivity. Minimal FOS immunoreactivity was observed in the GnRH neurons in the brains of rats from the 50 mg/kg (0.50±0.22) and contro l (0.50±0.40) groups.

In Experiment 3, LH surge and GnRH activ ation were decreased (p<0.05) by atrazine (200 mg/kg/day) on the final day of treatment, as seen in Experiment 1. LH and GnRH were still decreased after two days of at razine withdrawal compared to control, but were increased compared to the Day 0 concentrations in atrazi ne-dosed rats. By the f ourth day of atrazine withdrawal, peak LH concentra tions and the percentage of Gn RH neurons immunoreactive with
FOS returned to levels similar to control. Th ere were no differences in LH concentration and GnRH activation among the control groups at 0, 2, and 4 days after treatment.

These results demonstrate that acute atrazine exposure inhib its hormone-induced LH surge and GnRH neuronal activation, and these effects are transient and no longe r present after four days of
withdrawal from atrazine treatmen t. These results also support th e hypothesis that the brain is the primary target mediating the effects of atrazine on LH release in the rat.

In a second mechanistic study of female reproductive hormones, ( Foradori et al. , 2011 )
examined the role of adrenal activation on th e estrogen-/progesterone-induced LH surge in
ovariectomized rats to determine if LH inhibition after atrazine treatment was related to an
increase in corticosterone.

Female adult (60-90 days old) Wistar rats were housed in pairs, and provided food (commercial rodent diet) and water ad libitum . In Experiment 1, to determine th e effects of atrazine on plasma corticosterone concentrations, female rats (n =75) were ovariectomized and, after one week, orally dosed by gavage with atrazi ne (purity not reported) in 1% CMC sodium salt at 0 (vehicle), 50, or 200 mg/kg. Rats (3-5/group/time) were s acrificed, by decapitation under anesthesia, at 5, 15, or 30 minutes, and at 1, 3, 6, or 12 hours after the single gavage treatment. Trunk blood was
collected between 0800 and 1200 hours to avoid the diurnal increase in co rticosterone. Plasma was harvested, and stored at

− 80°C for corticosterone analysis by RIA.

In Experiment 2, to determine the effects of atr azine on the LH surge, female rats (n=70) were
ovariectomized with half of the rats further bi laterally adrenalectomized (ADX) 7-10 days after ovariectomy. ADX animals were injected ( s.c .) with sterile saline twice daily and provided water
with 0.9% NaCl to maintain sodium balance a nd plasma volume. A sham surgery was conducted on the remaining ovariectomized rats. One day after adrenalectomy, ra ts (7-11/group) were orally dosed by gavage with atrazine in 1% CMC sodium salt at 0 (vehicle), 10, 100 or 200 mg/kg/day for four consecutive days (b etween 1000 and 1100 hours). Starting Day 2 of atrazine dosing, rats were injected with EB ( s.c ., 40 μg/kg in safflower oi l) for three consecutive days (between 1000 and 1100 hours) and on Day 4 rats were injected with progesterone ( s.c .,ATRAZINE FINAL

Page 151 of 159 2 mg/animal in safflower oil) at 1100 hours to induce an afternoon LH surge. Blood samples were collected from free-moving rats by an atrial cannula at 1-hour interval s for 9 hours (starting at 1300 hours) after the progesterone treatment. Plasma was harves ted and stored frozen for LH
determinations with RIA. After the third blood sample was collected, red blood cells suspended
in saline were transfused back into the same rat to maintain blood volum e and hematocrit levels. To validate the completeness of the adrenalectomy, plasma from the first blood samples were analyzed for corticosterone concentration.

In Experiment 3, to evaluate the effects of atrazine on the pulsatile release pattern of LH, female rats (n=75) were ovariectomi zed and ADX as described for Experiment 2. One day after adrenalectomy, rats (9-13/group) were orally dose d by gavage with atrazine in 1% CMC sodium salt at 0 (vehicle), 50, or 200 mg/kg/day for four consecutive days (between 1000 and 1100 hours). At approximately three hours after the fi nal atrazine dose, blood was collected by atrial cannulation every 5 minutes over a three-hour period. Plasma was harvested and stored frozen for LH analysis with RIA; plasma from th e initial blood samples was also analyzed for
corticosterone concentration. Blood volume and hema tocrit levels were maintained as described previously.

A significant dose-dependent increase (p<0.0001) in plasma corticosterone concentration was observed after a single gavage treatment of atrazine. At the 20-minute post-treatment time,
corticosterone concentrations we re significantly increased (p<0.05) approximately 2- to 3-fold in both the 50 and 200 mg/kg dose groups. Corticoste rone concentrations in the 200 mg/kg group remained increased at all subsequent sampling times (1, 3, 6, and 12 hours); however, in the 50 mg/kg group, increases in corticosterone concentr ations were NS. Additionally, corticosterone concentrations in the 200 mg/kg group at the 3- and 6-hour post-treatme nt times also were significantly increased (p<0.05) relative to the 50 mg/kg group.

