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 74
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 

Page 2 of 74
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN AND THYROID 

PATHWAYS 

CHEMICAL: SIMAZINE 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

Page 3 of 74
SIMAZINE FINAL

 

 

ii 

TABLE OF CONTENTS 

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

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

I. Introduction.......................................................................................5 

II. Sources of Scientific Data and Technical Information......................................7 

A. EDSP Tier 1 Screening Assays............................................................7 

B. Other Scientifically Relevant Information (OSRI)......................................7 

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..................................................16 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................20 

IV. Committee’s Assessment of Weight of Evidence..........................................22 

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

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

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

B. Estrogen Pathway...........................................................................26 

C. Androgen Pathway.........................................................................28 

D. Thyroid Pathway...........................................................................28 

E. Conclusions.................................................................................29 

V. EDSP Tier 2 Testing Recommendations....................................................29 

VI. References.......................................................................................31 

LIST OF TABLES: 

 Table 1. Tier 1 Assays for Simazine.................................................................10 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Simazine.....................................13 

 Table 3. Androgenic/Anti-Androgenic Pathway for Simazine...................................17 

 Table 4. Thyroid Pathway for Simazine............................................................21 

APPENDIX 1. EDSP Tier 1 Screening Assays..........................................................36 

APPENDIX 2 Other Scientifically Relevant Information(OSRI).................................42 

APPENDIX 3. References Not Utilized in the Simazine WoE Analysis.........................65 

Page 4 of 74
SIMAZINE FINAL

 

 

iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ACTH Adrenocorticotropic hormone

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome 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

Page 5 of 74
SIMAZINE FINAL

 

 

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 

Methods 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

Page 6 of 74
SIMAZINE FINAL

 

 

v 

Abbreviation Terminology 

ML Medium low 

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

Page 7 of 74
SIMAZINE FINAL

 

 

vi 

Abbreviation Terminology 

SV Seminal Vesicles 

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

Page 8 of 74
SIMAZINE FINAL

 

 

Page 1 of 66 

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 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) perturbations. 

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 this assessment were the FIFRA Science Advisory Panel 

(SAP) reports on atrazine and the common mechanism grouping (CMG) of simazine with other 

similar triazines (http://www.epa.gov/pesticides/reregistration/atrazine/atrazine_update.htm) 

In determining whether simazine 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 simazine 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).

The weight of evidence approach (WoE) considered Tier 1 assay results, OSRI data, as well as 

the inclusion of simazine in the triazine common mechanism group (CMG) which relies on 

numerous SAPs on atrazine. 

For the estrogen pathway, although simazine was positive in the estrogen receptor (ER) binding 

assay by Tennant et al. (1994b), it was negative for ER binding in an assay conducted by Blair et 

al. (2000) using the rat uterine cytosol assay and negative in a cell proliferation assay (using 

MCF-7 cells) conducted by Connor et al. (1996). Additionally, simazine was also negative in 

the Tier 1 and OSRI ER transcriptional activation (ERTA) assays. Altered aromatase activity 

was reported in studies using JEG-3 cells by Sanderson et al. (2001). In a single dose 

steriodogenic study of simazine and its DACT and DIA metabolites, the levels of ACTH, 

progesterone, and/or corticosterone were increased (Laws et al., 2009). In the in vivo assays, 

simazine was negative for estrogenic activity in several OSRI uterotrophic assays. In the two 

OSRI female pubertal assays (Zorilla et al., 2010), delays in VO were seen at 25 mg/kg/day in 

both studies and at 50 mg/kg/day in the second study in the absence of overt toxicity; delays in 

Page 9 of 74
SIMAZINE FINAL

 

 

Page 2 of 66 

VO were also seen at high doses in both studies in the presence of overt toxicity. There were no 

estrogenic/anti-estrogenic effects noted in the Part 158 mammalian toxicity studies. 

There was a marginally significant (p=0.0512) reduction in female GSI at the high treatment 

level (3.4 mg/L) in the FSTRA; however, no effects on reproduction were observed. Female 

mallard ducks exposed to simazine in the diet presented a greater incidence of regressed ovaries 

and produced fewer eggs, fewer hatchling survivors, and fewer 14-day survivors at the highest 

treatment level; however, a slight decrease (10%) in female body weight was also observed at 

this dose. Decreases in eggs laid and possible embryo viability effects were observed in the quail 

study. 

Since simazine belongs to the triazine CMG, evidence of estrogen-related effects with atrazine 

and atrazine metabolites (Laws et al., 2000; 2003; SAP, 2000) were also considered. In studies 

by Laws et al. (2000), atrazine significantly delayed VO and reduced the weights of reproductive 

tissues (uterine and ovarian) associated with estrogenic responses in peripubertal rats. Similar 

effects were observed in peripubertal rats exposed to chlorinated metabolites of atrazine (Laws et 

al., 2003) indicating a similar mode of action for the metabolites. 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 resulting in delayed VO and alterations in estrogen-responsive tissues. 

For the androgen pathway, simazine was negative in the androgen receptor (AR) binding and AR 

transcriptional activation (ARTA) assays. Simazine was positive in the steriodogenesis assay by 

Sanderson et al. (2001). In a single dose steriodogenic study of simazine and its DACT and DIA 

metabolites, the levels of ACTH, progesterone, and/or corticosterone were increased (Laws et 

al., 2009). Simazine was negative in the Tier 1 in vivo Hershberger assay. All androgen-related 

effects in the male pubertal assay and Part 158 mammalian toxicity studies occurred in the 

presence of overt toxicity. There were no androgen-related effects in the FSTRA. Regressed or 

regressing testes were observed in 25% of male bobwhite quail at the highest treatment level in 

an avian reproduction study; decreases in embryo viability was observed in this study along with 

effect on offspring survival in the mallard study. 

Androgen-related effects noted with atrazine and its chlorinated metabolites (Stoker et al. 2000; 

2002) were also considered. 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 

Page 10 of 74
SIMAZINE FINAL

 

 

Page 3 of 66 

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. 

The available data do not provide convincing evidence of potential interaction of simazine with 

the thyroid pathway in mammals or 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 based on effects seen with atrazine. The POD of 2.56 

mg/kg/day based on a luteinizing hormone suppression with atrazine is regarded as a precursor 

event in the neuroendocrine mode of action of the triazines. This POD is used for deriving the 

chronic Reference Dose (RfD) used for chronic dietary risk assessment and calculating the 

Margin of Exposure (MOEs) for non-dietary risk assessment. 

In the EDSP in vivo Tier 1 male pubertal assay with atrazine, a delay in preputial separation 

(PPS) was seen at the lowest dose tested (12.5 mg/kg/day). In the EDSP in vivo female pubertal 

assay with simazine, delays in VO were seen at doses greater than 25 mg/kg/day and suppression 

of serum prolactin levels, suggesting that the mechanism of action of simazine is to alter 

hypothalamic regulation of pituitary hormone secretion (Zorrilla et al., 2010). Laws et al., 2000 

reported estrogen- related effects (i.e., delays in the onset of puberty and altered estrous 

cyclicity) with atrazine at doses greater than 50 mg/kg/day in the female pubertal assay with 

atrazine. The POD (2.56 mg/kg/day) is approximately 5-fold lower than the dose (12.5 

mg/kg/day that caused delay in PPS and approximately 8-fold lower than the dose (25 

mg/kg/day) that caused delay in VO. In addition, a dose of 6.25 mg/kg/day was reported to have 

no effect on the day of preputial separation when compared to controls in another male pubertal 

assay (Stoker et al., 2000). Therefore, the chronic RfD and the MOE are protective of the 

estrogen and androgen-related effects seen in the EDSP pubertal assays. 

With respect to determining whether additional studies are needed for ecological risk assessment 

based on the Tier 1 screen, the findings in the Tier 1 assays do not have an impact on the 

wildlife. For wildlife, the only effect on estrogen-, or androgen-related endpoints in the FSTRA 

was a marginally significant decrease in female GSI at 3.4 mg a.i./L, which was not associated 

with decreased reproduction at that treatment level. The most recent ecological risk assessment 

endpoints for chronic effects in freshwater fish and aquatic-phase amphibians are based on a 

120-day fish reproduction test with continuous exposure of fathead minnow to an 80% 

formulation of simazine, which yielded a NOAEC value of 0.96 mg a.i./L and a corresponding 

LOAEC value of 2.0 mg a.i./L (MRID 00043676, Mayer and Sanders 1975). The LOAEC value 

Page 11 of 74
SIMAZINE FINAL

 

 

Page 4 of 66 

is based on a 12% reduction in growth (length) in 30-day old fish. No effects on reproductive 

endpoints were observed in the fish full life cycle test. This fish data were not included in the 

WoE determinations of potential estrogen or androgen interaction because the test was conducted 

with a formulated product; however, the data are relevant here because they have previously 

been used as regulatory endpoints. For birds, Part 158 avian reproduction studies with both 

northern bobwhite quail and mallard duck are available for simazine. In both species, effects on 

reproductive parameters were observed. The type of data obtained from Part 158 avian 

reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential 

reproductive effects to birds from simazine exposure. Relative to the EDSP, additional testing is 

not recommended. 

Therefore, based on the WoE analysis, EDSP Tier 2 testing is not recommended for simazine 

since additional testing will not impact current EPA established regulatory endpoints for human 

health or ecological risk assessment. 

Page 12 of 74
SIMAZINE FINAL

 

 

Page 5 of 66 

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 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) perturbations. 

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 Science Advisory 

Panel (SAP) reports on atrazine (2009 – 2011) and the common mechanism grouping (CMG) of 

simazine with other similar triazines (refer to the Introduction section for more details). 

In determining whether a pesticide chemical 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 simazine 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).

Simazine, a chlorinated triazine herbicide, has a water solubility of 3.5 mg/L at 20oC. Laboratory 

absorption data show low water/soil partitioning for simazine, or ability to be absorbed into the 

soil. Laboratory studies indicate that simazine is able to persist in soil for months and possibly 

years in oxygen-deprived (anaerobic) aquatic systems, as it is not easily degraded by anaerobic 

aquatic microorganisms. If simazine is released at the soil surface and under direct sunlight, it 

undergoes more rapid degradation. Simazine is also stable to abiotic routes of degradation (e.g., 

hydrolysis and aqueous photolysis), increasing its likelihood to move into surface water. With a 

low vapor pressure (6.1×10−9

 mm Hg at 20°C) and low Henry’s Law constant (3.2×10−10

atm·m3

mol−1 at 25°C), losses of simazine from soil and water systems due to volatilization are 

expected to be insignificant compared to dissipation by chemical degradation and metabolism. 

The octanol-water partitioning coefficient is (Kow) 122 with bioconcentration factors of 4.92-7.30 

in whole fish. 

Page 13 of 74
SIMAZINE FINAL

 

 

Page 6 of 66 

The available information considered in determining the potential interaction of simazine 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 articles. 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. 

Other information considered in this WoE assessment include were the FIFRA SAP reports on 

atrazine and the CMG of simazine with other similar triazines. In 2002, the EPA concluded that 

the triazine-containing pesticides atrazine, simazine, and propazine and their three chlorinated 

degradates should be included in a CMG and considered through a cumulative risk assessment. 

EPA’s triazine cumulative risk assessment considered the combined effects of atrazine, simazine 

and propazine, closely related triazine herbicides. These chlorotriazine herbicides, along with 

respective three major chlorinated metabolites [i.e., desethyl-s-atrazine (DEA), desisopropyl-satrazine (DIA), and diamino chlorotriazine (DACT)], have been determined by 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) to share a common 

neuroendocrine mechanism of action which results in both reproductive and developmental 

alterations. These three chemicals (atrazine, simazine, and propazine) and their three major 

chloro metabolites constitute the triazine CMG. Studies have demonstrated that the CMG 

triazines can cause a disruption of the HPG axis in the 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 of the triazines. 

The grouping of the CMG triazines based on a common mechanism of action was presented to 

the FIFRA SAP in 2000 (http://www.epa.gov/scipoly/sap/meetings/2000/062700_mtg.htm). The 

SAP agreed with the Agency’s conclusion that there is sufficient evidence to support the 

proposed grouping for the neuroendocrine and neuroendocrine-related effects. For the purpose 

of estimating risks, the CMG triazines are all assumed to be equivalent in toxicity to atrazine. 

From 2009 – 2012, EPA presented information to several SAP meetings for independent 

scientific peer review of new studies on atrazine (see 

http://www.epa.gov/pesticides/reregistration/atrazine/atrazine_update.htm for links to specific 

SAP meetings). The overall conclusions from these meetings provided further support for a 

neuroendocrine mode of action (MOA) for the triazines with a decrease in luteinizing hormone 

as a precursor event to developmental and reproductive effects, which may be mediated through 

the estrogen and androgen pathways. 

Page 14 of 74
SIMAZINE FINAL

 

 

Page 7 of 66 

II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays and/or OSRI submitted to satisfy the Agency’s Test Order are shown below in 

Table 1. Executive summaries for each of the assays 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 simazine (USEPA 2010). Since then, the 

Agency has also conducted a more recent search of 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 

Scientific Advisory Panel (SAP) (USEPA, 2013). 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) other 

scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays 

consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were 

Page 15 of 74
SIMAZINE FINAL

 

 

Page 8 of 66 

designed specifically to evaluate a number of key biological events including potential effects on 

receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other 

effects on the HPG and HPT axes. OSRI may include published literature studies as well as 

studies conducted under USEPA (often referred to as Part 158 data) or 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, the consistency 

of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple 

endpoints are measured in each assay. 

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the 

relative degree of diagnostic utility of a 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. 

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 response 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. 

Page 16 of 74
SIMAZINE FINAL

 

 

Page 9 of 66 

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” (USEPA, 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 

extreme. In some instances, one parameter (i.e., death or >10% decrease in mammalian body 

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, , or 

Page 17 of 74
SIMAZINE FINAL

 

 

Page 10 of 66 

T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for 

each respective pathway (E, A or T) but are not reported in the table for E, A or T. 

A. EDSP Tier 1 Screening Assays 

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

in Table 1. 

