Regulations.Gov

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 DP Barcode: NA Decision No.: NA Registration No.: NA Petition No.: NA Regulatory Action: NA Risk Assessment Type: NA Case No.: NA TXR No.: See table, Attachment A CAS No.: NA MRIDNo.:NA 40CFR: NA Ver.Apr. 2010

FROM: Greg Akerman & ~ Health Effects Divison ::~:E£'~:::e f] g:ams d f 'v-- Environmental Fat~~fec~~~n ~ Office of Pesticide Program

THROUGH: Jess Rowland ~ S ~~~ Deputy Division Director Health Effects Division Office of Pesticide Programs

TO: Jolene Trujillo Chemical Review Manager Pesticide Re-Evaluation Division Office of Pesticide Programs

EPA has completed its Weight of Evidence (WoE) assessment evaluating results of the Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. The WoE documents for the 52 chemicals are listed in Attachment A along with the chemical and report identifiers.

Page I of2 Page 1 of 167 Attachment A. EDSP List 1 Chemicals Chemical Name PC Code TXR Number 2,4-D 030001 0057151 Abamectin 122804 0057152 Acephate 103301 0057153 Acetone 044101 0057154 Atrazine 080803 0057155 Benfluralin 084301 0057156 Bifenthrin 128825 0057157 Captan 081301 0057158 Carbaryl 056801 0057159 Carbofuran 090601 0057160 Chlorothalonil 081901 0057161 Chlorpyrifos 059101 0057162 Cyfluthrin 128831 0057163 Cypermethrin 109702 0057164 DCPA 078701 0057165 Diazinon 057801 0057166 Dichlobenil 027401 0057167 Dimethoate 035001 0057168 EPTC 041401 0057169 Esfenvalerate 109303 0057170 Ethoprop 041101 0057171 Fenbutatin-Oxide 104601 0057172 Flutolanil 128975 0057173 Folpet 081601 0057174 Glyphosate 417300 0057175 Imidacloprid 129099 0057176 Iprodione 109801 0057177 Isophorone 847401 0057178 Linuron 035506 0057179 Malathion 057701 0057180 Metalaxyl 113501 0057181 Methomyl 090301 0057182 Metolachlor 108801 0057183 Metribuzin 101101 0057184 MGK-264 057001 0057185 Myclobutanil 128857 0057186 Norflurazon 105801 0057150 o-Phenylphenol 064103 0057146 Oxamyl 103801 0057142 PCNB 056502 0057138 Permethrin 109701 0057149 Phosmet 059201 0057145 Piperonyl Butoxide 067501 0057141 Pronamide 101701 0057137 Propargite 097601 0057148 Propiconazole 122101 0057144 Pyriproxyfen 129032 0057140 Simazine 080807 0057136 Tebuconazole 128997 0057143 Tetrachlorvinphos (TCVP) 083701 0057147 Triadimefon 109901 0057139 Trifluralin 036101 0057135

Page 2 of 2 Page 2 of 167 EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID PATHWAYS

CHEMICAL: ATRAZINE

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

Page 3 of 167 ATRAZINE FINAL

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 Relevant 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-Estrogenic Pathway for Atrazine…………………….…………17 Table 3. Androgenic/Anti-Androgenic 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…..……..….…..…155

ii Page 4 of 167 ATRAZINE FINAL

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 Pathway AR Androgen Receptor Bmax Binding at maximum BROD Benzyloxyresorufin-O-dealkylase BUN Blood Urea Nitrogen CAR Constitutive Androstane Receptor CFR Code of Federal Regulations CG Cowper’s Gland ChE Cholinesterase ChEI Cholinesterase inhibition CMC Carboxymethyl cellulose CMG Common Mechanism Grouping CTA Comparative Thyroid Assay CV Coefficient of Variation CYP Cytochrome 450 DACT Diamino chlorotriazine DEA Desethyl-s-atrazine DER Data Evaluation Record DIA Desisopropyl-s-atrazine DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid DO Dissolved Oxygen DP Dorsolateral Prostrate E Estrogen hormonal pathway EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee EDRT Endocrine Disruptor Review Team EDSP Endocrine Disruptor Screening Program EE Ethinyl Estradiol ELISA Enzyme Linked Immunosorbent Assay EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat) ER Estrogen Receptor

iii Page 5 of 167 ATRAZINE FINAL

Abbreviation Terminology EROD Ethoxyresorufin-O-dealkylase (or deethylase) ERTA Estrogen Receptor Transcriptional Activation EtOH Ethanol F Female F1 First filial generation F2 Second filial generation Fcd Fecundity FIFRA Federal Insecticide, Fungicide and Rodenticide Act FOB Field Observation Battery FQPA Food Quality Protection Act Frt Fertility FSH Follicle Stimulating Hormone FSTRA Fish Short-Term Reproduction Assay FT Flutamide GD Gestation Day GGT Gamma-glutamyl Transpeptidase GnRH Gonadotropin-releasing hormone GP Glans Penis GSI Gonado-Somatic Index H High HLL Hind Limb Length HPG Hypothalamic-Pituitary-Gonadal Axis HPLC/MS/MS High Pressure Liquid Chromatography/Mass Spectroscopy HPT Hypothalamic-Pituitary-Thyroidal Axis I Inadequate IC50 Inhibitory Concentration at 50% of response ICCVAM Interagency Coordinating Committee on the Validation of Alternative Mhd Kd Equilibrium Dissociation Constant

KOW Octanol/Water Partition Coefficient L Low dose LABC Levator Ani-Bulbocavernosus LAGDA Larval Amphibian Growth and Development Assay LC50 Lethal Concentration in 50% of test organisms LD Lactation Day LH Luteinizing hormone LOAEC Lowest Observed Adverse Effect Concentration LOAEL Lowest Observed Adverse Effect Level LOQ Limit of Quantitation M Male MDL Minimum Detection Level MEOGRT Medaka Extended One Generation Reproduction Test MH Medium high ML Medium low

iv Page 6 of 167 ATRAZINE FINAL

Abbreviation Terminology MoA Mode of Action MOE Margin of Exposure MRID Master Record Identifier MROD Methoxyresorufin-O-dealkylase MTC Maximum Tolerated Concentration MTD Maximum Tolerated Dose N Negative NE Not examined/evaluated NF stage Nieuwkoop and Faber’s Staging Atlas NOAEC No Observed Adverse Effect Concentration NOAEL No Observed Adverse Effect Level NS Not Statistically Significant NR Not Reported OCSPP Office of Chemical Safety Pollution and Prevention OECD Organization for Economic Co-Operation and Development OPP Office of Pesticide Programs ORD Office of Research and Development OSCP Office of Science Coordination and Policy OSRI Other Scientifically Relevant Information P Positive P Parental generation PC Positive Control PC10 Positive Control at 10% of response

PC50 Positive Control at 50% of response PND Post-Natal Day POD Point of Departure PPS Preputial Separation PROD Pentaoxyresorufin-O-dealkylase (or depentylase) PXR Pregnane X receptor QC Quality Control RBA Relative Binding Affinity RBC Red Blood Cells RfD Reference Dose RPCmax Relative to Positive Control at maximum SAP Scientific Advisory Panel SC Solvent Control s.c Subcutaneous SDH Sorbitol dehydrogenase SDWA Safe Drinking Water Act SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley SVL Snout-to-Vent Length SV Seminal Vesicles

v Page 7 of 167 ATRAZINE FINAL

Abbreviation Terminology T Thyroid (hormonal pathway) T1WoERC EDSP Tier 1 Weight of Evidence Review Committee T3 Triiodothyronine T4 Thyroxine (tetraiodothyronine) TP Testosterone Propionate TR Thyroid Receptor TSH Thyroid Stimulating Hormone UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) VC Vehicle Control VO Vaginal Opening VP Ventral Prostate VTG Vitellogenin WoE Weight-of-Evidence

vi Page 8 of 167 ATRAZINE FINAL

Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) hormone pathways. This interaction includes agonism and antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as perturbations of the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. For atrazine, test order requirements for all eleven Tier 1 assays were satisfied by submissions of other scientifically relevant information (OSRI), comprised primarily of studies that were generally consistent with, 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 considered in this weight of evidence (WoE) evaluation. Also considered in the determination 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 responses, and the pattern of responses observed across studies, taxa, and sexes and levels of biological organization were considered. Additionally, the conditions under which effects occur were considered, in particular, whether or not endocrine- related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

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

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 provided additional lines of evidence to suggest potential downstream, estrogen pathway effects that may 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 induction occurred in MCF-7 cells. Increased aromatase activity in human granulosa-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

Page 1 of 159 Page 9 of 167 ATRAZINE FINAL effect on aromatase activity in rainbow trout brain and ovarian microsomes. However, atrazine acted as an inducer of 17β-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 pubertal assay, delays in estrus and vaginal opening, decreases pituitary and uterine weights were observed at the mid-dose in the absence of overt toxicity. There were no estrogen-related effects seen 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-occurred 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 water 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 snakes exposed to atrazine in the diet. In ovo exposure to atrazine resulted in no statistically significant effects in aromatase activity or sex steroid concentrations in chorionic fluid or on neonate gonadal histology. The percentage of female frogs was greater in atrazine-exposed treatments in one study, but other studies with frogs showed no effects on reproductive development in the absence of overt toxicity.

Although the available data reflect varying responses in estrogen-related endpoints across species and studies, the potential for interaction with the estrogen pathway is supported by the SAP conclusion that the chlorotriazines (including atrazine and its degradates) function through a neuroendocrine mode of action (MoA) that suppresses the hypothalamic release of GnRH and therefore LH, which may result in downstream 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 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 triazines in the CMG. In the in vitro Tier 1-like assays, atrazine was negative in the AR binding and ARTA assays. In the steriodogenesis assay, atrazine acted as an inducer of testosterone at 100 µM at two of five studies (USEPA, 2008c).

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

Page 2 of 159 Page 10 of 167 ATRAZINE FINAL

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 similar effects were not seen in all studies. Although the available data, especially the OSRI beyond the Tier 1 guideline-like studies, reflect varying responses in estrogen- and androgen-related endpoints across species and experiments, the potential for interaction with the estrogen and androgen pathways is supported by the SAP conclusion that the chlorotriazines [(including atrazine and its chlorinated degradates 2-chloro-4- isoproylamino-6-amino-s-triazine(DEA), chlordiamino-s-triazine(DACT), and 2-chloro-21 6- ethylamino-4-amino-s-triazine (DIA)] function through an upstream, neuroendocrine mode of action (MOA) that suppresses the hypothalamic release of gonadotropin releasing hormone (GnRH) and therefore luteinizing hormone (LH), which may result in downstream effects on estrogen and androgen signaling pathways. Therefore, based on a WoE analysis that also considered the triazine CMG and SAP reports on atrazine (http://www.epa.gov/pesticides/reregistration/atrazine/atrazine_update.htm), atrazine has the potential to interact with the estrogen and androgen pathways in mammals and other wildlife.

The available data for atrazine do not provide convincing 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 departure (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 mode of action (MOA) of the triazines. In a Tier 1-like male pubertal assay, 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 human health risk assessment. Estrogen-related effects (i.e., delays in the onset of puberty and altered estrous cyclicity) 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 mallard duck hatchling weight at 75 mg/kg diet and above, with reproductive effects (e.g., reduced number of hatchlings) observed only at higher treatment levels.

Therefore, based on the WoE analysis of EDSP Tier 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 ecological risk assessment.

Page 3 of 159 Page 11 of 167 ATRAZINE FINAL

I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) hormone pathways. This interaction includes agonism and antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as perturbations of the hypothalamic-pituitary-gonadal (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 systemic toxicity or overt toxicity.

Technical atrazine is relatively insoluble in water (33 mg/L at 20°C), with an octanol water −7 partition coefficient (Kow) of 501. The vapor pressure of atrazine is 3.0×10 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 (t½ = 14 hours), and the particulate-phase will be removed by wet or dry deposition. In soil, atrazine is subject to photolysis with half-lives ranging between 12 to 45 days; photolysis degradates include 2-chloro-4-isoproylamino-6-amino-s-triazine(DEA), chlordiamino-s- triazine(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 occur 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 content, with increased biodegradation (decreased half-lives) in wetter soils. 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 t½ =

Page 4 of 159 Page 12 of 167 ATRAZINE FINAL

335 days). A bioconcentration factor of up to 100 suggests that bioconcentration 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 studies and other published studies. These data are summarized in Section III.A through III.C. An analysis of the data submitted to the Agency, using the WoE approach outlined by the Agency (USEPA, 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 Sprague Dawley rats. Additional studies were conducted to determine the mechanisim of mammary carcinogenesis. Additionally, around the time of the evalution of atrazine for the Registration Eligibility Decision (RED), several studies concerning the potential effects 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 WoE 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 triazine-containing pesticides atrazine, simazine, and propazine and their three chlorinated degradates (i.e., DEA, DIA and DACT) should be included in a CMG1 (the triazine CMG) and considered through a cumulative risk assessment. EPA’s triazine cumulative risk assessment2 considered the combined effects of atrazine, propazine and simazine, which share a common neuroendocrine mechanism 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 decreases LH levels by suppression of gonadotrophin-releasing hormone (GnRH) from the hypothalamus. Suppression of LH is regarded as a precursor event in the neuroendocrine mode of action (MOA) of the triazines.

The grouping of the CMG triazines based on a common MOA was presented to the FIFRA SAP in 20003. The SAP agreed with the Agency’s conclusion that there is sufficient evidence to support the proposed grouping for the neuroendocrine-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/cumulative/common_mech_groups.htm 2 http://www.epa.gov/pesticides/cumulative/. 3 http://www.epa.gov/scipoly/sap/meetings/2000/062700_mtg.htm.

Page 5 of 159 Page 13 of 167 ATRAZINE FINAL presented information to several SAP meetings for independent scientific peer review of new studies on atrazine4. 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 effects within an adverse outcome 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 believed to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Order recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration (e.g., Part 158 Guideline studies). The Agency’s review of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team on atrazine (USEPA, 2010c). After reviewing the submitted OSRI, the Agency determined that the available OSRI fulfilled requirements for all the Tier 1 assays, with the exception of the Amphibian Metamorphosis Assay (AMA), as the existing amphibian metamorphosis studies did not examine the thyroid for histopathological changes. Therefore, additional data were requested 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 literature publications that were presented to the FIFRA SAP in May 20035, October 20076, and June 20127. The Agency has also conducted a more recent search of

4 See http://www.epa.gov/pesticides/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. Presented 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 Office of Pesticide Programs, Washington, DC. Docket ID: EPA-HQ- OPP-2012-0230.

Page 6 of 159 Page 14 of 167 ATRAZINE FINAL available scientific literature for any additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies considered but not utilized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

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

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 battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. 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 registrations. 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 individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a specific endpoint for discerning whether or not the chemical has interacted with the endocrine system, are considered. The collective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indication of potential interaction as measured by the study.

Page 7 of 159 Page 15 of 167 ATRAZINE FINAL

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

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide relevant, robust and consistent evidence in terms of complementarity and redundancy as well as biological plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biological organization? Finally, under what conditions did the responses occur including discussions regarding 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 effects specifically related to an endocrine-related effect from a non-endocrine toxic response.

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

For these WoE analyses, overt toxicity was generally 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 included, but were not limited to: mortality; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight decreases of ≥10% in mammals. Additionally, other effects including morphological (e.g., organ weights/histopathology), biochemical (e.g., blood chemistry), and other clinical signs (e.g., lethargy) were also considered when evaluating overt toxicity, especially if the effects were severe. In some instances, one parameter (i.e., death or >10% decrease in mammalian body

Page 8 of 159 Page 16 of 167 ATRAZINE FINAL weight) was sufficient to consider a dose/concentration to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decreases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these effects were generally considered as less severe forms of toxicity (e.g., changes in organ weights or blood chemistry). The circumstances for which a dose/concentration was considered overtly toxic for a particular study are described in Section IV.A.

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

The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4. These tables contain data that are considered scientifically and biologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

Page 9 of 159 Page 17 of 167 ATRAZINE FINAL

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

Test Tier 1 Assays Test Order Status Guideline ER Binding Assay (Rat uterine OSCPP Requirement satisfied by OSRI cytosol) 890.1250 (Tennant et al.; USEPA, 2009) OSCPP ERα Transcriptional Activation Requirement satisfied by OSRI 890.1300; Assay (Human cell line HeLa 9903) (Fujimoto and Honda, 2003) OECD 455 AR Binding Assay (Rat prostate OSCPP Requirement satisfied by OSRI (USEPA, 2008a) cytosol) 890.1150 OSCPP Steroidogenesis Assay (Human cell 890.1550; Requirement satisfied by OSRI (USEPA, 2008c) line H295R) OECD 456 Aromatase Assay (human OSCPP Requirement satisfied by OSRI recombinant microsomes) 890.1200 (Higley et al., 2010; USEPA, 2008b) OSCPP Requirement satisfied by OSRI Uterotrophic Assay (Rat) 890.1600; (Yamaski et al., 2000) OECD 440 OSCPP Requirement satisfied by OSRI Hershberger Assay (Rat) 890.1400; (Ashby et al., 2004: Yamaski et al., 2004) OECD 441 OSCPP Requirement satisfied by OSRI Pubertal Female Assay (Rat) 890.1450 (Laws et al., 2000)

OSCPP Requirement satisfied by OSRI Pubertal Male Assay (Rat) 890.1500 (Stoker et al., 2000) OSCPP Requirement satisfied by OSRI Fish Short-term Reproduction Assay 890.1350; (Battelle, 2005) OECD 229 OSCPP Amphibian Metamorphosis Assay Requirement satisfied by OSRI 890.1100; (MRID 47153501: Hosmer et al. 2007; Kloas et (Frog) OECD 231 al., 2009; MRID 48614801)

Page 10 of 159 Page 18 of 167 ATRAZINE FINAL

B. Effects on Hypothalamic-Pituitary-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 across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for estrogenic, 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 pathway 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 [3H]-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 negative 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 breast epithelial tumor (T47-D) cells transfected with human estrogen receptor-alpha (hERα) and/or human estrogen receptor-beta (hERβ) (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 tritium-release assay). This study demonstrated no direct effect on aromatase activity, but induction of aromatase activity at atrazine concentrations of 10 and 100 µM after 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 atrazine 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 occurred in MCF-7 cells. Similarly, atrazine exposure resulted in increased aromatase 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),

Page 11 of 159 Page 19 of 167 ATRAZINE FINAL

17β-estradiol production was induced at 100 µM and 10 µM atrazine in four out of five studies (results only reported 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 weights 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 increases in uterine epithelial 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 presence of overt toxicity (12% decrease in body weight). Decreases in uterine weight (without fluid) were significantly decreased at 100 and 200 mg/kg/day and and ovarian weights were decreased only at 200 mg/kg/day. Pituitary weights were significantly decreased in these two groups as well. Histopathological examination determined that corpus luteum development was decreased in the 200 mg/kg/day females, with increased atretic follicles and uterine hypoplasia. 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 approximately 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 seven 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. Terminal body weights were decreased 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 statistical significance (Ashby et al., 2002; MRID 45722401). Delays in the attainment of vaginal opening (VO) of 3 days were noted at the high dose in Wistart rats. In SD rats, 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 toxicity in rats, decreases in mean uterus and ovary weights in high- dose (1000 mg/kg/day) females were observed in the presence of overt toxicity. No corresponding gross or microscopic morphology findings were reported.

In a Part 158 two-generation rat reproduction toxicity study (MRID 40431303), no treatment- related effects were reported for any reproductive parameters up to the atrazine high dose of 38.7 mg/kg/day. Foradori et al. (2014) investigated the effects of atrazine treatment (bolus or diet) on estrous cyclicity and reproductive performance in SD and Long-Evans (LE) rats. The only treatment-related effects noted were in the bolus-treatment group. 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 all 100 mg/kg/day animals and the 50 mg/kg/day SD females. In

Page 12 of 159 Page 20 of 167 ATRAZINE FINAL addition, the percentage of females 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 day (GD) 6-10) to determine potential susceptibility to atrazine- induced 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 rats 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 mid dose (50 mg/kg/day; non-significant) and high dose (100 mg/kg/day; significant). Resporptions at these 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 decrease in the percentage of dams delivering after GD 21 at ≥50 mg/kg/day, and in SD rats and LE rats at 200 mg/kg/day.

In other OSRI, Davis et al., (2011) examined the effects of atrazine treatment in SD rats on female offspring reproductive indices (parental effects were not 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 decreases 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/reproductive 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 (decreased body weight). In another Part 158 developmental toxicity study in rats (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 this study increases in the mean number of resorptions and the percentage of post-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 the 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 17β-estradiol and pituitary weights were not affected. In

Page 13 of 159 Page 21 of 167 ATRAZINE FINAL addition, although single doses of atrazine did not affect ovulation, repeated dosing with 300 mg/kg/day prevented subsequent proestrus and ovulation.

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

In a chronic partial life-cycle study with the freshwater brook trout (Salvelinus fontinalis; MRID 00024377), statistically significant (p<0.01) decreases in F1 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 fecundity output for both controls and treatment groups was observed). Similarly, in a chronic full life-cycle study with the freshwater fathead minnows (Pimephales promelas; MRID 42547103), decreases in F0 and F1 growth (F0 length ↓5 – 10% and weight ↓19%; F1 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 were 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, respectively, reviewer calculated) and embryo mortality in the control group was relatively 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 histopathology, the study author reported, 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 controls. In a short-term reproduction assay (similar to FSTRA), Tillitt et al. (2010), 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 μg/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 estuarine/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 had mean body lengths and wet weights which were significantly (p<0.05) lower than controls by 17 and 46%, respectively, resulting in a NOAEC and LOAEC of 1.1 and 2.2 mg/L, respectively. For juvenile zebrafish (Plhalova et al. 2012), body weights and growth rates were reported to be significantly decreased (p<0.05) in fish in the

Page 14 of 159 Page 22 of 167 ATRAZINE FINAL high atrazine concentration group (90 µg/L; body 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 live embryos, and hatchlings per eggs set and weight gain in adult males at 675 mg/kg diet. All effects noted were statistically significant at p<0.05. In the bobwhite quail (Colinus virginianus; MRID 42547102), the number of eggs laid, viable embryos, live 3-week embryos, hatchlings, and 14-day old survivors generally were decreased. No treatment-related effects were observed for the numbers 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 signficiant effects on steroidogenesis (i.e., aromatase activity) were observed in hepatic or gonadal-adrenal mesonephros (GAM) complex tissues from neonatal alligators that were treated in ovo and incubated at a female-producing temperature. Decreases in egg chorioallantoic fluid (CAF) concentrations of 17β-estradiol and testosterone were observed, but did not reach statistical significance. No effects were seen on hepatic aromatase activity or gonadal histopathology. Maternal plasma 17β-estradiol concentration was not affected by dietary atrazine exposure in pregnant Northern water snakes (Nerodia sipedon; Neuman-Lee et al. 2014), but the proportion of live births 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 concomitant increases in progesterone (63%-104%, p<0.001) and testosterone (24%, p<0.01) concentrations in culture media, was observed with no effect on 17β-estradiol concentration.

Studies examining the potential effects of atrazine on amphibian development 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, gonadal development, sex ratio, and other aspects of sexual development under a range of atrazine test concentrations, experimental 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 support of each of the SAPs. Overall, the available data reflect varying responses in sexual and metamorphosis development and growth across species and studies; however, the evaluations conducted previously provide detailed analysis of study design elements that may have limited the utility of the various studies. In regards to the

Page 15 of 159 Page 23 of 167 ATRAZINE FINAL 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 concentrations 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. 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 effects 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).

