UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: June 29, 2015 

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

List 1 Chemicals 

PC Code: See table, Attachment A 

Decision No.: NA 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

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 

Ver.Apr. 2010 

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
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

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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

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ii 

TABLE OF CONTENTS 

Abbreviations...................................................................................................iii 

Executive Summary..........................................................................................1 

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

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

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

B. Other Scientifically 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 

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iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ACTH Adrenocorticotropic hormone 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome 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

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iv 

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 

K M hd d 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 

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v 

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

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vi 

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

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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 endocrinerelated 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 

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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. 

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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. 

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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. 

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).

Technical atrazine is relatively insoluble in water (33 mg/L at 20°C), with an octanol water 

partition coefficient (Kow) of 501. The vapor pressure of atrazine is 3.0×10−7 mm Hg at 20°C. If 

released to air, atrazine will exist in both the vapor and particulate phases. Vapor-phase atrazine 

will be degraded in the atmosphere by reaction with photochemically-produced hydroxyl radicals 

(t1⁄2 = 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-striazine(DACT) and 2-chloro-21 6-ethylamino-4-amino-s-triazine (DIA). Atrazine is expected to 

be mobile in soil, but volatilization from moist or dry soil surfaces, or from water surfaces, is not 

expected to be an important fate process. Biodegradation can 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 t1⁄2 = 

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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. 

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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-HQOPP-2012-0230.

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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. 

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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 nonendocrine 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 

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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. 

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A. EDSP Tier 1 Screening Assays 

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

in Table 1. 

Table 1. Tier 1 Screening Assays for Atrazine 

Tier 1 Assays Test 

Guideline Test Order Status 

ER Binding Assay (Rat uterine 

cytosol) 

OSCPP 

890.1250 

Requirement satisfied by OSRI 

(Tennant et al.; USEPA, 2009) 

ERα Transcriptional Activation 

Assay (Human cell line HeLa 9903) 

OSCPP 

890.1300; 

OECD 455 

Requirement satisfied by OSRI 

(Fujimoto and Honda, 2003) 

AR Binding Assay (Rat prostate 

cytosol) 

OSCPP 

890.1150 Requirement satisfied by OSRI (USEPA, 2008a) 

Steroidogenesis Assay (Human cell 

line H295R) 

OSCPP 

890.1550; 

OECD 456 

Requirement satisfied by OSRI (USEPA, 2008c) 

Aromatase Assay (human 

recombinant microsomes) 

OSCPP 

890.1200 

Requirement satisfied by OSRI 

(Higley et al., 2010; USEPA, 2008b) 

Uterotrophic Assay (Rat) 

OSCPP 

890.1600; 

OECD 440 

Requirement satisfied by OSRI 

(Yamaski et al., 2000) 

Hershberger Assay (Rat) 

OSCPP 

890.1400; 

OECD 441 

Requirement satisfied by OSRI 

(Ashby et al., 2004: Yamaski et al., 2004)

Pubertal Female Assay (Rat) OSCPP 

890.1450 

Requirement satisfied by OSRI 

(Laws et al., 2000) 

Pubertal Male Assay (Rat) OSCPP 

890.1500 

Requirement satisfied by OSRI 

 (Stoker et al., 2000) 

Fish Short-term Reproduction Assay 

OSCPP 

890.1350; 

OECD 229 

Requirement satisfied by OSRI 

(Battelle, 2005) 

Amphibian Metamorphosis Assay 

(Frog) 

OSCPP 

890.1100; 

OECD 231 

Requirement satisfied by OSRI 

(MRID 47153501: Hosmer et al. 2007; Kloas et 

al., 2009; MRID 48614801) 

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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 [3

H]-estradiol binding 

(Tennant et al., 1994b). Atrazine also was negative for ER transactivation in the MtT/E-2 cell 

line at concentrations up to 10−4 M (Fujimoto and Honda, 2003). Additional OSRI that were 

considered also supported these 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), 

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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 highdose (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 treatmentrelated 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 

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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 atrazineinduced resorptions in Fischer (F344), SD and LE rats. In the pilot study in F344 rats (100, 200 

and 300 mg/kg/day), there was a dose-related increase in the number of full litter resporptions at 

all doses; however, the effect occurred in the presence of overt toxicity (>10% decrease in body 

weights). In the main study, F334, SD and LE 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 

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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 

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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 

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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). 