Table 1. Tier 1 Screening Assays for Simazine 

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement satisfied by OSRI 

(Tennant et al., 1994b) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement satisfied 

(MRID 48619302) 

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement satisfied 

 (MRID 48619301) 

Steroidogenesis Assay (Human cell line 

H295R) 

OCSPP 890.1550; 

OECD 456 

Requirement satisfied by OSRI 

(Sanderson et al., 2001) 

Aromatase Assay (human recombinant 

microsomes) OCSPP 890.1200 Requirement satisfied by OSRI (Sanderson et al., 2001) 

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement satisfied by SORI 

(O’Connor et al., 2000 and 

Tennant et al., 1994a ) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement satisfied 

(MRID 48619303) 

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement satisfied by OSRI 

(Zorrilla et al., 2010) 

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement satisfied by OSRI 

(Stoker et al., 2005) 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement satisfied 

(MRID 48673602) 

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; 

OECD 231 

Requirement satisfied 

(MRID 48673601) 

Page 18 of 74
SIMAZINE FINAL

 

 

Page 11 of 66 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis 

1. Effects on Estrogen Pathway 

The potential for simazine to interact with the estrogen 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. 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. 

Simazine was positive in an OSRI estrogen receptor (ER) binding assay where simazine was 

weakly competitive with E2 for binding to the ER when pre-incubated at high concentrations 

(Tennant et al., 1994b); however, it was not clear whether simazine was acting as a competitive 

inhibitor or a non-competitive allosteric inhibitor. Blair et al. (2000) found no competitive 

interaction of simazine with the ER in a rat uterine cytosol assay. Simazine was negative in the 

Tier 1 ERTA assay as well as multiple ERTA assays considered as OSRI (O’Connor et al., 2000; 

Orton et al., 2009; Kojima et al., 2004; and Connor et al., 1996). In addition, simazine also did 

not induce proliferation in ER-responsive MCF-7 cells (Connor et al. 1996). 

In an in vitro steroidogenesis study considered as OSRI, Sanderson et al. (2001) reported that 

simazine caused concentration-dependent increases in aromatase activity in JEG-3 (placental 

choriocarcinoma) cells. In an in vivo study evaluating steroidogenesis, Laws et al. (2009) 

showed a single dose of simazine (188 mg/kg) administered to male rats elicited increases in 

adrenocorticotropic hormone (ACTH) and corticosterone, but not progesterone. 

In non-guideline uterotrophic assays considered as OSRI, simazine did not increase uterine 

weights in immature female rats (Conner et al., 1996) or ovariectomized rats (Tennant et al., 

1994a). In the Tennant et al. (1994a) study, uterine weights were decreased in rats coadministered simazine and estradiol (E2); however, the decreased weights were in the presence of 

overt toxicity. In another non-guideline uterotrophic assay in ovariectomized rats (O’Conner et 

al., 2000), no effects on uterine weights were observed; however all doses were determined to be 

overtly toxic (>10% body weight loss). 

Simazine demonstrated a potential anti-estrogenic response in the female pubertal rat assay; 

however, the responses were not clearly dose related and/or occurred at overtly toxic doses 

(Zorrilla et al., 2010). Two protocols were conducted in this study. In the first study, rats were 

dosed at 0, 12.5, 25, 50 or 100 mg/kg/day for 21 days. In the second study, rats were dosed at 0, 

12.5, 25, 50, 100, or 200 mg/kg/day for 41 days. The mean number of days until the first estrus 

was delayed at 100 mg/kg/day in both studies and at 200 mg/kg/day in the second study. In 

Study 1, VO was delayed by 4 days at 100 mg/kg/day where there was a significant decreased 

(↓13%) in body weight. In Study 2, VO was delayed (p<0.05) by 2.7, 2.9, 5.4 and 5.1 days at 

Page 19 of 74
SIMAZINE FINAL

 

 

Page 12 of 66 

25, 50, 100 and 200 mg/kg/day, respectively. However, overt toxicity was seen at both 100 and 

200 mg/kg/day on PND 33 (body weight ↓13.5 % and ↓17.6 %, respectively). Equivocal 

findings were noted for potential effects on prolactin levels. In Study 1, the decreased prolactin 

levels were not dose-related. In Study 2, there was a dose-dependent linear trend (not 

statistically significant by one-way ANOVA), however, the magnitude of change was slight at 

the lower doses (12.5 – 50 mg/kg/day) and at the higher doses (100 and 200 mg/kg/day) occurred 

in the presence of overt toxicity. There were no changes in estradiol, luteinizing hormone and 

progesterone levels. 

In the FSTRA, there were no significant effects in females fathead minnow, with the exception 

of a marginally significant (p=0.0512) decrease (↓23%) in GSI at the highest concentration tested 

(3.4 mg/L). There was a 41% decrease in fecundity at the middle treatment concentration (0.36 

mg a.i/L) that was not statistically significant and the percent reduction in fecundity from control 

at the highest treatment concentration was 1.2%. There were no significant effects on mortality 

and growth (length and weight) effect or clinical signs of toxicity in the study which was 

conducted at a concentration (3.4 mg a.i/L) close to the reported functional solubility (4.0 mg/L) 

of simazine. 

In the Part 158 rat subchronic toxicity study, absolute ovary weights were increased at the mid 

(142 mg/kg/day)) and high (276 mg/kg/day) dose levels; however, systemic and overt toxicity, 

presenting primarily as body weight loss/decreased body weights and decreased body weight 

gains, were noted at these dose levels. In a rabbit developmental toxicity study, abortions were 

seen at the high dose in the presence of maternal toxicity, presenting primarily as body weight 

losses. There were no estrogen-related effects noted in the other Part 158 mammalian studies. 

In a Part 158 avian reproduction toxicity study conducted with the mallard duck, there was a 

49% decrease in the number of eggs laid at the highest dose tested (450 mg/kg-diet), with 

concomitant decreases in the number of hatchlings (↓56%) and 14-day-old survivors (↓58%). 

Gross pathological observations included an increase from control in the number of regressing or 

regressed ovaries in the highest treatment concentration. These effects were observed with a 

significant decrease (10%) from control in female body weight. There were no treatment-related 

clinical signs of toxicity observed during the study. 

In the Part 158 study with northern bobwhite quail, there was an apparent reduction on the 

number of eggs laid and a possible effect on the number of viable embryos of eggs set at the 

highest treatment concentration (500 ppm) that were not statistically significant, but considered 

to be treatment-related. There were no clinical signs of toxicity or significant (p<0.05) effects on 

body weight observed during the test. 

Page 20 of 74
SIMAZINE FINAL 

Page 13 of 66 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Simazine

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Simazine1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen/Sex 

Steroid Hormones 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin (VTG) 

EDSP Tier 1 Assays 

ER Binding Requirement satisfied by OSRI (Tennant et al., 1994b; see below) ERTA (MRID 48619302) N Aromatase Requirement satisfied by OSRI (Sanderson et al., 2001; see below) Steroidogenesis Requirement satisfied by OSRI (Sanderson et al., 2001; see below) Uterotrophic Requirement satisfied by OSRI (O’Connor et al., 2000, and Tennant et al., 1994a; seFemale Pubertal Rat Requirement satisfied by OSRI (Zorrilla et al., 2010; see below) FSTRA (MRID 48673602) NE P(H) N N N N 

OSRI 

ER Binding (Blair et al., 2000) N ER Binding (Tennant et al., 1994b) P ER Binding/ERTA (O’Connor et al.; 2000) N N ERTA (Orton et al., 2009) N ERTA (Kojima et al., 2004) N 
SIMAZINE FINAL 

Page 14 of 66 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Simazine

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Simazine1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen/Sex 

Steroid Hormones 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin (VTG) 

ERTA/Cell Proliferation (Connor et al., 1996) N Aromatase/ Steroidogenesis (Sanderson et al., 2001) 

 P 

Steroidogenesis (Laws et al., 2009) E Female Pubertal (Zorrilla et al., 2010) P4 N N N N N 

↓2.7 d ↓2.9 d 

(ML, M) 

Uterotrophic (Tennant et al., 1994a) 

N 

Uterotrophic (Connor et al., 1996) N Subchronic Toxicity (Rat; MRID 00143265) 

N

E N N NE NE 

Subchronic Toxicity (Dog; MRID 00146655) 

N

E NE N NE NE 

Developmental Toxicity (Rat; MRID 40614403) N Developmental Toxicity (Rabbit; MRID 00161407) N N (H) Two-Generation Reproduction (Rat; MRID 41803601) 

N

E N N NE N 
SIMAZINE FINAL 

Page 15 of 66 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Simazine

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Simazine1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen/Sex 

Steroid Hormones 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin (VTG) 

Chronic Toxicity/ Oncogenicity (Rat; MRID 40614405) 

N

E N N NE 

Chronic Toxicity (Dog; MRID 40614402) 

N

E N N N 

Avian Reproduction (Duck; MRID 3576901) 

P5

(H) 

Avian Reproduction (Quail; MRID 0163134) 

P7

(H) 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High 

↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in 

weight are absolute unless otherwise indicated. BW= Body weight; FC= Food Consumption 

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. Prolactin decreased significantly at 50 mg/kg/day. 5. There was a 49% decrease in the number of eggs laid at the highest dose tested (450 mg/kg-diet), with concomitant decreases in th

(↓56%) and 14-day-old survivors (↓58%). Gross pathological observations included an increase from control in the number of re

ovaries in the highest treatment concentration. 

6. A significant decrease (10%) from control in female body weight at the high concentration. There were no treatment related clini

observed during the study. 

7. There was an apparent reduction on the number of eggs laid and a possible effect on the number of viable embryos of eggs set at t

concentration (500 ppm) that were not statistically significant, but considered to be treatment-related. 

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

E Equivocal NE Not examined 
SIMAZINE FINAL

 

 

Page 16 of 66 

2. Effects on Androgen Pathway 

The potential for simazine 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 for androgenic, anti-androgenicor 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. 

Simazine was negative in the Tier 1 in vitro androgen receptor (AR) binding assay. In a 

published AR transcriptional activation (ARTA) study by Orton et al. (2009), simazine was 

negative as an androgen agonist in an MDA-kb2 cell assay, but an anti-androgen effect was 

observed. However, in two other published ARTA studies (Birkhøj et al., 2004 and Kojima et 

al., 2004), simazine was negative for AR activation. In an in vivo study in male rats, Laws et al. 

(2009), reported increases (p<0.05) in ACTH, corticosterone and progesterone levels in the 

plasma of rats following a single dose of the simazine metabolite DACT. The ACTH and 

progesterone levels were increased (p<0.05) in a dose-dependent manner in the plasma/serum of 

rats dosed with the simazine metabolite DIA, and a single dose of simazine at 188 mg/kg elicited 

increases (p<0.05) in ACTH and corticosterone, but not progesterone, at 15 minutes post-dose. 

Simazine was negative in the Tier 1 in vivo Hershberger assay, as well as in an OSRI 

Hershberger assay performed by Birkhøj et al. (2004). In a male pubertal rat assay considered as 

OSRI (Stoker et al., 2005), rats were dosed at 0, 6.25, 25, 75, 150 or 300 mg/kg/day for 30 days. 

Lateral prostate, ventral prostate and seminal vesicle weights were decreased at 75 mg/kg and 

above. At 300 mg/kg, minimal to moderate testicular degeneration were observed in all animals, 

whereas testes weights were unaffected by treatment. However, all the androgen-related effects 

in the study occurred at doses (≥75 mg/kg//day) where overt toxicity (>10% terminal body 

weight decrement) was observed. 

In a Part 158 rat subchronic toxicity study and a dog subchronic toxicity study, decreased testes 

weights were noted; however, systemic and overt toxicity, presenting primarily as decreased 

body weights and body weight gains or body weight losses, were observed. There were no 

androgenic/anti-androgenic effects noted in the other Part 158 studies. 

In the FSTRA, there were no statistically significant effects determined for any endpoint in 

males. In the Part 158 avian studies, decreased hatchling survival was observed in the mallard 

study and in the bobwhite quail study, decreased embryo viability and regressed testes (gross 

pathology 4 birds) was observed. 

.

Page 24 of 74
SIMAZINE FINAL 

Page 17 of 66 

Table 3. Androgenic/Anti-Androgenic Pathway for Simazine

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Simazine1Study Type/ Literature Citation 

AR Binding/Activation 

Steroidogenesis 

Testosterone 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at PPS 

Vitellogenin (VTG) 

EDSP Tier 1 Assays 

AR Binding (MRID 48619301) N Steroidogenesis Requirement satisfied by OSRI (Sanderson et al., 2001; see below) Hershberger (MRID 48619303) NE N Male Pubertal Rat Requirement satisfied by OSRI (Stoker et al., 2005; see below) FSTRA (MRID 48673602) NE N N N N N 

OSRI 

ARTA (Birkhøj et al., 2004) N ARTA (Orton et al., 2009) P ARTA (Kojima et al., 2004) N Steroidogenesis (Sanderson et al., 

2001) 

 P N 

Steroidogenesis (Laws et al., 2009) P Hershberger (Birkhøj et al., 2004) N N 
SIMAZINE FINAL 

Page 18 of 66 

Table 3. Androgenic/Anti-Androgenic Pathway for Simazine

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Simazine1Study Type/ Literature Citation 

AR Binding/Activation 

Steroidogenesis 

Testosterone 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at PPS 

Vitellogenin (VTG) 

Male Pubertal (Stoker et al., 2005) N N N P (ML) N N N N Subchronic Toxicity (Rat; MRID 00143265) 

 N N NE N NE NE 

Subchronic Toxicity (Dog; MRID 00146655) 

 N N NE N NE 

Two-Generation Reproduction (Rat; MRID 41803601) 

N N N N 

Chronic Toxicity/ Oncogenicity (Rat; MRID 40614405) 

 N N NE N N 

Chronic Toxicity (Dog; MRID 40614402) 

 N N N N N 

Avian Reproduction (Duck; MRID 43576901) 

P4 

(H) 

Avian Reproduction (Quail; MRID 00163134) 

P5 

(H) 

P5 

(H) 
SIMAZINE FINAL 

Page 19 of 66 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High 

↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in t

weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prost

(DP) Lateral Prostate (LP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans pConsumption 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OS

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 

details see Section IV. A 

4. Decreased offspring survival 5. Decreased embryo viability and regressed testes P Positive finding N Negative finding (in vitro)/No effect (in vivo) 

NE Not examined 
SIMAZINE FINAL

 

 

Page 20 of 66 

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 data to detect changes in the HPT axis, i.e., the 

pubertal female and male rat assays, and the AMA (frog). 

The potential for simazine to interact with thyroid regulation are 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. 

Simazine decreased serum thyroxine (T4) levels in Study 1 of Zorrilla et al (2000). (data 

presented graphically) at an overtly toxic dose; thyroid stimulating hormone (TSH) levels were 

not evaluated. No effect on T4 or TSH levels was noted in Stoker et al. (2005. Thyroid weight 

and histopathology were not evaluated in the in vivo male (Stoker et al., 2005) or female pubertal 

(Zorrilla et al., 2010) assays. Pituitary weights were decreased by 16-22% in Zorrilla et al., and 

by 16-35% in Stoker et al.2005; however, overt toxicity, presenting primarily as decreased 

terminal body weights, were observed in these studies. There were no thyroid effects noted in the 

Part 158 mammalian studies. 