Page 16 of 159 Page 24 of 167 ATRAZINE FINAL

EDSP Tier 1 Assays

ER Binding Requirement satisfied by OSRI (Tennant et al., 1994b and USEPA, 2009)

ERTA Requirement satisfied by OSRI (Fujimoto and Honda, 2003)

Aromatase Requirement satisfied by OSRI (Higley et al., 2010 and USEPA, 2008b)

FSTRA Requirement satisfied by OSRI (Battelle, 2005)

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

Steroidogenesis Requirement satisfied by OSRI (USEPA, 2008c)

Uterotrophic Requirement satisfied by OSRI (Yamasaki et al., 2000) OSRI ER Binding (USEPA, 2009) N ER Binding (MRID 43598618; Tennant et al., N4 1994b) ER Binding (Blair et al., N 2000) ER Binding (O’Conner et al., N 2000)

Page 17 of 159 Page 25 of 167 ATRAZINE FINAL

ERTA (Kojima et al., 2004) N ERTA (Fujimoto and Honda, N 2003) ERTA (Balaguer et al., 1999) N

ERTA (Connor et al., 1996) N ERTA (Fukamachi et al., N 2004) ERTA (Legler et al., 2002) N

Aromatase (USEPA, 2008b) N Aromatase (Higley et al., N5 2010) Aromatase (Sanderson et al., P 2001) Aromatase (Sanderson et al., P 2002) Aromatase (Rainbow Trout; N Hinfrey et al., 2006) Aromatase (Holloway et al., P 2008) Aromatase (Tinfo et al., P 2011)

Page 18 of 159 Page 26 of 167 ATRAZINE FINAL

FSTRA (Battelle, 2005) N N N NR N N N N FSTRA (Bringolf et al., NE N N NR N N N N 2004) Fcd: Modified FSTRA ↓19- (Fathead Minnow; Tillitt et NE N P 39% N al., 2010) (L, M, H) Frt: NE: Modfied FSTRA (Japanese Fcd: Medaka; Papoulias et al., N N P N ↓36-42% 2014) (L, M, H) Age: ↑3.4, ↓16 wo/ 4.5 d %( Female Pubertal Rat fluid: P (M, X X NE N N L, (Laws et al., 2000) ↓31% (MH) MH); (H) (H) M (MH) BW: ↑ H) (M, MH) Steroidogenesis P (USEPA, 2008c) Uterotrophic N N N N (Yamasaki et al., 2000) X Uterotrophic ↓ X (M, (Tennant et al., 1994a) (M) (H) H)

Page 19 of 159 Page 27 of 167 ATRAZINE FINAL

Table 2: Estrogenic/Anti-Estrogenic Pathway for Atrazine Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Atrazine1 2 Study Type/ Literature 3 Citation ER Binding Binding ER ER Activation Steroidogenesis Estrogen level Weight Uterine Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity at & Weight Age VO 2° Sex Characteristics Fertility (Frt)/ Vitellogenin Systemic Toxicity Obsereved Overt Toxicity Observed Fecundity(Fcd) Uterotrophic N NE NE (Connor et al., 1996) Uterotrophic (Rat; MRID ↓41 X 45722401; Ashby et al., % NE N (H) 2002, Exp 1) (H) Uterotrophic (Rat; MRID ↓55 X 45722401; Ashby et al., % NE N (H) 2002, Exp 2) (H) Uterotrophic (Rat; MRID X X 45722401; Ashby et al., N N (H) (H) 2002, Exp 3) Uterotrophic (Rat; MRID ↑2.5, X 45722401; Ashby et al., N 3 day (M, N 2002, Exp 4) (M, H) H) X Uterotrophic (Rat; O’Conner X N (M, et al., 2000) (H) H) 21-Day Dermal Toxicity ↓44 ↓36 X X N N (Rabbit; MRID 42089902) % % (H) (H) Two-generation reproduction X toxicity (Rat; MRID X NE N N NE N (H) 40431303, DeSesso et al., (H)

2014) Reproductive toxicity, Exp 1- bolus (Rat; Foradori et al., NE P P N N 2014)

Page 20 of 159 Page 28 of 167 ATRAZINE FINAL

Table 2: Estrogenic/Anti-Estrogenic Pathway for Atrazine Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Atrazine1 2 Study Type/ Literature 3 Citation ER Binding Binding ER ER Activation Steroidogenesis Estrogen level Weight Uterine Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity at & Weight Age VO 2° Sex Characteristics Fertility (Frt)/ Vitellogenin Systemic Toxicity Obsereved Overt Toxicity Observed Fecundity(Fcd) Reproductive toxicity, Exp 1- X X diet (Rat; Foradori et al., NE N N (H) (H) 2014) Reproductive toxicity, Exp 2- bolus (Rat; Foradori et al., NE N N N 2014) Reproductive toxicity, Exp 2- X X diet (Rat; Foradori et al., NE N (H) (H) 2014) Frt: N; Reproductive toxicity, Pilot Fcd: X X NE (Rat; Narotsky et al., 2001) ↓63% (M) (M, H) (L) Reproductive toxicity, Single dose (Rat; Narotsky et al., NE N X X 2001) Reproductive toxicity, F344 X strain (Rat; Narotsky et al., NE N N (M, H) 2001) Reproductive toxicity, S-D X strain (Rat; Narotsky et al., NE N N (M, H) 2001) Reproductive toxicity, LE X strain (Rat; Narotsky et al., NE N N (H) 2001)

Page 21 of 159 Page 29 of 167 ATRAZINE FINAL

Table 2: Estrogenic/Anti-Estrogenic Pathway for Atrazine Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Atrazine1 2 Study Type/ Literature 3 Citation ER Binding Binding ER ER Activation Steroidogenesis Estrogen level Weight Uterine Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity at & Weight Age VO 2° Sex Characteristics Fertility (Frt)/ Vitellogenin Systemic Toxicity Obsereved Overt Toxicity Observed Fecundity(Fcd) Age: Reproductive toxicity, one ↑1.5 X X dose per day NE N N N day (H) (H) (Rat; Davis et al., 2011) (H) Age: Reproductive toxicity, two ↑1.9 X X doses per day NE N N N day (H) (H) (Rat; Davis et al., 2011) (H) Developmental reproductive X X toxicity, in utero exposure NE N (H) (H) (Rat; DeSesso et al., 2014) Developmental reproductive X X toxicity, Post-natal exposure NE N (H) (H) (Rat; DeSesso et al., 2014) Developmental toxicity (Rat; MRIDs 00143008, X X NE N 40566302; Scialli et al., (H) (H) 2014) Developmental toxicity (Rat; X X MRID 41065201; Scialli et NR N (H) (H) al., 2014) Developmental toxicity Frt: N; (Rabbit; MRIDs 00143006, Fcd: X X NE 4056630; Scialli et al., ↓33% (H) (H) 20142) (H) Chronic X X Toxicity/Carcinogenicity NE N N NE (MH, (MH, (Rat; MRID 40629302) H) H)

Page 22 of 159 Page 30 of 167 ATRAZINE FINAL

Table 2: Estrogenic/Anti-Estrogenic Pathway for Atrazine Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Atrazine1 2 Study Type/ Literature 3 Citation ER Binding Binding ER ER Activation Steroidogenesis Estrogen level Weight Uterine Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity at & Weight Age VO 2° Sex Characteristics Fertility (Frt)/ Vitellogenin Systemic Toxicity Obsereved Overt Toxicity Observed Fecundity(Fcd) X X Carcinogenicity (Mouse; NE NE N NE (MH, (MH, MRID 40431302) H) H) Chronic Toxicity (Dog; X X NE N N N MRID 40431301, 41293801) (H) (H) Frt: ↓ Avian Toxicity (Mallard (H); N Duck; MRID 42547101) Fcd: ↓ (H) Frt: ↓ Avian Toxicity (Bobwhite (H); N Quail; MRID 42547102) Fcd: ↓ (H) Avian Toxicity (Japanese N N Quail; Wilhelms et al., 2006) Fish Partial Life Cycle Toxicity (Brook trout; MRID. N N 00024377) Early Life-Stage Toxicity (Sticklebacks; Le Mer et al., N N N 2013) Fish Full Life-Cycle Toxicity (Fathead minnow, MRID N N 42547103) Sexual development, Study 1 (Zebrafish; Corvi et al., N N N 2012)

Page 23 of 159 Page 31 of 167 ATRAZINE FINAL

Table 2: Estrogenic/Anti-Estrogenic Pathway for Atrazine Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Atrazine1 2 Study Type/ Literature 3 Citation ER Binding Binding ER ER Activation Steroidogenesis Estrogen level Weight Uterine Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity at & Weight Age VO 2° Sex Characteristics Fertility (Frt)/ Vitellogenin Systemic Toxicity Obsereved Overt Toxicity Observed Fecundity(Fcd) Sexual development, Study 2 (Zebrafish; Corvi et al., N N N 2012) In ovo exposure, (Alligator; N N Crain et al., 1997) In ovo exposure, (Alligator; N N N Spiteri et al., 1999) Frt: Developmental toxicity NE; (Watersnake; Neuman-Lee et N N Fcd:↓ al., 2014) (M, H) In vitro ovulation (Frog; N P (L) Orton et al., 2009) Amphibian metamorphosis and reproductive development (Frog; Hosmer N N N et al., 2007 and Kloas et al., 2009) Amphibian reproductive ↑% development (Frog; Oka et N ♀ N N al., 2008) (H) Amphibian reproductive development (Frog; N N N Spolyarich et al., 2010) HPG Axis (Rat; Cooper et P X N N N al., 2000) (H) (H)

Page 24 of 159 Page 32 of 167 ATRAZINE FINAL

1. 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 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 −3 4. Competitive binding against estradiol (E2) was negative (up to 10 log M), but pre-incubation with excess atrazine inhibited E2 binding (IC50 = 20 µM). 5. Direct aromatase activity was not different from control at any concentration tested. Atrazine significantly induced indirect and combined aromatase activity. P Positive finding N Negative finding (in vitro)/No effect (in vivo) NE Not examined

Page 25 of 159 Page 33 of 167 ATRAZINE FINAL

2. Effects on Androgen Pathway

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

In an evaluation of androgenic activity 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) assays (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) binding 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 requirements for steroidogenesis (USEPA, 2008c), atrazine induced testosterone production ≥1.5 fold in tow labs, but produced increases of <1.5- fold at the remaining three labs. Male steroidogenesis was also evaluated in several literature studies. Pogrmic et al. (2009) investigated effects on steroidogenesis in Leydig cells harvested from peripubertal male Wistar rats. After atrazine treatment (50 or 200 mg/kg/day) for four weeks, testes, adrenal, and accessory sex organ weights were decreased. Serum androgen concentration [testosterone + dihydroetestosterone (DHT)] was decreased at the high dose, and in vitro basal and hCG-stimulated androgen production 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 atrazine treatment. Atrazine treatment increased basal and hCG-stimulated cAMP accumulation and demonstrated concentration- dependent effects on androgen production (with bi-directional effects on progesterone concentration). The effects of atrazine (232 µM) on Leydig cell androgenesis were reported to be hCG-concentration dependent. The results of the ex vivo studies showed increased concentrations of cAMP and androgen after one, three and five days of atrazine treatment in vivo, with peak concentrations at Day 3. Effects of atrazine treatment on intact or castrated male rats were investigated by Riffle et al. (2014). Serum progesterone and corticosterone concentrations generally were increased in all dose groups with increases in intact rats less than in castrated rats. Serum LH, androstenedione, and testosterone concentrations in intact rats produced non- monotonic dose-response. In contrast, LH concentrations were increased in the castrated males compared to the intact males, and androstenedione and testosterone concentrations were below the limit of detection. Finally, Jin et al. (2013) investigated effects of atrazine (up to 200 mg/kg/day) on hormone synthesis and the transcription of steroidogenic genes in male peripubertal mice. The absolute testis weights in the low- and high-dose groups were decreased.

Page 26 of 159 Page 34 of 167 ATRAZINE FINAL

Serum testosterone concentrations were decreased, and serum 17β-estradiol concentrations were increased, in all treated groups indicating that oral atrazine exposure during puberty affected the serum hormone homeostasis in male mice. Atrazine treatment had no effect on AR, ERα, or ERβ mRNA concentrations in the testes, with varied effects on hormone mRNA levels.

In the OSRI that satisfied the Tier 1 testing requirements 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 decreases 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 the 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 weights at PND 53 were decreased in the ≥50 mg/kg/day treatment groups in a dose-related 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 period. 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, histopathology, and testosterone 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 control 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 absolute 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 the twice daily dosing regimen. 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 mg/kg/day, with decreases 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 concentrations were decreased at atrazine concentrations ≥50 mg/kg/day on PND 60. Effects observed at 75 and 100 mg/kg/day occurred

Page 27 of 159 Page 35 of 167 ATRAZINE FINAL

in the presence of overt toxicity (>10% decrease in body weight). In another male reproductive toxicity study (Song et al., 2014), anti-androgenic effects were noted in the presence of overt toxicity at the atrazine high dose (154 mg/kg/day). Decreased serum testosterone and inhibin-B concentrations, increased FSH concentrations, disrupted testicular organization, 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 investigated. The incidence of abnormal sperm was increased and pituitary weights were 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 chlorinated metabolites (Stoker et al. 2000; 2002). There have been a number of studies demonstrating that atrazine delays male puberty 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 regard 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 reproductive 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 pituitary secretion of LH) and subsequent alterations in the hormonal control of pubertal development. Decreased LH secretion would lead to insufficient stimulation of the gonads during puberty to produce the necessary 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 alteration of the hypothalamic control of pubertal progression.

There were no androgen-related effects, 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 androgen-related effects observed in an additional FSTRA considered as OSRI (Bringolf et al., 2004). Hepatic VTG production was negative in male carp (Cyprinus carpio) hepatocytes exposed to atrazine at concentrations up to 30 µM (Sanderson et al., 2001). Alterations in gene expression 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 male 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 testicular lesions were reported. Tillitt et al. (2010) showed no clearly treatment-realted effects on androgen-relevant parameters in a short-term reproductions study with fathead minnow. A published study by Kroon et al. (2014) with juvenile barramundi

Page 28 of 159 Page 36 of 167 ATRAZINE FINAL

failed to demonstrate consistent atrazine treatment-related effects (up to 100 µg/L) on gill ventilation rate, growth, CYP19B and VTG expression, or concentrations of testosterone and 17β-estradiol in the catadromous fish. In freshwater 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 incidences of testicular oocytes were observed, with no dose dependence. In Le Mer (2013), no effects on sex ratio or VTG was observed.

In ovo exposure to atrazine was conducted with alligator 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 histopathology 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 chorionic fluid, on aromatase activity or on gonadal histology.

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

As discussed in the estrogen pathway, many studies 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 atrazine exposure in amphibians have primarily focused on potential effects on metamorphosis (discussed separately in the section on the thyroid pathway) and sexual/gonadal development. Sexual 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 reversal. Atrazine exposure in X. laevis [MRID 47153501; Hosmer et al., 2007; Kloas et al., 2009] from NF stage 46 to 66 at concentrations 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 and only a single incidence of testicular ovarian follicles.

Wilhelms et al. (2005) exposed different groups of male Japanese quail to atrazine 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 mg/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 consistent effects on testosterone, 17β-estradiol or LH throughout the experiments.

Page 29 of 159 Page 37 of 167 ATRAZINE FINAL

Table 3: Androgenic/Anti-Androgenic Pathway for Atrazine Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1

Study Type/

EDSP Tier 1 Asssays AR Binding Requirement satisfied by OSRI (USEPA, 2008a)

FSTRA Requirement satisfied by OSRI (Battelle, 2005)

Hershberger Requirement satisfied by OSRI (Ashby et al., 2004 and Yamasaki et al., 2004)

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

Steroidogenesis Requirement satisfied by OSRI (USEPA, 2008c)

OSRI AR Binding N (USEPA, 2008a) AR Binding (Fang et al., N 2003) Receptor Transactivation N (Kojima et al., 2004) FSTRA (Battelle, N N P NE N N N 2005) FSTRA (Bringolf NE N N N N N N et al., 2004) Modified FSTRA (Fathead minnow; NE N N N NE NE N Tillitt et al., 2010)

Page 30 of 159 Page 38 of 167 ATRAZINE FINAL

Table 3: Androgenic/Anti-Androgenic Pathway for Atrazine Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1

Study Type/

Page 31 of 159 Page 39 of 167 ATRAZINE FINAL

Table 3: Androgenic/Anti-Androgenic Pathway for Atrazine Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1

Study Type/

Page 32 of 159 Page 40 of 167 ATRAZINE FINAL

Table 3: Androgenic/Anti-Androgenic Pathway for Atrazine Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1

Study Type/

Page 33 of 159 Page 41 of 167 ATRAZINE FINAL

Table 3: Androgenic/Anti-Androgenic Pathway for Atrazine Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1

Study Type/

Orton et al., (M) 2009)

Page 34 of 159 Page 42 of 167 ATRAZINE FINAL

Table 3: Androgenic/Anti-Androgenic Pathway for Atrazine Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1

Study Type/

Page 35 of 159 Page 43 of 167 ATRAZINE FINAL

Table 3: Androgenic/Anti-Androgenic Pathway for Atrazine Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1

Study Type/

Literature 2 3 Citation AR Binding AR Activation Steroidogenesis level Testosterone Weight/GSI Testes and Staging Gonad Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Sex Accessory Weights/ Organ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) at Weight and Age PPS Vitellogenin Systemic Toxicity Observed Overt Toxicity Observed Male ↑31, 69, Steroidogenesis, 98% In vitro (Rat; (LM, Pogrmic-Majkic HM, H) et al., 2010) Male Steroidogenesis, In vivo (Rat; ↑ Pogrmic-Majkic et al., 2010) Male Steroidogenesis N X N (Rat; Riffle et al., (M,H) 2014) Male ↓ 25- ↓ Steroidogenesis X X 30% 10% (Mouse; Jin et al., (H) (H) (L, M) (L) 2013) 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 response. 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. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (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 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 P Positive finding

Page 36 of 159 Page 44 of 167 ATRAZINE FINAL

N Negative finding (in vitro)/No effect (in vivo) NE Not examined NR Not reported

Page 37 of 159 Page 45 of 167 ATRAZINE FINAL

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

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

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

In the OSRI that satisfied the requirements for the female pubertal rat assay (Laws et al., 2000), thyroid weights were not determined, but there were no effects on thyroid histopathology, serum total thyroxine (T4) or thyroid stimulating hormone (TSH) concentrations (up to 200 mg/kg/day). Pituitary weights were decreased 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 (T3) concentrations were increased in all treatment groups, with significance achieved only in the presence of overt toxicity (200 mg/kg/day). There were no significant change in T4 or TSH. Thyroid weights were not determined, but there were no effects on thyroid histopathology. In another literature study examining the HPG axis in the rat (Cooper et al., 2000), no treatment effects were noted on TSH concentration or pituitary weight in animals treated with atrazine up to 300 mg/kg/day.

There were no thyroid-related effects observed in the mammalian 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 treatment-related effects of atrazine in the follow-up thyroid tissue analysis from atrazine-treated frogs that had completed metamorphosis. There were also no treatment-related 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 dose dependent (also were observed in only the data from one of the two performing laboratories). 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 examined 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).

Page 38 of 159 Page 46 of 167 ATRAZINE FINAL

Table 4. Thyroid Pathway for Atrazine. Lines of Evidence Indicating Potential for Interaction with the Thyroid Pathway for Atrazine1 2

Study Type/

3 Levels Levels Literature Citation 4

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 ↓16, 16 X X NE N N N (Laws et al., 2000) (L, MH) (H) (H) Male Pubertal Rat X X NE N N N N (Stoker et al., 2000) (H) (H) 21-Day Dermal Toxicity X X (Rabbit; MRID N N N (H) (H) 42089902) Chronic X X Toxicity/Carcinogenicity N N NE (MH, H) (MH, H) (Rat; MRID 40629302) Carcinogenicity (Mouse; X X NE N NE MRID 40431302) (MH, H) (MH, H) Chronic Toxicity (Dog; X MRID 40431301, N N N N (H) 41293801) HPG Axis (Rat; Cooper X N N N et al., 2000) (H) BW: Amphibian P ↑12% X Metamorphosis (Toad; NE (ML, M, SVL: (H) Brodeur et al., 2013) MH) ↑4-5% (ML,M) BW: Amphibian ↓7% Metamorphosis (Frog; (L, M, H ) NE N N Hosmer et al., 2007; SVL: Kloas et al., 2009) ↓2-3% (L, M, H ) Amphibian Metamorphosis (Frog; NE N N Oka et al., 2008) Amphibian Metamorphosis (Frog; NE N N N Spolyarich et al., 2010)

Page 39 of 159 Page 47 of 167 ATRAZINE FINAL

Table 4. Thyroid Pathway for Atrazine. Lines of Evidence Indicating Potential for Interaction with the Thyroid Pathway for Atrazine1 2

Study Type/

3 Levels Levels Literature Citation 4

) or asynchronous, or asynchronous, ± HLL Serum TSH levels Pituitary Weight stage Developmental ( Thyroid Weight Weight Thyroid and Gross Thyroid: Histopathology Serum T Overt Toxicity Observed Growth Growth SVL) (BW, Systemic Toxicity HLL and Thyroid Histopathology of Archived Specimens from Hosmer et al. N5 N5 (2007) and Kloas et al. (2009) (MRID 48614801)4 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 response. 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 examined

Page 40 of 159 Page 48 of 167 ATRAZINE FINAL

IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the weight 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 recommendations 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 analysis in the context of the EDSP follows the Agency’s guidance (USEPA, 2011b) and is conducted on a case-by-case basis by first assessing the different lines of evidence (i.e., specific Tier 1 assays and OSRI), then performing an integrated analysis of those lines of evidence.

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

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

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and OSRI studies are described below. In addition to the endocrine-related effects described above for the in vivo Tier 1 assays and OSRI, other effects 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, more than one parameter was needed to determine overt toxicity. Effects which were considered to be signs of systemic toxicity were generally less severe forms of toxicity (e.g., changes in organ weights or blood chemistry).

Page 41 of 159 Page 49 of 167 ATRAZINE FINAL

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 were observed at higher doses 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 weights of male or female minnows were unaffected by treatment. Similarly, in a published study on adult fathead minnows by Bringolf et al. (2004), there were no effects of atrazine treatment on mortality, body length or weight, or larval survival.

The requirements for a Tier 1 Hershberger assay were satisfied by Ashby et al. (2004) and Yamasaki et al. (2004). In Ashby et al. (2004), body weights were decreased (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 rat 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 pituitary weights were decreased (↓16- 31%) 100 and 200 mg/kg/day. By ANCOVA analysis, 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 pubertal 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 modified 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 administered 100 mg/kg/day ate an average of 10% less food per day 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 feed- restricted vehicle group.

Page 42 of 159 Page 50 of 167 ATRAZINE FINAL

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 were 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<0.05) by 11% at 300 mg/kg/day. In rats co- administered 17β-estradiol, body weights were decreased (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 weights, body weight gains, organ weights) were not reported. In a published, multicomponent, modified uterotrophic study by Ashby and Tinwell (2002) [also see Ashby et al. (2002)], terminal body weights were 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 experiment (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, final 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 generally 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 decreases (p<0.05) in food consumption (↓28-67% males; ↓37-85% females) during Days 7-21. Relative (to body) and absolute spleen weights were increased in males and females, and may have been related to changes in hematocrit and red blood cell counts. 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 1000 mg/kg/day females (compared to 0 controls).

In a two-generation reproduction toxicity study in rats, mean premating body weights in the P and F1 males and females were decreased (p<0.05) at all measurement times (↓9-16%) at 500 ppm (equivalent to 25 mg/kg/day, calculated by reviewers). In addition, mean body weights in the P and F1 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) for the males and females in both generations, with recovery in females during gestation. Pup body weight decreases (p<0.05) on PND 21 at 50 (F2, ↓8%) and 500 (F1 and F2, ↓7 and 10%, respectively) ppm were considered to be related to treatment.

Page 43 of 159 Page 51 of 167 ATRAZINE FINAL

In a published female rat reproductive toxicity study by Foradori et al., (2014), two experiments were performed. In the first experiment, body weights 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, administration 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 weights 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 females at 50 mg/kg/day on Day 2 (↓14%), and on all days at 100 mg/kg/day (↓20-27%); food consumption also was decreased at 100 mg/kg/day in the LE animals on all treatment days (↓20-27%).

In another published female reproductive 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 dosed 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 weight 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 control (+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, respectively, compared to control (+1.6 g) during GD 6-7, with overall body weight decreases. 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 female offspring reproductive toxicity study in rats (Davis et al., 2011), conducted in conjunction with the investigation 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 administer equal amounts of atrazine. Postnatal mortality (birth to PND 4) was significantly increased (p<0.05) for both sexes at 100 mg/kg/day in Blocks 1 and 2. Body weights 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.

Page 44 of 159 Page 52 of 167 ATRAZINE FINAL

In a published male offspring reproductive 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 this dose level were further decreased (p<0.001) by GD 19-21. In contrast, the 100 mg/kg/day male offspring were significantly decreased (p<0.01) at birth (↓13 and 11%) and PND 4 (↓10 and 9%) with both dosing regimens, respectively. In Block 1, significantly decreased (p<0.01; ↓6-7%) body weights in male offspring 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 reproductive toxicity study in rats (Rosenberg et al., 2008), decreases in maternal body weight were observed from GD 18-21 at 75 and 100 mg/kg/day. Decreased mean daily food consumption during 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 differences 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/day 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 gestation 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. Additionally, 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 the high-dose group with survival 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 males and PND 14 for females at 125 mg/kg/day.

In the second of two special studies examining male reproductive development in rats (DeSesso et al., 2014), significant decreases (p<0.05) were observed in maternal food consumption and body weights and/or body weight gains during lactation and dosing in females at 125 mg/kg/day. Body weights were decreased (p<0.05) on PND 4 for male and female pups through PND 21 at 125 mg/kg/day. After weaning, body weights of the 125 mg/kg/day F1 males remained decreased through PND 63, but food consumption was comparable to controls.

Page 45 of 159 Page 53 of 167 ATRAZINE FINAL

In a prenatal developmental toxicity study in rats (Accession No. 00143008; MRID 40566302; Scialli et al., 2014), a mortality rate of approximately 78% was observed at 700 mg/kg/day (21/27). Clinical signs at this dose included salivation (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 were decreased (p<0.05) in the male (↓44%) and female (↓45%) pups.

In a second prenatal developmental toxicity study 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 high- dose dams was decreased (p<0.05) by 13%.

In a prenatal developmental toxicity study in rabbits, stool changes (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 control), 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. Treatment-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 and two 75 mg/kg/day females on GD 20 and 25, respectively.

In a combined two-year feeding/carcinogenicity 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, respectively) compared to 50% in the controls. Body weights were significantly decreased (p<0.01) at 500 ppm (↓8% males; ↓19% females) at Week 104. Body weights also were significantly decreased (p<0.01) for males and females at 1000 ppm through Week 104 (↓11-21 and 10-27%, respectively). Decreases 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 hemoglobin) were noted in females during the study, but were not significantly different at study termination. Absolute liver and kidney weights were decreased in the 1000 ppm males at the interim sacrifice. Gross pathology findings were minimal, but increased incidences of masses (e.g., abdominal, axillary and thoracic) were noted in females in all treatment groups except the low-concentration group. Histopathology findings in the 1000 ppm females included increased incidences of retinal degeneration, centrilobular necrosis in the liver, transitional epithelial hyperplasia in the bladder and the kidney, splenic extramedullary hematopoiesis and myeloid hyperplasia in both the femur and

Page 46 of 159 Page 54 of 167 ATRAZINE FINAL sternum bone marrow. Histopathology findings in the 1000 ppm males consisted of increased incidences of prostate epithelial hyperplasia, calculi in the kidney pelvis and acinar hyperplasia of the mammary gland.