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Table 2: Estrogenic/Anti-Estrogenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen level 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

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., 

1994b) 

N4

ER Binding (Blair et al.,2000) N ER Binding (O’Conner et al.,

2000) N 
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Table 2: Estrogenic/Anti-Estrogenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen level 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

ERTA (Kojima et al., 2004) N ERTA (Fujimoto and Honda, 2003) N ERTA (Balaguer et al., 1999) N ERTA (Connor et al., 1996) N ERTA (Fukamachi et al.,

2004) N 

ERTA (Legler et al., 2002) N Aromatase (USEPA, 2008b) N Aromatase (Higley et al., 

2010) N5

Aromatase (Sanderson et al., 2001) P Aromatase (Sanderson et al., 2002) P Aromatase (Rainbow Trout; Hinfrey et al., 2006) N Aromatase (Holloway et al., 2008) P Aromatase (Tinfo et al., 2011) P 
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Table 2: Estrogenic/Anti-Estrogenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen level 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

FSTRA (Battelle, 2005) N N N NR N FSTRA (Bringolf et al., 2004) NE N N NR N Modified FSTRA (Fathead Minnow; Tillitt et al., 2010) 

 NE N P 

Fcd: 

↓19-

39% 

(L, M, 

H) 

Modfied FSTRA (Japanese Medaka; Papoulias et al., 2014) 

N N P 

Frt: NE: 

Fcd: 

↓36-42% 

(L, M, H)

Female Pubertal Rat (Laws et al., 2000) NE 

wo/ 

fluid: 

↓31% 

(MH) 

N N 

↓16

%(

L, 

M

H) 

P 

(MH) 

Age: 

↑3.4, 4.5 d 

(M, 

MH); 

BW: ↑

(M, 

MH) 

Steroidogenesis (USEPA, 2008c) P Uterotrophic (Yamasaki et al., 2000) N N Uterotrophic (Tennant et al., 1994a) 

↓

(M) 
ATRAZINE FINAL 

Page 20 of 159 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen level 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Uterotrophic (Connor et al., 1996) N Uterotrophic (Rat; MRID 45722401; Ashby et al., 2002, Exp 1) 

↓41

% 

(H) 

NE 

Uterotrophic (Rat; MRID 45722401; Ashby et al., 2002, Exp 2) 

↓55

% 

(H) 

NE 

Uterotrophic (Rat; MRID 45722401; Ashby et al., 2002, Exp 3) 

N N 

Uterotrophic (Rat; MRID 45722401; Ashby et al., 2002, Exp 4) 

N 

↑2.5, 

3 day 

 (M, H) 

Uterotrophic (Rat; O’Conner et al., 2000) N 21-Day Dermal Toxicity (Rabbit; MRID 42089902) 

↓44

% 

↓36

% N N 

Two-generation reproduction toxicity (Rat; MRID 40431303, DeSesso et al., 2014) 

NE N N NE N 

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

 NE P P 
ATRAZINE FINAL 

Page 21 of 159 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen level 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Reproductive toxicity, Exp 1-diet (Rat; Foradori et al., 2014) 

 NE N N 

Reproductive toxicity, Exp 2-bolus (Rat; Foradori et al., 2014) 

 NE N 

Reproductive toxicity, Exp 2-diet (Rat; Foradori et al., 2014) 

 NE N 

Reproductive toxicity, Pilot (Rat; Narotsky et al., 2001) NE 

Frt: N; 

Fcd: 

↓63% 

(L) 

Reproductive toxicity, Single dose (Rat; Narotsky et al., 

2001) 

 NE N 

Reproductive toxicity, F344 strain (Rat; Narotsky et al., 

2001) 

 NE N 

Reproductive toxicity, S-D strain (Rat; Narotsky et al., 

2001) 