In the AMA, simazine increased (p<0.05) normalized 7-day hind limb length (HLL) by 6% and 

significantly (p<0.05) decreased 21-day normalized HLL by 14% in the highest treatment 

concentration (1.9 mg a.i/L). These differences in normalized HLL were not accompanied by 

any differences in Nieuwkoop-Faber (NF) stage. Significant (p<0.05) reductions from control 

were determined for Day 21 snout-vent length (SVL) and body weight measurements, with 

decreases of 7.2% and 23% at the 1.9 mg/L level for these endpoints, respectively. There was a 

slight increase in the incidence of animals with mild-to-moderate thyroid gland atrophy in the 

highest treatment group (10/20 vs. 4/20 control) and decreased thyroid follicular cell size (10/20 

vs. 6/20 control), which was considered to be treatment-related in the histopathology report. The 

study authors noted that observations of thyroid gland atrophy were generally associated with 

smaller-sized tadpoles regardless of treatment, suggesting that thyroid gland atrophy was 

secondary to smaller size at the high concentration. Overall, the results were not consistent with 

known patterns of thyroid agonism or antagonism in the AMA. 

Page 28 of 74
SIMAZINE FINAL

 

 

Page 21 of 66 

Table 4. Thyroid Pathway for Simazine

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Simazine1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Serum T4 Levels 

Serum TSH Levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, BL, SVL) Systemic Toxicity Observed2

Overt Toxicity Observed 3 

EDSP Tier 1 Assays 

Female Pubertal Rat Requirement satisfied by OSRI (Zorrilla et al., 2010; see below) 

Male Pubertal Rat Requirement satisfied by OSRI (Stoker et al., 2005; see below) 

AMA 

(Frog; MRID 48673601) 

P4

(H) 

HLL: 

↑6% Day 7; 

↓14% Day 21 

(H) 

Day 21:SVL: 

↓7%; 

 BW: ↓23% 

(H) 

 N 

OSRI 

Female Pubertal 

(Zorrilla et al., 2010) NE NE N NE N X 

(MH, H) 

X 

(MH, H) 

Male Pubertal 

(Stoker et al., 2005) NE NE N N N 

X 

(M, MH, 

H) 

X 

(M, MH, 

 H) 

Subchronic Toxicity (Rat; 

MRID 00143265) N X 

 (L, M, H) 

X 

 (M, H) 

Subchronic Toxicity 

(Dog; MRID 00146655) N X 

 (L, M, H) 

X 

(M, H) 

Subchronic Dermal 

Toxicity (Rabbit; MRID 

00057567) 

N N N N N 

Chronic Toxicity/ 

Carcinogenicity (Rat; 

MRID 40614405) 

N X 

 (H) 

X 

(M, H) 

Chronic Toxicity (Dog; 

MRID 40614402) N N N X 

(H) N 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high 

treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the 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. Slight increase in the incidence rate of mild-to-moderate thyroid gland atrophy in the highest treatment group 

(10/20 vs. 4/20 control) and decreased thyroid follicular cell size (10/20 vs. 6/20 control), The study authors 

noted that observations of thyroid gland atrophy were generally associated with smaller-sized tadpoles 

regardless of treatment, suggesting that thyroid gland atrophy was secondary to smaller size at the high 

concentration. 

P Positive finding 

N Negative finding 

NE Not examined 

Page 29 of 74
SIMAZINE FINAL

 

 

Page 22 of 66 

IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the WoE determination on the potential of simazine to 

interact with endocrine-mediated processes (i.e., E, A, or T hormonal pathways) as well as 

recommendations regarding Tier 2 testing. The results of the Tier 1 assays are considered, along 

with other scientifically relevant information (e.g., 40 CFR Part 158 test guidelines and 

published or publicly available peer-reviewed studies). WoE analysis in the context of the EDSP 

follows the Agency’s guidance (USEPA 2011) 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 WoE 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 nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 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). 

1. Tier 1 in vivo Assays 

Page 30 of 74
SIMAZINE FINAL

 

 

Page 23 of 66 

No treatment-related mortality was observed in the AMA study at test concentrations up to 1.9 

mg/L, and the only clinical sign noted was spinal curvature, which occurred with similar 

frequency in the control and low and mid-treatment concentrations. However, dose-dependent 

decreases in growth were noted, with the decrease attaining statistical significance (p<0.05) at 

1.9 mg/L. Compared to the negative control, the 1.9 mg/L concentration was overtly toxic based 

on decreases of 7.2 and 23.2% in SVL and wet weight at Day 21. 

No treatment-related mortality or clinical signs of toxicity were observed in the FSTRA study, 

and there was no evidence of systemic toxicity aside from a slight reduction in male body length 

(↓6%) at the mid-treatment concentration. 

In the Hershberger assay, all animals survived until scheduled termination, and there were no 

clinical signs of toxicity at doses up to 200 mg/kg/day. However, the high dose appeared to be 

overtly toxic based on the 10-11% decrease in terminal body weight in the androgenic portion of 

the assay, with no changes in food consumption. In addition, body weight gains were decreased 

by 36 and50% at the high dose in the agonist and antagonist portions of the assay, respectively 

2. OSRI 

In the published female pubertal studies by Zorrilla et al. (2010), no signs of clinical toxicity 

were observed, but body weights were decreased at the high dose levels. In the first study, body 

weight was decreased by 13.3% at VO (PND 32), but no significant decrease was noted at study 

termination (PND 42). In the second study, body weights were decreased by ≤8.6% at the low 

(25 mg/kg/day) and medium (50 mg/kg/day), and by 10.3 and 11.3% at medium-high (100 

mg/kg/day) and high (200 mg/kg/day) treatments, respectively, indicating that the two top doses 

were overtly toxic. In addition, absolute and relative adrenal weights were decreased at 100 

mg/kg/day in the first study. 

Limited information was available for the unpublished male pubertal study by Stoker et al. 

(2005), but no clinical signs of toxicity were reported. Terminal body weights were decreased in 

all simazine treatment groups, although the decreases were minor (↓≤5%) at doses ≤25 

mg/kg/day. Body weight were decreased by 10, 11 and 22% at doses of 75, 150 and 300 

mg/kg/day, respectively. 

In a uterotrophic assay by O’Connor et al. (2000), terminal body weights were decreased by 10, 

24 and 25% in ovariectomized rats following four days of intraperitoneal (i.p.) dosing with 

simazine at doses of 100, 200 and 300 mg/kg/day. In another modified uterotrophic study by 

Tennant et al. (1994a), terminal body weights were also decreased following 3 days of oral 

dosing with simazine at 100 (↓8%) and 300 (9-11%) mg/kg/day. 

Page 31 of 74
SIMAZINE FINAL

 

 

Page 24 of 66 

No information on systemic toxicity was provided in the study be Laws et al. (2009) examining 

time-course changes in hormones following a single dose of simazine at 188 mg/kg, or in uterine 

weight response study by Connor et al. (1996). 

In the subchronic toxicity study in rats, no treatment-related clinical signs of toxicity were 

observed during 13 weeks of dosing in the diet at simazine levels equivalent to ≤276 mg/kg/day. 

Reductions in feed consumption were noted for the 142 and 276 mg/kg/day groups throughout 

the 13 week study, and significant reductions in body weight gains and body weight also 

occurred in males and females in the mid and high dose groups. 

In the subchronic toxicity study in dogs, clinical signs of toxicity consisting of tremors beginning 

at Week 9 were observed in both sexes at the high dose (4000 ppm), and food consumption was 

reduced in both males and females at the mid dose (2000 ppm; ↓22-25%) and high dose (32-

36%). Reduced weight gains or weight losses indicate that the mid and high doses were overtly 

toxic. Selected blood parameters were also negatively affected in males and females at the mid 

and high doses. In addition, absolute heart weights were decreased in all treated males and high 

dose females, and relative heart weights were decreased in high-dose males and females. 

In a Part 158 Guideline developmental toxicity study in rats, there were no treatment-related 

clinical signs of toxicity observed during the study in females dosed up to 600 mg/kg/day during 

GD 6-15. Body weights were significantly decreased (p<0.05) in the 300 and 600 mg/kg/day 

females on GDs 10 (↓7% and ↓10%), 14 (↓11% and ↓14%) and 18 (↓6% and ↓9%); body weight 

corrected for gravid uterus weight was decreased in these groups as well (↓7% and ↓9%). Food 

consumption was decreased (p≤0.05) in mid- and high-dose animals during the dosing period 

and increased in these same animals after the dosing period (GDs 15-19). 

No treatment-related mortalities occurred in the Part 158 Guideline developmental toxicity study 

in rabbits. However there were treatment-related increases in tremors in the 75 and 200 

mg/kg/day dose groups (21 and 100%, respectively, vs. 0% in control). Significant body weight 

losses (p<0.05) during the dosing period were also noted at 75 (−243 g) and 200 (−456 g) 

mg/kg/day compared to a +230 g increase in controls. Body weights gains increased (p<0.05) 

during post-dosing (GD 19-29); however; overall (GD 0-29) body weight gains were decreased 

at 75 (↓74%) and 200 (↓75%) mg/kg/day. Body weight gains corrected for gravid uterine 

weights also displayed losses at 75 (−167 g) and 200 (−264 g) compared to a control weight gain 

of +3 g. Food consumption in the mid- and high-dose groups was also decreased (p<0.05) in the 

mid- and high-dose groups during the dosing period. 

In the two-generation reproduction study in rats, there were no treatment-related clinical signs of 

toxicity or deaths in any of the treated groups (10, 100 or 500 ppm), including adults and pups. 

For the 500 ppm parental (P) males, body weights were decreased (p<0.05) by 7-11% beginning 

on Day 14 and remain decreased throughout pre-mating in P males, and similar results were 

Page 32 of 74
SIMAZINE FINAL

 

 

Page 25 of 66 

observed for the 500 ppm first generation (F1) males at the first mating (↓12-13%). Body weight 

gains for the 500 ppm P and F1 males were also decreased (p<0.05) during the pre-mating, 

mating, and post-mating phases (8-51%) and during the rest phase for F1 males at second mating. 

Body weight gains for the 100 ppm P males were decreased during Days 0-70 (≈↓13%) and 

during Days 7-14 (≈↓11%) for the 100 ppm F1 males at first mating, but were increased from the 

rest period to term for F1 males at second mating. 

Body weights of the 500 ppm P females were decreased (p<0.05) by 10-13%, starting on Day 7 

and remaining decreased (p<0.05) through the gestational (↓11-14%) and lactational (↓7-12%) 

periods. Body weights for the 500 ppm F1 females were decreased during the first (↓11-15%) and 

second pre-mating periods, and remained decreased through the first (↓10-14%) and second 

(↓11-15%) gestational periods. Lactational body weights were decreased (p<0.05) during LD 0-

14 (↓7-11%) for the second generation (F2a) litters and only at LD 0 (↓11%) for the F2b litters. In 

the 100 ppm group, decreased body weights were observed during the premating phase in P and 

F1 first mating females (approximately 6%). In general, body weight gain for high-dose P and F1

females (both matings) was decreased (p≤0.05) during the pre-mating phase and increased 

(p≤0.05) during the lactational phase. For F1 females at first mating, body weight gain was 

decreased and then increased during pre-mating phase; it was also increased during the 

lactational phase. No treatment-related decreases in body weights were observed in F1 or F2

offspring on PND 14 or 21. Incidental clinical observations and gross malformations in pups 

were observed in both generations in all dose groups, including the control groups. 

In the chronic toxicity/carcinogenicity study in rats, there were treatment-related changes in the 

mortality rate during the second year. For the high-dose males (1000 ppm), the mortality rate 

was significantly decreased (p<0.01) compared to the controls (40% treated vs. 61% controls). 

For females, the mortality rate in the 100 (75%) and 1000 (80%) ppm groups was significantly 

greater (p<0.01) than controls (66%), with a significant increasing trend. At the 1000 ppm high 

dose [(45.8/63.1 mg/kg/day (M/F)], There was hair loss (i.e., alopecia) in 1000 ppm females and 

swollen appendages in the 1000 ppm males. Body weights in the 1000 ppm males and females 

were significantly decreased (p<0.05) beginning on Day 7 and continuing through termination 

(↓22% and ↓23% for males and females, respectively); body weight gains were also significantly 

decreased (p<0.05) in these animals throughout the study (overall ↓27% and ↓28% for males and 

females). Females also had decreased body weights at different intervals at the mid dose (100 

ppm); however, overall body weight gain was not significantly decreased at study termination. 

Food consumption was significantly decreased (p<0.05) in the 1000 ppm males from Day 7 

through 700 (4 weeks prior to sacrifice); for the 1000 ppm females, food consumption was 

significantly decreased (p<0.05) from Day 7 through 560 but not during the final 6 months of the 

study. 

Hematological parameters were also affected in the high dose rats. In the high dose female rats, 

red blood cell (RBC) count, hemoglobin (HGB), hematocrit (HCT) and lymphocytes were 

Page 33 of 74
SIMAZINE FINAL

 

 

Page 26 of 66 

decreased (p<0.01), and mean corpuscular hemoglobin (MCH), white blood cell (WBC) count 

and neutrophils were significantly increased (p<0.05) at most time points of sampling. In the 

high dose males, mean corpuscular hemoglobin concentration (MCHC) was increased (Day 361) 

and WBC was decreased (Day 537). In addition, the incidence of liver and spleen blood 

production (hematopoiesis) and hematopoiesis were significantly increased (p<0.05) in the 1000 

ppm females (7% and 14% respectively) compared to controls (0% and 4%, respectively). 

In the chronic toxicity study in dogs, there were no mortalities, but cachexia (weakness) was 

observed at the high dose (1250 ppm) in 1 male and 1 female from Week 14 through Week 20 or 

22. Body weight gain was decreased (p<0.05) in the 1250 ppm males on Days 7 and 14, and in 

the 1250 ppm females on Days 7-224 compared to controls. Body weights of the 1250 ppm 

females were decreased compared to controls on Days 63 through 140. Food consumption in 

these animals was also decreased frequently during the first 20 weeks of the study. In the 1250 

ppm males, there was a transient nominal decrease (not statistically significant) in RBC and 

HGB at Days 86 and 177, while HCT was significantly decreased (p<0.05) at Day 86; on Day 

359, these values were nominally higher or equivalent to control values. Platelets were 

significantly increased (p<0.05) in the 1250 ppm males after 86 (142%), 177 (170%), and 359 

(155%) days of dosing. In the 1250 ppm females, there was a statistically significant but 

transient decrease (p<0.05) in RBC, HGB, and HCT at 86 and 177 days; these values were only 

nominally decreased at 359 days compared to controls, but not when compared to pre-dose 

values in high-dose females. MCHB was slightly but significantly increased (p<0.05) in the1250 

ppm females at 359 days. No treatment-related, consistent changes were noted in the clinical 

chemistry or urinalysis results. 