After four days of gavage treatment, the EB+ progesterone-induced LH surge was inhibited by atrazine treatment (100 or 200 mg/kg/day) in both sham and ADX animals compared to the respective controls. The peak amplitude in the LH rise and AUC for the LH surge were significantly decreased (p<0.05) in both sham and ADX rats in the 100 and 200 mg/kg treatment groups. Additionally, no interacti on between adrenalectomy and at razine treatment was observed for peak LH concentration or AUC, and no e ffect of adrenalectomy alone on peak LH
concentration or AUC was observe d in vehicle-gavaged rats.

Finally, LH pulses with increased (p<0.05) pulse amplitude and pulse period were observed in sham-operated rats treated with atrazine at 200 mg/kg compared to sham control. ADX animals showed no effect of atrazine on LH pulse amp litude or pulse period at either tested dose compared to ADX control. There was no effect of adrenalectomy alone on the LH pulse amplitude or pulse period in vehicle-gavaged rats . These data indicate th at atrazine selectivelyATRAZINE FINAL

Page 152 of 159 affects the LH pulse generator th rough alterations in adrenal horm one secretion, and that adrenal activation does not play a role in suppression of the LH surge due to atrazine treatment.

Mechanistic Study: HPG Axis, Female (Rat)

In a published study by Cooper et al . (2000) , female SD and LE rats we re exposed to atrazine (97.1% purity) in aqueous 1% methylcellulose by oral gavage to examine the effects on estrogen-induced surges of LH and prolactin in ovariectomized rats. Three separate experiments
were conducted.

In the first experiment, the effect s of atrazine after a single ( 1X ) or multiple day (3 days= 3X or 21 days= 21X ) exposure were evaluated. To eval uate the immediate effects, the 1X females in
each strain were ovariectomized and received an estrogen implant on Day 0. On Day 3, groups of ten females were gavaged with 0, 50, 100, 200, or 300 mg/kg atrazine, and then killed by decapitation at 0, 1, 3, or 6 hours post-gavage. Trunk blood was collected and serum was assayed for LH, FSH, prolactin, TSH, and 17 β -estradiol. The pituitaries we re removed and the neural
lobe was dissected free and discarded. The rema ining anterior lobe wa s weighed, sonicated, and frozen immediately at

− 70°C until assayed for LH, FSH, prolactin, TSH, and 17 β -estradiol. In
the 3X females, groups (n=10) of ovariectomi zed , implanted LE and SD females were administered atrazine as above on Days 1, 2, an d 3, and then killed at 0, 1, 3, and 6 hours postgavage on Day 3. The blood and pituitaries we re processed as described above for the 1X
females. In the 21X females, groups of LE and SD fema les were ovariectomized on Day 0 and received atrazine at 0, 75, 150, or 300 mg/kg/da y. On Day 21, females received an estrogen implant. On Day 24, the rats were killed by decapitation at the time at which peak LH concentrations were routinely observed. The bloo d and pituitaries were processed as described
for the 1X females.

In SD rats, the prolactin surge was significantl y reduced (p<0.05) after three daily doses of 300 mg/kg atrazine. However, the secretion of thes e two hormones in SD females was not different from controls at any of the other time points or doses of atrazine examined. In contrast, after a single dose of 300 mg/kg atrazine, the amplitudes of the LH and prolactin surges were decreased at 1, 3, and 6 hours. Three daily doses of 100, 200, or 300 mg/kg atrazine completely suppressed the estrogen-induced surges of both hormones. The onset of the LH surge in control LE females occurred at 1 hour, with peak c oncentrations occurring in most an imals at the 3-hour time point. Although surges of both LH and pr olactin were observed in the LE females administered three doses of 50 mg/kg atrazine, the onset of the surg e was delayed as evidenced by the presence of
baseline values at 1 and 3 hours and a peak appearing at 6 hours. In the 3X LE females, atrazine exposure inhibited the drop in pituitary prolactin concentration that was observed in control females during the observation period. In the 21X females of both strains, atrazine resulted in a dose-dependent suppression (p<0.05) of serum LH a nd prolactin. In addition, in both strains, the
concentration of LH and prolactin in the pituitary also was significantly decreased (p<0.05) after exposure to atrazine. In the 21-day-treated fem ale s, the amount of both LH and prolactin presentATRAZINE FINAL

Page 153 of 159 in the pituitary was generally greater in both rat strains. Body weight was unaffected in the
females administered atrazine fo r 1 or 3 days. Similarly, atrazine did not affect body weight in
the 21X females at 75 and 150 mg/kg. However, the ra te of body weight gain in those females administered 300 mg/kg was signifi cantly decreased (p<0.05, data not presented). Mean pituitary weight was not affected in any of the groups evaluated. The concen tration of serum or pituitary
TSH and FSH was not different from control at any of the doses or time points examined, and

serum 17 β -estradiol concentrations were also similar in control and treated groups.