 NE N 

Reproductive toxicity, LE strain (Rat; Narotsky et al., 

2001) 

 NE N 
ATRAZINE FINAL 

Page 22 of 159 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen level 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Reproductive toxicity, one dose per day (Rat; Davis et al., 2011) 

 NE N 

Age: 

↑1.5 

day 

(H) 

N N 

Reproductive toxicity, two doses per day (Rat; Davis et al., 2011) 

 NE N 

Age: 

↑1.9 

day 

(H) 

N N 

Developmental reproductive toxicity, in utero exposure (Rat; DeSesso et al., 2014) 

 NE N 

Developmental reproductive toxicity, Post-natal exposure (Rat; DeSesso et al., 2014) 

 NE N 

Developmental toxicity (Rat; MRIDs 00143008, 40566302; Scialli et al., 2014) 

 NE N 

Developmental toxicity (Rat; MRID 41065201; Scialli et al., 2014) 

 NR N 

Developmental toxicity (Rabbit; MRIDs 00143006, 4056630; Scialli et al., 

20142) 

 NE 

Frt: N; 

Fcd:

↓33% 

(H) 

Chronic Toxicity/Carcinogenicity (Rat; MRID 40629302) 

 NE N N NE 
ATRAZINE FINAL 

Page 23 of 159 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen level 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Carcinogenicity (Mouse; MRID 40431302) NE NE N NE Chronic Toxicity (Dog; MRID 40431301, 41293801) NE N N N Avian Toxicity (Mallard Duck; MRID 42547101) 

Frt: ↓

(H); 

Fcd: ↓

(H) 

Avian Toxicity (Bobwhite Quail; MRID 42547102) 

Frt: ↓

(H); 

Fcd: ↓

(H) 

Avian Toxicity (Japanese Quail; Wilhelms et al., 2006) N Fish Partial Life Cycle Toxicity (Brook trout; MRID. 00024377) 

N 

Early Life-Stage Toxicity (Sticklebacks; Le Mer et al., 2013) 

 N 

Fish Full Life-Cycle Toxicity (Fathead minnow, MRID 42547103) 

N 

Sexual development, Study 1 (Zebrafish; Corvi et al., 2012) 

N N 
ATRAZINE FINAL 

Page 24 of 159 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Atrazine1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen level 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Sexual development, Study 2 (Zebrafish; Corvi et al., 2012) 

N N 

In ovo exposure, (Alligator; Crain et al., 1997) N N In ovo exposure, (Alligator; Spiteri et al., 1999) N N N Developmental toxicity (Watersnake; Neuman-Lee et al., 2014)

N 

Frt: 

NE; 

Fcd:↓

(M, H) 

In vitro ovulation (Frog; Orton et al., 2009) N P (L) Amphibian metamorphosis and reproductive development (Frog; Hosmer et al., 2007 and Kloas et al., 

2009) 

N N 

Amphibian reproductive development (Frog; Oka et al., 2008) 

N 

↑%

♀

(H) 

Amphibian reproductive development (Frog; Spolyarich et al., 2010) 

N N 

HPG Axis (Rat; Cooper et al., 2000) N N P (H) 
ATRAZINE FINAL 

Page 25 of 159 

1. Key to responses: Lowest treatment (L), Medium-low treatment (ML), Low-medium treatment (LM), Medium treatment (M), H

(HM), Medium-high treatment (MH), High test treatment (H). Arrows (↓ or ↑) indicate the direction of the response. A shaded cenot 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 O

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 

details see Section IV. A 

4. Competitive binding against estradiol (E2) was negative (up to 10−3 log M), but pre-incubation with excess atrazine inhibited E2 b

5. Direct aromatase activity was not different from control at any concentration tested. Atrazine significantly induced indirect and co

activity. 