In the avian reproduction study on mallard ducks, there was no treatment-related mortality and 

no clinical signs of toxicity at dietary concentrations up to 450 ppm. Mean terminal body weight 

of hens in the 450 ppm group was significantly decreased (p<0.05) by 10.2% compared to 

controls, with food consumption being decreased in the high-dose group only during Week 1. 

No signs of systemic or overt toxicity were noted in the following published or guideline studies: 

the Hershberger study by Birkhøj et al. (2004) at a dose of 25 mg/kg/day; subchronic dermal 

toxicity study in rabbits at doses up to 1000 mg/kg/day; and the avian reproduction study on 

bobwhite quail at dietary concentrations up to 500 ppm. 

B. Estrogen Pathway 

The weight of evidence approach (WoE) considered Tier 1 assay results, OSRI data, as well as 

the inclusion of simazine in the triazine common mechanism group (CMG) which relies on 

numerous SAPs on atrazine. 

Page 34 of 74
SIMAZINE FINAL

 

 

Page 27 of 66 

In the in vitro Tier 1 assays, although simazine was positive in the ER binding assay by Tennant 

et al. (1994b), it was negative for ER binding in an assay conducted by Blair et al. (2000) using 

the rat uterine cytosol assay and negative in a cell proliferation assay (using MCF-7 cells) 

conducted by Connor et al. (1996). Additionally, simazine was also negative in the Tier 1 and 

OSRI ERTA assays. Altered aromatase activity was reported in studies using JEG-3 cells by 

Sanderson et al. (2001). In a single dose steriodogenic study of simazine and its DACT and DIA 

metabolites, the levels of ACTH, progesterone, and/or corticosterone were increased (Laws 

2009). 

In the in vivo assays, simazine was negative for estrogenic activity in several uterotrophic assays. 

In the OSRI female pubertal assays, delays in VO were seen at 25 mg/kg/day in both studies and 

at 50 mg/kg/day in the second study in the absence of overt toxicity; delays in VO were also seen 

at high doses in both studies in the presence of overt toxicity. There were no estrogenic/antiestrogenic effects noted in the Part 158 mammalian toxicity studies. 

There was a marginally significant (p=0.0512) reduction in female GSI at the high treatment 

level (3.4 mg/L) in the FSTRA; however, no effects on reproduction were observed. Female 

mallard ducks exposed to simazine in the diet presented a greater incidence of regressed ovaries 

and produced fewer eggs, fewer hatchling survivors, and fewer 14-day survivors at the highest 

treatment level; however, a slight decrease (10%) in female body weight was also observed at 

this dose. Decreases in eggs laid and possible embryo viability effects were observed in the quail 

study. 

Evidence of estrogen-related effects have been noted with atrazine and atrazine metabolites 

(Laws et al., 2000; 2003; SAP, 2000). In studies by Laws et al. (2000), atrazine significantly 

delayed VO and reduced the weights of reproductive tissues (uterine and ovarian) associated 

with estrogenic responses in peripubertal rats. Similar effects were observed in peripubertal rats 

exposed to chlorinated metabolites of atrazine (Laws et al., 2003) indicating a similar mode of 

action for the metabolites. 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 resulting in delayed VO and alterations 

in estrogen-responsive tissues. 

Page 35 of 74
SIMAZINE FINAL

 

 

Page 28 of 66 

C. Androgen Pathway 

In the in vitro assays, simazine was negative in the AR binding and ARTA assays. Simazine was 

positive in the steriodogenesis assay (Sanderson et al., 2001). In a single dose steriodogenic 

study of simazine and its DACT and DIA metabolites, the levels of ACTH, progesterone, and/or 

corticosterone were increased (Laws et al., 2009). 

Simazine was negative in the Tier 1 in vivo Hershberger assay. All androgen-related effects in 

the male pubertal assay and Part 158 mammalian toxicity studies occurred in the presence of 

overt toxicity. 

There were no androgen-related effects in the FSTRA. Regressed or regressing testes were 

observed in 25% of male bobwhite quail at the highest treatment level in an avian reproduction 

study; decreases in embryo viability was observed in this study along with effect on offspring 

survival in the mallard study. 

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. For non-mammalian taxa, while no androgen-related effects 

were seen in fish, regressing testes were observed in some birds exposed to simazine. 

D. Thyroid Pathway 

There was no convincing evidence of potential interaction of simazine with the thyroid pathway 

in mammals or wildlife.

Page 36 of 74
SIMAZINE FINAL

 

 

Page 29 of 66 

E. Conclusions 

For the estrogen pathway, while there was some evidence of potential interaction in the in vitro

assays (aromatase, steroidogenesis), there is limited evidence of potential interaction in vivo. 

Delays in VO were observed in the female pubertal assay, however, there were no other 

estrogen-related effects observed in other mammalian studies in the absence of overt toxicity. 

Additionally, while decreased GSI was observed in the FSTRA, there were no other estrogenrelated effects observed including alterations in reproduction. Regressed ovaries were observed 

an avian study, along with decreases in egg production and embryo viability/offspring survival. 

For the androgen pathway, in vitro effects on steroidogenesis were observed. However, there 

were no androgen-related effects observed in mammals or were observed in the presence of 

toxicity. Additionally, there were no androgen-related effects observed in the FSTRA. In the 

avian reproduction studies, regressed testes were observed. 

Nevertheless, the potential for interaction with the estrogen and androgen pathway is supported 

by the SAP conclusion that the chlorotriazines (including simazine) 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. 

V. EDSP Tier 2 Testing Recommendations 

There is evidence for potential interaction with the estrogen and androgen pathway for the 

chlorotriazine, including simazine. 

The current point of departure (POD) used for human health risk assessment for the triazine 

CMG is based on effects seen with atrazine. The POD of 2.56 mg/kg/day based on a luteinizing 

hormone suppression with atrazine is regarded as a precursor event in the neuroendocrine mode 

of action of the triazines. This POD is used for deriving the chronic Reference Dose (RfD) used 

for chronic dietary risk assessment and calculating the Margin of Exposure (MOEs) for nondietary risk assessment. 

In the EDSP in vivo Tier 1 male pubertal assay with atrazine, a delay in preputial separation 

(PPS) was seen at the lowest dose tested (12.5 mg/kg/day). 

In the EDSP in vivo female pubertal assay with simazine, delays in VO were seen at doses 

greater than 25 mg/kg/day and suppression of serum prolactin levels, suggesting that the 

mechanism of action of simazine is to alter hypothalamic regulation of pituitary hormone 

secretion (Zorrilla et al., 2010). Laws et al., 2000 reported estrogen- related effects (i.e., delays 

in the onset of puberty and altered estrous cyclicity) with atrazine at doses greater than 50 

mg/kg/day in the female pubertal assay with atrazine. 

Page 37 of 74
SIMAZINE FINAL

 

 

Page 30 of 66 

The POD (2.56 mg/kg/day) is approximately 5-fold lower than the dose (12.5 mg/kg/day that 

caused delay in PPS and approximately 8-fold lower than the dose (25 mg/kg/day) that caused 

delay in VO. In addition, a dose of 6.25 mg/kg/day was reported to have no effect on the day of 

preputial separation when compared to controls in another male pubertal assay (Stoker et al.,

2000). Therefore, the chronic RfD and the MOE are protective of the estrogen and androgenrelated effects seen in the EDSP pubertal assays. 

With respect to determining whether additional studies are needed for ecological risk assessment 

based on the Tier 1 screen, the findings in the Tier 1 assays do not have an impact on the 

wildlife. For wildlife, the only effect on estrogen-, or androgen-related endpoints in the FSTRA 

was a marginally significant decrease in female GSI at 3.4 mg a.i./L, which was not associated 

with decreased reproduction at that treatment level. The most recent ecological risk assessment 

endpoints for chronic effects in freshwater fish and aquatic-phase amphibians are based on a 

120-day fish reproduction test with continuous exposure of fathead minnow to an 80% 

formulation of simazine, which yielded a NOAEC value of 0.96 mg a.i./L and a corresponding 

LOAEC value of 2.0 mg a.i./L (MRID 00043676, Mayer and Sanders 1975). The LOAEC value 

is based on a 12% reduction in growth (length) in 30-day old fish. No effects on reproductive 

endpoints were observed in the fish full life cycle test. This fish data were not included in the 

WoE determinations of potential estrogen or androgen interaction because the test was conducted 

with a formulated product; however, the data are relevant here because they have previously 

been used as regulatory endpoints. 

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard 

duck are available for simazine. In both species, effects on reproductive parameters were 

observed. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) 

are considered sufficient for evaluating potential reproductive effects to birds from simazine 

exposure. Relative to the EDSP, additional testing is not recommended. 

Based on the WoE analysis, EDSP Tier 2 testing is not recommended for simazine since 

additional testing will not impact current EPA established regulatory endpoints for human health 

or ecological risk assessment. 

Page 38 of 74
SIMAZINE FINAL

 

 

Page 31 of 66 

VI. References 

Arthur, A (1984) A teratology study of simazine technical in New Zealand white rabbits. CGSCiba Geigy Corp., Summit, NJ. Laboratory Report No. 62-83, March 29, 1984. Unpublished. 

(MRID 00161407) 

Beavers, J., and Breslin, J. (1986) Simazine technical: a one-generation reproduction study with 

the bobwhite (Colinus virginianus). Wildlife International Ltd., St. Michaels, MD. Laboratory 

Project No. 108-245, completion date not provided. Unpublished. (MRID 00163134) 

Beavers, J., Foster, J., Mitchell, L., et al. (1994) Simazine technical: a reproduction study with 

the mallard. Wildlife International Ltd., Easton, MD. Laboratory Project No. 108-356, 

completion date not provided. Unpublished. (MRID 43576901) 

Bier, C., and Oliveira, P. (1980) 21-day subacute dermal toxicity study in albino rabbits 

administered test article simazine. Bio-Research Laboratories Ltd., Canada. Laboratory Project 

No. 12017, January 14, 1980. Unpublished. (MRID 00057567) 

Birkhøj, M., Nellemann, C., Jarfelt, K., et al. (2004) The combined anti-androgenic effects of 

five commonly used pesticides. Toxicol. Appl. Pharmacol. 201: 10-20. (MRID 48205101) 

Blair, R., Fang, H., Branham, W., et al. (2000) The estrogen receptor relative binding affinities 

of 188 natural and xenochemicals: structural diversity of ligands. Toxicol. Sci. 54: 138-153. 

Connor, K., Howell, J., Chen, I., et al. (1996) Failure of chloro-S-triazine-derived compounds to 

induce estrogen receptor-mediated responses in vivo and in vitro. Fundam. Appl. Toxicol. 30: 93-

101. (MRID 48203001) 

Epstein, D., Hazelette, J., and Yau, E. (1991) Simazine technical: two-generation reproductive 

toxicology study in rats. Ciba-Geigy Corp., Summit, NJ. Laboratory Project Report 882095, 

February 12, 1991. Unpublished. (MRID 41803601) 

Friedmann, A.S (2002) Atrazine inhibition of testosterone production in rat males following 

peripubertal exposure. Reprod.Toxicol. 6(3): 275-279. 

Kojima, H., Katsura, E., Takeuchi, S., et al. (2004) Screening for estrogen and androgen receptor 

activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. 

Environ. Health Perspect. 112 (5): 524-531. (MRID 48033008). 

Laws, S.C, Ferrell, J.M., Stoker, T.E., et al. (2000) The effects of atrazine on female Wistar rats: 

an evaluation of the protocol for assessing pubertal development and thyroid function. Toxicol. 

Sci. 58:366-376 

Page 39 of 74
SIMAZINE FINAL

 

 

Page 32 of 66 

Laws, S.C., Ferrell, J.M., Stoker T.E., et al. (2003) Pubertal development in female Wistar rats 

following exposure to propazine and atrazine biotransformation by-products, diamino-Schlorotriazine and hydroxyatrazine. Toxicol. Sci. 76(1):190-200. 

Laws, S., Hotchkiss, M., Ferrell, J., et al. (2009) Chlorotriazine herbicides and metabolites 

activate an ACTH-dependent release of corticosterone in male Wistar rats. Toxicol Sci. 

112(1):78-87. 

Mayer, F., and Sanders, H. (1975) Effects of Aquazine(R) on daphnids and fathead minnows 

under continuous and usage-pattern exposures: prelim. report. U.S. Fish and Wildlife Service, 

Fish- Pesticide Research Laboratory, AK. MRID 00043676. Unpublished. 

Mayer, F., and Sanders, H. (1975) Effects of Aquazine(R) on daphnids and fathead minnows 

under continuous and usage-pattern exposures: prelim. report. U.S. Fish and Wildlife Service, 

Fish- Pesticide Research Laboratory, AK. MRID 00043676. Unpublished. 

Mainiero, J., Wimbert, K., Wright, J., et al. (1986) Simazine technical: a teratology study in rats. 

CGU-Ciba-Geigy Corp., Summit, NJ. Laboratory Study No. B6/110, April 7, 1986. 

Unpublished. (MRID 40614403) 

McCormick, G., and Green, M. (1988) Simazine technical: a 52-week oral feeding study in dogs. 

CGS-Ciba-Geigy Corp., Summit NJ. Laboratory Study No. A7/17, March 28, 1988. 

Unpublished. (MRID 40614402) 

McCormick, G., Arthur, A., and Green, M. (1988) Simazine-technical: 104-week oral chronic 

toxicity and carcinogenicity study in rats. CGS-Ciba-Geigy Corp., Summit NJ. Laboratory Study 

No. 2-011-09, April 12, 1988. Unpublished. (MRID 40614405) 

O’Connor, J., Plowchalk, D., Van Pelt, C., et al. (2000) Role of prolactin in chloro-S-triazine rat 

mammary tumorigenesis. Drug and Chemical Toxicology 23(4): 575-601. (MRID 48203004) 

Orton, F., Lutz, I., Kloas, W., et al. (2009) Endocrine disrupting effects of herbicides and 

pentachlorophenol: in vitro and in vivo evidence. Environ. Sci. Technol: 43: 2144-2150. 

Rayner, J.L., Enoch, R.R., Wolf, D.C., et al. (2007) Atrazine-induced reproductive tract 

alterations after transplacental and/or lactational exposure in male Long-Evans rats. Toxicol. 