There was an increased incidence of mammary adenocarcinoma in the medium-low, medium- high and high-concentration females, as well as an increase in fibroadenoma in the high- concentration females, in the carcinogenicity subgroup. Some females developed multiple mammary tumors. There also was an increase (NS) 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.

In a mouse carcinogenicity study, there was a significant increase in mortality (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/day, respectively). At Week 91, body weight gain was significantly decreased (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 consumption were observed in the 1500 ppm males and 3000 ppm animals. Organ weight changes were minimal, with decreased kidney weights noted in the 3000 ppm females. Gross pathology findings included enlarged atria/atrium of the heart and tan lesions of the heart in the 1500 and 3000 ppm animals. The incidence 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 males 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 incidences attributed to myocardial degeneration 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 were 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 decreases of 22% in the males and 20% in the females at study termination. Absolute heart weights were decreased 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 myolysis and focal atrophy of the heart were noted at 1000 ppm.

Page 47 of 159 Page 55 of 167 ATRAZINE FINAL

In a special study by Fukamachi et al. (2004), the potential modifying 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 susceptible to DMBA-induced carcinogenesis. Final mean body weight in female rats fed 500 ppm atrazine was decreased (p<0.01; ↓14%) compared to control, and food intake was significantly decreased (p<0.05; ↓10%).

In a mechanistic study in peripubertal 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 group remained significantly decreased (p<0.01) 24 hours after the final dose.

The relative roles of adrenal and testis hormones after 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 treatment groups within each intact or castrated category. However, the overall mean body weights were decreased (significance not reported) in the castrated groups (339.0 g) compared to the intact groups (370.4 g). After three days of treatment in both groups, weight losses (significance 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 atrazine 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-dependent 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. Similarly, 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 significantly 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 affected. 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 dietary concentrations up to 675 ppm.

In a published study by Wilhelms et al. (2005), a series of seven experiments 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 Experiment 3, 1000 ppm atrazine significantly decreased (p<0.05) body

Page 48 of 159 Page 56 of 167 ATRAZINE FINAL weight gain by 64% with no effect on liver weight or liver-somatic index. In contrast, body weight gain and liver weight, but not liver-somatic index, were reduced by 91 and 26% in pair- fed 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 development. In Experiment 5, atrazine administered by silastic implant had no effect on indices of growth and reproductive development. Atrazine significantly decreased (p<0.05) body weight gains and feed intake 32% and 14%, respectively, in Experiment 6. In Experiment 7, addition of 1000 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 investigating the effects of atrazine (>98% purity) on sexual maturation in female Japanese quail were conducted. Dietary administration of atrazine up to 1000 ppm had no effect on mortality, average body weight gain (ADG) or feed intake. Subcutaneous or implanted atrazine had no effect 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 atrazine delivered either by s.c. injection or implants had no effect on plasma concentrations of corticosterone or aldosterone.

In a published study by Hermanutz et al. (1973; Accession No. 00057846) on fathead minnow embryos exposed to atrazine for 45 weeks, survival of parental fish was decreased (p<0.05) in the mean-measured 63.5 µg/L treatment group (↓98%). The parental fish length and the F1 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 unexposed parents incubated at 63.5 µg/L atrazine was similar to that of control embryos, but all larvae died 5-8 days after hatching. There was 100% mortality of 1-day-old larvae from unexposed parents incubated at 63.5 µg/L atrazine within 24 hours. Survival of 3-day-old hatchlings in the medium-high concentration group was decreased (p=0.05) approximately 50%.

In a early life-stage toxicity study in sheepshead minnow embryos exposed to atrazine for 33 days, the total length and wet weights were adversely 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 decreases in body length (↓17-25%) and weight (↓56-63%) compared to the negative (water) control. Growth of the F1 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 of 39% to 45% (p<0.01) were noted in length and weight, respectively, at atrazine concentrations ≥0.24 mg/L.

Page 49 of 159 Page 57 of 167 ATRAZINE FINAL

In a published non-Guideline study by Le Mer et al. (2013) on larval threespine sticklebacks, mortality was <11% for the fish exposed to atrazine 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 generation, 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 post- hatch in the F1 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 decreased 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 ventilation rate, total length 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 either study. In Study I, significantly decreased (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, significantly 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 females 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 weights and growth rates were significantly decreased (p<0.05) at study termination in fish in the 90 µg/L group. Hepatotoxicity (based on histology) 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 registrant 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 assessing overt/systemic toxicity.

In an investigation of reproductive toxicity/metamorphosis in frogs by Oka et al. (2008), survival rates in two experiments were determined. 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.

Page 50 of 159 Page 58 of 167 ATRAZINE FINAL

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 signs of overt toxicity.

In another developmental frog study by Spolyarich et al. (2010) in developing 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 attributed to handling stress. Atrazine had no observable effect on the development of the spotted 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 pathway, the rich dataset of supporting OSRI provided additional lines of evidence to suggest potential downstream, estrogen pathway effects that may 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 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 induction occurred in MCF-7 cells. Increased aromatase activity in human granulosa-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 brain and ovarian microsomes. However, atrazine acted as an inducer of 17β-estradiol in the steriodogenesis assay.

Page 51 of 159 Page 59 of 167 ATRAZINE FINAL frogs was greater in atrazine-exposed treatments in one study, but other studies with frogs showed no effects on reproductive development in the absence of overt toxicity.

Although the available data reflect varying responses in estrogen-related endpoints across species and studies, the potential for interaction with the estrogen pathway is supported by the SAP conclusion that the chlorotriazines (including atrazine and its DEA, DIA and DACT degradates) function through a neuroendocrine MOA that suppresses 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 guideline-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 triazines in the CMG. In the in vitro Tier 1-like assays, atrazine was negative in the AR binding and ARTA assays. In the steriodogenesis assay, atrazine acted as an inducer of testosterone at 100 µM at two of five studies (USEPA, 2008c).

Page 52 of 159 Page 60 of 167 ATRAZINE FINAL

D. Thyroid Pathway

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

E. Conclusions

Based on the weight-of-evidence analysis, atrazine has the potential to interact with the estrogen and androgen pathways in mammals and other wildlife. 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 departure (POD) of 2.56 mg/kg/day is based on a luteinizing hormone suppression with atrazine which is regarded as a precursor event in the neuroendocrine mode of action of the triazines. In a Tier 1-like male pubertal assay, atrazine caused a delay in preputial separation at the lowest dose tested (12.5 mg/kg/day). This dose is approximately five-fold greater (i.e., less sensitive) than the POD of 2.56 mg/kg/day used for human health risk assessment. Estrogen-related 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).

Therefore, based on the WoE analysis of EDSP Tier 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 ecological risk assessment.

Page 53 of 159 Page 61 of 167 ATRAZINE FINAL

VI. References

Arthur, A. (1984) A teratology study of atrazine technical in New Zealand White rabbits. Safety Evaluation Facility, Ciba-Geigy Corp., Summit, NJ. Laboratory Project No. 68-84, September 18, 1984. Unpublished. (MRID 00143006)

Ashby, J., Tinwell, H., Stevens, J., et al. (2002) The effects of atrazine on the sexual maturation of female rats. Regulatory Toxicology and Pharmacology 35(3):468-473.

Ashby, J. and Tinwell, H. (2002) The effects of atrazine on the sexual maturation of female Alderley Park-Wistar and Sprague-Dawley rats. Central Toxicology Laboratory, Alderley Park MacClesfield, Cheshire, UK. Laboratory Study No. 177502, July 3, 2002. Unpublished. (MRID 45722401)

Ashby, J. (2004) Atrazine: studies on atrazine using the Hershberger castrated rat assay for anti-androgens. Alderley Park MacClesfield, Cheshire, UK. Laboratory Report No. CTL/026222, November 5, 2004. Unpublished. (MRID 48098801)

Balaguer, P., Francois, F., Comunale, F., et al. (1999) Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci. Total Environ. 233:47-56.

Battelle. (2005) Draft Final Report WA 2-29 on multi-chemical evaluation of the short-term reproduction assay with the fathead minnow. Report to U.S. Environmental Protection Agency, Endocrine Disruptor Screening Program. http://www.epa.gov/oscpmont/oscpendo/pubs/edmvac/draft fish repro multichemical.pdf

Blair, R.M., Fang, H., Branham, W.S., et al. (2000) The Estrogen Receptor Relative Binding Affinities of 188 Natural and Xenochemicals: Structural Diversity of Ligands. Toxicol. Sci. 54:138-153.

Bringolf, R.B., Belden, J.B., and Summerfelt, R.C. (2004) Effects of Atrazine on Fathead Minnow in a Short-Term Reproduction Assay. Environmental Toxicology and Chemistry 23(4):1019-1025.

Brodeur, J.C., Sassone, A., Hermida, G.N., et al. (2013) Environmentally-relevant concentrations of atrazine induce non-monotonic acceleration of developmental rate and increased size at metamorphosis in Rhinella arenarum tadpoles. Ecotoxicology and Environmental Safety. 92:10- 17.

Cafarella, M.A. (2005) Atrazine (G-30027) - early life-stage toxicity test with sheepshead minnow (Cyprinodon variegates). Springborn Smithers Laboratories, Wareham, MA. Laboratory Project No. 1781.6642, September 8, 2005. Unpublished. (MRID 46648203)

Page 54 of 159 Page 62 of 167 ATRAZINE FINAL

Choung, C.B., Hyne, R.V., Mann, R.M., et al. (2011) Developmental toxicity of two common corn pesticides to the endangered southern bell frog (Litoria raniformis). Environ. Pollul. 159:2648-2655. (ECOTOX Ref No. 153858).

Connor, K., Howell, J., Chen, I., et al. (1996) Failure of chloro-S-triazine-derived compounds to induce estrogen receptor-mediated responses in vivo and in vitro. Fundam. Appl. Toxicol. 30:93- 101.

Cooper, R.L., Stoker, T.E., Tyrey, L., et al. (2000) Atrazine disrupts the hypothalamic control of pituitary-ovarian function. Toxicol. Sci. 53(2):297-307.

Cooper, R.L., Laws, S.C., Das, P.C., et al. (2007) Atrazine and reproductive function: mode and mechanism of action studies. Birth Defects Res. (Part B) 80(2):98-112.

Corvi, M.M., Stanley, K.A., Peterson, T.S., et al. (2012) Investigating the impact of chronic atrazine exposure on sexual development in Zebrafish. Birth Defects Res. B Dev. Reprod. Toxicol. 95(4):276-288.

Crain, D.A., Guillette, Jr., L.J., Rooney, A.A., et al. (1997) Alterations in steroidogenesis in alligators (Alligator mississippiensis) exposed naturally and experimentally to environmental contaminants. Environ. Health Perspect. 105(5):528-533.

Davis, L.K., Murr, A.S., Best, D.S., et al. (2011) The effects of prenatal exposure to atrazine on pubertal and postnatal reproductive indices in the female rat. Reprod. Toxicol. 32(1):43-51.

DeSesso, J. M., Scialli, A. R., White, T. E., et al. (2014) Multigeneration reproduction and male developmental toxicity studies on atrazine in rats. Birth Defects Res. B Dev. Reprod. Toxicol. 101(3):237-253.

Dionne, E. (1992) Atrazine technical – chronic toxicity to the fathead minnow (Pimephales promelas) during a full life-cycle exposure. Springborn Laboratories, Inc., Wareham, MA. SLI Report No. 92-7-4324. Unpublished. (MRID 42547103)

Fang, H., Tong, W., Branham, W.S., et al. (2003) Study of 202 natural, synthetic, and environmental chemicals for binding to the androgen receptor. Chem. Res. Toxicol. 16:1338-1358.

Foradori, C.D., Hinds, L.R., Hanneman, W.H., et al. (2009) Effects of atrazine and its withdrawal on gonadotropin-releasing hormone neuroendocrine function in the adult female Wistar rat. Biol. Reprod. 81(6):1099-1105.

Page 55 of 159 Page 63 of 167 ATRAZINE FINAL

Foradori, C.D., Hinds, L.R., Quihuis, A.M., et al. (2011) The differential effect of atrazine on luteinizing hormone release in adrenalectomized adult female Wistar rats. Biol. Reprod. 85(4):684-689.

Foradori, C. D., Sawhney, C. P., Tisdel, M., et al. (2014) The effect of atrazine administered by gavage or in diet on the LH surge and reproductive performance in intact female Sprague- Dawley and Long Evans rats. Birth Defects Res B Dev. Reprod. Toxicol. 101(3):262-275.

Fraites, M.J., Narotsky, M.G., Best, D.S., et al. (2011) Gestational atrazine exposure: effects on male reproductive development and metabolite distribution in the dam, fetus, and neonate. Reprod. Toxicol. 32(1):52-63.

Friedmann, A.S. (2002) Atrazine inhibition of testosterone production in rat males following peripubertal exposure. Reprod. Toxicol. 6(3):275-279.

Fujimoto, N. and Honda, H. (2003) Effects of environmental estrogenic compounds on growth of a transplanted estrogen responsive pituitary tumor cell line in rats. Food Chem. Toxicol. 41(12):1711-1717.

Fukamachi, K., Han, B.S., Kim, C.K., et al. (2004) Possible enhancing effects of atrazine and nonylphenol on 7,12-dimethylbenz[a]anthracene-induced mammary tumor development in human c-Ha-ras proto-oncogene transgenic rats. Cancer Sci. 95(5):404-410.

Giknis, M. (1989) Atrazine - a teratology (segment II) study in rats. Ciba-Geigy Corp., Summit, NJ. Laboratory Study No. 882049, February 23, 1989. Unpublished. (MRID 41065201)

Hayes, T.B., Collins, A., Lee, M., et al. (2002) Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proc. Natl. Acad. Sci. USA 99(8):5476-5480.

Hayes, T.B., Stuart, A.A., Mendoza, M., et al. (2006) Characterization of atrazine-induced gonadal malformations in African clawed frogs (Xenopus laevis) and comparisons with effects of an androgen antagonist (cyproterone acetate) and exogenous estrogen (17beta-estradiol): Support for the demasculinization/feminization hypothesis. Environ. Health Perspect. 114 Suppl:134- 141.

Hayes, T.B., Khoury, V., Narayan, A., et al. (2010) Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis). Proceedings of the National Academy of Science. www.pnas/cgi/10.1073/pnas.0909519107

Page 56 of 159 Page 64 of 167 ATRAZINE FINAL

Hazelette, J.R. and Green, J.D. (1987) Atrazine technical: 91-week oral carcinogenicity. Division of Toxicology/Pathology, Ciba-Geigy Corp. Laboratory Report No. 842120, October 30, 1987. Unpublished. (MRID 40431302)

Higley, E., Newsted, J., and Zhang, X. (2010) Assessment of chemical effects on aromatase activity using the H29SR cell line. Environ. Sci. Pollut. Res. 17(5):1137-1148.

Hinfray, N., Porcher, J.M., Brion, F. (2006) Inhibition of rainbow trout (Oncorhynchus mykiss) P450 aromatase activities in brain and ovarian microsomes by various environmental substances. Comp. Biochem. Physiol. Part C. 144(3):252-262.

Holloway, A.C., Anger, D.A., Crankshaw, D.J., et al. (2008) Atrazine-induced changes in aromatase activity in estrogen sensitive target tissues. J. Appl. Toxicol. 28(3):260-270.

Hosmer, A., Kloas, W., Lutz, I., et al. (2007) Atrazine: response of larval Xenopus laevis to atrazine exposure: assessment of metamorphosis and gonadal morphology. Leibniz Institute of Freshwater Biology and Inland Fisheries (IGB), Wildlife International, Ltd., and Experimental Pathology Laboratories, Inc. Unpublished. (MRID 47153501)

Huber, K.R. (1989) Atrazine technical: 21-day dermal toxicity study in rabbits. Division of Toxicology/Pathology, Ciba-Geigy Corp. Laboratory Report No.882035, December 1, 1989. Unpublished. (MRID 42089902)

Infurna, R. (1984) A teratology study of atrazine technical in Charles River rats. Safety Evaluation Facility, Ciba Geigy Corp., Summit, NJ. Laboratory Project No. 60-84, September 18, 1984. Unpublished. (MRID 00143008)

Jin, Y., Wang, L., and Fu, Z. (2013) Oral exposure to atrazine modulates hormone synthesis and the transcription of steroidogenic genes in male peripubertal mice. Gen. Comp. Endocrinol. 184:120-127.

Kloas, W., Lutz, I. Urbatzka, R., et al. (2009) Does atrazine affect larval development and sexual differentiation of South African clawed frogs? Toxicol. Sci. 107(2):376-384.

Kojima, H., Iida, M., Katsura, E., et al. (2003) Effects of a diphenyl ether-type herbicide, chlornitrofen, and its amino derivative on androgen and estrogen receptor activities. Environ. Health Perspect. 111:497–502.

Kojima, H., Katsura, E., Takeuchi, S., et al. (2004) Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. Environ. Health Perspect. 112 (5):524-531.

Page 57 of 159 Page 65 of 167 ATRAZINE FINAL

Kroon, F. J., Hook, S. E., Jones, D., et al. (2014) Effects of atrazine on endocrinology and physiology in juvenile Barramundi (Lates calcarifer, Bloch). Environ. Toxicol. Chem. 33(7):1607-1614.

Laws, S.C., Ferrell, J.M., Stoker, T.E., et al. (2000) The effects of atrazine on female Wistar rats: an evaluation of the protocol for assessing pubertal development and thyroid function. Toxicol. Sci. 58: 366-376.

Laws, S.C., Ferrell, J.M., Stoker T.E., et al. (2003) Pubertal development in female Wistar rats following exposure to propazine and atrazine biotransformation by-products, diamino-S- chlorotriazine and hydroxyatrazine. Toxicol. Sci. 76(1):190-200.

Laws, S.C., Hotchkiss, M., Ferrell, J., et al. (2009) Chlorotriazine herbicides and metabolites activate an ACTH-dependent release of corticosterone in male Wistar rats. Toxicol. Sci. 112(1):78-87.

Legler, J., Dennekamp, M. Vethaak, A.D., et al. (2002) Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays. Sci. Total Environ. 293(1-3):69-83.

Le Mer, C., Roy, R.L., Pellerin, J., et al. (2013) Effects of chronic exposures to the herbicides atrazine and glyphosate to larvae of the threespine stickleback (Gasterosteus aculeatus). Ecotoxicol. Environ. Saf. 89:174-181.

Lenkowski, J.R, Sanchez-Bravo, G., and McLaughlin, K.A. (2010) Low concentration of atrazine, glyphosate, 2, 4-dischlorophenoxyacetic acid, and triadimefon exposures have diverse effects on Xenopus laevis organ morphogenesis. J. Environ. Sci. 22(9):1305-1308.

Lutz, I., Kloas, W., Springer, T., et al. (2008) Development, standardization, and refinement of procedures for evaluating effects of endocrine active compounds on development and sexual differentiation of Xenopus laevis. Anal. Bioanal. Chem. 390(8):2031-48.

Mainiero J., Youreneff, M., and Ginknis, M. (1987) Two-generation reproduction study in rats: atrazine technical. Research Department, Ciba-Geigy Corp. Laboratory Study No. 852063, November 17, 1987. Unpublished. (MRID 40431303)

Macek, K.J., Buxton, K.S., Sauter, S., et al. (1976) Chronic toxicity of atrazine to selected aquatic invertebrates and fishes. Bionomics, EG&G, Inc. Duluth, MN. Unpublished. (MRID 00024377)

Mayhew, D.A., Taylor, G.D., Smith, S.H., et al. (1986) Twenty-four month combined oral toxicity and carcinogenicity study in rats utilizing atrazine technical. American Biogenics Corp., Decatur, IL. Laboratory Report No. 410-1102, April 29, 1986. Unpublished. (MRID 40629302)

Page 58 of 159 Page 66 of 167 ATRAZINE FINAL

Narotsky, M.G., Best, D.S., Guidici, D.L., et al. (2001) Strain comparisons of atrazine-induced pregnancy loss in the rat. Repro. Toxicol. 15(1):61-69.

Neuman-Lee, L. A., Gaines, K. F., Baumgartner, K. A., et al. (2014) Assessing multiple endpoints of atrazine ingestion on gravid Northern Watersnakes (Nerodia sipedon) and their offspring. Environ. Toxicol. 29(9):1072-1082.

O'Conner, D.J., McCormick, G.C., and Green, J.D. (1987) Atrazine technical - 52-week feeding study in dogs. Research Dept., Ciba-Geigy. Laboratory Report No. 852008, October 27, 1987. Unpublished. (MRID 40431301)

O'Connor, J.C., Plowchalk, D.R., Van Pelt, C.S., et al. (2000) Role of prolactin in chloro-S- triazine rat mammary tumorigenesis. Drug Chem. Toxicol. 23(4):575-601.

Oka, T., Tooi, O., Mitsui, N., et al. (2008) Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis. Aquatic Toxicology 87:215-226.

Orton, F., Lutz, I., Kloas, W., et al. (2009) Endocrine disrupting effects of herbicides and pentachlorophenol: in vitro and in vivo evidence. Environ. Sci. Technol. 43:2144-2150.

Papoulias, D. M., Tillitt, D. E., Talykina, M. G., et al. (2014) Atrazine reduces reproduction in Japanese Medaka (Oryzias latipes). Aquat. Toxicol. 154:230-239.

Pedersen, C.A. and DuCharme, D. R. (1992). Atrazine technical: toxicity and reproduction study in bobwhite quail. Bio-Life Associates, Ltd., Neillsville, WI. Laboratory Project No. 102-012-07. Unpublished. (MRID 42547102)

Pedersen, C.A. and DuCharme, D.R. (1992) Atrazine technical: toxicity and reproduction study in mallard ducks. Bio-Life Associates, Ltd., Neillsville, WI. Laboratory Project ID No. 102-013- 08. Unpublished. (MRID 42547101).

Plhalova, L., Blahova, J., Mikulikova, I., Stepanova, S., Dolezelova, P., Praskova, E., Marsalek, P., Skoric, M., Pistekova, V., Bedanova, I., and Svobodova, Z. (2012). Effects of subchronic exposure to atrazine on zebrafish (Danio rerio). Pol J Vet Sci. 15:417–423.

Pogrmic, K., Fa, S., Dakic, V., et al. (2009) Atrazine oral exposure of peri-pubertal male rats downregulates steroidogenesis gene expression in Leydig cells. Toxicol. Sci. 111(1):189-197.

Pogrmic-Majkic, K., Fa, S., Dakic, V., et al. (2010) Upregulation of peripubertal rat Leydig cell steroidogenesis following 24 h in vitro and in vivo exposure to atrazine. Toxicol. Sci. 118(1):52- 60.

Page 59 of 159 Page 67 of 167 ATRAZINE FINAL

Reylea, R.A. (2009) A cocktail of contaminants: how mixtures of pesticides at low concentrations affect aquatic communities. Oecologia 159, 363-376.

Rayner, J.L., Enoch, R.R., Wolf, D.C., et al. (2007) Atrazine-induced reproductive tract alterations after transplacental and/or lactational exposure in male Long-Evans rats. Toxicol. Appl. Pharmacol. 218(3):238-248.

Riffle, B. W., Klinefelter, G. R., Cooper, R. L., et al. (2014) Novel molecular events associated with altered steroidogenesis induced by exposure to atrazine in the intact and castrate male rat. Reprod. Toxicol. 47:59-69.

Rosenberg, B.G., Chen, H., Folmer, J., et al. (2008) Gestational exposure to atrazine: effects on the postnatal development of male offspring. J Androl. 29(3):304-311.

Sanderson, J.T., Letcher, R., Heneweer, M., et al. (2001) Effects of chloro-S-triazine production in male carp hepatocytes. Environ. Health Perspect. 109(10):1027-1031.

Sanderson, J.T., Boerma, J., Lansbergen, G.W.A., et al. (2002) Induction and inhibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicol. Appl. Pharmacol. 182:44-54.

Scialli, A. R., DeSesso, J. M., and Breckenridge, C. B. (2014) Developmental toxicity studies with atrazine and its major metabolites in rats and rabbits. Birth Defects Res. B Dev. Reprod. Toxicol. 101(3):199-214.

Song, Y., Jia, Z. C., Chen, J. Y., et al. (2014) Toxic effects of atrazine on reproductive system of male rats. Biomed. Environ. Sci. 27(4):281-288.

Spiteri, I.D., Guillette, Jr., L.J., and Crain, D.A. (1999) The functional and structural observations of the neonatal reproductive system of alligators exposed in ovo to atrazine, 2,4-D, or estradiol. Toxicol. Ind. Health 15:181-186.

Spolyarich, N., Hyne, R., Wilson, S., et al. (2010) Growth, development and sex ratios of spotted marsh frog (Limnodynastes tasmaniensis) larvae exposed to atrazine and a herbicide mixture. Chemosphere 78:807-813.

Stoker, T.E., Laws, S.C., Guidici, D.L., et al. (2000) The effect of atrazine on puberty in male Wistar rats: an evaluation in the protocol 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.

Page 60 of 159 Page 68 of 167 ATRAZINE FINAL

Storrs-Méndez, S.I., Tillitt, D.E., Rittenhouse, T.A.G., et al. (2009) Behavioral response and kinetics of terrestrial atrazine exposure in American toads (Bufo americanus). Arch. Environ. Contam. Toxicol. 57:590-597.

Storrs-Méndez, S.I. and Semlitsch, R.D. (2009) Intersex Gonads in Frogs: Understanding the Time Course of Natural Development and Role of Endocrine Disruptors. J. of Exp. Zool. (Mol. Dev. Evol.) 312B:1-11.

Tennant, M.K., Hill, D.S., Eldridge, J.C., et al. (1994a) Possible anti-estrogenic properties of chloro-S-triazines in rat uterus. Toxicol. Environ. Health 43(2):183-196.