P Positive finding N Negative finding (in vitro)/No effect (in vivo) NE Not examined 
ATRAZINE FINAL 

Page 26 of 159 

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 concentrationdependent 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 nonmonotonic 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 34 of 167
ATRAZINE FINAL 

Page 27 of 159 

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 35 of 167
ATRAZINE FINAL 

Page 28 of 159 

in the presence of overt toxicity (>10% decrease in body weight). In another male reproductive 

toxicity study (Song et al., 2014), anti-androgenic 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 36 of 167
ATRAZINE FINAL 

Page 29 of 159 

failed to demonstrate consistent atrazine treatment-related effects (up to 100 μg/L) on gill 

ventilation rate, growth, CYP19B and VTG expression, or 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 37 of 167
ATRAZINE FINAL 

Page 30 of 159 

Table 3: Androgenic/Anti-Androgenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone level 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at 

PPS 

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 (USEPA, 2008a) N AR Binding (Fang et al., 2003) 

N 

Receptor Transactivation (Kojima et al., 

2004) 

 N 

FSTRA (Battelle, 2005) N N P NE N FSTRA (Bringolf et al., 2004) NE N N N N Modified FSTRA (Fathead minnow; Tillitt et al., 2010) 

NE N N N NE 
ATRAZINE FINAL 

Page 31 of 159 

Table 3: Androgenic/Anti-Androgenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone level 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Modified FSTRA (JMedaka; Papoulias et al., 

2014) 

N N 

P 

(L, M, 

H) 

Frt: NE 

Vitellogenin (Sanderson et al., 

2001) 

Hershberger (Ashby et al., 2004) 

LABC:, 

↓16-18% 

(MH, H)

GP:↑10% 

(H) 

Hershberger (Yamasaki et al., 

2004) 

N 

Male Pubertal Rat (Stoker et al., 2000) 

N N N N↓

VP: ↓

(M, HM, 

MH) 

N 

Modified Male Pubertal (Rat; Trentacoste et al., 2001) 

↓34 

(H) NR NE NE NE NE 

SV: 

↓32 (H); 

VP: 

↓26 

(H) 

Age: 

↑3 day 

(H); BW: 

NR 

Steroidogenesis (USEPA, 2008c) P 21-Day Dermal Toxicity (Rabbit; MRID 42089902) 

N N N N N N 
ATRAZINE FINAL 

Page 32 of 159 

Table 3: Androgenic/Anti-Androgenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone level 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Two-generation reproduction toxicity (Rat; MRID 40431303, DeSesso et al., 

2014) 

N N NE N NE N N 

Reproductive toxicity, once daily dosing (Rat; Fraites et al., 2011) 

N N NE N NE 

↓7, 8, 

12% 

(ML, 

MH, 

H) 

N N 

Reproductive toxicity, twice daily dosing (Rat; Fraites et al., 2011) 

N N NE N NE N N N 

Reproductive toxicity (in uteroexposure in Rat; Rosenberg et al., 

2008) 

↓

(M) N NE NE NE NE N N 

Reproductive toxicity (Rat; Song et al., 2014) 

↓10% 

(M) N P (M) N NE NE N ↓Sperm (M) 
ATRAZINE FINAL 

Page 33 of 159 

Table 3: Androgenic/Anti-Androgenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone level 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Developmental reproductive toxicity, in uteroexposure (Rat; DeSesso et al., 

2014) 

N N P (MH) N NE N PR: N; SV: N 

Developmental reproductive toxicity, Postnatal exposure (Rat; Desesso et al., 2014) 

N ↓7, 6% (H) P (NS;H) ↓12% (H) NE N PR: N; SV: N 

Chronic Toxicity/ Carcinogenicity (Rat; MRID 40629302) 

N N N N NE PR: N 

Carcinogenicity (Mouse; MRID 40431302) 

N N NE N NE NE 

Chronic Toxicity (Dog; MRID 40431301, 41293801) 

N N N N N NE 

Reproductive development in males (Japanese Quail (Wilhelms et al., 2005) 

↑200%

 (H) 

↑46% 

(L) 

↑49% 

(L) 

N 
ATRAZINE FINAL 

Page 34 of 159 

Table 3: Androgenic/Anti-Androgenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone level 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Steroidogenesis, in vivo (Juvenile Barramundi; Kroon et al., 2014) 