Appl. Pharmacol. 218(3):238-248. 

Rosenber, B.G., Chen, H., Folmer, J. et al. (2008) Gestational exposure to atrazine: effects on the 

postnatal development of male offspring. J. Androl. 29(3):303-311. 

Page 40 of 74
SIMAZINE FINAL

 

 

Page 33 of 66 

Sanderson, J, Seinen, W, Giesy, J, et al. (2000) 2-Chloro-S-triazine herbicides induce aromatase 

(CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for 

estrogenicity? Toxicol. Sci. 54: 121–127. 

Sanderson, J., Letcher, R., Heneweer, M., et al. (2001) Effects of chloro-S-triazine herbicides 

and metabolites on aromatase activity in various human cell lines and on vitellogenin production 

in male carp hepatocytes. Environ. Health Perspect. 109(10): 1027-1031. (MRID 48322301) 

Sawhney Coder, P. (2011). Simazine - Hershberger Bioassay in Orchidoepididymectomized 

Rats. WIL Research Laboratories, LLC, Ashland, OH. Laboratory Report No.: WIL-639137, 

December 2, 2011. Unpublished. (MRID 48619303) 

Schneider, S., Kendall, T., and Krueger, H. (2012) Simazine: amphibian metamorphosis assay 

for the detection of thyroid active substances. Wildlife International, Ltd., Easton, MD. 

Laboratory Report No. 528A-227, November 8, 2012. Sponsored by Syngenta Crop Protection, 

LLC, Greensboro, NC. Unpublished. (MRID 46873601) 

Schneider, S., Martin, K., Kendall, T., et al. (2012) Simazine: fish short-term reproduction assay 

with the fathead minnow (Pimephales promelas). Wildlife International, Ltd., Easton, MD. 

Laboratory Report No. 528A-226, November 8, 2012. Submitted by Syngenta Crop Protection, 

LLC, Greensboro, NC. Unpublished. (MRID 48673602) 

Stoker, T.E., Laws, S.C., Guidici, D.L., et al (2000) The effect of atrazine on puberty in male 

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

function. Toxicol. Sci. 58:50-59. 

Stoker, T.E., Guidici, D.L., Laws, S.C. et al (20002) The effect of atrazine metabolites on 

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

Stoker, T., Buckalew, A., Farrell, J., et al. (2005) Effect of simazine on male reproductive 

development in the rat. Society Study Reprod. Abstract M274. 38th Annual Mtg. July 24-

27,2005. Quebec City, Quebec, Canada. 

Tai, C., Breckenridge, C., and Green, J. (1985) Simazine technical: subacute ora1 13-week 

toxicity study in dogs. Ciba-Geigy Corp., Summit, NJ. Laboratory Report No. 85022, April 12, 

1985. Unpublished. (MRID 00146655) 

Tai, C., Breckenridge, C., and Green, J. (1985) Simazine technical: subacute oral 13-week 

toxicity study in rats. CibaGeigy Corp., Summit, NJ. Laboratory Report No. 85018, April 10, 

1985. Unpublished. (MRID 00143265) 

Tennant, M., Hill, D., Eldridge, C., et al. (1994a) Possible anti-estrogenic properties of chloro-Striazines in rat uterus. J. Toxicol. Environ. Health, Part A. 43: 183-196. (MRID 43598617) 

Page 41 of 74
SIMAZINE FINAL

 

 

Page 34 of 66 

Tennant, M., Hill, D., Eldridge, C., et al. (1994b) Chloro-S-triazine antagonism of estrogen 

action: limited interaction with estrogen receptor binding. J. Toxicol. Environ. Health, Part A. 

43: 197-211. (MRID 43598618) 

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. 

Andro.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. (2007). Risks of Simazine Use to California Red‐legged Frog (Rana aurora draytonii). 

Environmental Fate and Effects Division, Office of Pesticide Programs, Washington, D.C. 20460 

USEPA. (2007). Risks of Simazine Use to Barton Springs Salamander (Eurycea sosorum). 

Environmental Fate and Effects Division, Office of Pesticide Programs, Washington, D.C. 20460 

USEPA (2010) Simazine - Report of the Endocrine Disruptor Review Team; Test Order# EDSP080807-130; 131 and 132. December 16, 2010. Office of Chemical Safety and Pollution 

Prevention. U.S. Environmental Protection Agency, Washington, D.C. 

USEPA (2011) 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. U.S. Environmental Protection Agency, Washington, DC. 

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. 

Willoughby, J. (2011) Simazine: androgen receptor binding (rat prostate cytosol). CeeTox Inc., 

Kalamazoo, MI. Laboratory Study No. 9047V-100328ARB, December 5, 2011. Unpublished. 

(MRID 48619301) 

Willoughby, J. (2011) Simazine: estrogen receptor transcriptional activation (human cell line 

(HeLa-9903)). CeeTox, Inc., Kalamazoo, MI. Laboratory Report No. 9047V-100328ERTA, 

December 5, 2011. Unpublished. (MRID 48619302) 

Willoughby, J. (2012) Supplemental Information: laboratory proficiency data for ERTA assays 

and saturation binding data for AR and ER binding assays for assorted chemicals. CeeTox, Inc., 

Kalamazoo, MI. July 19, 2011. Unpublished. (MRID 48843501) 

Page 42 of 74
SIMAZINE FINAL

 

 

Page 35 of 66 

Zorrilla L., Gibson E., and Stoker T. (2010) The effects of simazine, a chlorotriazine herbicide, 

on pubertal development in the female Wistar rat. Reproductive Toxicology. 29(2010): 393-400. 

(MRID 48205102) 

Page 43 of 74
SIMAZINE FINAL

 

 

Page 36 of 66 

APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA): (OCSPP 890.1100) 

The 21-day assay (MRID 48673601) of simazine on amphibian metamorphosis of African 

clawed frog (Xenopus laevis) was conducted under flow-through conditions. Amphibian larvae at 

Nieuwkoop-Faber (NF) stage 51 (80 per control and treatment group) were exposed to nominal 

concentrations of simazine (97.8% purity) at 0 (negative control), 0.020, 0.20, and 2.0 mg a.i./L; 

mean-measured concentrations were <0.0100 (<LOQ; control), 0.019, 0.18, and 1.9 mg a.i./L. 

The test system was maintained at 21.4 to 22.6°C and a pH of 8.0 to 8.2. 

Unless otherwise indicated, all effects are reported based on comparison to the negative (clean 

water) control. A single mortality was observed in the low treatment group prior to Day 7; no 

additional non-incidental mortalities were observed throughout the remainder of the test. Clinical 

signs (spinal curvature) were observed in the control (8%), low (6%), and mid (9%) treatment 

groups and were not considered to be treatment related. 

Simazine significantly increased (p<0.05) normalized (for snout-vent length) Day 7 hind-limb 

length (HLL) by 6% at the high treatment level and significantly decreased Day 21 normalized 

HLL by 14% in the same treatment group; the response was concentration-dependent in both 

cases. There was no significant acceleration or delay of median NF developmental stage at Day 7 

or 21 in any treatment group. Further, no asynchronous development was observed throughout 

the test. A single late-stage tadpole was observed in the negative control (NF=61) and in the low 

and mid treatment groups (NF=62). These tadpoles were excluded from analysis of growth 

endpoints and normalized HLL. Concentration-dependent reductions in growth were observed on 

Day 21 for snout-vent length (SVL) and wet weight, with significant decreases of 7 and 23% at 

the high treatment level for SVL and wet weight, respectively relative to the negative control 

(p<0.05; Jonckheere-Terpstra). 

Changes in thyroid gland histopathology were observed in all treatment groups including the 

control; however, the observation of a slight increase in the incidence of animals with mild to 

moderate thyroid gland atrophy in the highest treatment (10/20) compared to the negative control 

(4/20) was reported to be treatment-related. The study authors noted that thyroid gland atrophy 

was generally associated with smaller sized tadpoles regardless of treatment, suggesting that 

thyroid gland atrophy was secondary to reductions in size at the high concentration. 

Androgen Receptor (AR) Binding Assay; (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48619301), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment, 

which measured the binding of a single concentration of [3

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

Page 44 of 74
SIMAZINE FINAL

 

 

Page 37 of 66 

increasing concentrations (10−10 to 10−3

 M) of simazine (96.9% purity, Lot# 429546). DMSO 

was used as the vehicle at a final assay concentration of ~3.2%. A total of three independent 

runs were performed, and each run included dexamethasone as a weak positive control, and 

R1881 as the ligand reference standard. 

Saturation binding data were not originally provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. As separate cytosolic preparations were used for each of the 

three competitive binding runs, the data from all three saturation binding experiments were 

evaluated. For the cytosolic preparation made on March 30, 2011, the mean dissociation 

constant (Kd) for [3

H]-R1881 was 0.614 (±0.065) nM, and the estimated Bmax was 1.080 (±0.109) 

fmol/100 μg protein. For the cytosolic preparation made on July 23, 2011, mean Kd for 

[

3

H]-R1881 was 1.080 nM (±0.690), and the estimated Bmax was 1.071 (±0.167) fmol/100 μg 

protein. For the cytosolic preparation made on September 24, 2011, the mean Kd for [3

H]-R1881 

was 0.613 nM (±0.041), and the estimated Bmax was 0.817 fmol/100 μg protein (±0.049). The 

mean and individual Kd values were generally below the range reported in the EPA validation 

program (0.685 to 1.57 nM), although two individual values were within the range and one value 

was above the range. Confidence in these numbers is generally high based on the goodness of fit 

(R2

 = 0.880-0.992) and the small variation in R2 and Kd estimates among runs (although greater 

variability was observed in the July 23, 2011 saturation binding data). 

In the competitive binding experiment, precipitation of simazine was visually observed at the 

highest concentration tested (10−3 M) in the first and second runs. Therefore, only data from 

concentrations ≤10−4 M were used for data assessment, and the highest concentration tested in 

the third run was 10−4 M. Specific binding of [3H]-R1881 was 87.9-100.1% at simazine 

concentrations of 10−10 to 10−4 M in each run, with the exception of the 10−6 M concentration in 

Run 3 that had binding of 70.6%. However, this decrease in binding was not concentration 

dependent as binding was 92.1-92.7% at concentrations of 10−5 and 10−4 M in the same run. 

Because specific binding was not reduced to ≤50% at any simazine concentration, an IC50 and 

relative binding affinity (RBA) could not be calculated for simazine. 

The estimated average log IC50s for R1881 (−9.3 M) and the weak positive control, 

dexamethasone (−4.4 M), were within the expected ranges, and the mean RBA for the weak 

positive control was 0.0024%. Confidence in these numbers was moderate due to variation in 

IC50 values for the reference standards. However, the performance criteria were generally met. 

Based on the results from the three runs, simazine is classified as a Non-Binder in the Androgen 

Receptor Binding Assay. 

Page 45 of 74
SIMAZINE FINAL

 

 

Page 38 of 66 

Aromatase Assay; (OCSPP 890.1200) 

The requirement for a Tier 1 aromatase assay is satisfied by the in vitro assays reported by 

Sanderson et al. (2001). A summary of this study is presented in Appendix 2. 

Estrogen Receptor (ER) Binding Assay; (OCSPP 890.1250) 

The requirement for a Tier 1 ER binding assay is satisfied by the in vitro assay reported by 

Tennant et al. (1994b). A summary of this study is presented in Appendix 2. 

ERα Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48619302), hERα-HeLa-9903 

cells cultured in vitro were exposed to simazine (96.9% a.i., Lot# G27692U) in 0.1% DMSO at 

logarithmically increasing concentrations from 10−11 to 10−4

 M for 24±2 hours in two 

independent runs. The experiments were performed using 96-well plates and each simazine 

concentration was tested in replicates of 6 wells/plate in each run. Cells were exposed to 

simazine to induce reporter (luciferase) gene products. Luciferase expression in response to 

activation of the estrogen receptor was measured upon addition of a luciferase substrate and 

detection with a luminometer with acceptable sensitivity. 

Simazine was tested up to the limit of solubility, 10−4 M. The mean RPCMax for simazine was 

3.5%, with an associated PCMax of 10−7 M in the first run; the mean RPCMax was <0% for the 

second run at concentrations up to 10−4

 M. Performance criteria values were generally below the 

validated ranges, indicating that the current assay was more sensitive than during the validation 

experiments. Therefore the performance criteria deviations do not affect then interpretation of 

the study. Because the RPCMax < PC10 in both runs, simazine was considered negative for 

estrogen receptor transcriptional activation in this test system. 

Fish Short Term Reproduction Assay (FSTRA); (OCSPP 890.1350) 

The 21-day short-term reproduction assay (MRID 48673602) of EPTC with fathead minnows 

(Pimephales promelas) was conducted under flow-through conditions. Adult fish, 16 spawning 

groups (2 males and 4 females in each group; 6 months old) were exposed to EPTC (>99.9% 

purity) at nominal concentrations of 0 (control), 0.08, 0.80, and 8.0 mg a.i./L; mean-measured 

concentrations were <LOQ (<0.014), 0.069, 0.69, and 6.9 mg a.i./L. The test system was 

maintained at 24 to 25oC and a pH of 6.7 to 7.4. 

Page 46 of 74
SIMAZINE FINAL

 

 

Page 39 of 66 

Mean survival was 100, 100, 75, and 13% for males and 94, 100, 38 and 6% for females in the 

control, low, mid, and high treatment groups, respectively; overall survival (both sexes) was 96, 

100, 50, and 9% respectively. Due to the high mortality at the high concentration, these data 

were not used for statistical analysis. Mortality was significantly different in the mid and high 

treatment groups compared to the negative control (p<0.05; Fisher’s Exact Test). There were no 

notable observations with regards to behavior or changes in appearance, specifically 

color/banding, ovipositor appearance, or size of dorsal nape pad, in the control or treated groups. 

There were no notable observations of secondary sex characteristics or clinical signs for any 

treatment group compared to the controls. Male and female growth parameters (wet weight and 

total length) were not significantly different from the control. 

Spawning in each replicate occurred at least every 4 days and mean fecundity in controls was 22 

eggs/female/day/replicate (range 18-25 E/F/D); fertilization success in the control group 

averaged 96%. Fecundity was significantly (p<0.05; Dunnett’s) reduced by 75% at the mid 

treatment concentration. 

There were no significant differences between controls and the low and mid treatment groups for 

male or female gonado-somatic index (GSI). Male tubercle scores was marginally significantly 

(p=0.057; Mann-Whitney) reduced compared to the control (26 vs 41 in control) at the 0.69 mg 

a.i./L treatment level and was approaching significance (p=0.086) in the 0.069 mg ai/L treatment 

level (32 vs. 41); none of the surviving females were found to have tubercles. 