Tennant, M.K., Hill, D.S., Eldridge, J.C., et al. (1994b). Chloro-S-triazine antagonism of estrogen action: limited interaction with estrogen receptor binding. J. Toxicol. Environ. Health 43:197-212.

Tillitt, D.E., Papoulias, D.M., Whyte, J.J., et al. (2010) Atrazine reduces reproduction in fathead minnow (Pimephales promelas). Aquat. Toxicol. 99(2):149-159.

Tinfo, N.S., Hotchkiss, M.G., Buckalew, A.R., et al. (2011) Understanding the effects of atrazine on steroidogenesis in rat granulosa and H295R adrenal cortical carcinoma cells. Reprod. Toxicol. 31(2):184-193.

Trentacoste, S.V., Friedmann, A.S., Youker, R.T., et al. (2001) Atrazine effects on testosterone levels and androgen-dependent reproductive organs in peripubertal male rats. J. Androl. 22(1):142-148.

USEPA (2002) The Grouping of a Series of Triazine Pesticides Based on a Common Mechanism of Toxicity. Health Effects Division, Office of Pesticide 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 Disruptor Screening Program. Revision of February 18, 2008.

Page 61 of 159 Page 69 of 167 ATRAZINE FINAL

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 validation of an estrogen receptor assay using rat uterine cytosol as source of receptor as a potential screen in the 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-evaluation of the Human Health Effects of Atrazine: Review of Experimental Animal and In Vitro Studies and Drinking Water Monitoring Frequency. Office of Chemical Safety 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-evaluation of the Human Health Effects of Atrazine: Review of Non-cancer Effects and Drinking Water Monitoring 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; and -4. December 16, 2010. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection 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-evaluation 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 Disruptor Screening Program – Weight of Evidence: evaluating results of EDSP Tier 1 screening to identify the need for Tier 2 testing. Office of Chemical Safety and Pollution Prevention, Office of Science 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 Associated with “Problem Formulation for the Reassessment of Ecological Effects from the Use of Atrazine”. Office of Chemical Safety and Pollution Prevention, U.S. Environmental Protection Agency, Washington, DC.

Page 62 of 159 Page 70 of 167 ATRAZINE FINAL

USEPA (2013) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting held July 30 - August 1, 2013 on Weight-of-Evidence: Evaluating Results of EDSP Tier 1 Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

Weber, G. J., Sepúlveda, M. S., Peterson, S. M., et al. (2013) Transcriptome alterations following developmental atrazine exposure in Zebrafish are associated with disruption of neuroendocrine and reproductive system function, cell cycle, and carcinogenesis. Toxicol. Sci. 132(2):458-466.

Wilhelms, K.W., Cutler, S.A., Proudman, J.A., et al. (2005) Atrazine and the hypothalamo- pituitary-gonadal axis in sexually maturing precocial birds: studies in male Japanese quail. Toxicological Sciences 86(1):152–160.

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 satisfy 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 tissues available from Kloas et al. studies. Experimental Pathology Laboratories, Inc., Sterling, VA. Laboratory 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 the 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 exposure affects growth, body condition and liver health in Xenopus laevis tadpoles. Aquat. Toxicol. 104:243–253.

Page 63 of 159 Page 71 of 167 ATRAZINE FINAL

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, provide 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 determination on the potential of atrazine to interact with the thyroid and HPT axis. Therefore, the AMA test order requirement is satisfied by the available data.

Androgen Receptor (AR) Binding 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.

ERα Transcriptional Activation 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.

Page 64 of 159 Page 72 of 167 ATRAZINE FINAL

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.

Page 65 of 159 Page 73 of 167 ATRAZINE FINAL

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 conditions at nominal concentrations of 0 (negative control), 0.01, 0.1, 10, 25 and 100 µg/L. A positive control, 17β-estradiol (0.2 µg/L), also was included. Separate studies were conducted at two laboratories [i.e., Wildlife International, Inc. (WLI), and the Leibniz Institute of Freshwater Ecology 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 control and atrazine treatment groups, with 25 larvae per replicate. Exposures continued through completion of metamorphosis (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 conducted with a dissecting stereomicroscope, and extra-gonadal anomalies were determined. Phenotypic sex was identified as male, female, or mixed; final sex determinations were made based on histopathology.

Mortality and time to metamorphosis were not affected 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 17β-estradiol positive control group 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 (data and significance not reported) compared to the negative control. In addition, atrazine exposure concentrations did not result in any consistent effect on size at metamorphosis, shift 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 morphological and histological endpoints, with effects occasionally noted in the positive control and atrazine treatment groups. No frogs displaying intersex or testicular oocytes were observed in these experiments. The frequencies of morphological and histological changes tended to be low, and none of the measurements exhibited consistent responses across laboratories or dose-dependent 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.

Page 66 of 159 Page 74 of 167 ATRAZINE FINAL

This study is consistent with recommendations made by the Agency regarding study design elements needed to conduct studies to determine 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 subsequent 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 tissues were analyzed for thyroid pathology and growth parameters (hind-limb length, snout-vent length, and wet weight) were measured by image analysis. Tissue samples from amphibian larvae (100 frogs from the control and each treatment group) were evaluated from the original study. These archived samples were generally all of NF stage 66 frogs 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 points in the OCSPP Guideline 890.1100 AMA.

Normalized hind-limb length (HLL) was 3, 3, and 5% greater in the low-, medium-, and high- concentration groups at NF stage 66 than in negative controls (p<0.05). There were no effects detected on snout-to-vent length or dry weight endpoints. Since all frogs were at NF 66 at test termination, no effects on NF stage were reported. 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 effect. 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 validation 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 requirement to identify chemicals capable of binding to the AR. Atrazine was selected to be a negative chemical in the validation 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 facilities; the performance of atrazine in the AR binding assay was determined in four of the five testing facilities.

Page 67 of 159 Page 75 of 167 ATRAZINE FINAL

Ventral prostate cytosol from SD rats 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 [3H]-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 Saturation Binding Experiments, the mean dissociation constant (Kd) for 3 [ H]-R1881 was 0.613±0.041 nM and the mean estimated Bmax was 0.817±0.049 fmol/100 μg protein for a standard batch of prostate cytosol that was tested. The overall ranges for Kd and Bmax values were 0.685 to 1.57 nM and 6.67 to 15.6 fmol/100 μg protein, respectively.

In the inter-laboratory Competitive Binding Experiments, the mean log IC50 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 affinity (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 [3H]-ligand over the concentration range of −11 −3 10 to 10 M; therefore, a log IC50 and RBA could not be calculated for atrazine as it did not result in 50% reduction in binding at any concentration. Consistent 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-binder” 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, antiestrogens, flutamide derivatives, bisphenol A derivatives, alkylphenols, parabens, alkyloxyphenols, phthalates, siloxanes, phytoestrogens, DDTs, PCBs, pesticides, and organophosphate insecticides 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 [3H]-R1881 (1 nM) in the presence of increasing concentrations (logarithmic 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.

3 In the Saturation Binding Experiment, the mean dissociation constant (Kd) for [ H]-R1881 was 2.03±0.15 nM. The mean estimated Bmax was not reported. In the Competitive Binding −9 −10 Experiment, the mean log IC50 value for R1881 was 3.07×10 ± 9.12×10 M.

Page 68 of 159 Page 76 of 167 ATRAZINE FINAL

Precipitation and/or cytotoxicity with atrazine were not reported. Atrazine had no effect on specific binding of the [3H]-ligand over the concentration range of 4.28×10−9 to 4.28×10−5 M; −5 therefore, a log IC50 = >4.28×10 M was determined for atrazine. The RBA could not be calculated for atrazine. Atrazine was classified as a “non-binder” with the AR. The nonchlorinated triazine pesticides (including atrazine) were all non-binders; these results are considered to be consistent with an endocrine 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 requirement 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 except that the protein activity is higher in the latter system. Atrazine was one of two compounds selected as a negative (non-inhibitor) chemical in the validation of an aromatase 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 aromatase assay was conducted with four testing 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 concentration 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 background 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 tritiated androstenedione ([1β-3H(N)]-androst-4-ene-3,17-dione; [3H]- 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, two of the four testing laboratories modified the atrazine concentrations selected for testing in Runs 2 and 3, and two laboratories used the same eight concentrations for all three runs. The modifications (addition of mid-level concentrations) were made in an attempt to characterize 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.

Page 69 of 159 Page 77 of 167 ATRAZINE FINAL

For atrazine, the percentage of control aromatase 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 microsomal 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 concentrations ranging from 0.01-100 µM for 48 hours. The culture medium was removed and analyzed for 17β-estradiol and testosterone concentrations, after which the live 3 cells were subjected to a H2O -release assay for determination of aromatase activity. Based on these results, three concentrations (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 rinsed with PBS, and aromatase activity determined; ii) combined aromatase activity with cells exposed as described in the indirect assay, and the same atrazine concentrations were added again during the conduct of the tritium-release assay to evaluate the combined effects of pre-exposure and direct interaction with catalytic enzyme activity; and iii) direct aromatase activity with atrazine added to untreated cells during the conduct of the tritium- release assay at the same concentrations as described in the combined aromatase assay to assess direct interaction with the enzyme.

Atrazine induced indirect (p<0.001) and combined (p<0.05) aromatase activity, but direct aromatase activity was not different from control at any concentration tested. Aromatase activity measured in the indirect and combined aromatase 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 increased (p<0.05) compared to the solvent control starting at 10 µM. Atrazine increased (p<0.05 or 0.01) 17β-estradiol production in a dose- dependent manner at concentrations ≥1 µM. 17β-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 magnitude of the change was less than observed for 17β-estradiol (maximum fold change 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 choriocarcinoma), and MCF-7 (breast cancer) cells. The cell lines were exposed to atrazine or the metabolites at concentrations of 0, 0.3, 1.0, 3.0, 10.0, or 30 µM.

Atrazine and the metabolites were able to induce the catalytic activity of aromatase in a concentration-dependent manner to an apparent maximum of just over 2-fold (data presented graphically) in H295R cells. Additionally, in H295R cells treated with 30 µM atrazine or metabolites, tritiated water release and estrone production were increased in a 1:1 ratio. The mechanism of induction of aromatase activity appeared to involve the induction of CYP19

Page 70 of 159 Page 78 of 167 ATRAZINE FINAL mRNA, because atrazine and the metabolites were able to increase mRNA concentrations for CYP19 relative to control.

In JEG-3 cells, atrazine induced aromatase activity in a concentration-dependent manner, producing increases (p<0.05) in activity above control at concentrations 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 aromatase activity was undetectable, and atrazine was not able to induce the activity to detectable 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 effects on the catalytic activity and mRNA expression on aromatase. To investigate the mechanism of aromatase induction in H295R cells, the ability 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 present study, atrazine increased (NS) cAMP concentrations in a time-dependent manner in H295R cells, producing a time-response course with an apparent 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 concentrations from 10 nM to 100 µM, and 3 aromatase activity measured by the H2O -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 E2 (PGE2) and vomitoxin were included as positive control groups. The medium 3 was removed and aromatase activity was measured by the H2O-release assay. Additionally, a cell-free human recombinant aromatase assay was conducted 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 across 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 PGE2 (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,

Page 71 of 159 Page 79 of 167 ATRAZINE FINAL suggesting that treatments had no effect on cell number and that atrazine, at the concentrations studied, was not cytotoxic to either 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 expressing 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 effect of atrazine on aromatase activity was tested with a cell-free aromatase assay. Atrazine at 1 μM had no effect on human recombinant microsomal aromatase activity. By comparison, testosterone, used as a positive control in this study, competitively inhibited dibenzyfluorescein (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 17β- estradiol and progesterone was determined in the media, and aromatase activity was measured by 3 the H2O-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 17β-estradiol production as compared with control. Atrazine did not alter 17β-estradiol production over the 24-hour exposure period. However, these experiments demonstrated the ability of atrazine to alter the production of progesterone. Cells treated with atrazine (10 and 30 µM) increased (p<0.05) progesterone production 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 chemicals immediately after plating. Again, no effects were observed on 17β-estradiol production determined after 24 or 48 hours of atrazine; however, atrazine increased progesterone production after 24-hour (significance not reported; 30 µM) and 48-hour (p<0.05; 10 and 30 µM) exposure in granulosa cells.

The production of 17β-estradiol and progesterone, as well as aromatase activity, were determined in granulosa cells during a 1-hour period after 24-hour exposure. Concentrations of 17β-estradiol were significantly increased for atrazine (p<0.05; 10 and 30 µM) when evaluated over the 1-hour period. To investigate the possible mechanism behind the increase in 17β-estradiol observed after treatment with atrazine, aromatase activity 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 demonstrate 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 17β-estradiol production was similar to that observed for the increase in aromatase activity after atrazine treatment. In addition, progesterone production was increased (p<0.05) during the 1-hour post-exposure period for atrazine (1, 10, and 30 µM).

Page 72 of 159 Page 80 of 167 ATRAZINE FINAL

Finally, H295R cells were exposed to 1, 10, or 30 µM atrazine prior to determining the concentrations of estrone, 17β-estradiol, and progesterone in the culture medium. Atrazine (10 and 30 µM) increased (p<0.05) the concentrations of estrone, 17β-estradiol, and progesterone. These studies confirm previous reports that atrazine treatment stimulates estrone and 17β-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 validation of an estrogen receptor (ER) binding assay (USEPA, 2009), an in vitro rat uterine cytosol ER binding 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 inter- laboratory validation of the ER binding assay was conducted with three testing 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, albeit 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 Competitive Binding Experiment measured the binding of a single concentration of [3H]-17β-estradiol (1 nM) in the 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 control, octyltriethoxysilane); and 17β-estradiol as the ligand reference standard.

In the second set of inter-laboratory Saturation Binding Experiments, the mean dissociation 3 constant (Kd) for [ H]-17β-estradiol ranged from 0.1137 to 0.6588 nM and the mean estimated Bmax ranged from 25.85 to 89.97 fmol/100 μg protein for the site-specific batches of prostate cytosol that were prepared and tested.

In the second set of inter-laboratory Competitive Binding Experiments, the mean log IC50 values for 17β-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 were 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 assay, the reasons for these conclusions were not reported. This category covers compounds which appear to be interacting with the receptor at

Page 73 of 159 Page 81 of 167 ATRAZINE FINAL 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 atrazine (as well as other chloro-s-triazines and metabolites) to bind to rat uterine 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 lyophilized pools of uterine cytosol prepared from intact adult female SD rats. Determination of ER binding in uteri of triazine-dosed rats was conducted with freshly prepared cytosols from individual ovariectomized female SD rats.

Binding was determined by incubating cytosol, 5 nM [3H]-estradiol, and competitor (e.g., atrazine, unlabeled estradiol, or blank) at room temperature (25°C), refrigerated at 4°C, or in an ice-water bath. All incubations were complete with 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 suspensions of 50 or 300 mg/kg by oral gavage for two days. The uteri were dissected at necropsy, sliced, and incubated in modified Eagle’s medium 3 with 10 nM [ H]-estradiol for 30 minutes at 37°C in 95% O2/5% CO2. 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), dithiothreitol (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 [3H]-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 [3H]-estradiol-ER binding was assessed by pre-incubating 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 [3H]-estradiol. Under conditions that strongly favored the competitor, binding of [3H]-estradiol was reduced by atrazine, but to a much lesser extent than the reduction caused by estradiol. Reduction of radioligand binding to a 50% extent required approximately 20 µM atrazine, which was four to five orders of magnitude greater than the approximate IC50 of estradiol.

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

Page 74 of 159 Page 82 of 167 ATRAZINE FINAL

Administration of atrazine at 50 mg/kg/day reduced estrogen binding by 18% (NS). These results demonstrated that, in the presence of a high dose 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 rat uterine ER in vitro in a competitive binding assay was examined. The in vitro binding studies were conducted with ER- enriched uterine cytosol prepared from intact adult female SD rats.

The [3H]-estradiol (1×10–9 M final assay concentration) was incubated with increasing concentrations of radio-inert competitor, uterine 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% hydroxylapatite (HAP) slurry was added to each tube, followed by incubation in an icewater bath for 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 each tube to extract the radiolabeled estradiol from the HAP. The resulting extracted supernatants were counted by liquid scintillation counter (LSC).

Atrazine (and other test chemicals) 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 number of chemicals tested, the ER competitive- binding 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 (Tier 1). If a test compound exhibited binding to the ER, then a second 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 concentrations which bracketed the IC50 observed in the Tier 2 assay) was conducted if necessary. Chemicals which failed to bind the ER were designated as “non-binders.” All assays were replicated a minimum of two times and IC50 values of positive chemicals are the means of the replicates.

Atrazine was negative in the ER competitive binding assay (Tier 1), with an estimated IC50 of >1×10−4 (the highest concentration tested).

In a published study by O’Connor et al. (2000), the potential for atrazine (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 incubated with ER homogenates and the appropriate radioligand (0.3 nM [125I]-estradiol), and the percent binding inhibition was calculated. Atrazine was negative in the ER competition binding assay.

Page 75 of 159 Page 83 of 167 ATRAZINE FINAL

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 (hERα and hERβ) and one human AR (hAR) in transactivation assays in Chinese hamster ovary cells (CHO- K1 cells). Cells were grown in DMEM/F-12 (Dulbecco’s modified Eagle medium plus Ham’s F-12 nutrient mixture) supplemented with 10% fetal bovine serum (FBS) and antibiotics at a temperature of 37°C in an atmosphere of 5% CO2 under saturating humidity. Atrazine was dissolved in DMSO to a final concentration 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 hERα and hAR expression vectors (pcDNAERα and pZeoSV2AR, respectively) were constructed as described previously (Kojima et al., 2003). The hERβ expression vector was constructed by cloning the hERβ cDNA with reverse transcriptase-polymerase chain reaction from human placental RNA. The sequence was verified and inserted into the mammalian expression vector pcDNA3.1Zeo(-) creating pcDNAERβ. The construction of the estrogen or androgen responsive elements containing reporter plasmid (pGL3-tkERE and pIND-ARE, respectively) was described previously (Kojima et al., 2003). pRL-SV40 containing the Renilla luciferase gene was used as an internal control for transfection efficiency.

Host CHO cells were plated into 96-well microtiter plates at a density of 8,400 cells/well in phenol red-free DMEM/F-12 containing 5% CD-FBS (complete medium) one day before transfection. For detection of hERα or hERβ activity, cells were transfected with 5 ng pcDNAERα or pcDNAERβ, 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 concentrations of test compounds (95-100% purity) or with 0.1% DMSO (vehicle control) in complete medium. For −11 −10 measurement of antagonistic activity to hERα, hERβ and hAR, either 10 or 10 M E2 (17β- estradiol, > 97% purity) or 10−10 M DHT (5α-dihydrotestosterone, 95% purity) was added to the cell cultures along with the test compound, respectively. 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 MiniLumat LB 9506 luminometer and was normalized based on the activity of co-transfected pRL-SV40. At least three independent experiments were conducted for each compound.

Agonistic activities were evaluated by relative activity expressed as REC20 (20% relative effective concentration). The REC20 is the concentration of the compound showing 20% of the −10 −9 M −9 activity of 10 M E2, 10 E2, or 10 M DHT for ERα, ERβ and AR, respectively. Antagonistic activities were evaluated by relative activity expressed as RIC20 (20% relative inhibitory concentration). The RIC20 is the concentration of the compound showing 20% of the

Page 76 of 159 Page 84 of 167 ATRAZINE FINAL

−10 −9 M −9 inhibition of the activity of 10 M E2, 10 E2 or 10 M DHT for ERα, ERβ 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. When the activity of the test compound was higher than the REC20 the pesticide was judged to be positive for activity. When the activity of the test compound was higher than the RIC20 the pesticide was judged to be positive for inhibitory activity. The statistical significance of differences was evaluated with a two-tailed Student’s t test with a nominal level of significance of p<0.05.

Atrazine exhibited no activity against hERα, hERβ, 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 proliferation, was used to investigate the ability of atrazine to activate the ER. Intact 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 transfected with an ERE-luciferase reporter gene and exposed to atrazine at the same concentrations to see if luciferase activity was stimulated. In a third experiment, six week-old surgically ovariectomized F344 rats were inoculated with MtT/E-2 cells at two sites within the subcutaneous fat pads and administered atrazine in the diet at dose levels of 0, 5, 50, or 500 mg/kg. The resulting tumors were allowed to grow to 1-2 cm diameter, then the rats were euthanized and the pituitary glands and uteri were collected and weighed. Blood samples were collected for serum prolactin concentration determination.

Atrazine had no effect on cell proliferation, induction of luciferase activity, tumor promotion, pituitary or uterus weights, or serum prolactin.

In a published study by Balaguer et al. (1999), MCF-7 cells were stably transfected with an ERE-luciferase reporter gene, and HeLa cells were stably transfected with an ERE-luciferase reporter gene and then ERα or ERβ 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 atrazine (>97% purity) to determine if atrazine could activate the ER. MCF-7 cells were treated with atrazine at concentrations of 10 nM to 10 µM in the presence and absence of 17β-estradiol to investigate ER-mediated cell growth; MCF-7 cells were transiently transfected with Gal4-ER chimera and a Gal4-luciferase reporter construct to measure atrazine effects on reporter gene activity in the presence or absence of 17β-estradiol; and the recombinant estrogen-dependent yeast strain PL3 was exposed to 10 µM atrazine to measure ER-mediated growth in the absence of 17β-estradiol. Additionally, a gel electrophoretic mobility shift assay for progesterone receptor (PR) - progesterone responsive element (PRE) complex formation was conducted on nuclear extracts from 17β-estradiol, atrazine, and 17β-estradiol + atrazine treated MCF-7 cells.

Page 77 of 159 Page 85 of 167 ATRAZINE FINAL

Atrazine did not alter MCF-7 cell proliferation with or without 17β-estradiol at any concentration tested. Similarly, atrazine alone did not induce reporter gene activity in transiently transfected MCF-7 cells, and atrazine did not inhibit or enhance 17β-estradiol -induced reporter gene activity in these cells. 17β-estradiol caused a 3.5-fold increase in PR-PRE band intensity, whereas atrazine did not induce the formation of the PR-PRE complex. In cells co-treated with 17β- estradiol + atrazine, neither compound affected the intensity of 17β-estradiol -induced PR-PRE complex bands relative to E2 alone. Finally, atrazine did not stimulate growth of 17β-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 three 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 luciferase reporter gene (ER-CALUX) assay were examined. The ER-CALUX assay uses T47-D human breast adenocarcinoma cells expressing endogenous ERα and ERβ, 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 luciferase 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-term 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-term reproductive fitness in fish.

In a 21-day FSTRA study conducted 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 control (acetone) not specified], 25, and 250 µg/L. Mean-measured concentrations were <2.5 (

One female in the control group died during the assay; overall survival was 100% in all atrazine treatment groups. No treatment-related effects on secondary sex characteristics or clinical signs were reported, although secondary sex characteristics were reportedly assessed as part of the daily observations to determine overall health. Body weights of male or female minnows did not demonstrate any treatment-related differences 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 total fecundity were noted during the pre-exposure or assay periods between the study groups, although total fecundity was decreased (NS) by 17%

Page 78 of 159 Page 86 of 167 ATRAZINE FINAL 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, respectively in the treated groups (NS; ↓21%). Fertilization success was 100% in all replicates, except for a single replicate at the low concentration. In addition, no treatment-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 observed at any treatment concentration. Sex-steroid concentrations (estradiol and testosterone) were measured in male and female fish; no treatment- related 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-concentration group having a lower proportion of cells in Stage 2B, compared to control. Finally, 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 for 21 days under static conditions. An additional group exposed to 0.5 µg/L 17β-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 post- hatch). 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 observation 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 under a dissecting microscope. Plasma VTG concentrations were quantified with a commercially-available ELISA method. Fixed gonads were processed and stained with hematoxylin and eosin.

Fish survival was 100% during the exposure period. 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 atrazine was slightly decreased (NS) compared to controls. A rapid divergence in fecundity was evident in the 17β-estradiol treatment group; no

Page 79 of 159 Page 87 of 167 ATRAZINE FINAL eggs were produced in the 17β-estradiol treatment group after the seventh day of the exposure period.

Fertilization success was not significantly 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 hours post-hatch. Male and female GSI and gonad maturation were reduced by exposure to 17β-estradiol but no statistically signficiant differences from controls were observed for atrazine-treated animals. No mixed gonads (testis-ova) were identified in fish in any of the treatment groups.

Atrazine did not have an apparent effect on the secondary sexual characteristics of male fish, although the effects of 17β-estradiol exposure were evident. Exposure to atrazine did not reduce the number of breeding tubercles compared with control fish; however, fish exposed to 17β- estradiol did not have breeding tubercles. It was not known if the fish exposed to 17β-estradiol had breeding tubercles that regressed in response to the 17β-estradiol exposure or if the tubercles failed to develop. No obvious effects of atrazine on coloration of male fish were observed, although all males exposed to 17β-estradiol lacked vertical banding patterns on their lateral body surfaces. Plasma VTG concentrations were increased (p<0.0001 and 0.05, respectively) in male and female fish exposed to 17β-estradiol compared to all other treatments. Plasma VTG concentrations were greater in male fish exposed to both atrazine treatment concentrationsand in solvent control group males, as compared to negative controls. Although apparently greater than in negative controls (statistical 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 17β-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 atrazine 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 the metabolites at 0, 0.3, 1.0, 3.0, 10.0, or 30 µM in the presence and absence of 17β-estradiol.