↑

(L, M) 

Early Life-Stage Toxicity (Sticklebacks; Le Mer et al., 2013) 

N 

Reproductive development (Zebrafish; Corvi et al., 2012) 

N N 

Reproductive development (in ovo exposure in Alligator; Crain et al., 1997)

N N N 

Reproductive development (in ovo exposure in Alligator; Spiteri et al., 1999)

N N N N 

In vitro ovulation assay (Frog; Orton et al., 

2009) 

↑24% 

(M) 
ATRAZINE FINAL 

Page 35 of 159 

Table 3: Androgenic/Anti-Androgenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone level 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Amphibian metamorphosis and reproductive development (Frog; Hosmer et al., 2007 and Kloas et al., 

2009) 

 N N 

Amphibian reproductive development (Frog; Spolyarich et al., 2010) 

 N 

Amphibian reproductive development (Frog; Oka et al., 2008) 

 N ↑%♀ (H) 

Tumor Development (Rat; Fukamachi et al., 2004) 

N 

Male Steroidogenesis (Rat; Pogrmic et al., 2009) 

N ↓8% (L) 

SV: 

↓30% (L) 

VP: 

↓22% (L) 
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Table 3: Androgenic/Anti-Androgenic Pathway for Atrazine 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Atrazine1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone level 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Male Steroidogenesis, In vitro (Rat; Pogrmic-Majkic et al., 2010) 

↑31, 69, 

98% 

(LM, 

HM, H)

Male Steroidogenesis, In vivo (Rat; Pogrmic-Majkic et al., 2010) 

↑

Male Steroidogenesis (Rat; Riffle et al., 2014) 

N 

Male Steroidogenesis (Mouse; Jin et al., 

2013) 

↓ 25-

30% 

(L, M) 

↓

10% 

(L) 

1. Key to responses: Lowest treatment (L), Medium-low treatment (ML), Low-Medium (LM), Medium treatment (M), High-Mediu

treatment (MH), Highest treatment (H). Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is para

evaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated. Abbreviations for androgen 

Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididygland (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 O

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 

details see Section IV. A 

P Positive finding 
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N Negative finding (in vitro)/No effect (in vivo) 

NE Not examined NR Not reported 
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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). 

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Table 4. Thyroid Pathway for Atrazine.

Lines of Evidence Indicating Potential for Interaction with the Thyroid Pathway for Atrazine1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage (± or asynchronous, HLL)

Growth (BW, SVL) Systemic Toxicity2 

Overt Toxicity Observed3

EDSP Tier 1 Assays 

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

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

OSRI

Female Pubertal Rat 

(Laws et al., 2000) NE N N N ↓16, 16

 (L, MH)

X 

(H) 

X 

(H) 

Male Pubertal Rat 

(Stoker et al., 2000) NE N N N N X 

(H) 

X 

(H) 

21-Day Dermal Toxicity 

(Rabbit; MRID 

42089902) 

N N N X 

(H) 

X 

(H) 

Chronic 

Toxicity/Carcinogenicity 

(Rat; MRID 40629302) 

N N NE X 

(MH, H)

X 

(MH, H)

Carcinogenicity (Mouse; 

MRID 40431302) NE N NE X 

(MH, H)

X 

(MH, H)

Chronic Toxicity (Dog; 

MRID 40431301, 

41293801) 

N N N N X 

(H) 

HPG Axis (Rat; Cooper 

et al., 2000) N N X 

(H) N 

Amphibian 

Metamorphosis (Toad; 

Brodeur et al., 2013) 

 NE 

P 

(ML, M, 

MH) 

BW: 

↑12% 

SVL: 

↑4-5% 

(ML,M) 

X 

(H)

Amphibian 

Metamorphosis (Frog; 

Hosmer et al., 2007; 

Kloas et al., 2009) 

 NE N 

BW: 

↓7% 

(L, M, H ) 

SVL:

↓2-3% 

(L, M, H ) 

 N 

Amphibian 

Metamorphosis (Frog; 

Oka et al., 2008) 

NE N N 

Amphibian 

Metamorphosis (Frog; 

Spolyarich et al., 2010) 

NE N N N 

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Table 4. Thyroid Pathway for Atrazine.