In a second experiment to determine the effect of a single dose of at razine on ovulation and ovarian cycling, groups of int act LE females (20/dose group) di splaying regular 4-day estrous cycles were gavaged with 0, 75, 150, or 300 mg/kg at razine on the day of vaginal proestrus. The
effect of this treatment on ovarian function was de termined in half of the animals in each dose group by examining the vaginal smear daily for th ree additional weeks after injection. In the remaining animals, the effect of the proestrus dose of atrazine on ovulation was determined by collecting and counting oocytes on the day of vaginal estrus.

The 300 mg/kg atrazine dose markedly attenuated (data not reported) the amplitude of both the estrogen-induced LH and prolactin surges in the LE females, but had no effect on ovulation. Examination of the vaginal smears in a different group of rats treated w ith the high-dose on the day of vaginal proestrus revealed that 7 out of 9 females became pseudopregnant. The lower
doses of atrazine either were without effect on the number of oocytes shed (measured the morning after dosing), the day of estrus in the su bsequent cycle, or on su bsequent ovarian cycles as determined by vaginal smears. Continued dosi ng with 300 mg/kg/day atrazine for three days, beginning on vaginal diestrus Day 1, blocked the appearance of subsequent proestrus and
ovulation.

In the third experiment, three studi es were conducted to examine the site of action of atrazine. In the first study, to determine whethe r atrazine alters pituitary prolac tin secretion by a direct effect on the lactotrophs, the pituitary of seve n LE female rats was removed from the sella tursica and implanted under the kidney capsule. The rats were allowed 4 weeks to recover. On the test day, two baseline tail-blood samples were obtained at 0 and 1 hour prior to administration of 300 mg/kg atrazine by gavage. Si x hourly post-dosing tail-blood samples were obtained from each female and the blood was processed for prolactin analysis. In the second study, LH
secretion after intravenous GnRH administrati on was examined. LE rats were ovariectomized and implanted, and three daily doses of 300-mg /kg atrazine were administered by gavage; control females received vehicle only. Immediatel y after the third dose, the females were fitted with in-dwelling cardia c catheters. Two hours after placemen t of the catheters, a baseline blood sample was taken and replaced with heparinized saline. Ten minutes later, another baseline blood sample was obtained and, immediately thereaf ter, 50 ng/kg GnRH dissolved in saline was administered through the catheter. Additional blood samples were collected at 5, 15, 30, 45, and
60 minutes after the bolus of GnRH. In the third study, th e effect of atrazine on in vitro prolactin and LH release was determined by pituitary perfusion. After removi ng the pituitaries, the neuralATRAZINE FINAL

Page 154 of 159 lobe was immediately dissected fr ee and the anterior l obe was then hemisected along the rostralcaudal plane. One-half (randomly selected left or right portion) wa s weighed, sonicated, and frozen for subsequent assays for LH and prol actin. The remaining half was quartered, weighed, and placed in a sterile, cooled microplate well. After carefully tr ansferring tissue fragments to the perifusion chambers, the tissue was perifused for an initial 2-hour period to allow sufficient time for hormone secretion to reach baseline. Sample s were collected at10-minute intervals. After three 10-minute baseline samples, the hemi-pituitaries were expos ed to two 30-minute challenges (spaced 60 minutes apart) with a combination of GnRH and thyroid releasing hormone (TRH). Sixty minutes after the second ch allenge, tissues were exposed once more (10 minutes) with a depolarizing stimulus of 60 mM potassium chlori de (KCl) to assess tissue viability, and sample
collections were continued for an additional 40 minutes.