VTG concentrations in unexposed or dimethylsulfoxide (DMSO)-exposed male carp hepatocytes were undetectable. A lowest observed- effect concentration of 17β-estradiol of about 2 nM produced a detectable amount of VTG of about 100–400 ng/mg cellular protein; the EC50 of 17β- estradiol-induced VTG concentrations to 4,000–6,000 ng/mg protein. Exposure of male carp hepatocytes to various concentrations (0–30 μM) of the atrazine did not induce VTG production. None of the compounds could produce a concentration-dependent anti-estrogenic response in the presence of 100 nM 17β-estradiol.

Page 80 of 159 Page 88 of 167 ATRAZINE FINAL

Hershberger Assay

In a Hershberger antagonist assay (Ashby, 2004), six-week old male rats [Alpk:APfSD (AP)] were castrated, acclimated for 24 hours, and randomized into treatment groups (6/group) based on body weight. The rats were dosed by oral gavage 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 blocking agent), or finasteride (FIN; inhibitor of the enzyme testosterone-5α-reductase). The vehicle for all treatments was 0.5% (w/v) carboxymethylcellulose (CMC) sodium salt in deionized water. Each treatment group included a s.c. injection of 0.4 mg/kg/day testosterone propionate (TP) in tocopherol-stripped corn oil at a dose volume of 1 mL/kg. The first study included a vehicle control group, four atrazine dose groups (10, 25, 50, or 100 mg/kg/day), and the positive control groups FLU (3 mg/kg/day) and FIN (25 mg/kg/day). The second study included a vehicle control group, 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/day). 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 glands, and accessory sex tissues (AST; Cowper’s glands, LABC, seminal vesicles [with coagulating gland], ventral prostate, and glans penis).

Body weights in the high-dose atrazine group showed small decreases (NS) compared to the controls in all three studies. A similar decrease was also observed in the medium-high dose group in the first study. Based on this, the study author concluded that higher doses would not have been tolerated. In the first study, the LABC weights were decreased (p<0.05) in the high- dose group. In the second study, the LABC weights were decreased in the 50 mg/kg/day (p<0.01) and 100 mg/kg/day (p<0.05) atrazine dose groups. LABC weights also were decreased (p<0.01) in the high-dose group, and the glans penis 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 three 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 OPPTS 890.1400 Guideline, as it was conducted prior to Guideline publication. In addition, the study 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 castrated and acclimated for 14 days. Rats were randomized into treatment groups (6/group) based on body weight. The rats were dosed by oral gavage for ten consecutive days with vehicle control (olive oil) or atrazine (purity 99.9%) at 3.2, 16, or

Page 81 of 159 Page 89 of 167 ATRAZINE FINAL

80 mg/kg/day in the agonist portion of the study. In the antagonist portion of the study, the vehicle and atrazine doses were administered 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 activity 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% purity) in aqueous 1% methyl cellulose 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 treatment 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/day 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, kidney, adrenal, ovary, uterus, and pituitary weights were recorded, and serum T3, T4, 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 microscopically. 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 estrus 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 treatment 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 groups, 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, and 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 was

Page 82 of 159 Page 90 of 167 ATRAZINE FINAL 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 were similar between the pair- fed control and the 200 mg/kg/day rats, and were decreased 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 decreased 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 group, and pituitary weights were decreased by 13% and 16% in the medium-high and high-dose groups.

In the high-dose group, seven females 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 weights 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 T3, T4, 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, decreased 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 cycle was delayed (p<0.05) by 6.9 days and 9.6 days in the 100 and 200 mg/kg/day groups, 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 cycles at 200 mg/kg/day vs. 2.7 cycles for control). A similar response was noted in the pair-fed control (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 pair-fed control group.

Page 83 of 159 Page 91 of 167 ATRAZINE FINAL

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 male rats in each treatment group 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 pair- fed control group (n=18) in which the rat’s diet was restricted to match the dietary intake of the rats in the 200 mg/kg/day dose group. Pups were initially assigned 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 groups 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 recovery of reproductive tract weights and hormone 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 pituitary, testes, ventral and lateral prostates, epididymides, and seminal vesicles with coagulating gland (with fluid) were determined. Serum and the anterior pituitary were stored at −80°C for hormone analysis. The epididymides, left testis, and thyroid were collected for histological evaluation. The right testis was stored at −80°C for subsequent LH receptor assays. LABC weights were not evaluated in this study. Hormone assays at PNDs 45 and 53 included serum and pituitary LH, prolactin (PRL), and TSH concentrations. Serum testosterone, estradiol, estrone, and total T3 and T4 concentrations were also determined.

Body weights for the 200 mg/kg/day (high-dose) group were decreased (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 recovery 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 the 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 other reproductive organ weights were similar to control.

Page 84 of 159 Page 92 of 167 ATRAZINE FINAL

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 significant (p=0.07), and not dose- related. PPS for the pair-fed control was delayed (p<0.05) compared to control (2 days) and this was shorter (p<0.05) compared to the high-dose group (3 days).

Serum testosterone concentrations were not different from control in any treatment group at PND 53, or in the high-dose group at PND 45, although concentrations were decreased (NS) by 16-49%. Intratesticular testosterone was decreased (p<0.05; ↓34%) in the high-dose group on PND 45 and decreased (NS) by 30% on PND 53. Serum 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 decreased (NS) in a dose-dependent manner in all treatment groups, and serum PRL concentrations were decreased (NS) in the 150 and 200 mg/kg/day dose groups. Serum LH and PRL concentrations were similar for the vehicle control and pair-fed control. No differences in mean pituitary LH or PRL concentrations were observed.

T4 and TSH serum concentrations in the treated groups were not different from vehicle control. However, serum T3 concentrations were increased in a generally dose-dependent manner in all treated groups, but only the 200 mg/kg/day dose group was significantly increased (p<0.05). Serum concentrations of T3, T4 and TSH were similar for the vehicle and pair-fed controls.

No effect on the number of testicular LH receptors 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 sections for the vehicle control and high-dose groups showed no treatment-related differences.

The results of this study demonstrate that atrazine delays puberty in male rats dosed from PNDs 23 to 53, with a LOAEL of 12.5 mg/kg/day, the lowest 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 atrazine 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 through PND 47 (26 days). Animal weights were recorded on every other day of treatment (PNDs 22-46; control 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 sacrifice, trunk blood was collected and serum was

Page 85 of 159 Page 93 of 167 ATRAZINE FINAL 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 weight after removal of the coagulating gland).

In a second portion of the study designed to examine the effects of food intake/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 daily 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 generally 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 were decreased (p<0.05) in the 100 and 200 mg/kg/day dose groups by 34 and 32%, respectively, and interstitial testosterone concentrations 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 decreases 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 the study (feed deprivation examination), rats administered atrazine at 100 mg/kg/day ate an average of 10% less food per day than the vehicle- gavage 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 concentrations, and ventral prostate and seminal vesicle weights, were observed to generally similar extents as well in the treated and food-restricted dose groups. One noted difference was an approximately two-fold increase in control serum testosterone concentration in the second portion of the study compared to the first with a similar response in the 100 mg/kg/day dose group between the two portions of the study. The serum testosterone concentration in the food-restricted vehicle group also was decreased (p<0.05), but was similar to the control concentration of testosterone 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 mean body weight).

Page 86 of 159 Page 94 of 167 ATRAZINE FINAL

Steroidogenesis Assay

In the multi-laboratory validation of the H295R steroidogenesis assay (USEPA, 2008), an in vitro H295R human adrenocarcinoma cell 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% CO2 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% CO2 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 responded properly to positive control agents at two concentrations. The positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production.

Atrazine did not demonstrate cytotoxicity. One laboratory reported a 2.4-fold increase and another laboratory reported a 1.5-fold increase in testosterone production relative to solvent control (SC) treatment with atrazine at the high concentration (100 µM). The remaining three laboratories reported a less than 1.5-fold increase. A less than 1.5-fold increase is generally considered to be a negative response. Atrazine acted as an inducer of estradiol at 10 and 100 µM (approximately 5- and 10- to 20-fold, respectively) relative to SC in four of the laboratories; the magnitude of the increase was expressed graphically, but not numerically, in the report. Atrazine produced interference with the testosterone assay that was considered likely to have an impact on the final result in one of the laboratories. No laboratories reported interference with hormone measurements in the estradiol 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 administered 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 seven days. A second set of rats were treated as above and co-administered 17β-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 anesthesia approximately 6 hours after the last dose administration. The 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.

Page 87 of 159 Page 95 of 167 ATRAZINE FINAL

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 17β-estradiol after six days, and uterine fluid was present in all rats administered 17β-estradiol for three or seven days. In rats administered atrazine only, uterine 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 17β-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 administered atrazine (97.7% purity) in aqueous 0.5% CMC by oral gavage at doses 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 17β-estradiol by s.c. injection on Days 2 and 3 of treatment. Rats were 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 co- administered 17β-estradiol, body weights were decreased (p<0.05) by 7% in the 100 and 300 mg/kg/day atrazine group. Uterine weights of ovariectomized female rats were not increased by atrazine at doses up to 300 mg/kg/day. In ovariectomized rats co-administered 17β-estradiol, atrazine decreased (p<0.05) the 17β-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 two vehicle control groups and n=4 per treatment group) were administered atrazine (>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 17β-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, blotted, and cut into halves such that each half contained a uterine horn. The uterine 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 centrifugation step were re-suspended, clarified by centrifugation, and uterine peroxidase activity of the supernatant was determined.

Atrazine alone did not induce uterine wet weights, cytosolic PR binding, or uterine peroxidase activity compared to control. Rats treated with 10 µg/kg/day 17β-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 observed in uterine PR binding activity (↓43 and 44 for the medium- and high-dose groups) and peroxidase activity (↓40, 58, and 95 for the low-, medium- and high-dose groups) after atrazine treatment. Co-treatment of rats with 17β-estradiol plus atrazine did not affect 17β-estradiol -induced uterine wet weight increases, PR binding, or

Page 88 of 159 Page 96 of 167 ATRAZINE FINAL uterine peroxidase activities after co-administration of atrazine relative to animals treated with 17β-estradiol alone; however, these were 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-releasing hormone (GnRH) antagonist Antarelix (2.5 mg/kg/day) were included to examine atrazine interference with GnRH signaling. Interference with the GnRH signaling pathway results in inhibition of LH release from the pituitary. The studies evaluated the effects of atrazine treatment on the timing of uterine growth and VO. Uterine weights (blotted and dry) were recorded for AP rats 24 hours after 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 monitored 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 experiments were conducted. The first two involved determination of uterine weight on either PND 30 (start of puberty) or on PND 33 (uterine growth assumed to have been completed) for AP rats. The third experiment 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 determined (PND 43). The fourth experiment involved dosing SD rats until completion of VO in the vehicle control group, at which time the animals were terminated (PND 46) and uterine and body weights recorded.

In the first experiment with AP female rats, there was a dose-related decrease in uterine weight (both blotted and dry) after atrazine exposure on PND 22-29 (termination 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 were 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 atrazine 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 weight (blotted 20.2 mg/dry 4.0 mg) of naive control animals at PND 22 with uterine weights (blotted 23.1 mg/dry 5.2 mg) in Antarelix- treated rats on PND 30 shows a lack 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 were comparable to control.

Page 89 of 159 Page 97 of 167 ATRAZINE FINAL

In the third experiment with AP female rats, there also was a dose-related decrease in uterine weight (both blotted and dry) after atrazine exposure on PND 22-42 (termination PND 43). Decreases (NS) similar to Experiment 1 at the medium-dose (↓18/19%) were observed, with a further decrease of 21/23% in the high-dose animals (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 (NS) of 13% in body-weight gain. Body weight and body-weight gain in the Antarelix-treated rats 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 atrazine exposure on PND 22-45 (termination PND 46). Decreases (NS) were only observed 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 gain, respectively. Antarelix was not administered to SD rats.

Assessments of VO also were conducted during Experiments 3 and 4. There was a delay (p<0.01) in the age at VO in AP females dosed from PND 22 to PND 42. Although not different than control, the standard deviation of the age at VO in the medium-dose females was increased compared to the other dose groups and the number 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, respectively) 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. Ovariectomized, six-week-old, female Crl:CD BR rats were acclimated for one week prior to study initiation. Atrazine (purity not reported) in 0.25% methylcellulose vehicle was administered at doses of 50, 150, and 200 mg/kg/day. Atrazine doses were administered by i.p. injection three times daily (⅓ of the total daily dose was administered at each of the three dosing intervals) at approximately 8-hour intervals (0800, 1600, and 2300 hours) for four days in order to facilitate detection of weak endocrine activity. The i.p. route of administration was selected in order to enhance the sensitivity of the assay and to facilitate potency comparisons. Between 0600 and 0900 hours on the morning of test day

Page 90 of 159 Page 98 of 167 ATRAZINE FINAL

(Day 5), rats were anesthetized with CO2 and euthanized by exsanguination. The presence of fluid in the uterine horns was recorded. Processed tissues were embedded in paraffin, sectioned nominally at 5 µm, and fixed to slides for staining. 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 conversion. Blood was collected at the time of sacrifice (under CO2 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, final body weights were decreased (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 uterine 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 the 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 atrazine (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 the study. Treatment-related clinical observations in the high-dose males and females on Test Days 4-21 included sporadic 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 consistent with significant decreases (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 absolute spleen weights were increased (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 were

Page 91 of 159 Page 99 of 167 ATRAZINE FINAL observed. Significant decreases also were noted in mean uterus (p<0.01; ↓44%) and ovaries (p<0.05; ↓36%) weights in high-dose females, without 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 (compared to 0 in control animals).

The LOEL is 1000 mg/kg/day based on decreased 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 reproduction 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 article consumption between generations; the F0 and F1 males had similar test article consumption during the 70-day premating period as did the F0 and F1 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 F0 and F1 generation females during their gestation period did not vary greatly between generations. Mean compound consumption for both generations were 0, 0.66, 3.33 and 35.43 mg/kg/day

Parental body weights, body weight gain, and food consumption were statistically significantly reduced at the 500 ppm dose (highest dose tested) in both sexes and both generations throughout the study. Compared to controls, body weights for F0 high-dose males and females at 70 days into the study were decreased by 12% and 15%, respectively while F1 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 occurred in both generations at the high dose. There did not appear to be any reproductive effects from compound exposure. 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 consumption. 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 males and 3.7 mg/kg/day in females.

Page 92 of 159 Page 100 of 167 ATRAZINE FINAL

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-day- old) Sprague Dawley (SD) and Long-Evans (LE) rats 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 determine the stage of the estrous 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 administered continually in the diet (distributed dose) or daily for four days by gavage (bolus dose) to intact female 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 deionized water) at a volume of 5 mL/kg. Animals assigned to the gavage treatment groups continued to receive the vehicle daily by gavage, and animals assigned to the dietary groups did not receive further gavage administrations; 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 concentrations in the diet were 30, 100, or 500 ppm for SD females and 160, 660, or 1460 for LE females. The mean-measured atrazine 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 animals per treatment group were used. Vaginal lavages were conducted daily just after lights on during the treatment period. On the last day of treatment, 250 μL of blood was collected in pre-chilled heparinized tubes from each animal by jugular vein puncture at 6-, 11-, 13-, and 18-hours post-lights on. The 18:00-hour sample (4-hours post-lights out) was collected under 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 new 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 smears

Page 93 of 159 Page 101 of 167 ATRAZINE FINAL were again obtained the next morning, and the female was continued in the 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 slide. The ampulla was opened to allow exit of the eggs within into saline for counting. Ovaries 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 dietary groups in the first experiment were a subset of animals from another experiment 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 first experiment. No apparent effect on body weight occurred due to gavage administration, but body weights were decreased (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 the 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, respectively. 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 decreased (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 concentrations were decreased (p<0.0001) in bolus-treated, SD females in the 50 and 100 mg/kg/day dose group; decreases in the bolus-dosed LE groups were not significant. Administration of atrazine in the diet had no effect on peak LH concentrations at any dose for SD or LE females.

The mean numbers of corpora lutea 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 concomitant

Page 94 of 159 Page 102 of 167 ATRAZINE FINAL decreases (p<0.05) in the number of ova per animal (↓40 and 43%, respectively). Non-dose- dependent decreases (p<0.05) were noted in the LE females administered atrazine by oral gavage. The number of corpora lutea were decreased in the 3 and 100 mg/kg/day groups (↓46 and 56%, respectively), with concomitant decreases (p<0.05) in the number of ova per animal (↓50 and 66%, respectively). No treatment effects 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 reproductive performance also was evaluated. Animal husbandry and pretreatment preparations were described 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 reduction in the pre-ovulatory LH surge affected fertility. A subgroup of LE females were administered 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 plug) 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 lavage was taken and the stage of estrous was recorded. If the female was in estrus, the female was separated from the male without further opportunity for mating. Finally, if the female was not in estrus on the morning after the initial pairing, treatment continued for an additional day and the female was paired with a second male. In the absence of mating on the second day, the stage of the estrous cycle was recorded and the female was separated from the male without further opportunity for mating. Pregnant females were euthanized on GD 20, and the gravid uterine weights 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 second experiment. Administration 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) on

Page 95 of 159 Page 103 of 167 ATRAZINE FINAL 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 effect 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 administration. Likewise, there was no effect of atrazine treatment on the mean number of fetuses or fertility per litter at the termination of pregnancy. An increase (p<0.05) in post-implantation loss (↑175%) was observed in the 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 lutea 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 evaluated to determine susceptibility to atrazine-induced resorption. In addition, three 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 rats were used for the study. The day that evidence of mating was detected (copulatory plug or vaginal sperm) was designated GD 0. All animals were individually housed in polycarbonate cages; animals 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, the 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, independent 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 deliver were stained with 10% (v/v) ammonium sulfide to enhance detection of resorption sites.

Page 96 of 159 Page 104 of 167 ATRAZINE FINAL

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 during the treatment period at all three doses, and mortality in 7/12 dams in the 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, respectively. The 200 mg/kg/day dams that successfully maintained their litters 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 establish an experimental model with a shorter dosing period that would cause a high rate of pregnancy loss, as well as to assess the effect of exposure after the LH-dependent period of pregnancy. Maternal 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 studies in each strain included atrazine administration at 0, 25, 50, and 100 mg/kg/day.

Maternal toxicity was observed in both atrazine-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-treatment periods, respectively; no clear signs of gavage trauma were found. For dams dosed on GD 8-10, full litter resorption was seen in three of eight (38%) dams. This incidence (NS) was attributed to atrazine 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 marginally 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%, respectively). Among the surviving litters, parturition was delayed (p<0.05) for the F344 dams 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 observed in all three strains. 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.

Page 97 of 159 Page 105 of 167 ATRAZINE FINAL

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 occurrence 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 maternal 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 (NS) of 24 and 101%, respectively.

All SD dams exposed to atrazine at ≤100 mg/kg maintained their litters. 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 non- significant body weight decreases. 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. Maternal weight loss after the 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 administered 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 effects of atrazine on male offspring reproductive indices (Fraites et al., 2011). Timed-pregnant SD rats (70-90 days old) were individually housed 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. Atrazine (97.1% purity) dosing solutions 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 oral gavage from GD 14 to 21. The b.i.d. dosing was included due to the rapid metabolism of atrazine, and was expected to produce a more consistent exposure of atrazine relative to the single daily dose. Additional timed-pregnant dams (5/dosing regimen) that did 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, sexed, counted, and weighed collectively 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 on

Page 98 of 159 Page 106 of 167 ATRAZINE FINAL

PND 7. At weaning (PND 21 for Block 1 or PND 22 for Block 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-weaning and uterine implantation sites were counted. Female offspring were examined daily beginning on PND 28 to determine the day VO was completed. Female offspring were weighed daily 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 images 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, the distance between the 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 assigned a score on a scale of 1-4, based on the degree of development (a score of 1 represented poor development 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 exposure resulted in premature emergence of reproductive senescence, estrous cyclicity of offspring was examined through PND 272. After all female rats achieved VO, estrous cycles were examined 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 diestrus) or non-cycling indicating persistent estrus (>4 consecutive days of estrus). The number of animals in each 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 the second block, and prenatal and neonatal mortality in these litters were unremarkable, indicating that there was no association with treatment. No differences among groups were found 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 mortality were seen from PND 4 to 21. AGD on PND 7 was similar to control for all atrazine treatment groups.

All dams in Block 2 (b.i.d.) in the vehicle control, and low 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. Gestation lengths (21-22 days) were not different between groups, but postnatal mortality was significantly increased (p<0.05) at PND 4 in the

Page 99 of 159 Page 107 of 167 ATRAZINE FINAL high-dose group; no differences in postnatal mortality were seen from PND 4 to 21. Litter size, sex distribution, and AGD were similar to control for all atrazine 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 control. Body weights of offspring from the high- dose group remained decreased (p<0.5; ↓15%) at PND 4, but recovered by PND 21. At PND 35, body weights of offspring from the low-dose group 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 delayed (p<0.01) in female offspring from the high-dose group (PND 34.9±0.4) compared with vehicle control (PND 33.3±0.4). In Block 2, VO also was significantly delayed (p<0.05) in the high-dose female offspring (PND 34.9±0.3 vs. PND 33.0±0.3 control). Body weights on the day of VO were 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 mounts. 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 studies 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 cycling 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 treatment on female offspring at doses <100 mg/kg/day and few differences in the measured parameters between the two dosing regimens. Treatment-related effects occurred only in the high-dose group (i.e., offspring body weights decreased at birth, mortality increased shortly after birth, and decreased VO). No differences in mammary gland development at PND 45 or in estrous cyclicity through PND 272 were observed.

Page 100 of 159 Page 108 of 167 ATRAZINE FINAL

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 offspring 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 through 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 daily during PPS evaluations and weighed weekly afterwards.

At PND 59, two male offspring from each litter were randomly selected for necropsy to assess AGD, serum testosterone concentration, ex vivo testosterone production by the testis, and androgen-sensitive organ weights. To examine the ex vivo testicular testosterone 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 bicarbonate, and supplemented with bovine serum albumin (0.2%) insulin-transferrin-selenium mix sodium pyruvate 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 rough-and-tumble play behavior. Cage-mates (littermates; one pair per litter) were separated for approximately 24 hours prior to testing, reunited in their home cage at the onset of the dark phase of the light cycle, and observed under red light for a 10-minute period between 1 and 4 hours after the onset of the dark cycle. Both rough-and-tumble play behavior and anogenital sniffing, as a measure of social interaction, were scored. Play bouts were scored for both duration (number of seconds the pair spent engaged in a play bout during the 10-minute scoring period) and frequency (the total number of bouts that occurred during the scoring period). Social interactions were scored for frequency only; female vehicle control animals were scored (one pair per litter) to provide a comparison to the males for this sexually dimorphic behavior.

At study termination, two males per litter were sacrificed by decapitation and trunk blood was collected in serum-separation tubes. The pituitary, testes, ventral prostate, left epididymis, and seminal vesicles with coagulating gland (with fluid) were removed and weighed. Serum was

Page 101 of 159 Page 109 of 167 ATRAZINE FINAL harvested and divided into two aliquots, and stored at −80°C. The whole brain and pituitary were frozen separately on dry ice and stored at −80°C for subsequent analysis. The right testis and epididymis from one male from each litter was fixed in Bouin's fixative for subsequent histological analysis. The right testis from the second male of the litter was immediately processed for interstitial fluid extraction. The 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 resultant interstitial fluid was stored at −80°C. Serum and media testosterone, and LH, analyses 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 first day of dosing (GD 14). Maternal body weights in this group were significantly decreased relative to control (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 groups 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 weights were also similar to the vehicle control group. In contrast, high-dose male 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, offspring 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 either 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 production 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 interstitial fluid testosterone and serum LH concentrations were not different between the control and any dose group in either dosing regimen. Testosterone production in stimulated or unstimulated testis 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 weight was decreased in male offspring in the s.i.d. regimen in the medium-low and medium-high dose groups (p<0.05; ↓7 and 8%, respectively) and in the high-dose males (p<0.01; ↓12%). No treatment-related effects on

Page 102 of 159 Page 110 of 167 ATRAZINE FINAL 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 dosage 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 given litter were pooled. Intra-testicular testosterone was assayed by RIA. With the remaining seven to eight dams per dosage group, the number of pups per dam was culled to eight (four males and four females) on PND 2. The body weights and AGD were measured for each pup on PND 21. The AGD index 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. After 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 which 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 weighed individually. Intra-testicular fluid, which included fluid from both the seminiferous tubules and interstitial space, was collected and frozen in liquid nitrogen. For testosterone 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 seminal vesicles were weighed.

Maternal body weights were comparable in all 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-high and high-dose groups were observed from GD 18-21. Decreased mean daily food consumption during gestation was observed in the medium, medium-high, and high-dose groups; consumption was decreased by approximately 33, 38, and 47%, respectively.

The average number of pups born to individual dams in the control and atrazine groups ranged from 11 to 14, with no treatment-related differences. However, there were dose-dependent differences in pup survival. With increasing atrazine 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.

Page 103 of 159 Page 111 of 167 ATRAZINE FINAL

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 delayed by approximately 1 day in the medium and medium-high dose groups (p<0.05 and 0.01, respectively) and by 2.5-3 days in the high-dose group (p<0.004).

No differences were noted in body or testis weights 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 seminal vesicle or ventral prostate weights. Serum testosterone concentrations were decreased compared to control in the medium, medium- high and high-dose groups, and intra-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 weights were recorded at study initiation and termination. Organ weights determined at sacrifice included the testes, epididymides, prostate, and seminal vesicles. Testes were fixed, sections, 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 dehydrogenase (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 were 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 atrazine treatment. Rats in the control and low-dose group had compact and regular arrangement of cells in the seminiferous tubules, and nearly all stages of spermatogenesis were present. In the high-dose group, normal spermatogenic cell organization was disrupted with visible holes among the cells in the tubules; cell 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 seminiferous tubules in the medium-dose group was present, but less severe.