There were no treatment-related histopathologic observations in males. In female fish, there was 

an increase in incidence of minimal to moderate oocyte atresia at the mid treatment level 

compared to the control; however, the smaller sample size (n=6) prevented further interpretation 

of this finding. There were no treatment-related effects at any test level for gonadal staging. 

There were no other significant effects on other endpoints. For male vitellogenin (VTG), the 

reported control values were unusually high (comparable to control female values) even after reanalysis; the values in the other treatment groups were also comparable to female VTG values. 

No obvious reasons for these high values were reported and as such, these data are not 

considered reliable for interpretation. Sex steroids were not measured in this study. 

Hershberger Assay; (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48619303) screening for androgenic activity, simazine (96.9% 

purity, Batch # 429546) in 0.5% (w/v) carboxymethylcellulose (CMC) was administered daily 

via oral gavage (5 mL/kg) to groups of six 56- to 57-day old (approximate), castrated male 

Sprague Dawley rats at dose levels of 0 (vehicle), 65, or 200 mg/kg/day. An androgenic positive 

control group consisted of six castrated rats exposed to 0.2 mg/kg/day of testosterone propionate 

(TP) by subcutaneous (s.c.) injection plus vehicle (0.5% CMC) by oral gavage (5 mL/kg). 

Page 47 of 74
SIMAZINE FINAL

 

 

Page 40 of 66 

To screen for potential anti-androgenic activity, simazine in 0.5% CMC was administered daily 

for ten days via oral gavage (5 mL/kg) to groups of six 56- to 57-day old (approximate), 

castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 20, 65, or 200 mg/kg/day in 

conjunction with a daily dose of reference androgen TP at 0.2 mg/kg/day by s.c. injection. The 

anti-androgenic positive control group consisted of six castrated rats exposed to 0.2 mg/kg/day 

TP and 3 mg/kg/day flutamide (FT). TP alone was used as the anti-androgenic negative control. 

All animals survived until scheduled termination. No animals exhibited any dose-related clinical 

signs of toxicology and there were no treatment-related gross pathological findings. Animals 

were sacrificed approximately 24 hours after the final dose administration. At necropsy, the five 

androgen-dependent tissues and liver were collected and weighed. 

In the androgen agonist assay, body weights for the 200 mg/kg/day group were decreased 

(p<0.05) by 8-10% on Days 7, 8 and 11 compared to controls, resulting in a 50% decrease 

(p<0.05) in overall (Days 1-11) body weight gain. Overall body weight gain was also decreased 

(p<0.05) by 25% in the 65 mg/kg/day group. Body weights in the positive control group were 

comparable to controls throughout the duration of the study. Overall food consumption was 

similar across all groups. There were no treatment-related effects on the weights of accessory 

sex organs or liver for any dose group. Animals in the positive TP control group had increased 

(p<0.01) accessory sex organ weights as follows: 347% in seminal vesicles; 484% in ventral 

prostate; 174% in levator ani-bulbocavernosus (LABC); 308% in Cowper’s gland; and 58% in 

glans penis. Liver weights in the positive control group were comparable to the vehicle control. 

There were no significant weight increases in androgen-dependent tissues under the conditions of 

this assay. 

Percent CVs for the vehicle control and the simazine treatment groups were compared to the 

performance criteria in the Guidelines. All %CV values were less than the maximum 

recommended values, with the exception of Cowper’s glands in the control group (CV = 68.0%; 

maximum recommended CV = 55%). 

In the anti-androgen assay, overall body weight gain was decreased (p<0.05) by 36% in the 200 

mg/kg/day group. Body weights in the 20 and 65 mg/kg/day dose group and positive control 

group were comparable to controls throughout the duration of the study. Overall food 

consumption was similar across all groups. There were no treatment-related effects on the 

weights of accessory sex organs or liver for any dose group. Animals dosed with TP + FT 

(positive control) had decreased (p<0.01) accessory sex organ weights as follows: 69% in 

seminal vesicles; 75% in ventral prostate; 47% in LABC; 74% in Cowper’s gland; and 20% in 

glans penis. Liver weights in the positive control group were comparable to the vehicle control. 

The performance criteria indicated that this assay was performing as expected. There were no 

significant weight decreases in androgen-dependent tissues under the conditions of this assay. 

Page 48 of 74
SIMAZINE FINAL

 

 

Page 41 of 66 

Based on the observed decreases in body weight gain of 36-50% at 200 mg/kg/day, the rats used 

in this assay were dosed adequately. 

Statistically significant changes were not seen in two or more of the five androgen sensitive 

tissues. Simazine was negative for androgenicity and anti-androgenicity in the Hershberger 

assay. 

Female Pubertal; (OCSPP 890.1450) 

The requirement for a Tier 1 female pubertal assay is satisfied by the study reported by Zorrilla 

et al. (2010). A summary of this study is presented in Appendix 2. 

Male Pubertal; (OCSPP 890.1500)

The requirement for a Tier 1 male pubertal assay is satisfied by the study reported by Stoker et 

al. (2005). A summary of this study is presented in Appendix 2. 

Steroidogenesis; (OCSPP 890.1550) 

The requirement for a Tier 1 steroidogenesis assay is satisfied by the in vitro assays reported by 

Sanderson et al. (2001). A summary of this study is presented in Appendix 2. 

Uterotrophic; (OCSPP 890.1600) 

The requirement for a Tier 1 uterotrophic assay is satisfied by the studies reported by O’Connor 

et al. (2000) and Tennant et al. (1994a). A summary of these studies is presented in Appendix 2. 

Page 49 of 74
SIMAZINE FINAL

 

 

Page 42 of 66 

APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Published literature and Part 158 general toxicity studies considered relevant for determining the 

potential interaction of simazine with the endocrine system are summarized below. Given the 

breadth of toxicity data available on simazine, the only Part 158 studies discussed in this 

appendix are those that fulfill current guideline requirements and are directly relevant to 

assessing potential endocrine effects (i.e., multiple-generation rodent reproductive studies, 

development studies, chronic toxicity studies, etc.). 

AR Transcriptional Activation (ARTA) 

In a multi-component published study, Birkhøj et al. (2004) examined the potential antiandrogenic effects of simazine using an AR reporter gene assay in vitro. Simazine, deltamethrin, 

methiocarb, prochloraz, tribenuron-methyl and a mixture of the five pesticides were tested. 

Simazine (98.5% purity) was dissolved in DMSO as a 10 mM stock solution. The AR agonist 

R1881 and the positive control hydroxyflutamide (OHF) were dissolved in ethanol as stock 

solutions at 10 μM and 5 mM, respectively. The AR reporter gene assay was performed using 

CHO cells transfected with the expression vector pSV AR0 and the luciferase reporter plasmid 

MMTV-LUC. Twelve concentrations (0.025-50 μM) of simazine with or without R1881 

(0.1 nM) were tested in quadruplicate. The mixture experiment combined 10 μM of each of the 

five pesticides (including simazine) to make a 50 μM stock solution. OHF was included in every 

experiment as a positive control. Cells and test compounds were incubated, the dosing medium 

was removed, and the cells were lysed; substrate containing 1 mM luciferin and 1 mM ATP in 

lysis buffer were added, and chemiluminescence was measured. Cytotoxicity measurements were 

determined by replacing pSV AR0 in the procedure with pSV AR13 (active AR vector) that 

lacks the ligand binding domain. 

The data for the positive control flutamide were not reported, but simazine had no antagonistic 

effect on R1881-induced activation of the AR. 

Receptor Activation Assays 

In a published, non-Guideline study by Orton et al. (2009), several chemicals (>97% pure), 

including simazine, were tested for endocrine-disrupting effects using two in vitro assays. A 

recombinant yeast screen was used to detect receptor mediated (anti-) estrogenic and (anti-) 

androgenic activity, and cultured Xenopus oocytes were used to measure effects on the ovulatory 

response and ovarian steroidogenesis. 

Briefly, for the yeast estrogen assay, stimulation of the transfected human estrogen or androgen 

receptor causes a color change in the media, which is measured by absorbance at 540 nm. Plates 

were also measured at 620 nm to measure cell growth (turbidity) to check for any cytotoxic 

effects. In the ovulation assay, ovaries from sexually mature Xenopus laevis were removed, 

Page 50 of 74
SIMAZINE FINAL

 

 

Page 43 of 66 

cultured in medium, and cut into 10 oocyte fragments. The oocytes were incubated with 6.25, 

12.5, 25, and 50 IU of human chorionic gonadotropin (hCG) or control media for 20 h. The 

media was sampled and hormone analysis was performed by RIA. After incubation, a 

submaximal concentration of hCG (~60% of maximum ovulatory response) was chosen for coincubation with simazine at concentrations of 6.25 and 62.5 μM. Pesticides that had no effect or 

only an effect at 62.5 μM, were retested at the same concentrations. In addition, the 3β-HSD 

inhibitor epostane was tested at 0.001, 0.01, 0.1, and 1 μM, to verify inhibition of ovulation via 

inhibition of steroid hormone synthesis. 

In the yeast screen, none of the pesticides tested were agonistic in the estrogen or androgen 

screen. However, simazine was found to be anti-androgenic (125 μM, p<0.03, with an increase 

of approximately 17% above the control) in the anti-androgen assay, although it did not have a 

stimulatory effect on the androgen receptor in the absence of testosterone. 

In the ovulation assay, testosterone concentrations were elevated by simazine at 62.5 μM. 

In a published, non-Guideline study, Kojima et al. (2004) examined the potential of simazine 

(≥95% purity) to interact with two human estrogen receptor subtypes (hERα and hERβ) and one 

human androgen receptor (hAR) in transactivation assays using Chinese hamster ovary cells 

(CHO-K1 cells) in a luciferase reporter assay. For measurement of antagonistic activity to hERα, 

hERβ and hAR, either 10−11 or 10−10 M 17β-estradiol (E2) or 10−10 M 5α-dihydrotestosterone 

(DHT) was added to the cell cultures along with the test compound, respectively. Agonistic 

activities were evaluated by relative activity expressed as 20% relative effective concentration 

(REC20), 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 20% relative inhibitory concentration (RIC20), the concentration 

of the compound showing 20% inhibition of the activity of 10−10 M E2, 10−9 M E2, or 10−9 M 

DHT for ERα, ERβ, and AR, respectively. Simazine did not have agonist or antagonist activity 

for ERα and ERβ, or agonist activity for AR in this study. 

ERTA/ER Binding/Cell Proliferation 

In a published study, Blair et al. (2000) simazine was tested in an ER competitive binding assay 

to determine ER affinity. The source of the ER for this assay was rat uterine cytosol. Chemicals 

were first screened at a high concentration and those that were positive for binding were further 

tested by using a range of concentrations to generate a binding curve and calculate the IC50 for 

each chemical. Simazine (99% purity) was reported to be negative for ER binding at a 

concentration of 3.33×10−5

 M. 

In a multi-component published study, Connor et al. (1996) tested simazine in a MCF-7 cell 

proliferation assay, a reporter gene assay with MCF-7 cells transfected with a Gal4 ER chimera 

and β-galactosidase reporter, and a yeast assay. Simazine was negative for both agonism and 

Page 51 of 74
SIMAZINE FINAL

 

 

Page 44 of 66 

antagonism in the cell proliferation assay when tested at 0.01, 0.1, 1.0 and 10 μM. Simazine 

showed no ER agonism when tested in the reporter gene assay from 10–9 to 10–5 M. The cell 

proliferation assay and transcriptional activation assay with the chimeric receptor are considered 

to be inferior test systems to transcriptional activation assays using natural full length receptors. 

Simazine failed to induce any luciferase activity in the ER-CALUX assay, indicating it has no in 

vitro estrogenic properties detectable by this assay. 

In a multi-component published study, O’Connor et al. (2000) examined the effects of simazine 

(purity not reported) in vitro using estrogen/biogenic amine receptor competition binding assays 

and a yeast-expressed human estrogen receptor transcription system. Binding of simazine to the 

human ER was examined using a yeast reporter gene assay. Yeast was incubated with simazine 

and β-galactosidase induction was measured. For competitive inhibition studies, an approximate 

EC50 concentration of 17β-estradiol was added to the wells and percent inhibition was evaluated. 

Finasteride was included as a model compound for this assay. 

For the receptor competitive binding assays, simazine concentrations of 10−7 M, 10−5 M and 

10−3

 M were incubated with the biogenic amine and estrogen receptors alpha 1 adrenergic, alpha 

2 adrenergic, beta, dopamine 1, dopamine 2, serotonin 1 and serotonin 2 and the appropriate 

radioligand. Percent binding inhibition of the 17β-estradiol response was calculated. 

In the yeast transcription assays, simazine did not interact with the estrogen receptor or produce 

transcriptional activation. No competition was observed for any of the receptors tested in the 

vitro receptor competition binding assays with simazine. The positive controls, 17β-estradiol and 

finasteride, induced a dose-dependent increase and decrease in activation, respectively. 

In another published, non-Guideline study, Tennant et al. (1994b) investigated in vitro and in 

vivo competitive binding of three chemicals, including simazine and its metabolite 

diaminochlorotriazine (DACT; >96 % purity for both compounds) to the ER; several different 

experiments were performed utilizing extracted cytosolic ER from adult female Sprague-Dawley 

rats. Experiments observed the displacement of radiolabeled estradiol by simazine and DACT 

under both equilibrium (simultaneous exposure with radiolabeled estradiol at 4°C) and at 

disequilibrium conditions (pre-exposure at 25°C for 30 min. prior to exposure to radiolabeled 

estradiol). Results of equilibrium experiments indicated that simazine and DACT did not 

displace any radiolabeled estradiol whereas the positive control exhibited a dose-dependent 

displacement of the radiolabeled estradiol. Under disequilibrium conditions in a time-course 

experiment, uterine cytosolic extracts were first incubated with simazine or DACT at 100 μM for 

30 min. prior to exposure to radiolabeled estradiol for 5-150 minutes. With time, radiolabeled 

estradiol displaced simazine and DACT that had bound during the initial exposure. In a doseresponse experiment under disequilibrium conditions, uterine cytosolic extracts were first 

incubated with simazine or DACT at concentrations of 10−9

 to 10−3 M or unlabeled estradiol at 

Page 52 of 74
SIMAZINE FINAL

 

 

Page 45 of 66 

10−11 to 10−7

 M prior to exposure to radiolabeled estradiol. The IC50 for unlabeled estradiol was 

approximately 10−9 M whereas the IC50 was 100 μM for simazine and DACT. 