In the first study, serum prolac tin concentrations remained unaltered at all the time points examined, indicating that secreti on of prolactin by the pi tuitary is not altered by atrazine if the gland is removed from the influence of CNS fact ors. In the second study, LH secretion remained
at basal levels in atrazine + saline-treated fema les. However, an LH surge was observed in the atrazine + GnRH females. In fact, serum LH co ncentrations in the atrazine + GnRH females were comparable to the estrogen-induced LH surg e observed in control females, indicating that
the effect of atrazine on LH secr etion is not the result of a direct impairment of LH release from the gonadotrophs. In the third study, no differences in either LH or prolac tin release were noted from the pituitaries of untreate d females exposed to atrazine in vitro. Similarly, no changes in
basal or GnRH-stimulated LH release or TRH-st imulated prolactin release were noted in the pituitaries obtained from females dosed with atr azine for three days. In these same pituitaries, there was an initial increase in prolactin re lease from LE pituitaries that may have been
associated with a modest elevation in pituitary prolactin concentrations.ATRAZINE FINAL

Page 155 of 159 APPENDIX 3: References Not Utilized in the Atrazine WoE Analysis

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

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

The following published and unpublished references were considered for use in the WoE analysis for Atrazine but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the com pound of interest was not us ed in the study; 3) the test material was not adequately described; 4) a formulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only da ta on invertebrates.

Aït-Aïssa, S., Laskowski, S., Laville, N., et al . (2010) Anti-androge nic activities of environmental pesticides in the MDA-kb2 reporter cell line. Toxicol. In Vitro. 24:1979-1985.

Ashby, J. and Lefevre, P.A. (2000) The Peripubertal Male Rat Assay as an Alternative to the Hershberger Castrated Male Rat Assay for the Detection of Anti-androgens, Oestrogens and
Metabolic Modulators. J. Appl. Toxicol. 20:35–47.

Brodeur, J.C., Svartz, G., Perez-Coll, C.S., et al . (2009) Comparative suscep tibility to atrazine of three developmental stages of Rhinella arenarum and influence on metamorphosis; Nonmonotonous acceleration of the time to c limax and delayed tail resorption. Aquat Toxicol

. 91:161-170.ATRAZINE FINAL

Page 156 of 159 Brown, T.M. (2009) The effects of pesticide ex posure on gonadal deve lopment, phonotaxis, and calling in the African Clawed Frog, Xenopus laevis . Ph.D. Dissertation, Univ ersity of California, Berkeley, CA.

Carr, J.A., Gentles, A., Smith, E

. E., et al . (2003) Response of larval Xenopus laevis to atrazine: assessment of growth, metamorphosis, and gonadal and laryngeal morphology

. Environ. Toxicol.
Chem. 22(2): 396-405

.

Casas, E., Bonilla, E., Ducolomb, Y., et al . (2010) Differential effects of herbicides atrazine and fenoxaprop-ethyl, and insecticides diazinon and malathion, on viability and maturation of porcine oocytes in vitro. Toxicol. In Vitro. 24(1):224-230.

de la Casa-Resino, I., Valdehita, A., Soler, F., et al . (2012) Endocrine disr uption caused by oral administration of atrazine in European quail ( Coturnix coturnix coturnix ). Comp. Biochem. Physiol C. 156(3-4):159-165.

de la Casa-Resino, I., Navas, J.M., and Ferná ndez-Cruz, M.L. (2014) Ch lorotriazines do not activate the aryl hydrocarbon receptor, the oestrogen receptor or the thyroid receptor in in vitro

assays. Altern. Lab Anim. 42(1):25-30.

Eldridge, J.C., Stevens, J.T ., and Breckenridge, C.B. (2008) Atrazine interaction with estrogen expression systems. Rev. Environ. Contam. Toxicol. 196:147-160.

FAO/WHO Pesticide Residues in Food (2007) 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 Geneva, Switzerland, 18–27 September 2007.

FAO/WHO Pesticide Residues in Food (2007) 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. Evaluations, PartII - Toxicological Geneva, Switzerland, 18–27 September 2007.

Forgacs, A.L., Ding, Q., Jaremba, R.G., et al . (2012) BLTK1 murine Leydig cells: a novel steroidogenic model for evaluating the effects of reproductive and deve lopmental toxicants.
Toxicol. Sci . 127(2):391-402.

Forgacs, A.L., D'Souza, M.L., Huhtaniemi, I.T., et al . (2013) Triazine he rbicides and their chlorometabolites alter steroidogenesi s in BLTK1 murine Leydig cells. Toxicol. Sci

. 134(1):155-167.

Gammon, D., Aldous, C., Carr, W.J., et al . (2005) A risk assessment of atrazine use in California: human health and ecological aspects. Pest. Manag. Sci. 61(4):331-355.ATRAZINE FINAL

Page 157 of 159 Ghodageri, M.G. and Katti, P. (2013) In vi tro induction/inhibition of germinal vesicle breakdown (GVBD) in frog ( Euphlyctis cyanophlyctis ) oocytes by endocrine active compounds. Drug and Chem. Toxicol. 36(2):217-223.

Gunderson, M.P., Veldhoen, N., Skirrow, R.C., et al . (2011) Effect of low dose exposure to the
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