Page 104 of 159 Page 112 of 167 ATRAZINE FINAL

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 percentages of the total number of spermatozoa abnormalities were increased in a dose-dependent manner. The percentage of double-headed and hookless spermatozoa were increased (p<0.05) by 44 and 41%, respectively, in the medium-dose males, and all abnormalities (including amorphous and coiled-tailed spermatozoa) were increased by ≥100% in the high-dose males.

The activity of the four testicular marker enzymes examined decreased in a generally dose- dependent manner. Activities for ACP and LDH were 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 high- dose groups. The total antioxidant capacity in the testes was decreased (p<0.05 or 0.01) in all atrazine treatment groups in a dose-dependent manner. Mean testicular GSH concentration was decreased (p<0.05) and MDA concentration was 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 concentrations were increased in male rats in the medium- and high-dose groups (p<0.05 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 examining 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 and 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 weaning and necropsied. After weaning, the remaining F1 male offspring were group-housed. At PND 70, 25 males per dose group were randomly selected for sperm evaluation. Thirty-five of the remaining F1 males were randomly selected to remain on study through PND 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 vesicles, and testes were weighed. The left testis and left cauda epididymis were separately minced and homogenized in 0.9% NaCl with 0.5% Triton X-100. Homogenization-resistant spermatids or spermatozoa in the testes and epididymides, respectively, were counted in a hemocytometer. Spermatozoa obtained from the ductus deferens were stained with 2% eosin and assessed morphologically with a light microscope at 40×

Page 105 of 159 Page 113 of 167 ATRAZINE FINAL magnification. Plasma testosterone concentrations were determined in duplicate with an ELISA method.

There were no treatment-related effects on clinical signs or mortality 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 increase in body weight gain occurred in the high- dose group; overall mean body weight was not different from control during lactation. The maternal LOAEL is 25 mg/kg/day, based on decreased 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 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 in the high-dose group (↓39%). Postnatal pup survival also was 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 other treatment groups. Body weights 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 was no effect on pup weight at doses ≤25 mg/kg/day. There were no treatment-related effects on survival or body weight in the subset of male pups selected for developmental studies post-weaning.

Due to excessive pre- and postnatal 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 utero resulted in decreased (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 testes, spermatozoa counts in the epididymides, or plasma testosterone concentrations on PND 70 or PND 170 at any dose. Increases (p<0.05) in the percentage of abnormal sperm were observed on PND 70 in the high- dose 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 administered 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 animals, female offspring, and, where possible, one male pup per litter were euthanized at weaning and necropsied. After weaning, the remaining F1

Page 106 of 159 Page 114 of 167 ATRAZINE FINAL male offspring were group-housed. At PND 70, 25 males per dose group were randomly selected for sperm evaluation. Thirty-five of the remaining F1 males were randomly selected to remain on study through PND 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.

There were no treatment-related effects on clinical signs or mortality in pregnant female rats administered atrazine up to 125 mg/kg/day 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. The 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 F1 males remained decreased through PND 63, but food consumption and clinical observations for F1 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 F1 males. By PND 170, there were no differences in epididymis weight, but testis 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 F1 males on PNDs 70 or 170. An increase (p<0.05) in the percentage 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 lactational exposure to atrazine was 25 mg/kg/day.

Developmental Toxicity (Rat, OCSPP 870.3700)

In a prenatal developmental 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 containing 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 lutea, 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 the 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 salivation (13/27), oral/nasal discharge (12/27), ptosis (11/27),

Page 107 of 159 Page 115 of 167 ATRAZINE FINAL 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; alopecia occurred in the medium- and high-dose groups, but was not considered biologically 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-dose 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 female 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 fetal 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, bifurcated, or cervical.

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

In a second prenatal developmental toxicity study in rats (MRID 41065201; Scialli et al., 2014), pregnant COBS CD (SD) BR rats (26/dose group) were administered atrazine (purity not reported) in 3% aqueous corn starch 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, implantations, and resorptions were recorded. Fetuses were examined for external abnormalities, sexed, and weighed. Visceral examination was performed on approximately one half of the fetuses in each litter. 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. Salivation was observed in 18/26 high-dose females, with no other signs in any dose 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 high-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.

Page 108 of 159 Page 116 of 167 ATRAZINE FINAL

No deleterious 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, 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 high-dose group. No skeletal malformations were observed. Increases (p<0.05) in the fetal and litter incidence of incomplete ossification of the hyoid, occipitals, and parietals were observed in the high-dose pups, and increases (p<0.05) in the fetal and litter incidences of incomplete ossification 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 administered atrazine (96.3% purity) in 3% aqueous corn starch containing 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, implantations, and resorptions were recorded. Fetuses were examined for external abnormalities, sexed, and weighed. Each fetus underwent a visceral examination, followed by an examination of skeletal abnormalities by the alizarin red S method.

All animals survived, with the exception of three 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 similar extent, but were increased (p<0.01) in the high-dose does (↑111%; 19/19). Blood on the vulva or in the cage also was increased (<0.05) in the high-dose group (4/19 vs. 0/19 control).

Decreases (p<0.01) in body weight were observed in the high-dose dams on GD 14, 19, 21, 25, and 29 (↓12, 19, 18, 10, and 8%, respectively). In addition, body weight gains in the high-dose group were significantly decreased (p<0.01) at GD 7-14 (↓562%) and 14-19 (↓270%), with significantly increased (p<0.01 or 0.05) body weight gains at GD 21-25 (↑1348%) and 25-29 (↑319%). The overall body weight gain for the high-dose group for the entire 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 changes in food consumption were observed in the high-dose group only, with decreases ((p<0.01) from GD 7-20, and increases (p<0.05) from GD 24-28. Three abortions occurred during the study; a medium-dose female on GD 21 and two high-dose females on GD 20 and 25, respectively.

Page 109 of 159 Page 117 of 167 ATRAZINE FINAL

The maternal LOAEL is 75 mg/kg/day, based on clinical signs and decreased 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 resorptions was increased (p<0.05) in the high-dose group by 269%, and the percent post-implantation 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, fetal weights were decreased (p<0.05) by 23 and 19% in males and female pups, respectively. Skeletal findings indicated a slight increase in skeletal variations concerning delayed ossification.

The developmental LOAEL is 75 mg/kg/day based on increased resorptions, decreased live fetuses, decreased fetal weight, and skeletal variations. The developmental NOAEL is 5 mg/kg/day.

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300)

In a combined two-year feeding/carcinogenicity study (MRID 40629302) atrazine (98.9% purity) was administered to SD rats (20/sex/treatment in the chronic 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 weight and food consumption were determined pretreatment, weekly through Week 13 of treatment, and monthly for the remainder of the study. Ophthalmoscopic examinations were conducted pretreatment, at the 12- or 13-month interim sacrifices, and prior to study termination. Organs that were weighed included the liver, kidneys, adrenals, thyroids, epididymides, prostate, testes, and ovaries. Gross and microscopic pathological examinations were conducted on these tissues as well as the seminal vesicle, uterus/cervix, urinary bladder, vagina, pituitary, 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-concentration animals, respectively. Survival was increased (p<0.01) in the high-concentration males (67%) compared to control (44%) at study termination. Body weights were decreased (p<0.01) for males and females in the medium- high 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 concentration 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 Weeks 26 and 52 in the medium-high and high- concentration males, respectively. 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 treatment- related ophthalmic changes were observed during the study. Decreases (p<0.05 or 0.01) in hematology parameters (e.g., ↓RBC, hematocrit, and hemoglobin) were noted in females during

Page 110 of 159 Page 118 of 167 ATRAZINE FINAL the study, but were not significantly different at study termination. No hematological effects were noted in males.

Absolute liver and kidney weights were decreased (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 significantly different from control. Gross pathology findings were minimal, but increased incidences of masses (e.g., abdominal, axillary, and thoracic) were noted in females in all treatment groups except the low-concentration group. Histopathology findings in high-concentration females included increased incidences of retinal degeneration (p<0.05; ↑83%); centrolobular necrosis in the liver (p<0.01; ↑300%); degeneration of the rectus-femoris 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 hyperplasia in both the femur (p<0.01; ↑108%) and sternum bone marrow (p<0.01; ↑119%). Histopathology findings in high-concentration 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 endocrine-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, medium- high, and high-concentration females, as well as an increase in fibroadenoma in the high- concentration females, in the carcinogenicity subgroup. Some females developed multiple mammary tumors. There also was an increase (NS) 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 (NS) in interstitial cell tumors at the high dose.

The LOEL is 500 ppm (25 mg/kg/day) based 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 reported) was administered to CD-1 mice (58-60/sex/treatment) in the diet at nominal concentrations 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 animals were observed daily, and body weight and food consumption were determined pretreatment, weekly through Week 12 of treatment, biweekly during Weeks 14-25, and every four weeks for the remainder of the study.

Page 111 of 159 Page 119 of 167 ATRAZINE FINAL

Ophthalmoscopic examinations were conducted 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 high- concentration) 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 only 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 treatment- related 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 two highest treatment groups. Treatment-related decreases (significance not reported) in food consumption were observed in medium-high concentration males and high-dose animals, with sporadic changes noted in medium-high concentration females and medium-low concentration animals; no changes were noted in the low treatment group. No ophthalmic changes were observed during the study.

Organ weight changes were minimal, with decreased (p<0.01) kidney weights (↓16%) weights noted in the high-concentration females. Gross pathology findings included 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-concentration males (p<0.05; 9/23) and medium-high (p<0.01; 11/33) and high-concentration (p<0.05; 24/45) females, respectively. The incidence of cardiac thrombi in surviving animals 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 endocrine-related endpoints were 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 and 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 increase 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 concentration 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, or

Page 112 of 159 Page 120 of 167 ATRAZINE FINAL

1000 ppm for 52 weeks. The mean-measured daily doses were identical for the low and medium- concentration groups (0.48 and 4.97 mg/kg/day, respectively) and were similar for the high- concentration group (33.65 and 33.80 mg/kg/day for males and females, respectively). The animals were observed daily, and body weight and food consumption were determined pretreatment, weekly through Week 13 of treatment, 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). Organs that were weighed included the liver, kidney, pituitary, thyroid/parathyroid, ovary, and testis with epididymis. Gross and microscopic pathological examinations were conducted on these tissues as well as the adrenals, uterus, urinary bladder, prostate, vagina, and mammary gland.

Three animals were sacrificed during the study in a moribund condition: one medium- concentration male on Day 75, and one high-concentration female and male on Days 113 and 250, respectively. The death in the medium-concentration dose group was not considered to be related to treatment; the deaths in the high-concentration group were either treatment-related (female) or treatment may have been a contributing factor (male). Treatment-related clinical observations were noted at low incidences in the high-dose group that were attributed to atrazine- induced myocardial degeneration. These included ascites (one high-dose male and female), cachexia (one female), and labored/shallow breathing (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 heart rates). These treatment-related clinical signs were correlated with pathologic findings attributed to atrazine administration.

Mean body weights at Week 52 were decreased (NS) in the high-concentration 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 males and decreased (NS) by 40% in the high- concentration females. Males and females in the high-concentration group also had reduced food consumption throughout the course of the study (decreases were NS after the first quarter). At study termination, mean food consumption was decreased by 22% in males and 20% in females. No compound-related ocular changes 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 group 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 survivors (4/5 males and 5/5 females) and consisted mainly of moderate to severe dilatation of right and/or left atria. Fluid-filled pericardium (1/5) and an enlarged soft heart 2/5) also were noted in high-concentration males. Minimal atrial

Page 113 of 159 Page 121 of 167 ATRAZINE FINAL 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 the heart also was observed in 2/6 males and 5/6 females in the high-concentration group, with no similar findings in the control group. Two high- and one medium-treatment animals were sacrificed in moribund condition during the course of the study. Moribundity in the high-concentration animals may have been due to compound- related cardiac dysfunction. No changes in endocrine-related endpoints were 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 alterations as well as microscopic and macroscopic cardiac abnormalities). The NOEL is 150 ppm (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 atrazine (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. Each treatment group consisted 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 during 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 behavior. 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 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 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 housed 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 all concentrations tested, with decreases ranging from 5.3 to 12.3% at 75 to 675 mg a.i./kg-diet, respectively. At a concentration of ≥225 mg a.i./kg-diet, there were effects on egg production and mean food consumption while live embryos and hatchlings per eggs set and male 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 consumption values. Examination of one-half of the adult

Page 114 of 159 Page 122 of 167 ATRAZINE FINAL birds at study termination found no abnormal findings. Unless otherwise specified, all differences were considered statistically 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 nominal concentrations of 75, 225 and 675 ppm for up to 20 weeks, with a three-week 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 during 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 behavior. 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. Feed 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 weighed at hatching and at Day 14 post-hatch. Eggshell thickness was determined in eggs collected on seven separate occasions.

One male in the medium-concentration group and eleven females (1 control, 4 low-, 4 medium-, and 2 high-concentration) died during the study. Post-mortem examinations showed abnormal findings, but none of the deaths were attributed to the test substance. 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 weight throughout the study. Feed consumption was higher during Week 12 in the medium-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 considered 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) was decreased by 6 and 10%, respectively, in the 675 ppm group, compared to the control. There were no` differences in egg shell thickness. Although changes were seen in body weights of hatchlings 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 experiments investigating the effects of atrazine (99.9% purity) on sexual maturation in male Japanese quail were conducted.

Page 115 of 159 Page 123 of 167 ATRAZINE FINAL

In the first experiment, groups of 12 male quail (six-week-old) were administered atrazine at 0, 10, 100, or 1000 ppm in the diet. A positive control of 17β-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 the diet. The third experiment investigated the effect of 0 or 1000 ppm atrazine on growth and sexual maturation in six-week- old birds with an additional group of pair-fed birds (n=7). Pair-fed birds were used to differentiate the effects of reduced feed consumption and those due to exposure to atrazine. In experiment 4, atrazine in propylene glycol was administered 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) controls 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 E2, 1000 ppm atrazine, and combinations of 100 ppm 17β-estradiol plus 100 ppm tamoxifen and 100 ppm 17β-estradiol plus 1000 ppm atrazine were administered in the diet for two weeks (n=8). In experiment 7, silastic implants of testosterone (positive 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. However, atrazine had no effect on liver weights or the liver-somatic index. In contrast, 100 ppm 17β-estradiol reduced (p < 0.05) body weight gains and feed intake by 21% each, respectively, and increased (p<0.05) liver weights and liver- somatic index by 50% and 58%, respectively.

Atrazine, up to 1000 ppm, had no effect on testes weight or the ratio of the left-to-right testis weight. In contrast, 100 ppm 17β-estradiol decreased (p<0.05) testes 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 circulating concentrations of LH. However, atrazine at 1000 ppm increased (p<0.05) circulating concentrations of testosterone by 3-fold compared to the negative control. In addition, atrazine at 10 ppm increased (p<0.05) circulating concentrations of 17β-estradiol by 3.6-fold compared to control. There were no changes in circulating 17β-estradiol at the higher concentrations of atrazine tested.

In the pair-fed examination, the high concentration 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 the study (p<0.05).. Neither atrazine nor feed restriction in the pair-fed birds had any effect on testes weights, GSI, or circulating

Page 116 of 159 Page 124 of 167 ATRAZINE FINAL concentrations of 17β-estradiol and testosterone. Similarly, 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 estradiol-3-benzoate decreased (p<0.05) both testes weights and GSI by 47% and 43%, respectively. However, the estrogen control had no effect on the other parameters analyzed. Atrazine administered by silastic implant had no effect on indices of growth and reproductive development. In contrast, the estrogen control delivered by silastic implant increased (p<0.05) liver weights and liver-somatic index 44% and 50%, respectively. Furthermore, estrogen delivered by implant decreased (p<0.05) testes weights and GSI by 77% and 83%, respectively.

Atrazine and pair-feeding had no effect on the stage 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 other concentration of atrazine (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 17β-estradiol to the diet decreased (p<0.05) feed intake by 16% and tended to decrease body weight gains. Furthermore, 17β-estradiol increased (p<0.05) the liver- somatic index by 27%. In contrast, concurrent administration of 100 ppm 17β-estradiol and 1000 ppm atrazine in the diet decreased (p<0.05) body weight gains and feed intake by 63% and 29%, respectively. Addition of tamoxifen (an anti-estrogen) to the diet marginally inhibited the deleterious effects of 17β-estradiol on growth, feed intake, and liver-somatic index.

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

In Experiment 7, administration of 1000 ppm atrazine in the diet or systemic administration of testosterone via an implant did not influence growth, feed intake, or liver weights versus control. However, concurrent administration of 1000 ppm atrazine and testosterone 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 weight and circulating concentrations of LH by 92% and 69%, respectively. Administration of atrazine in the diet in the presence of testosterone decreased (p<0.05) testes weights by 96%. This decrease was observed to be 49% more than the effect of testosterone alone (p<0.05). However, the combination of atrazine and testosterone had no additive effect on circulating concentrations of LH.

Page 117 of 159 Page 125 of 167 ATRAZINE FINAL

In a second study by Wilhelms et al. (2006), a series of five experiments 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 (17β-estradiol or estradiol -3-benzoate) were dissolved in chloroform, 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 birds/treatment and in Experiment 2, there were eight birds/treatment. In Experiment 3, atrazine and 17β-estradiol were weighed and directly mixed into feed to yield doses of 0, 10, 100, or 1000 ppm atrazine and 100 ppm 17β-estradiol (n=8). The alternative feed was used due to limited availability of the starter diet.

Experiments 4 and 5 compared the effects of systemically administered atrazine to the effects of dietary atrazine (n=8). In Experiment 4, vehicle (propylene glycol), atrazine (1 and 10 mg/kg) or 17β-estradiol (1 mg/kg) was administered by daily s.c. injection. In Experiment 5, quail received atrazine, 17β-estradiol or a negative control (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 17β-estradiol positive control 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 17β-estradiol (100 ppm) increased (p<0.001) liver weight by 53%. Additionally, estradiol-3-benzoate increased (p<0.05) oviduct weight by 44% at 1 ppm and 17β-estradiol increased (p<0.05) oviduct weight by 100-fold at 100 ppm.

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

Atrazine did not influence circulating 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 corticosterone or aldosterone.

Fish Early Life-stage Toxicity (Sheepshead minnow)

In an unpublished, early life-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 concentrations of 0 (dilution water control), 0.20, 0.40, 0.80, 1.6, or 3.2 mg/L, under flow-through conditions for 33 days. Each treatment level included two replicate aquaria with 60 embryos per tank (120 embryos/treatment group). Mean-measured concentrations were

Page 118 of 159 Page 126 of 167 ATRAZINE FINAL

<0.028 (

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 reported. At study termination (28-days post-hatch), the total length and wet weights were adversely 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 LOAEC 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, percent hatchability, and growth (length and wet weight) and survival of the F1 generation at 90 days.

Atrazine exposure in the F0 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 F0 generation on number of eggs spawned, fertilization or hatchability at atrazine concentrations up to 0.72 mg/L. However, growth of the F1 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 the reproductive biomarkers VTG and spiggin8 (SPG) as endpoints in exposed larvae. The tested concentrations were 0.1, 1, 10, and 100 μg/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)

Page 119 of 159 Page 127 of 167 ATRAZINE FINAL for atrazine, and the tests included a seawater control, an acetone (5 μg/L) control, a positive control of 17α-ethinylestradiol (0.05 μg/L), and a positive control of DHT (3 μg/L). The three lowest atrazine concentrations were considered to be representative of environmental concentrations found in estuarine and coastal waters of eastern North America. Atrazine was initially dissolved in acetone and then diluted with deionized water such that the final concentration of acetone in exposure containers was 0.0005%. Measured concentrations of atrazine were ≥78% of nominal.

The larvae utilized for testing were obtained from 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 replicates, with 11-13 fish per replicate. Fish were exposed throughout the embryo-larvae and juvenile life stages (from hatching to Day 42 post-hatching) until fish had a mean length of ~20 mm. Larvae were fed once per day with Artemia nauplii; water was changed every day (>80% replacement) before feeding. Dissolved oxygen, temperature, and fish mortality were monitored daily.

At the end of the 42-day exposure period, surviving fish were sacrificed and total body lengths (L) and wet weights (W) were determined, and a condition factor (CF) was calculated as CF = (W/L3)100. Half of the fish from each replicate, selected randomly, were frozen at –80°C for VTG and SPG determinations, and the remaining fish were preserved for intersex evaluations; a subset of these fish were fixed in phosphate-buffered formalin, dehydrated, embedded in paraffin, sectioned, and stained (hematoxylin and eosin) for histological evaluation. VTG was isolated from the whole-body homogenates of juvenile 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 gonad was determined to be an ovary when the entire gonad was packed with oocytes, and was determined to be a testis when it contained spermatogonia- like germ cells packed in lobules and no oocytes. Gonads with an appearance intermediate between an ovary and testis, containing at least one oocyte but showing a testis-like structure or spermatogonia-like germ cells, was determined as ovotestis.

The tests were conducted over two 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 differences in wet weight, body length, or condition of juvenile fish between atrazine-exposed fish and controls (acetone or seawater, respectively) in either test year.

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

Page 120 of 159 Page 128 of 167 ATRAZINE FINAL or atrazine, or fish in the negative 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 μg/L. The proportion of mixed-sex individuals was higher in the positive controls (17α-ethinylestradiol, DHT) compared to the negative controls (seawater, acetone). The proportion of female individuals did not differ among treatments, but tended to be higher in the fish exposed to 17α-ethinylestradiol in the experiment run concurrent with atrazine treatments. The study authors concluded that atrazine treatment did not demonstrate estrogenic or androgenic 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 atrazine (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 aquaria 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 aquaria). Twenty five larvae from each embryo incubation group were randomly selected and distributed to growth chambers in each aquarium (four groups per treatment level). Two growth chambers were used to facilitate handling of fry for 30- and 60-day measurements by a photographic 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, total length, and wet weight after 274 days. At approximately 140 days, two spawning groups, consisting of one male and two females 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 for determination of percentage hatch. As F1 embryo groups hatched, two groups of 25 newly-hatched fry were established in each aquarium, one from each spawning group. After 30-days post-hatch, each larval group was terminated, and percentage survival and individual body length and wet weights were determined. Parental fish (F0) were terminated when no spawning had occurred 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 significant effect on hatching success, survival or reproduction (number of eggs/spawn, total number of eggs, number of spawns/female, or number

Page 121 of 159 Page 129 of 167 ATRAZINE FINAL of eggs/female) at concentrations up to 2.0 mg/L. However, a significant effect was noted on larval growth. For the parental generation (F0), 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 F1 generation, the differences between solvent control and negative control were not statistically significant; therefore, the reviewer compared treatment results to the negative (dilution water) control. Total length in atrazine-treated F1 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 growth, the LOAEC was the lowest level tested 0.15 mg/L, and a NOEAC was not observed.

Fish Endocrine (Steroidogenesis) 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 abundance, plasma hormone (testosterone and 17β- 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 glass 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 tank 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 the experiment was conducted. Fish were acclimated for seven days prior to the two-day exposure period. After acclimation, fish (5/group) were randomly assigned to the following groups: negative control, solvent control (ethanol; SC), positive control (17α-ethynylestradiol), and atrazine (analytical standard, purity not reported; 0.1, 0.5, 5, 50, and 100 µg/L). The SC and atrazine-treatment aquaria contained 10 µL/L ethanol.

All fish were observed for gill ventilation rate (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 heparinized 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 solutions on Day 0, and immediately after removal of fish on Day 2. Samples were stored at 4°C prior to analysis.

Page 122 of 159 Page 130 of 167 ATRAZINE FINAL

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 subsequently 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. Quantitative PCR analyses on brain and liver samples were conducted by investigators under blinded conditions. Expression levels of CYP19B and VTG were normalized to β-actin concentration. Plasma concentrations of testosterone and 17β- 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 herbicides were detected in the water samples.

Expression of CYP19B and VTG in juvenile barramundi did not exhibit 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). Atrazine treatment did not affect CYP19B or VTG expression compared to the negative (water) control.

Concentrations of testosterone and 17β-estradiol were detectable in all fish. Mean plasma testosterone concentrations were not affected by the solvent or positive control treatments. Plasma testosterone concentrations were increased (p<0.05) in the atrazine low- and medium- concentration groups compared to the water control, but this increase was not dose-dependent (testosterone concentrations in the low-, medium-low, and medium-concentration groups were similar, with lower mean testosterone concentrations at the higher atrazine treatments). Mean plasma 17β-estradiol concentrations were not affected in the SC, but were increased (p<0.05) in the positive control group. Exposure to atrazine in the medium-concentration group yielded increased (p<0.05) plasma 17β-estradiol concentrations compared to the water control, but there was no dose-dependent effect noted. The ratio of testosterone/17β-estradiol was not affected by any treatment. Finally, no treatment-related effect on gill ventilation rate (biomarker for oxidative stress) was observed 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, respectively) 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:water solution. The fish were obtained from the Columbia

Page 123 of 159 Page 131 of 167 ATRAZINE FINAL

Environmental Research Center stock with initial 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 which 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 included 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 minimize 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 measured weekly with an ELISA method, and confirmatory analysis of atrazine stock preparations and selected tank water samples were conducted using gas chromatography with a nitrogen-phosphorous detector (GC/NPD).

Adult mortality and egg production were determined daily. Eggs were collected from each tank (by stripping eggs from females or by examining 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 assays; for all tanks, fish were collected for euthanasia during the same time period (19:00 to 24:00) to minimize the diurnal effects of reproductive hormones. The brain, liver and gonads were harvested, and the 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 17β-estradiol and testosterone concentrations in eviscerated carcasses were measured by RIA, and the estradiol to testosterone (E/T) ratio was calculated; GSI also was calculated.