An additional experiment under disequilibrium conditions was performed to mimic a Scatchard 

type analysis using a constant molar excess (100X estradiol or 10,000X simazine) relative to the 

radiolabeled estradiol (0.2 nM, 0.5 nM, 1.5 nM, and 5.0 nM). Results indicated simazine 

competed with the radioligand better at lower concentrations of tracer. In the Scatchard-type 

plot, dissociation constants were produced; 0.5 nM in the presence of 100-fold molar excess of 

estradiol and 1 nM in the presence of 10,000 fold molar excess of simazine. The x-intercepts of 

the plot were approximately equal (210 fmol/mg) in the presence of estradiol and simazine 

indicating competitive binding. 

Making the assumption that living rats contain the activated form of the ER, in order to test 

whether simazine and DACT competed more effectively against the transformed ER, 

ovariectomized rats were dosed with 50 or 300 mg/kg/day simazine or DACT for 2 days prior to 

uterine dissection and competitive binding experiments. The binding of radiolabeled estradiol 

was reduced by an average of 39% and 24% for simazine and DACT, respectively (p<0.05 vs. 

vehicle controls) in the 300 mg/kg/day group. In the 50 mg/kg/day group, binding of 

radiolabeled estradiol was reduced (not statistically significant) by 21 and 13%. 

Overall the results indicate that simazine and DACT do exhibit some competitive binding with 

estradiol but only under conditions which favor triazine binding. 

In another published study, Sanderson et al. (2001; MRID 48322301) investigated the effect of 

simazine on human aromatase CYP19 activity in three cell lines: H295R (adrenocortical 

carcinoma), JEG-3 (placental choriocarcinoma) and MCF-7 (breast cancer). Receptor-mediated 

induction of vitellogenin was tested in primary hepatocyte cultures of adult male carp. For the 

aromatase experiments, cells were plated at ca. 1-2 × 105

 cells/well in 24-well culture plates, and 

exposed to 0, 0.3, 1.0, 3.0, 10.0 or 30 μM simazine or its metabolites des-isopropyl-s-atrazine 

(DIA) and DACT for 24 hours. Positive control cells were exposed to 100 μM of 8-bromo-cyclic 

adenosine monophosphate (8Br-cAMP) in DMSO. Negative controls included treatment with 

DMSO and no treatment; all plates contained all controls and all treatments were performed in 

quadruplicate. CYP19 expression was detected by RT-PCR, and aromatase activity was detected 

using a method based on estrone production of tritiated water. Cells exposed to the positive 

control 8Br-cAMP produced a significant increase in aromatase activity and levels of CYP19 

mRNA compared to controls. 

Male carp hepatocytes were isolated and cells were seeded in 96-well plates at a density of 1 × 

106

 cells/mL. For the estrogenicity study, cells were exposed to 17β-estradiol (0.06-6 μM) or 

simazine at the same concentrations as above; for the anti-estrogenicity study, cells were exposed 

to the same simazine conditions but in the presence of 100 nM 17β-estradiol (approximate EC50). 

Page 53 of 74
SIMAZINE FINAL

 

 

Page 46 of 66 

Positive controls added to every plate were either 100 nM 17β-estradiol (for estrogenicity) or the 

known estrogen receptor antagonist tamoxifen at 0.1, 1.0 and 10 μM (for anti-estrogenicity). 

Estrogenicity was quantified by vitellogenin production using indirect competitive ELISA. Cell 

viability was assessed and found to be acceptable. Vitellogenin concentrations in unexposed or 

DMSO-exposed male carp hepatocytes were undetectable. The lowest-observed effect 

concentration of 17β-estradiol of ca. 2 nM produced about 100-400 ng/ml cellular protein and 

the EC50 concentration of 17β-estradiol produced ca. 4000-6000 ng/mg protein. Co-exposure of 

hepatocytes to 100 nM 17β-estradiol and 0.1, 1 or 10 μM tamoxifen inhibited 17β-estradiolinduced vitellogenin synthesis by 54%, 89%, and 91% respectively. 

Simazine and its metabolites induced aromatase activity concentration-dependently, producing 

statistically significant increases in activity above control at concentrations above 1 μM (Student 

t-test, p < 0.05) in JEG-3 cells. In a previously published study by this author (Sanderson et al., 

2000), simazine also increased aromatase activity and CYP19 mRNA levels in H295R cells. 

Simazine did not induce or inhibit the induction of vitellogenin in male carp hepatocytes. 

Hershberger (OCSPP 890.1400)

In a multi-component published study, Birkhøj et al. (2004) examined potential anti-androgenic 

effects of simazine (99.0% purity) using a Hershberger assay in vivo. Simazine, deltamethrin, 

methiocarb, prochloraz, tribenuron-methyl and a mixture of the five pesticides were tested. Fourweek old male Wistar rats were castrated and shipped to the laboratory after one week. On 

arrival, rats were weighed and randomized by body weight into nine experimental groups having 

similar weights among groups (n=6). After one week acclimation, the rats were dosed for seven 

days. Group 1, the vehicle control, received 5 mL/kg peanut oil only. All remaining groups 

received s.c. doses of 0.5 mg/kg/day testosterone propionate (TP). Group 2 was administered TP 

only and was used for comparison to treated groups. Group 3, the positive control, received 20 

mg/kg/day flutamide (2 mL/kg) subcutaneously. Groups 4–8 (deltamethrin, methiocarb, 

prochloraz, simazine, and tribenuron-methyl) received oral doses of 2.5, 2.5, 25, 25 and 25 

mg/kg/day, respectively. Group 9 received a mixture of the five chemicals at the combined dose 

of 80 mg/kg/day. TP was administered 2 to 10 minutes after the test compounds and the last 

dosing was performed on the day of sacrifice. 

Following the last dose, rats were anaesthetized followed by exsanguination. Blood from each rat 

was collected and serum was stored at -80°C until hormone analysis. The ventral prostate, 

seminal vesicles/coagulation gland, levator ani/bulbocavernosus (LABC) muscle, bulbourethral 

glands, liver, thyroid gland, paired adrenal glands, and paired kidneys were dissected and 

weighed. Serum testosterone was analyzed by time-resolved fluorescence using FIA kits; 

thyroxin was analyzed using kits; prolactin was analyzed using enzyme immunoassay; 

luteinizing hormone (LH) was analyzed using an immunofluorimetric assay. For ventral 

prostates, total RNA was isolated and cDNA was produced using reverse transcriptase 

Page 54 of 74
SIMAZINE FINAL

 

 

Page 47 of 66 

polymerase chain reaction (RT-PCR). Real-time RT-PCR with online detection of the PCR 

reaction based on fluorescence monitoring was used to quantify the relative gene expression in 

ventral prostates. 

Simazine alone had no effect on body weights, organ weights or hormone levels when tested for 

seven days in castrated male Wistar rats. The mixture treatment group had significantly increased 

adrenal weights and reduced LABC weights. The positive control, flutamide, had significantly 

reduced reproductive organ weights and LH hormone levels. Prostate binding protein subunit C3 

mRNA and ornithine decarboxylase mRNA levels were significantly reduced for the simazine, 

flutamide, and mixture groups. Testosterone-repressed prostatic message 2 (TRPM-2) mRNA 

levels were decreased in the simazine group compared to the testosterone control. The flutamide 

TRPM-2 mRNA levels were significantly increased compared to the testosterone control. 

The authors concluded that the in vivo gene expression changes following simazine exposure 

were either anti-androgenic or regulated by factors independent of AR function. 

Female Pubertal (OCSPP 890.1450) 

In a published study, Zorrilla et al. (2010) evaluated the effects of simazine (98.3% purity) on 

pubertal development in female Wistar rats using the EDSP Tier 1 Female Peripubertal Rat 

Protocol. Twenty timed-pregnant Wistar rats were allowed to deliver naturally; the day of birth 

was designated as PND 0. On PND 3 the litters were standardized to 8-10 pups. On PND 21 

female pups were weaned, weighed, weight ranked and assigned to treatment groups with similar 

body weight means and variations with only one pup per litter in each treatment group. 

Dosing solutions were prepared in corn oil (vehicle control) and the doses were selected based 

on earlier studies with atrazine in weanling rats. Doses were administered by oral gavage at a 

volume of 0.5 mL/100 g bw to groups of 10 females beginning on PND 22. Two studies were 

conducted, the first followed the EDSP protocol and the second was a modified EDSP protocol 

of longer duration. The first study consisted of 5 simazine treatment groups (0, 12.5, 25, 50 and 

100 mg/kg/day) and animals were dosed until PND 42/43 (21 days). In the second study, groups 

of females received the same 5 doses as in Study 1 with an additional group at 200 mg/kg/day. 

Dosing in this study continued until PND 62/65 (41 days) in order to examine the effects of 

treatment on ovarian cycling. 

Animals were examined for VO throughout the dosing period, and the age of complete VO was 

recorded. On the day of VO, daily vaginal smears were collected and observed for the presence 

of leukocytes (diestrus), nucleated epithelial cells (proestrus) or cornified epithelial cells (estrus). 

In the first study, vaginal cytology (VC) was continued daily until scheduled sacrifice. In the 

second study, VC was also conducted daily and sacrifice was on the first day of estrus after PND 

62. 

Page 55 of 74
SIMAZINE FINAL

 

 

Page 48 of 66 

Two hours after the final dose, animals were sacrificed by decapitation, trunk blood was 

collected, and liver, kidney, adrenals, ovaries, uterus and pituitary were removed and weighed. 

Immediately after weighing, uteri and ovaries were fixed in 10% buffered formalin for 24 h, 

rinsed and stored in 70% ethanol. Cross-sections of the uterus and ovary were prepared in 

paraffin, stained with hematoxylin and eosin and a cross-section of the uterine horn was scored 

subjectively for luminal and glandular epithelial height, and stromal thickness. Sections of ovary 

were evaluated for corpora lutea count (all stages), combined count of primordial and primary 

follicles, atretic follicle count, and the presence or absence of antral follicles. Blood was allowed 

to clot, centrifuged to collect serum and stored at −80oC for subsequent hormone assays for T4

estradiol (E2), progesterone (P4, Study 2 only) luteinizing hormone (LH) and prolactin (PRL). 

Anterior pituitaries were frozen on dry ice and stored at -80 ̊C for PRL and LH assays. Serum E2, 

P4 and T4 were measured by radioimmunoassay. LH and PRL RIAs were performed using 

materials supplied by the National Hormone and Pituitary Agency (Torrance, CA). 

No clinical signs of toxicity were observed in any of the treatment groups. In Study 1, mean 

body weights on the day of VO (PND 32) were significantly lower at 100 mg/kg/day relative to 

controls (↓13.3 %); by PND 42 (necropsy), there was no significant difference. In Study 2, body 

weights were decreased at 100 and 200 mg/kg on Day 33 (↓13.5 % and 17.6 %, respectively); at 

Days 62-65 body weights were decreased in the 25 (↓8.6 %), 50 (↓8.0 %), 100 (↓10.3 %) and 

200 (↓11.3 %) mg/kg/day groups. The age at VO was delayed (p<0.05) in Study 1 at 25 and 100 

mg/kg, but no significant difference was noted at 12.5 or 50 mg/kg/day simazine. Mean VO 

occurred on PND 32.1 ± 0.4 in the control group and PND 34.4 ± 0.7 and 36.1 ± 0.4 in the 25 

and 100 mg/kg groups, respectively; delayed VO coincided with reduced body weight at 100 but 

not at 25 mg/kg. In Study 2, VO was delayed (p<0.05) in all simazine dose group; VO occurred 

at 31.6 ± 0.2 days in control animals and at 34.3 ± 0.6, 34.5 ± 0.6, 37.0 ± 0.7 and 36.7 ± 0.8 days 

in the 25, 50, 100 and 200 mg/kg/day groups, respectively. In these animals, delayed VO 

coincided with reduced body weight at 100 and 200 mg/kg/day but not at 25 and 50 mg/kg/day. 

In Study 1, absolute and relative pituitary and adrenal weights were significantly lower in the 

100 mg/kg/day group; absolute but not relative adrenal weights were also lower at 50 mg/kg/day. 

Relative (but not absolute) liver weights were higher at 100 mg/kg/day. In Study 2 absolute (but 

not relative) pituitary weights were significantly lower at 100 and 200 mg/kg/day. Absolute (but 

not relative) liver weights were lower in the 12.5 and 25 mg/kg/day groups; however liver 

weights from the higher dose groups were not different from controls. Relative ovarian weights 

were significantly greater in the 200 mg/kg/day group. 

In Study 1, decreased serum T4 levels were observed at 100 mg/kg/day; however, in Study 2, 

serum T4 levels were unaffected at any dose. Simazine did not alter serum E2 in either study. In 

Study 1, serum PRL levels were decreased at 25 (77.7 %), 50 (82.3 %) and 100 (80.4 %) 

mg/kg/day, but the decrease was significant only at 50 mg/kg/day. In Study 2, there was a dose 

dependent linear trend in decreased serum PRL by linear trend analysis (p ≤ 0.01) that was not 

Page 56 of 74
SIMAZINE FINAL

 

 

Page 49 of 66 

significant by ANOVA. No changes in baseline serum LG, pituitary LG or pituitary PRL were 

observed in either study. 

In Study 1, the mean number of days until the first estrus was significantly delayed at 100 mg/kg 

relative to controls (PND 42.6 ± 0.6 vs 37.9 ± 0.4, respectively). The number of days in estrus 

was also significantly decreased at 100 mg/kg, but the number of days in proestrus and diestrus 

were not changed; however as a result of the short time between VO and sacrifice only 1.0 ± 

0.15 estrus cycles were observed in the control animals, and fewer in the treated. 

In Study 2, dosing was extended 20 days longer than in the EDSP protocol to evaluate the effect 

of simazine on estrus cyclicity for a longer time period. There was a significant delay in the 

number of days until first estrus in the 100 and 200 mg/kg dose groups (PND 44.4 ± 1.1 and 43.7 

± 0.5 days, respectively) compared with control (PND 39.5 ± 0.73). The number of days in 

proestrus was significantly decreased only in the 50 mg/kg group. Although there appeared to be 

a slight decrease in the number of cycles in the 25 and 200 mg/kg groups, neither was 

significantly different from controls. 

Histology showed no effects of simazine on uterine morphology, epithelial cell height, stromal 

thickness or estrus cyclicity. Additionally, no apparent differences existed in the number of 

corpora lutea, primordial, primary or atretic follicles. Antral follicles were present in all rats. 

The most sensitive endpoint in this study was age at VO, as was shown by a delay of 2.3 days 

(25 mg/kg/day) and 4.0 days (100 mg/kg/day) in the 21-day exposure study, and by 2.7, 2.9, 5.4 

and 5.1 days (25, 50, 100 and 200 mg/kg/day, respectively) in the 41-day exposure study. 