Lines of Evidence Indicating Potential for Interaction with the Thyroid Pathway for Atrazine1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage (± or asynchronous, HLL)

Growth (BW, SVL) Systemic Toxicity2 

Overt Toxicity Observed3

HLL and Thyroid 

Histopathology of 

Archived Specimens 

from Hosmer et al. 

(2007) and Kloas et al. 

(2009) 

(MRID 48614801)4

 N5 N5

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 

 

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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 nonendocrine 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).

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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. 

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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 coadministered 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. 

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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. 

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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. 

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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 highdose 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 

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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, mediumhigh and high-concentration females, as well as an increase in fibroadenoma in the highconcentration females, in the carcinogenicity subgroup. Some females developed multiple 

mammary tumors. There also was an increase (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. 

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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 

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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 pairfed birds. In Experiment 4, atrazine administered at 1 and 10 mg/kg/day by s.c. injection had no 

effect on indices of growth or reproductive 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. 

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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 posthatch 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. 

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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. 

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 

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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). 

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. 

The data synthesized in this WoE evaluation reflect varying responses in androgen-related 

endpoints across species and studies. Data from OSRI beyond the Tier 1-like provide 

complementarity and redundancy of findings with respect to steroidogenesis (in vitro and in 

vivo) and gonadal histology, though similar effects were not seen in all studies. Overall, the 

potential for interaction with the androgen 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.

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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). 

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. 

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with altered steroidogenesis induced by exposure to atrazine in the intact and castrate male rat. 

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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.

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with atrazine and its major metabolites in rats and rabbits. Birth Defects Res. B Dev. Reprod. 

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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, 

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Wistar rats: an evaluation in the protocol for the assessment of pubertal development and thyroid 

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puberty and thyroid function in the male Wistar Rat. Toxicol. Sci. 67:198-206. 

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kinetics of terrestrial atrazine exposure in American toads (Bufo americanus). Arch. Environ. 

Contam. Toxicol. 57:590-597. 

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Time Course of Natural Development and Role of Endocrine Disruptors. J. of Exp. Zool. (Mol. 

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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. 

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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. 

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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. 

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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. 

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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 hypothalamopituitary-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. 

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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. 

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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. 

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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. 

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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 highconcentration 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. 

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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 [3

H]-R1881 (1 nM) in the presence of increasing concentrations 

(logarithmic increase from 10−11 to 10−3

 M) of atrazine (98% purity). A total of three runs were 

conducted, and each run included dexamethasone as a weak positive control, and R1881 as the 

ligand reference standard. 

In the inter-laboratory 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 

10−11 to 10−3 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 [3

H]-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. 

In the Saturation Binding Experiment, the mean dissociation constant (Kd) for [3

H]-R1881 was 

2.03±0.15 nM. The mean estimated Bmax was not reported. In the Competitive Binding 

Experiment, the mean log IC50 value for R1881 was 3.07×10−9 ± 9.12×10−10 M. 

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Precipitation and/or cytotoxicity with atrazine were not reported. Atrazine had no effect on 

specific binding of the [3

H]-ligand over the concentration range of 4.28×10−9 to 4.28×10−5 M; 

therefore, a log IC50 = >4.28×10−5 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 interlaboratory 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 ([1β3H(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. 

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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 

cells were subjected to a 3

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 tritiumrelease 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 dosedependent 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 

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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 

aromatase activity measured by the 3

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 

was removed and aromatase activity was measured by the 3

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, 

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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 17βestradiol and progesterone was determined in the media, and aromatase activity was measured by 

the 3

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). 

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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 interlaboratory 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 [3

H]-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 

constant (Kd) for [3

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 

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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 [3

H]-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 

with 10 nM [3H]-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 [3

H]-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 [3

H]-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 [3

H]-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. 

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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 ERenriched uterine cytosol prepared from intact adult female SD rats. 