Histological examinations of gonads were conducted to evaluate reproductive stage and pathological lesions. For each post-exposure collection 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 ovaries were stained with hematoxylin and eosin, and testes sections were stained with Giemsa or hematoxylin/eosin, for histological analysis with a compound microscope. Two ovary sections from each fish were evaluated for oogenic stage: pre- vitellogenic (stage II), early vitellogenic [stage III; central germinal 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 counted, and the percentages and average were determined for each fish based on the two sections. The testis sections (serial sections of at least one-half of each testis) were evaluated for developmental stage and pathological lesions. The testis reproductive stages also were evaluated: tubules containing spermatogonia singly or in clusters predominate (stage II); spermatocytes prevalent (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 for

Page 124 of 159 Page 132 of 167 ATRAZINE FINAL all stages of mitotic spermatogonia but primarily at anaphase and telophase. The percent of cells with chromosomal abnormalities was calculated for 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 water 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 mortality was observed during the study.

Egg production was decreased in all atrazine-exposed groups compared to control. The study authors reported significant decreases in total egg production, weekly egg production, but not for daily egg production. According to the reviewers’ analysis, cumulative 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 percent mortality (±SD) of embryos 24 hours after collection (38±18 in control vs. 44±23, 41±19, and 45±20 for the 0.5, 5.0 and 50 µg/L, respectively. It is noted that the magnitude of the decreased 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 staging, 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 maximum of one out of three sampled fish at Day 14 were at stage IV, and two of three sampled fish at Day 38 were at stage IV. The percent of abnormal mitotic events based on aberrant spermatogonia was significantly 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 germ 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 aromatase protein content at 14 or 38 days. There were significantly fewer (p<0.01) pre-vitellogenic oocytes and more vitellogenic oocytes in the low- and medium-concentration atrazine 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 four-

Page 125 of 159 Page 133 of 167 ATRAZINE FINAL month chronic exposure studies were conducted in 2009 (Study I) and 2010 (Study II) which targeted sensitive gonadal developmental windows during sexual maturity.

Adult wild-type zebrafish were housed in polycarbonate tanks filled with fish water (reverse- osmosis water containing 0.6% Instant Ocean) and equipped with a recirculating system. Fish were group-spawned, and embryos were collected at 2 to 3 hours post-fertilization. Embryos were bleached, rinsed with water, and incubated 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 optimum dissolved oxygen concentrations.

A 20 mg/L stock solution of atrazine (97.6% purity) in fish water was prepared every third day, and a 1 mg/mL 17β-estradiol ethanol stock solution was diluted in fish water to produce the working stock solution. A solvent (ethanol) control was not used in this study as ethanol comprised only ≤0.00005% of the final 17β-estradiol exposure 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 17β-estradiol at 1 nM (0.27 µg/L). Each treatment included eight replicate tanks, and solutions were freshly prepared and renewed every third day; control tanks contained untreated water only. Fish were 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 supplemented 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 post- fertilization) were collected every third day 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 euthanized, and wet weight and total body length were determined. Fish were fixed in Dietrich's fixative for 60 hours under continuous agitation. Fish were transferred to buckets containing Dietrich's fixative with 5% trichloroacetic acid and further agitated for 18 hours to decalcify bones. Finally, fish were rinsed and stored in 70% ethanol until histologic preparation. Concentrations of atrazine and 17β-estradiol were determined every third day, during solution renewals, during the chronic toxicity studies. Atrazine concentrations were determined with a commercial enzyme immunoassay (EIA) kit during Study I and with a liquid chromatography tandem mass spectroscopy (LC/MS/MS) method during Study II. For both studies, 17β-estradiol concentrations were determined with a commercial EIA kit.

Page 126 of 159 Page 134 of 167 ATRAZINE FINAL

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 evaluation. Gonads were categorized as: female (100% ovarian tissue); male (100% testicular tissue); testicular 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 defined); or abnormal male [male fish with immature testicular development (e.g., the presence of spermatocytes but no spermatids), testicular degeneration, reduced amount of testicular tissue, or ectopic oocyte in a male fish]. Gonadal developmental abnormalities were defined as any deviation from mature male or female gonadal tissue.

In Study I, mean measured atrazine treatment concentrations were 0.088, 0.88, and 8.9 μM (19, 190 and 1,920 μg/L) in the nominal 0.1, 1, and 10 μM (0, 21.6, 216, and 2160 µg/L) treatment groups, respectively, and in Study II, mean measured concentrations were 0.096, 0.92, and 9.4 μM (20.7, 198, and 2,027 μg/L, respectively. For the 1 nM 17β-estradiol treatment, mean- measured concentrations were 0.72 nM for Study I and 0.32 nM for Study II; no explanation was provided for the decreased concentration in Study II. Atrazine was detected in some Study I control samples. It was detected at 0.00028-0.022 μM (0.06 – 4.7 μg/L) on seven sampling days, and at concentrations above the upper range of the calibration curve (>0.023 μΜ; >5.0 μg/L) on three sampling days. The detectable atrazine contamination 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 μM) 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, overall (across treatments and controls) weights and lengths were decreased compared to Study II weights and lengths; the study authors attributed the differences to changes in feeding protocols and tank design 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 weights for males and females exposed to 17β- estradiol or the low concentration of atrazine, and females exposed to the medium concentration of atrazine, were not different compared to control. Atrazine or 17β-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 17β-estradiol (↑26%) was significantly increased (p<0.01); however, mean body weight of females exposed to 17β-estradiol was similar to control. Significantly decreased (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 females exposed to the low concentration of atrazine, and females exposed to the medium concentration of atrazine, were not different

Page 127 of 159 Page 135 of 167 ATRAZINE FINAL 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 17β- estradiol-exposed males were increased (p<0.01) by 5%, and body lengths of females exposed to 17β-estradiol were decreased (p<0.01) by 5%.

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

In Study I, three fish in the 17β-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 sections prepared. The percentage of fish, in either treatment, with unidentifiable gonads was not different from control in Study I. In Study II, fish exposed to 17β-estradiol had a significantly increased (p<0.01) number of abnormal males (12 fish) and fish with indistinguishable gonads (6 fish) 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 undifferentiated 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) phenotype; 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) phenotype. Pre-vitellogenic oocytes were observed in fish in the low- and medium-dose atrazine groups and the 17β-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 observed with mixed gonad features including testes- ova and ova-testes in the 17β-estradiol treatment group. There were no differences observed in the incidence of TO for Study I or II fish when compared to untreated fish.

Limited spawning data were obtained from Study 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 17β-estradiol tanks. The age, number, and quality of offspring were estimated, and no statistical analyses were conducted on these data. The results only show that spawning occurred for atrazine-exposed and unexposed zebrafish in a similar manner. The

Page 128 of 159 Page 136 of 167 ATRAZINE FINAL results of these investigations demonstrate that chronic atrazine 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-day- old) zebrafish were randomly distributed (40/tank) into 30-L glass aquariums; 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 μg/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 amounts 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%, and 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 carbon dioxide, weighed, and the tank average growth rate was calculated (the slope of the line determined by the change in the averaged log of fish weights over time). Ten fish from each treatment group were used for histopathological examination of the gills, kidneys, and liver. Tissues 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 changes in behavior were noted except for the 90.0 μg/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 solvent 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 decreased (p<0.05) in fish in the high atrazine concentration group; body weight data were not provided. Growth rates also were significantly decreased (p<0.05) in fish in the high-concentration group.

Histopathological examinations revealed no pathological lesions in atrazine-exposed fish, except at the highest test concentration. In the high-concentration group, moderate dystrophic lesions of the hepatocytes were observed, and there were signs of initial cell injury as indicated by the hydropic to vacuolar degeneration of the hepatocytes, the dilatation of the capillaries, and hyperaemia, compared to the negative control. All fish examined from the 90 µg/L group,

Page 129 of 159 Page 137 of 167 ATRAZINE FINAL exhibited the same intensity of morphological changes in the liver throughout the organ. The study authors reported a NOEC of 30.0 μg/L and a LOEC of 90.0 μg/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 Refuge, Florida. Eggs were transported to the laboratory and placed in an incubator at 30°C, and one egg from each clutch was opened to stage the embryos. Five days after collection (prior to sex determination during the temperature- sensitive period), eggs were separated into two groups; half of the eggs from one clutch were incubated at 30°C (female-producing temperature) and half at 33°C (male-producing temperature). Eggs were maintained at approximately 90% humidity with sphagnum moss as incubation material. Within each incubation group, 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 either water or 95% ethanol, used as a test substance solvent. A positive control group, 17β-estradiol, also was included with the same treatment concentrations in addition to a low dose of 0.014 ppm. Treatments were applied topically to the eggshell in 50 μL 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. Total 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 housed for ten days prior to tissue collection, and sacrificed after the collection of blood from the dorsal postcranial sinus. The right gonadal-adrenal mesonephros (GAM) complex was immediately removed for the aromatase bioassay. Aromatase activity was measured indirectly based on the release of tritium (3H) from 1β-3H-androstenedione during aromatization of the substrate into estrogen. GAMs from three additional control female alligators were used to determine the specificity of the aromatase assay.

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

There were no differences in results among controls with and without the solvent. The 17β- estradiol and testosterone concentrations were determined in plasma and CAF of all hatchlings that provided sufficient fluid volume. Radio-immuno assays (RIAs) for 17β-estradiol and testosterone were conducted. Cross-reactivities of the 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,17β-diol, 2.5%; -4-androstenedione, 2%; 5β-androstan-3β,17β-diol, 1.5%;

Page 130 of 159 Page 138 of 167 ATRAZINE FINAL estradiol, 0.5%; all other ligands <0.2%. Cross-reactivities of the 17β-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 expressed 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 concentrations, and GAM aromatase activity. Total (100%) sex reversal (male to female) was noted for all doses of 17β-estradiol. Atrazine treatment did not result in sex reversal. Atrazine treatment had no statistically 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 housed for ten days prior to tissue collection, and sacrificed after the collection of blood from the dorsal postcranial sinus. The right GAM complex was immediately removed for the aromatase bioassay (Crain et al., 1997). Histology was conducted to determine histological sex in order to document induction of sex reversal. The left GAM complex was preserved in Bouin's fixative, serial sectioned at 7 μm after paraffin embedding, and stained. Gonads were inspected and scored as testis or ovary by two independent researchers. In brief, criteria for testis included reduced cortex and medullary sex cord diameter and proliferation, and criteria for ovaries included hypertrophied cortex, medullary reduction, the presence of lacunae in the medulla, and germ cells in the cortex. In addition, both Mullerian duct epithelial cell height in the ovaries and the degree of degeneration 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 17β-estradiol produced female hatchlings at the male- producing temperature. Epithelial cell height of the Mullerian duct was significantly increased (p<0.001) in the high-concentration 17β-estradiol treatment groups in both male and female temperature regimes. The regression of the ovarian medulla was not different. The study authors reported slight reductions (NS) in sex-cord diameter in the atrazine-treated males.

Developmental Reproductive Toxicity in Reptiles (Water snake)

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

Page 131 of 159 Page 139 of 167 ATRAZINE FINAL

A total of 25 female and 12 male watersnakes were collected from two lakes in Illinois (previous exposure history unknown). Each snake was housed 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 selected 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 intramuscular injections of 0.1 mL of atrazine solution per gram of fish. The amount of fish with each solution was mass-corrected for each snake on a weekly basis such that each snake received the identical atrazine concentration. These concentrations were meant to represent potential environmental levels of atrazine, not necessarily within the fish. After injection, the fish were immediately offered to the respective snake for a period of not more than 1 hour to avoid 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 continuously exposed to the appropriate atrazine concentration throughout gestation. Food intake was standardized, with pregnant females fed 5% of their body mass twice weekly.

At approximately Week 12, mothers were observed for signs of parturition. Stillborn offspring were measured (weight and SVL), probed for sex determination, marked uniquely, and frozen at −20°C. Live born offspring were measured (weight and SVL), probed for sex determination, and photographed for identification by pattern. All neonates, regardless 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 included 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 linked immunosorbent assay (ELISA) evaluation of 17β-estradiol concentration after solid-phase extraction. A known concentration of 17β-estradiol was extracted to determine the percent recovery.

Neonates were measured for asymmetry to assess possible aberrations in development. All offspring that had full scalation were used in the analyses of these characteristics. The left and right dorsal scales extending 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 offspring were produced to analyze left and right jaw

Page 132 of 159 Page 140 of 167 ATRAZINE FINAL 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 offspring. In addition, there was no interaction between the number of stillborn neonates, sex, and treatment, and no treatment- related 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 decreased in all atrazine treatment groups in a non- dose-dependent manner compared to control. Atrazine treatment was also associated with effects on scale row symmetry at the mid-body, with 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 fragments. The oocytes were incubated with 6.25, 12.5, 25, or 50 IU of human chorionic gonadotropin (hCG) 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 pesticides. 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 3β-HSD inhibitor (epostane) was tested at 0.001, 0.01, 0.1, and 1 µM to verify inhibition of ovulation by inhibition of steroid hormone synthesis. Incubations were conducted for 20 hours, and media samples were stored frozen at −80°C. Hormone analyses in the media samples (i.e., progesterone, testosterone, and 17β-estradiol) were conducted by RIA methods.

In the ovulation assay, nine pesticides, including atrazine, had an effect on the ovulatory response or hormone production. Atrazine exposure was associated with apparent stimulatory ((↑50% at 6.25 µM; p<0.05) and inhibitory (↓68% at 0.0625 µM; p<0.01) effects on ovulation at different exposure levels. In response to atrazine, progesterone levels were higher at 6.25 5 µM (↑63%) and at 62.5 µM (↑104%) (p<0.001). Testosterone levels were higher) at 6.25 µM (↑24%, p<0.01) but not statistically different at 62.5 µM. No effects on estrogen levels were detected.

Page 133 of 159 Page 141 of 167 ATRAZINE FINAL

In Vivo (Frog)

In an investigation of reproductive/metamorphosis in frogs by Oka et al. (2008), the potential for atrazine to affect sex differentiation 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-chlorinated tap water that has been 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-type male were injected with 500 i.u. per 100 g body weight of gonadotropin. They were allowed to mate semi-naturally 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 deionized 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 were 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 tadpoles 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 comprised of duplicate (for atrazine exposure concentrations) or triplicate (control group) test tanks. In Experiment II, a single tank per test treatment was used. Atrazine (97.4% purity) and 17β-estradiol were dissolved 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 because previous studies have demonstrated that NF stages 49-56 are critical stages 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 metamorphosis (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) 17β-estradiol for a positive control was exposed from NF stages 49 to 66. In both experiments, control tadpoles were maintained in test medium containing the vehicle (0.01% ethanol). Developing larvae were staged every week in Experiment I. In both experiments, froglets were collected upon completion of metamorphosis (NF stage 66), and total length and time to metamorphosis of the tadpoles were recorded. Any dead tadpoles found during exposure were

Page 134 of 159 Page 142 of 167 ATRAZINE FINAL removed and recorded. Chemical analysis of atrazine in the test medium was conducted with GC-MS according to the standard guidelines.

Froglets were collected at NF stage 66, anaesthetized, 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 characteristics 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 atrazine) 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 were 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) froglets in Experiment I and from 30 (control and atrazine groups) and 28 (17β-estradiol group) froglets in Experiment II. The body of each froglet, including gonads, was fixed in Bouin’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-sections 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 PCR amplifications were conducted in triplicate. In Experiment II, five gonads of male or feminized froglets from each treatment were used to examine the expression levels of aromatase mRNA. Total RNA was isolated, followed by DNase I treatment. The cDNA was synthesized from purified total RNA, but PCR amplifications were conducted in a single assay.

The livers from control froglets and atrazine- or 17β-estradiol-exposed froglets were removed and weighed. For VTG determinations, livers were individually homogenized, centrifuged, and the supernatants were collected and frozen at −70°C until analyzed with an ELISA assay. For the in vitro VTG investigation, hepatocytes were isolated from adult male X. laevis. Cells were inoculated at 4×104 cells/well into 96-well tissue culture plates and incubated in air at 22°C. After incubation for 48 hours, the cells were exposed to a test compound by addition of the test compound-containing medium. Atrazine and 17β-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 17β-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 the volume. Three days after medium renewal, the

Page 135 of 159 Page 143 of 167 ATRAZINE FINAL 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 observed 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 study. 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 froglets in each group, and sex ratios ascertained on the basis of gross morphology were corroborated 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-reversal 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 17β-estradiol as positive control. Twenty-one feminized (female) frogs and seven hermaphroditic frogs were confirmed in the 17β-estradiol group.

The expression level of P450 aromatase in gonad-kidney complexes was significantly 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 17β-estradiol.

There were no differences in VTG concentrations of NF stage 66 froglets between males and females in any experimental group including the control group; 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 (Experiment I) and all-male cohorts (Experiment II). On the other hand, hepatic VTG was induced in the all-male ZZ froglets exposed to 0.27 ppb 17β-estradiol. A concentration-dependent increase of VTG in culture media was observed after primary-cultured hepatocytes were exposed to 17β-estradiol. However, no VTG was observed in hepatocyte cultures exposed to atrazine concentrations up to 1000 ppb, indicating no estrogenic

Page 136 of 159 Page 144 of 167 ATRAZINE FINAL activity of atrazine under the study conditions. Atrazine did not inhibit 17β-estradiol-induced hepatocyte VTG synthesis after co-exposure with 17β-estradiol in vitro.

On Day 0, actual concentrations in the 0.1, 1, 10, and 100 ppb atrazine 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 17β- estradiol concentrations 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 acceleration 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 wild-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 replicate, 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 solvent; the final acetone concentration in AS was 0.02% (v/v). The study also included a control group exposed to AS only; a solvent control group exposed to AS containing 0.02% (v/v) acetone; and a positive control group exposed to 100 µg/L of 17β-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 malformations, 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 positive 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 concentration-response, with the greatest effect occurring at the 10 µg/L concentration. Compared to control, the median 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 100

Page 137 of 159 Page 145 of 167 ATRAZINE FINAL

µg/L. The effect of 17β-estradiol at 100 µg/L was similar to the 1 and 100 µg/L atrazine concentrations, decreasing the median times to each stage by 2.9, 4.3 and 4.7 days, respectively. Atrazine at 1-100 µg/L and 17β-estradiol at 100 µg/L had no effect on the intervals between each stage of development, suggesting that the acceleration in development occurred primarily between stages 25 and 39.

A similar U-shaped response was noted for effects 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 17β-estradiol group having similar increases in body weight (18%) and SVL (5%). In addition, the body mass of the metamorphs at specific SVLs was significantly increased by approximately 10% at at 1, and 10 µg/L atrazine and at 100 µg/L 17β-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 other 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 collected from the wild (previous exposure history unreported) and the resulting larvae were reared in the laboratory in aquaria. Exposures were started at early limb bud emergence (Gosner stage 28) and continuing until the majority of larvae underwent tail resorption (Gosner 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 weeks, with each treatment consisting of three replicates (10 tadpoles/replicate, n=30). Each replicate was contained in 10 L of water within 14.5-L glass aquaria fitted with a glass lid to limit evaporation. Each aquarium was aerated and was on a 12 hour:12 hour light:dark cycle. Spiking took place after every water renewal after every third or fourth day of exposure. 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 Tests 1, 6, 7 (atrazine only) were examined histologically for the presence of oocytes. Body lengths were measured from the snout to the tip of the tail.

Mortality was limited (2.3%; 18/800 animals) across all experiments, and occurred mainly during the first week of exposure and was attributed to handling stress. Atrazine had no observable effect on the development of the spotted marsh frog larvae at concentrations up to 30 µg/L. Atrazine treatments varied from the control 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,

Page 138 of 159 Page 146 of 167 ATRAZINE FINAL 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 μg/L atrazine treatments in Test 1, but the authors noted that this trend was not strong in the 3 μg/L atrazine treatment in Tests 6 and 7. No changes in male gonadal histology were noted after atrazine exposure, with the exception of two males in one experiment. Testicular ovarian follicles were observed in one male at Gosner stage 44 after 4 weeks of exposure to atrazine at 3 μg/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 the 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 developed more quickly than the treatment with the combined pesticides. For the definitive test, gosner stage 26 tadpoles (ca. 6 weeks post hatch; obtained from laboratory-reared spawns) were exposed to a 25 µg/L atrazine concentration along with a blank (filtered, aged Sydney Australia tap water) control and a mixture of 25 and 10 µg/L atrazine and terbufos sulfone, respectively, for 10 weeks to encompass complete metamorphosis completely. There were three replicates (glass aquariums) for each treatment with 10 tadpoles in each replicate (10L solutions); test vessels aerated and maintained at 25ºC under a 12:12 light:dark period. Test solutions renewed twice weekly along with measured test concentrations (duplicate samples) and environmental quality (dissolved oxygen, electrical conductivity, pH, and temperature). 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. The results presented will focus on the definitive test. There were three mortalities overall, one in terbufos and two in combined, with all other tadpoles completing metamorphosis. There was no significant difference in survival, SVL, mass, or metamorphosis between the control and atrazine-only group.

In Weber et al. 2013, evaluation of trascriptomic and protein alterations in zebrafish embyros was examined after exposure to atrazine (technical 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 strain; 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 results not included here). Results showed alterations in gene expression at all atrazine 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 development, function, and disease; cell cycle control; and carcinogenesis. Two of these genes (CYP17A1 and SAMHD1) found to be altered at all three exposure concentrations.

Page 139 of 159 Page 147 of 167 ATRAZINE FINAL

Tumor Development (Rat)

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 (isoflavone-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. Palpation 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 ether anesthesia at the end of Week 8 for Tg females and Week 20 for non-Tg females and 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 tumor 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 identified) occurred prior to study termination that were considered unrelated to atrazine treatment. Final mean body weight in female rats fed 500 ppm atrazine was significantly decreased (p<0.01; ↓14%) compared to control. There were no consistent differences noted in the weights of liver, kidneys, ovaries/testes, and uterus. The daily food intakes of groups fed the 5 and 50 ppm atrazine-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 were observed at four weeks 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 single

Page 140 of 159 Page 148 of 167 ATRAZINE FINAL 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 text, but no summary tabular data was included for a atrazine- treated, non-Tg group. In female Tg rats, the adenoma and adenocarcinoma incidences for rats fed 5 ppm atrazine were significantly increased (p<0.01 and p<0.05, respectively) compared to the Tg control (↑50 and 50%, respectively). In addition, the 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 contrast, skin tumors (squamous cell papillomas and carcinomas) were induced only in males. Initial appearances were at Week 10 in a control male, Week 11 in Tg rats fed 5 or 50 ppm atrazine, and Week 15 in Tg rats fed 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 peripubertal 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 administered. Body weights were determined on the first day of dosing and every other day through PND 51 (prior to sacrifice). Organ weights were determined for the testis, seminal vesicle, ventral prostate, dorsal prostate, 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 3βHSD activity (typically >95%). Purified Leydig cells obtained individually from four to eight rats were pooled, and each pool/group was cultured in three to eight replicates 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 steroid substrates: 22-OH cholesterol, pregnenolone, progesterone, or D4-androstenedione. Additionally, in each experiment, purified Leydig cells were plated in 50-mm Petri dishes (2.5×106 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 saturated dose (10 ng/mL) of hCG. Purified Leydig cells were stimulated with different concentrations of 22-OH cholesterol (1 and 50 µM) for two hours, and the cells were plated in 50-mm Petri dishes (1×106 cells/2-mL culture medium/dish) and progesterone and androgen concentrations 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), androgen, and progesterone concentrations; cell lysates were used for real-time PCR analysis.

Page 141 of 159 Page 149 of 167 ATRAZINE FINAL

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 experiments were run in one assay/hormone. The amounts of cAMP secreted into the culture medium were measured by a cAMP EIA kit.

Total RNAs from purified rat Leydig cells were isolated with the RNAqueous 4PCR kit. Concentrations of RNA, measured by absorbance at 260 nm, and purity estimated by the 260/280-nm absorbance ratio, were determined spectrophotometrically. 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 mixture 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 the 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, 3βHSD, CYP17A1, and 17βHSD.

Mean body weights in atrazine-treated rats were decreased in the high-dose group beginning on PNDs 27-29 (p<0.05), with further decreases on PNDs 31-35 (p<0.01) and PNDs 37-49 (p<0.001). Mean terminal body weight in the high-dose group remained significantly decreased (p<0.01) 24 hours after the final dose. Atrazine treatment also resulted 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) and significantly decreased (p<0.01) in the high-dose group (↓10 and 34%, respectively). Seminal vesicle and dorsal prostate weights were decreased (p<0.001 and NS, respectively) in the low-dose group (↓30 and 13%, respectively) and significantly decreased (p<0.001) in the high-dose group (↓60 and 37%, respectively). In addition, adrenal gland weights were significantly increased (p<0.01 and 0.001, respectively) after atrazine treatment in the low- and high-dose groups (↑57 and 94%, respectively).

The serum androgen (T + DHT) and LH concentrations were determined in samples taken 24 hours after the final administration of atrazine. No difference was observed in serum LH concentrations after atrazine treatment, 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 significantly decreased (p<0.001) in a dose-dependent manner across a range of hCG (0.25-20 ng/well); similar results generally were noted for Leydig

Page 142 of 159 Page 150 of 167 ATRAZINE FINAL cells from the low-dose animals. Progesterone production by Leydig cells obtained from atrazine-treated rats was unaltered under basal conditions, 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, atrazine treatment resulted in significant dose-dependent decreases (p<0.001) in extracellular 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 cholesterol, pregnenolone, progesterone, and Δ4- 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 Δ4-androstenedione as a substrate.

The transcript levels of the majority of the genes examined (i.e., LHR, SR-B1, StAR, SF-1, PDE4B, CYP17A1, and 17βHSD) were significantly decreased (p<0.05) in a dose-dependent manner by in vivo atrazine treatment. TSPO, a translocater protein, was decreased in the high- dose group only, and the expression of the gene for 3βHSD, 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/kg/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 from all rats as described above.

For in vitro experiments, Leydig cells were pooled, allowed three hours to attach (1×106 cells/2 mL culture medium/dish), and treated for 24 hours with atrazine (0.001, 1, 10, 20, or 50 µM) in DMSO. Culture medium was collected at the end of the incubation period, and cells were stimulated with different concentrations of hCG, progesterone, or Δ4-androstenedione for two hours. After treatment, cell-free media were collected 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 incubated 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 reflected the activity of CYP17A1 and/or 17βHSD, also was measured. Additionally, the conversion of Δ4-androstenedione to testosterone, which reflected the activity of 17βHSD, was measured. Leydig cells were exposed to atrazine for 24 hours and challenged with progesterone or Δ4-androstenedione in a concentration-dependent manner.

For each in vivo experiment, Leydig cells obtained individually from four to five rats were pooled, and each pool/group was cultured in four to eight replicates per experiment. Cells (2.5×106 cells/2 mL culture medium/dish) were allowed to attach for three hours and cultured in

Page 143 of 159 Page 151 of 167 ATRAZINE FINAL

M199-0.1% BSA without or with a saturated dose of hCG (10 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 concentrations. In addition, the high concentration of atrazine (20 µM) significantly increased (p<0.05) hCG-stimulated cAMP accumulation during a further 2-hour incubation.

In parallel experiments, the concentration-dependent effects of atrazine on basal and hCG- stimulated steroidogenesis were investigated. Basal progesterone production was bi-directionally affected by atrazine; progesterone concentration was significantly 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 significantly increased (p<0.05) by atrazine in a concentration- dependent 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 higher atrazine concentrations (20 and 50 µM), and significant concentration-dependent increases (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 stimulatory 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 androgen production generally was unchanged 10-34%.

Expression of mRNA transcripts for genes involved 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 17βHSD genes (↑153%). In contrast, the transcript levels for LHR, SR-B1, TSPO, 3βHSD, PDE4B, and CYP19A1were not affected by atrazine treatment.

Conversion of progesterone to testosterone was significantly increased (p<0.05) when Leydig cells were challenged with higher concentrations of atrazine (20 and 50 µM; 4.3 and 11 ppm) for 24 hours and incubated in the presence of progesterone and atrazine for an additional two hours. The increased conversion of progesterone to testosterone in the presence of these two concentrations of atrazine was similar regardless of concentrations of progesterone or atrazine (average ↑227%). Conversion of Δ4-androstenedione to testosterone was also stimulated in the presence of atrazine. Androgen concentration was increased in the presence of 0.25 µM Δ4- androstenedione at all concentrations of atrazine except 0.001 µM; higher concentrations of Δ4-

Page 144 of 159 Page 152 of 167 ATRAZINE FINAL androstenedione (0.5 and 10 µM) increased (p<0.05) androgen conversion only in the presence of 20 µM atrazine.

The results of the ex vivo studies demonstrated significantly increased (p<0.05) concentrations of cAMP and T + HDT under both basal and hCG-stimulated conditions after one, three, and five days of atrazine treatment in vivo. These experiments revealed similar pattern of changes in basal and hCG-stimulated cAMP accumulation and androgen 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 transient stimulatory action on cAMP signaling pathway in Leydig cells, leading to facilitated androgenesis.

The relative roles of adrenal and testis hormones after intact or castrated male rats were exposed to atrazine with and without androgen 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 groups, 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 administered a daily oral gavage dose of atrazine (97.1% purity) at 0 (1% methyl cellulose in water), 5, 25, 75, or 200 mg/kg/day for three days. The rats were dosed with the vehicle or the test chemical between 0700 and 0900 hours, and sacrificed by decapitation 30 minutes after dosing on Day 3. Body weights were recorded daily.

At sacrifice, trunk blood was collected and processed to isolate serum and plasma; the portions were stored at −80°C prior to analysis. Adrenal weights were recorded at necropsy. The androstenedione, corticosterone, progesterone, testosterone, estrone, and estradiol concentrations in the serum were determined with commercial radioimmunoassay kits. The LH concentrations were determined with a dissociation-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)-dimethylammonio]-1- propanesulfonate, n-octyl-β-glucopyranoside, and phenylmethylsulfonyl fluoride. The testes extracts were assayed for protein, and analyzed quantitatively with two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE). The proteins for which expression was significantly altered by treatment or significantly correlated to testosterone production were isolated from the 2D gels and identified with matrix-assisted laser desorption/ionization coupled mass spectrometry and commercial proteomics and rat species protein database software. The proteins that were altered with atrazine 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 the

Page 145 of 159 Page 153 of 167 ATRAZINE FINAL

Western blot procedure, the proteins were subjected to electrophoresis under standard conditions and transferred onto nitrocellulose membranes. The nitrocellulose membranes were then incubated with both primary and secondary antibodies and washed, and the membranes were scanned with an infrared imaging system.

The mean body weights were similar among all treatment groups within each intact or castrated category. However, the overall mean body weights were decreased (significance not reported) in the castrated groups (339.0 g) compared to the intact groups (370.4 g). After three days of treatment in both groups, weight losses (significance not reported) were observed in the intact and castrate 75 mg/kg (1.3% and 2.4% loss, respectively) and 200 mg/kg (4.3% and 5.4% loss, respectively) groups. No differences in adrenal weights were observed for 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 rats 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 concentrations in intact rats were significantly increased (p<0.05) by approximately 370, 550, and 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 castrated rats by 180 and 260%, respectively. The serum progesterone and corticosterone concentrations were significantly (not reported) correlated with one another in both the intact and castrated rats.

The serum LH, androsenedione, and testosterone concentrations for intact rats were each observed with a similar non-monotonic dose-response. Significant increases (p<0.05) in LH and testosterone concentrations were observed in the medium-low dose group (2.9-fold increases). The androstenedione concentrations followed the same pattern as LH and testosterone in intact males, with the highest (NS) concentrations observed in the medium-low dose group. Medium- high and high-dose concentrations were decreased relative to the medium-low dose group, with high-dose concentrations generally similar to control. 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 testosterone concentrations across all atrazine treatment groups were below the limit of detection in the castrated rats. Concentration data for estrone and estradiol were not reported.

In the adrenals of intact rats, the expressions of six different proteins were significantly decreased (p<0.05) as a result of atrazine exposure. The expression of one protein, PGRMC1, was correlated in the intact rats with the non-monotonic increase in serum testosterone and the

Page 146 of 159 Page 154 of 167 ATRAZINE FINAL expression of another protein, HSPD1. In the adrenals of castrated rats, the expression of one protein, HSPD1, was correlated with the serum progesterone and corticosterone concentrations. The expression of PGRMC1 was correlated to the HSPD1 expression levels in the castrated rats. The correlations between HSPD1 and 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 rats, the HSPD1 expression was up-regulated and the PGRMC1 expression was down-regulated. Western blot analyses confirmed the upregulation of PGRMCl and the down-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 different proteins were correlated with the non- monotonic increase in serum testosterone 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 atrazine (>97% purity; vehicle not described) at doses of 0, 50, 100, or 200 mg/kg/day for three weeks. On the last day of treatment, all mice were euthanized, and serum, liver, and testes were collected. Testosterone and 17β-estradiol concentrations and aromatase activity were determined. 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 examined 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 were 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 significantly decreased (p<0.05) in all treated groups. Serum 17β-estradiol concentrations increased 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 E2/T ratio was increased in all treated groups in a dose-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.

Page 147 of 159 Page 155 of 167 ATRAZINE FINAL

Atrazine treatment had no effect on AR, ERα, or ERβ mRNA concentrations in the testes. In addition, mRNA concentrations 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 atrazine 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 the testes were significantly decreased (p<0.05) by 33% and 37% after exposure in the medium- and high-dose groups, respectively. No differences in the testicular mRNA concentrations of 3β-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 17α, the testicular mRNA concentrations were significantly decreased (p<0.05) in treated groups, with decreases of 38%, 48%, and 30% after treatment with 50, 100 and 200 mg/kg/day, respectively. Testicular 17β-HSD mRNA concentrations were significantly decreased (p<0.05) by approximately 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-/progesterone-induced LH surge in ovariectomized rats was examined to determine if LH inhibition was concomitant with decreased gonadotrophin-releasing hormone (GnRH) neuronal activation.

Female adult (60-90 days old) enhanced green fluorescent protein (eGFP)-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 17β-estradiol-3-benzoate (EB) in safflower oil (s.c., between 1000 and 1100 hours) for three consecutive days to induce a LH surge beginning 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 free-moving rats by an atrial cannula (implanted on Day 2) at 1-hour intervals for 9 hours, starting on the afternoon of the final day of atrazine dosing. Red blood cells (collected from the previous sample 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.

Page 148 of 159 Page 156 of 167 ATRAZINE FINAL

Concentrations of GnRH and the Finkel-Biskis-Jinkins osteosarcoma (cFOS) were determined in every fourth section. A second group of ovariectomized rats was treated (as described) with atrazine at 0 and 50 mg/kg/day without EB and progesterone injections to determine if atrazine alone has a stimulatory effect on GnRH neuronal activation. On the afternoon of Day 4, rats were euthanized and brains collected (as above) for FOS analysis.

In Experiment 3, to evaluate the effects of atrazine withdrawal on GnRH neuronal activation and LH surge, ovariectomized rats (4-13/group) were 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 (as described above). For LH surge induction, rats received EB injections three consecutive days starting on the second day of atrazine dosing, the final day of atrazine dosing, or two days after atrazine dosing. On the final day of EB injections, rats were injected with progesterone and blood samples were collected 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 collection. Close to the LH surge peak (1700 hours), rats were perfused with paraformaldehyde and brains were collected (as described for Experiment 2) for histological examination.

Blood and/or brain samples from the 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 location 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 double labeled if eGFP-positive cytoplasm surrounded the FOS immunoreactivity nucleus through multiple fields.

The EB+ progesterone -induced LH surge was significantly decreased (p<0.05) to a similar extent by atrazine at all doses. Significant decreases (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 concentrations >2 standard deviations (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) surge 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 control group (4/4) had peak FSH concentrations >2 SD above the baseline, but a lower proportion of rats responded in the atrazine-treatment groups [50 mg/kg (5/6), 100 mg/kg (4/8), and 200 mg/kg (3/5)].

The number of activated GnRH neurons present during the afternoon after EB+ progesterone priming was decreased in atrazine-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 significantly decreased (p<0.02) with atrazine

Page 149 of 159 Page 157 of 167 ATRAZINE FINAL treatment at 100 and 200 mg/kg/day (↓59 and 74%, respectively). There were no atrazine- treatment effects on the number of GnRH immunoreactive cells. Finally, atrazine treatment alone (50 mg/kg without EB+ progesterone) 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 control (0.50±0.40) groups.

In Experiment 3, LH surge and GnRH activation 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 atrazine withdrawal compared to control, but were increased compared to the Day 0 concentrations in atrazine-dosed rats. By the fourth day of atrazine withdrawal, peak LH concentrations and the percentage of GnRH neurons immunoreactive with FOS returned to levels similar to control. There 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 inhibits hormone-induced LH surge and GnRH neuronal activation, and these effects are transient and no longer present after four days of withdrawal from atrazine treatment. These results also support the 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 the 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 the effects of atrazine on plasma corticosterone concentrations, female rats (n=75) were ovariectomized and, after one week, orally dosed by gavage with atrazine (purity not reported) in 1% CMC sodium salt at 0 (vehicle), 50, or 200 mg/kg. Rats (3-5/group/time) were sacrificed, 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 corticosterone. Plasma was harvested, and stored at −80°C for corticosterone analysis by RIA.

In Experiment 2, to determine the effects of atrazine on the LH surge, female rats (n=70) were ovariectomized with half of the rats further bilaterally 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 and plasma volume. A sham surgery was conducted on the remaining ovariectomized rats. One day after adrenalectomy, rats (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 (between 1000 and 1100 hours). Starting Day 2 of atrazine dosing, rats were injected with EB (s.c., 40 µg/kg in safflower oil) for three consecutive days (between 1000 and 1100 hours) and on Day 4 rats were injected with progesterone (s.c.,

Page 150 of 159 Page 158 of 167 ATRAZINE FINAL

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 intervals for 9 hours (starting at 1300 hours) after the progesterone treatment. Plasma was harvested 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 volume 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 ovariectomized and ADX as described for Experiment 2. One day after adrenalectomy, rats (9-13/group) were orally dosed 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 final 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 the initial blood samples was also analyzed for corticosterone concentration. Blood volume and hematocrit 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 were significantly increased (p<0.05) approximately 2- to 3-fold in both the 50 and 200 mg/kg dose groups. Corticosterone 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 concentrations were NS. Additionally, corticosterone concentrations in the 200 mg/kg group at the 3- and 6-hour post-treatment 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 interaction between adrenalectomy and atrazine treatment was observed for peak LH concentration or AUC, and no effect of adrenalectomy alone on peak LH concentration or AUC was observed 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 amplitude 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 that atrazine selectively

Page 151 of 159 Page 159 of 167 ATRAZINE FINAL affects the LH pulse generator through alterations in adrenal hormone 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 were 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 effects of atrazine after a single (1X) or multiple day (3 days=3X or 21 days=21X) exposure were evaluated. To evaluate 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 17β-estradiol. The pituitaries were removed and the neural lobe was dissected free and discarded. The remaining anterior lobe was weighed, sonicated, and frozen immediately at −70°C until assayed for LH, FSH, prolactin, TSH, and 17β-estradiol. In the 3X females, groups (n=10) of ovariectomized , implanted LE and SD females were administered atrazine as above on Days 1, 2, and 3, and then killed at 0, 1, 3, and 6 hours post- gavage on Day 3. The blood and pituitaries were processed as described above for the 1X females. In the 21X females, groups of LE and SD females were ovariectomized on Day 0 and received atrazine at 0, 75, 150, or 300 mg/kg/day. 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 blood and pituitaries were processed as described for the 1X females.

In SD rats, the prolactin surge was significantly reduced (p<0.05) after three daily doses of 300 mg/kg atrazine. However, the secretion of these 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 concentrations occurring in most animals at the 3-hour time point. Although surges of both LH and prolactin were observed in the LE females administered three doses of 50 mg/kg atrazine, the onset of the surge 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 and 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 females, the amount of both LH and prolactin present

Page 152 of 159 Page 160 of 167 ATRAZINE FINAL in the pituitary was generally greater in both rat strains. Body weight was unaffected in the females administered atrazine for 1 or 3 days. Similarly, atrazine did not affect body weight in the 21X females at 75 and 150 mg/kg. However, the rate of body weight gain in those females administered 300 mg/kg was significantly decreased (p<0.05, data not presented). Mean pituitary weight was not affected in any of the groups evaluated. The concentration of serum or pituitary TSH and FSH was not different from control at any of the doses or time points examined, and serum 17β-estradiol concentrations were also similar in control and treated groups.

In a second experiment to determine the effect of a single dose of atrazine on ovulation and ovarian cycling, groups of intact LE females (20/dose group) displaying regular 4-day estrous cycles were gavaged with 0, 75, 150, or 300 mg/kg atrazine on the day of vaginal proestrus. The effect of this treatment on ovarian function was determined in half of the animals in each dose group by examining the vaginal smear daily for three 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 with 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 subsequent cycle, or on subsequent ovarian cycles as determined by vaginal smears. Continued dosing 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 studies were conducted to examine the site of action of atrazine. In the first study, to determine whether atrazine alters pituitary prolactin secretion by a direct effect on the lactotrophs, the pituitary of seven 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. Six 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 administration 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. Immediately after the third dose, the females were fitted with in-dwelling cardiac catheters. Two hours after placement 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 thereafter, 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, the effect of atrazine on in vitro prolactin and LH release was determined by pituitary perfusion. After removing the pituitaries, the neural

Page 153 of 159 Page 161 of 167 ATRAZINE FINAL lobe was immediately dissected free and the anterior lobe was then hemisected along the rostral- caudal plane. One-half (randomly selected left or right portion) was weighed, sonicated, and frozen for subsequent assays for LH and prolactin. The remaining half was quartered, weighed, and placed in a sterile, cooled microplate well. After carefully transferring 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. Samples were collected at10-minute intervals. After three 10-minute baseline samples, the hemi-pituitaries were exposed to two 30-minute challenges (spaced 60 minutes apart) with a combination of GnRH and thyroid releasing hormone (TRH). Sixty minutes after the second challenge, tissues were exposed once more (10 minutes) with a depolarizing stimulus of 60 mM potassium chloride (KCl) to assess tissue viability, and sample collections were continued for an additional 40 minutes.

In the first study, serum prolactin concentrations remained unaltered at all the time points examined, indicating that secretion of prolactin by the pituitary is not altered by atrazine if the gland is removed from the influence of CNS factors. In the second study, LH secretion remained at basal levels in atrazine + saline-treated females. However, an LH surge was observed in the atrazine + GnRH females. In fact, serum LH concentrations in the atrazine + GnRH females were comparable to the estrogen-induced LH surge observed in control females, indicating that the effect of atrazine on LH secretion is not the result of a direct impairment of LH release from the gonadotrophs. In the third study, no differences in either LH or prolactin release were noted from the pituitaries of untreated females exposed to atrazine in vitro. Similarly, no changes in basal or GnRH-stimulated LH release or TRH-stimulated prolactin release were noted in the pituitaries obtained from females dosed with atrazine for three days. In these same pituitaries, there was an initial increase in prolactin release from LE pituitaries that may have been associated with a modest elevation in pituitary prolactin concentrations.

Page 154 of 159 Page 162 of 167 ATRAZINE FINAL

APPENDIX 3: References Not Utilized in the Atrazine WoE Analysis

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

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies previously submitted to the agency for registration/reregistration, published literature articles and/or Tier 1 assays. The agency also conducted a more recent search (2009 to 2014) of available scientific literature for any additional relevant information for their weight of evidence (WoE) evaluations. These articles were evaluated in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 (http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species _reregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance for considering and Using Open Literature Toxicity Studies to Support Human Health Risk Assessment (http://www.epa.gov/pesticides/science/lit-studies.pdf).

The following published and unpublished references were considered for use in the WoE analysis for Atrazine but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the compound of interest was not used in the study; 3) the test material was not adequately described; 4) a formulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequately described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapter and does not contain primary study data; 9) insufficient information was available to adequately assess the validity of the study results; 10) the 40 CFR Part 158 guideline study was classified as unacceptable/inadequate; 11) the study dealt only with non-EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) the study contained only data on invertebrates.

Aït-Aïssa, S., Laskowski, S., Laville, N., et al. (2010) Anti-androgenic 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 susceptibility to atrazine of three developmental stages of Rhinella arenarum and influence on metamorphosis; Non- monotonous acceleration of the time to climax and delayed tail resorption. Aquat Toxicol. 91:161-170.

Page 155 of 159 Page 163 of 167 ATRAZINE FINAL

Brown, T.M. (2009) The effects of pesticide exposure on gonadal development, phonotaxis, and calling in the African Clawed Frog, Xenopus laevis. Ph.D. Dissertation, University 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 disruption 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 Fernández-Cruz, M.L. (2014) Chlorotriazines 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.

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 developmental toxicants. Toxicol. Sci. 127(2):391-402.

Forgacs, A.L., D'Souza, M.L., Huhtaniemi, I.T., et al. (2013) Triazine herbicides and their chlorometabolites alter steroidogenesis 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.

Page 156 of 159 Page 164 of 167 ATRAZINE FINAL

Ghodageri, M.G. and Katti, P. (2013) In vitro 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 herbicide atrazine and its metabolite on cytochrome P450 aromatase and steroidogenic factor-1 mRNA levels in the brain of premetamorphic bullfrog tadpoles (Rana catesbeiana). Aquat. Toxicol. 102(1-2):31-38.

Hannas, B. R., Wang, Y.H., Thomson, S., et al. (2011) Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna). Aquat. Toxicol. 101(2):351- 357.

Hyne, R.V., Spolyarich, N., Wilson, S.P., et al. (2009) Distribution of frogs in rice bays within an irrigated agricultural area: links to pesticide usage and farm practices. Environ. Toxicol. Chem. 28(6):1255-1265.

Jin, Y., Zhang, X., Shu, L., et al. (2010) Oxidative stress response and gene expression with atrazine exposure in adult female Zebrafish (Danio rerio). Chemosphere. 78:846-852.

Jin, Y., Wang, L., Chen, G., et al. (2014) Exposure of mice to atrazine and its metabolite diaminochlorotriazine elicits oxidative stress and endocrine disruption. Environ. Toxicol. Pharmacol. 37(2):782-790.

Jowa, L. and Howd, R. (2011) Should atrazine and related chlorotriazines be considered carcinogenic for human health risk assessment? J. Environ. Sci. Health C Environ. Carcinog. Ecotoxicol. Rev. 29(2):91-144.

Kucka, M., Pogrmic-Majkic, K., Fa, S., et al. (2012) Atrazine acts as an endocrine disrupter by inhibiting cAMP-specific phosphodiesterase-4. Toxicol. Appl. Pharmacol. 265(1):19-26.

Langerveld, A.J., Celestine, R., Zaya, R., et al. (2009) Chronic exposure to high levels of atrazine alters expression of genes that regulate immune and growth –related function in developing Xenopus laevis tadpoles. Environ. Res. 109:379-389.

Langlois, V.S., Carew, A.C., Pauli, B.D., et al. (2010) Low levels of the herbicide atrazine alter sex ratios and reduce metamorphic success in Rana pipiens tadpoles raised in outdoor mesocosms. Environ. Health Perspec. 118(4):552-557.

Page 157 of 159 Page 165 of 167 ATRAZINE FINAL

Li, Y., Sun, Y., Yang, J., et al. (2014) The long-term effects of the herbicide atrazine on the dopaminergic system following exposure during pubertal development. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 763:23-29.

Mitsumori, K., Shimo, T., Onodera, H., et al. (2000) Modifying effects of ethinylestradiol but not methoxychlor on N-ethyl-N-nitrosourea-induced uterine carcinogenesis in heterozygous p53- deficient CBA mice. Toxicologocal Sciences (58):43-49.

Palma, P., Palma, V.L., Matos, C., et al. (2009) Assessment of the pesticides atrazine, endosulfan sulphate and chlorpyrifos for juvenoid-related endocrine activity using Daphnia magna. Chemosphere. 76(3):335-340.

Pogrmic-Majkic, K., Kaisarevic, S., Fa, S., et al. (2012) Atrazine effects on antioxidant status and xenobiotic metabolizing enzymes after oral administration in peripubertal male rat. Environ. Toxicol. Pharmacol. 34(2):495-501.

Raldúa, D. and Babin, P.J. (2009) Simple Rapid Zebrafish Larva Bioassay for Assessing the Potential of Chemical Pollutants and Drugs to Disrupt Thyroid Gland Function. Environ. Sci. Technol. 43:6844-6850.

Rey, F., González, M., Zayas, M.A., et al. (2009) Prenatal exposure to pesticides disrupts testicular histoarchitecture and alters testosterone levels in male Caiman latirostris. Gen. Comp. Endocrinol. 162:286-292.

Rohr, J.R. and McCoy, K.A. (2010) A qualitative meta-analysis reveals consistent effects of atrazine on freshwater fish and amphibians. Environ. Health Perspect. 118(1):20-32.

Rohr, J.R., Sesterhenn, T.M., and Stieha, C. (2011) Will climate change reduce the effects of a pesticide on amphibians?: partitioning effects on exposure and susceptibility to contaminants. Global Change Biology. 17:657–666.

Shenoy, K. (2014) Prenatal exposure to low doses of atrazine affects mating behaviors in male guppies. Horm. Behav. 66(2):439-448.

Solomon, K.R., Carr, J.A., Du Preez, L.H., et al. (2008) Effects of Atrazine on Fish, Amphibians, and Aquatic Reptiles: A Critical Review. Critical Reviews in Toxicology 38:9, 721- 772.

Spolyarich, N., Hyne, R.V., Wilson, S.P., et al. (2011) Morphological abnormalities in frogs from a rice-growing region in NSW, Australia, with investigations into pesticide exposure. Environ. Monit. Assess. 173(1-4):397-407.

Page 158 of 159 Page 166 of 167 ATRAZINE FINAL

Van der Kraak, G.J., Hosmer, A.J., Hanson, M.L., et al. (2014) Effects of atrazine in fish, amphibians, and reptiles: An analysis based on quantitative weight of evidence Crit. Rev. Toxicol, 44(S5):1–66.

Victor-Costa, A.B., Bandeira, S.M., Oliveira, A.G., et al. (2010) Changes in testicular morphology and steroidogenesis in adult rats exposed to Atrazine. Reprod. Toxicol. 29(3):323- 31.

Xing, H., Zhang, Z., Yao, H., et al. (2014) Effects of atrazine and chlorpyrifos on cytochrome P450 in common carp liver. Chemosphere 104:244-250.

Yang, L. Zha, J., Zhang, X., et al. (2010) Alterations in mRNA expression of steroid receptors and heat shock proteins in the liver of rare minnow (Grobiocypris rarus) exposed to atrazine and p,p’-DDE. Aquat. Toxicol. 98:381-387.

Yi, K.D., Breckenridge, C.B., and Simpkins, J.W. (2010a) Cytotoxicity of atrazine and its metabolites. Unpublished study prepared by Syngenta Crop Protection, Inc., Report Number: TK0025544. (MRID 47972810)

Yi, K.D., Breckenridge, C.B., and Simpkins, J.W. (2010b) Evaluation of the potential mechanisms of actions underlying the effect of triazines on aromatase expression in vitro – relevance to man and the environment. Unpublished study prepared by Syngenta Crop Protection, Inc., Report Number: TK0025529. (MRID 479728109)

Page 159 of 159 Page 167 of 167