The [3

H]-estradiol (1×10–9 M final assay concentration) was incubated with increasing 

concentrations of radio-inert competitor, 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 competitivebinding assays were conducted in a tiered design. Unless known to bind to the ER, test chemicals 

were initially run at only two high concentrations spanning 3 log concentrations (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. 

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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 (CHOK1 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 

measurement of antagonistic activity to hERα, hERβ and hAR, either 10−11 or 10−10 M E2 (17βestradiol, > 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 

activity of 10−10 M E2, 10−9 M E2, or 10−9

 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 

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inhibition of the activity of 10−10 M E2, 10−9 M E2 or 10−9 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. 

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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 17βestradiol + 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 (<MDL; control), 24.5±18.9, and 223.7±156.6 μg/L. The test system 

was nominally maintained at 24-26°C; pH was not reported. 

One female in the control group died during the 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% 

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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 treatmentrelated effects were noted in either sex for sex-steroid concentrations. 

Treatment-related effects on gonadal histopathology were not observed in female fish. In male 

fish, a difference in testicular staging was observed with both treatment groups having a greater 

proportion of cells in Stage 2A, and the low-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 posthatch). On Day 21, coloration of male fish was evaluated in situ based on the presence or 

absence of light and dark vertical bands on the body surface, and the presence or absence of an 

ovipositor was noted for female fish. After 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 

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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 17βestradiol 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 17βestradiol-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. 

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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 highdose 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 

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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 

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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 pairfed 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. 

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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 pairfed 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. 

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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 doserelated. 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 

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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 vehiclegavage control group (420 g vs. 463 g control) with concomitant decreases in daily mean body 

weight and decreased (p<0.05) terminal mean body weight (~↓10%). Terminal mean body 

weight was decreased (p<0.05) to a similar extent (~↓10%) in the food- restricted vehicle group. 

Decreases in serum testosterone and LH 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). 

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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. 

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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 coadministered 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 

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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 Antarelixtreated 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. 

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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 (1⁄3 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 

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(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 

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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. 

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Reproductive Toxicity in Females 

In a female reproductive toxicity study (Foradori et al., 2014), the effect of orally administered 

or dietary atrazine on estrous cyclicity in rats was examined. Young adult female (60- to 90-dayold) Sprague Dawley (SD) and Long-Evans (LE) 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 

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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 

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decreases (p<0.05) in the number of ova per animal (↓40 and 43%, respectively). Non-dosedependent 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 

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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. 

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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. 

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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 nonsignificant 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 

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PND 7. At weaning (PND21 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 

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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 highdose 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. 

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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 

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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 

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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. 

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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, mediumhigh 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. 

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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 dosedependent 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 highdose 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× 

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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 highdose 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 postimplantation loss (↑455%) and a decrease (p<0.05) in the mean number of pups per litter at birth 

in the high-dose group (↓39%). Postnatal pup survival also 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 highdose group (↑80%) and on PND 170 in the medium-high dose group (↑32%). Therefore, the 

NOEL for male developmental toxicity was 5 mg/kg/day. 

In the second of two special studies examining male reproductive development (DeSesso et al., 

2014), groups of 27-31 time-mated female Wistar rats were assigned to five experimental 

groups. After delivery, atrazine (97.2% purity) was 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

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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), 

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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. 

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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. 

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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 mediumhigh concentration group through Week 78 (↓7-14 and 6-21%, respectively) with decreases (NS 

or p<0.05) of 8 and 19%, respectively, at Week 104. Body weights also were decreased (p<0.01) 

for males and females in the high 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 highconcentration 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 treatmentrelated 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 

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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, mediumhigh, and high-concentration females, as well as an increase in fibroadenoma in the highconcentration females, in the carcinogenicity subgroup. Some females developed multiple 

mammary tumors. There also was an increase (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. 

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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 highconcentration) received a daily atrazine dose of approximately 25% higher compared to males in 

these treatment groups. There was an increase in mortality (p<0.05) in high-concentration 

females, but not males, with 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 treatmentrelated clinical signs observed during the study. Mean body weight gain decreases were reported 

at Weeks 12 and 91 for the medium-high and high-concentration animals. At Week 91, body 

weight gain was decreased (p<0.05) by 25 and 24%, respectively, in males and decreased by 

10% (NS) and 52% (p<0.01) in females in the 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 

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1000 ppm for 52 weeks. The mean-measured daily doses were identical for the low and mediumconcentration groups (0.48 and 4.97 mg/kg/day, respectively) and were similar for the highconcentration 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 mediumconcentration 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 atrazineinduced 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 highconcentration 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 

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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 highand 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 compoundrelated 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 

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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. 

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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-weekold 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 liversomatic 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 

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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 liversomatic 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. 

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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 

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<0.028 (<LOQ, control), 0.15, 0.30, 0.57, 1.1, and 2.2 mg/L, respectively. The test system was 

maintained at 24-27°C and a pH of 7.8-8.2. 

No treatment-related effect was observed on hatching (71-79%) or fry survival (≥97%), and no 

abnormal behavior or other clinical signs of toxicity were 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) 

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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 spermatogonialike 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 

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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 

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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 17βestradiol) 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. 

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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 17βestradiol 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 mediumconcentration 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 

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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: previtellogenic (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 

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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 fourPage 133 of 167
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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 (reverseosmosis 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 postfertilization) 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. 

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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, meanmeasured concentrations were 0.72 nM for Study I and 0.32 nM for Study II; no explanation was 

provided for the decreased concentration in 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 17βestradiol 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 

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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 17βestradiol-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 testesova 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 

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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-dayold) 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, 

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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 temperaturesensitive 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 (3

H) from 1β3H-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 17βestradiol 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%; 

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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 maleproducing 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. 

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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 

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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 treatmentrelated effect on mortality was observed in any treatment group. No effects were seen on 

maternal estradiol. However, atrazine-treated females displayed an increased incidence of 

infection which led to death (2-3 treated vs. 1 control). 

The proportions of live born offspring were decreased in all atrazine treatment groups in a nondose-dependent manner compared to control. Atrazine treatment was also associated with 

effects on scale row symmetry at the mid-body, 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. 

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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 

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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 

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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 

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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 17βestradiol 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 

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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, 

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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. 

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Tumor Development (Rat) 

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 protooncogene with its own promoter region, which is highly susceptible to DMBA-induced 

carcinogenesis. 

Human c-Ha-ras proto-oncogene transgenic (Tg) and non-Tg rats at 7 weeks of age were 

maintained in plastic cages at 22±2°C and 55±10% humidity. The animals were allowed access 

to pellet basal diet (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 

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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 atrazinetreated, 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. 

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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 

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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 highdose 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 

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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 hCGstimulated 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 concentrationdependent manner (up to 98% increase at 50 μM (11 ppm)). Atrazine treatment in the presence 

of hCG (0.125-10 ng/mL) demonstrated similar effects. Progesterone concentrations were 

decreased (p<0.05) at the 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

-

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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 

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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. Mediumhigh 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 

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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 nonmonotonic 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. 

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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. 

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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 

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treatment at 100 and 200 mg/kg/day (↓59 and 74%, respectively). There were no atrazinetreatment 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., 

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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 

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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 postgavage 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 

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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 

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lobe was immediately dissected free and the anterior lobe was then hemisected along the rostralcaudal 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. 

 

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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; Nonmonotonous acceleration of the time to climax and delayed tail resorption. Aquat Toxicol. 

91:161-170. 

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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. 

FAO/WHO Pesticide Residues in Food (2007) Report of the Joint Meeting of the FAO Panel of 

Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment 

Group on Pesticide Residues. Evaluations, Part II - Toxicological Geneva, Switzerland, 18–27 

September 2007. 

Forgacs, A.L., Ding, Q., Jaremba, R.G., et al. (2012) BLTK1 murine Leydig cells: a novel 

steroidogenic model for evaluating the effects of reproductive and 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. 

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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. 

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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.

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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) 

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