Although body weight changes are known to possibly affect onset of puberty, the delays in VO 

observed at 25 mg/kg/day in this study do not appear to be affected by the reduced weight gain 

following simazine exposure. The study demonstrates that simazine delays VO at doses ≥25 

mg/kg/day and suppresses PRL secretion, suggesting that the mechanism of action of simazine is 

to alter hypothalamic regulation of pituitary hormone secretion. 

Male Pubertal (OCSPP 890.1500)

In a published abstract, Stoker et al. (2005) conducted a male rat pubertal screen using 

procedures consistent with the study design that was eventually adopted by the EDSP as OPPTS 

890.1500. Groups of intact 23 day old weanling male Wistar rats were administered simazine at 

dose levels of 0, 6.25, 25, 75, 150, and 300 mg/kg/day for 30 days. Preputial separation was 

monitored from PND 33 onwards and the animals were necropsied on PND 53. The pituitary, 

testes, ventral prostate, lateral prostate, epididymides, and seminal vesicles with coagulating 

gland were removed and weighed. Tissue samples were examined by histology. Blood was 

analyzed for TSH, T4, T3, LH, PRL, androgen and estrogens. Additionally, LH and PRL were 

determined in the anterior pituitaries. 

Page 57 of 74
SIMAZINE FINAL

 

 

Page 50 of 66 

A non-monotonic dose response was found for the onset of puberty, with an advancement at the 

low doses (6.25 mg/kg by 1.3 days and 25 mg/kg by 2.5 days) and a non-significant rise at the 

three highest doses (data not provided). Dose-dependent decreases (NS) were noted at 75 mg/kg 

and above in the right lateral prostate (↓12-35%; p<0.05 at 300 mg/kg), left lateral prostate (↓5-

41%; p<0.05 at 150 mg/kg and above), ventral prostate (↓12-33%; p<0.05 at 300 mg/kg), 

seminal vesicle (↓11-31%; p<0.05 at 300 mg/kg), and anterior pituitary (↓16-35%; p<0.05 at 150 

mg/kg and above). Non-dose-dependent decreases were also observed in liver (↓5-16%) and 

paired kidney (↓10-18%). However, these decreases corresponded with decreases (p<0.05) in 

terminal body weights of 10-22% at 75 mg/kg and above. Serum testosterone levels were 

increased by 100-167% (p<0.05 at 6.25 mg/kg only) at all dose levels in an manner inverse to 

dose. Alterations in serum T3, T4, and TSH were found, but the occurred in a manner unrelated to 

dose. 

Simazine at 300 mg/kg caused minimal to moderate testicular degeneration in 11/11 (100%) 

testes examined. The affected testes were as large as or slightly larger than the testes from the 

control rats or rats in the other treated groups. Mild to moderately severe amounts of germinal 

epithelium were present in the epididymal duct of 11/12 (92%) epididymides from the 300 

mg/kg dose group rats. Mild to severe hypospermia was present in the epididymal ducts of 11/12 

(92%) epididymides from the 300 mg/kg dose group rats. The increased amounts of germinal 

epithelium and the hypospermia occurred in the same individual 300 mg/kg dose group rats that 

had testicular degeneration. The epithelial cells lining the epididymal ducts from all of the 

epididymides examined were not remarkable, indicating that the testicular degeneration was 

responsible for the changes in the epididymal fluid and that the epididymides were not affected 

by simazine. 

Pituitary/Adrenal Hormone Effects (Rat)

In a published non-Guideline study, Laws et al. (2009) characterized the dose response and time 

course for effects of chlorotriazine herbicides, including simazine (98% purity), and its 

chlorinated metabolites, DIA (91% purity) and DACT (97% purity) on pituitary and adrenal 

hormone secretion in male rats. In addition, plasma concentrations of the test chemicals were 

measured to provide estimates of the parent compounds and metabolites. All test chemicals were 

prepared as a suspension in 1% methyl cellulose in water. Male Wistar rats (50–60 days old) 

were maintained under controlled conditions, ranked by body weight, and assigned to treatment 

groups so that the mean body weights for all groups were similar. Rats received a single oral 

dose by gavage of simazine at 188 mg/kg; DIA at 4, 10, 40, 80, or 160 mg/kg; or DACT at 3.37, 

33.7, 67.5, or 135 mg/kg. The control group was dosed with 1% methyl cellulose only. The rats 

were dosed with the vehicle or the test chemical at 0900 h and sacrificed by decapitation at 

designated times: 5, 15, 30, 60, or 180 minutes after dosing. At sacrifice, blood was processed 

for serum and plasma, and aliquots were stored for determination of adrenocorticotropic 

hormone (ACTH), corticosterone (CORT), progesterone (P4), and prolactin levels by 

Page 58 of 74
SIMAZINE FINAL

 

 

Page 51 of 66 

radioimmunoassay or for analytical chemistry analyses for the test chemical concentrations. 

Additionally, one group of rats was placed in adequately ventilated plexi-glass injection 

restrainers for 15 min just prior to necropsy at 0900 h to serve as positive controls for the acute 

stress response. Extraction and analysis of rat plasma samples for levels of the metabolites DIA 

and DACT were conducted using modifications of previously described gas 

chromatography/mass selective detection methods. 

Following a single dose of the metabolite DACT, ACTH levels were significantly increased in 

the plasma of rats dosed at 33.7 and 135 mg/kg at 30 minutes, and at 67.5 mg/kg at 60 minutes, 

compared to controls. Compared to controls, significant increases were observed for CORT in rat 

serum with DACT dosing at 33.7 and 67.5 mg/kg at 30 minutes, and 33.7, 67.5, and 135 mg/kg 

at 60 minutes, and for progesterone at 67.5 and 135 mg/kg at 30 and 60 minutes. 

ACTH and P4 levels were significantly increased in a dose-dependent manner in the 

plasma/serum of rats dosed with DIA at 40 and 80 mg/kg at 15 and 30 minutes, compared to 

controls; maximum increases for ACTH were observed in the 80 mg/kg at 15 (3.9 fold) and 30 

(3.7 fold) minutes. The CORT response was significantly increased in rat plasma with DIA 

dosing at 10, 40, and 80 mg/kg at 15 minutes, and 40 and 80 mg/kg at 30 minutes, compared to 

controls. 

Compared to controls, a single dose of simazine at 188 mg/kg elicited significant increases in 

ACTH and CORT, but not P4, at 15 minutes. 

Metabolite concentrations in the plasma of rats following a single dose of DIA or DACT were 

measured; uptake of DIA was rapid with the metabolite undergoing dealkylation yielding similar 

concentrations of DACT. Plasma concentrations of DACT in rats following a single dose of 

DACT were dose responsive and increased with post-dosing time. Plasma concentrations of 

simazine were not measured; however, 15 minutes post-dosing with simazine, both metabolites 

were found in plasma. 

The study authors reported that significant increases in plasma ACTH were observed within 15 

minutes following exposure to simazine or the metabolite DIA. Dose-dependent elevations in 

CORT and P4 were also observed at 15 and 30 minutes post-dosing with these compounds, 

indicating an activation of adrenal steroidogenesis. Measurement of the plasma concentrations of 

the parent compounds and metabolites confirmed that simazine is rapidly metabolized to DACT. 

The authors noted that although DACT had only minimal effects on ACTH and steroid release, 

dosing with this metabolite resulted in plasma DACT concentrations that were 8-fold greater 

than an equimolar dose of DIA, indicating that DACT is not likely the primary inducer of ACTH 

release. Thus, the rapid release of ACTH and subsequent activation of adrenal steroidogenesis 

following a single exposure to simazine or DIA may reflect chlorotriazine-induced changes at 

the level of the brain and/or pituitary. 

Page 59 of 74
SIMAZINE FINAL

 

 

Page 52 of 66 

Uterotrophic (OCSPP 890.1600)

In a multi-component published study, O’Connor et al. (2000) examined the effects of simazine 

(purity not reported) in vivo using 42-day-old ovariectomized (Day 41) female Crl:CD BR rats. 

The animals were weighed and randomly assigned to experimental groups using a stratified 

design to ensure weight similarities among groups (five groups, n=20), and maintained under 

standard laboratory conditions. The biochemical/hormonal measurements used groups of 14 rats, 

and the remaining 6 rats per group were designated for cell proliferation/morphometric 

evaluation. The rats were dosed by intraperitoneal injection three times per day for 4 days (ca. 

1/3 of the daily dose for each injection) at nominal simazine doses of 100, 200 and 300 

mg/kg/day in 0.25% methylcellulose. On Day 5, rats were anesthetized followed by 

exsanguination. The presence of uterine fluid in the uterine horns was recorded and uterine 

stumps were saved in formalin and examined to confirm the absence of ovarian tissue. For the 

cell proliferation subset, the rats were anesthetized and s.c. osmotic pumps loaded with 20 mg/ml 

BrdU were implanted on test Day -1. On test Day +5, these rats were euthanized, uteri were 

trimmed and fixed for two hours in Bouin’s solution; processed tissues were imbedded in 

paraffin, and stained for immuno-histochemical analysis of BrdU incorporation into DNA. Onethousand uterine endometrial stromal cells were counted. The same six rats were used to 

determine uterine epithelial cell heights by measuring hematoxylin and eosin stained 5 μM 

sections using an image analyzer. Estrous cycling was determined by examining daily vaginal 

washes in the 14 rats used for biochemical/hormonal evaluations. Blood was collected from all 

animals to determine serum prolactin levels by radio-immunoassay. 

Final body weights were significantly reduced in the 100 (↓10%), 200 (↓24%), and 300 (↓25%) 

mg/kg/day groups. No effects on uterine weight, uterine fluid imbibition, or stromal cell 

proliferation were observed. Estrus conversion was not affected by treatment. Uterine epithelial 

cell heights were significantly increased in the 200 and 300 mg/kg/day groups (p≤0.05). 

Simazine serum prolactin levels were similar to the control. 

In a published, non-Guideline study, Tennant et al. (1994a) conducted three in vivo assays to 

investigate the estrogenic effects of three chemicals, including simazine and its metabolite 

DACT (purity > 96%), on uterine weight stimulation/suppression, progesterone receptor binding, 

and thymidine incorporation into uterine DNA in female Sprague-Dawley rats. Only the results 

for simazine are discussed here. 

Young adult ovariectomized SD rats were used for the uterine weight and progesterone receptor 

studies; for the thymidine incorporation study, sexually immature intact female Sprague-Dawley 

rats were randomly assigned to treatment groups at an age of 23 days. In the uterine weight 

stimulation/suppression studies, rats were dosed by oral gavage for three consecutive days with 

simazine at 100 or 300 mg/kg/day or with a vehicle control, and in co-exposure experiments with 

2 μg estradiol (E2) on Day 2 and 3, to investigate the inhibition of estrogen-stimulated uterine 

Page 60 of 74
SIMAZINE FINAL

 

 

Page 53 of 66 

weight gain. Body weights were recorded on the first day of treatment and just prior to sacrifice. 

Uteri were dissected, trimmed, and weighed fresh. 

For the progesterone receptor (PgR) study, ovariectomized rats were dosed by oral gavage for 

three consecutive days with simazine at 0 or 300 mg/kg/day to groups of 15 rats, and at 50 or 300 

mg/kg/day followed by s.c. injection of E2 on Days 2 and 3 to groups of 12-15 rats (50 

mg/kg/day groups) or 12-20 rats (300 mg/kg/day groups). E2 was administered two hours after 

each simazine treatment. Body weights were recorded on the first day of treatment and just prior 

to sacrifice. Uteri were dissected at sacrifice. 

In the thymidine incorporation study, rats were dosed by oral gavage for 2 consecutive days with 

simazine at 0, 1, 10, 50, 100, or 300 mg/kg/day. E2 was administered by s.c. injection to each 

group on Day 2. Additional groups of rats at the 0 or 300 mg/kg/day dose levels received no E2

treatment. Animals were weighed prior to Day 1 of treatment and prior to sacrifice. The 

incorporation of radiolabeled thymidine into uterine DNA was determined and expressed as cpm 

radioactivity per μg DNA. Animals were sacrificed 24 hours following treatment, and the uteri 

were dissected. 

In ovariectomized rats, no significant increase in uterine weights were observed compared to the 

vehicle controls. For ovariectomized rats injected with E2, uterine weights were significantly 

decreased in the 300 mg/kg group compared to the vehicle controls receiving E2. Day 3 body 

weights were significantly lower in the 100 and 300 mg/kg groups (↓8 and 11%) in the uterine 

weight stimulation study (no E2). For the suppression study, the Day 3 body weights were 

significantly lower in the 300 mg/kg group (↓9%). 

The PgR binding capacity was significantly suppressed for the 300 mg/kg but not 50 mg/kg 

treatments plus E2. For treatments without E2 injections, PgR was significantly suppressed at 300 

mg/kg compared to the vehicle control. The study authors reported that PgR study rats received 

13 times more estradiol than the thymidine study rats, which could have caused the difference in 

inhibitory response at 50 mg/kg. 

Incorporation of radiolabeled thymidine was significantly inhibited (p≤0.01) in the 50, 100 and 

300 mg/kg groups with co-exposure to E2, but not at 1 or 10 mg/kg, compared to the E2 control. 

The 300 mg/kg treatments without E2 showed significantly less (p≤0.05) thymidine incorporation 

than the control without E2 and the 300 mg/kg treatment with E2. 

The results of the three studies indicate no estrogen-like activity and weak estrogenic antagonist 

activity for simazine and DACT using the rat uterine model system. 

In a multi-component published study, Connor et al. (1996) investigated three estrogen-induced

in vivo responses, including increases in uterine wet weight, progesterone receptor (PR) binding 

activity, and uterine peroxidase activity. Groups of 4 immature SD female rats received 50, 150 

Page 61 of 74
SIMAZINE FINAL

 

 

Page 54 of 66 

or 300 mg/kg/day simazine, dissolved in DMSO and suspended in an appropriate volume of 

water and 5% hydroxypropyl cellulose, via gavage for 3 days beginning at 21 days of age. Other 

groups of 4 rats that received simazine at the same doses also received 10 μg/kg/day of 17βestradiol (E2) in corn oil by intraperitoneal injections on the same treatment days (21-23). 

Control groups consisted of: 10 μg/kg/day E2 alone; the vehicle; or corn oil. 

Simazine alone did not increase uterine wet weight at any dose, but did cause significant 

(p<0.05) decreases in PR binding levels and uterine peroxidase activity. E2 alone caused 6.2-, 

8.9-, and 9.7-fold increases in uterine wet weight, PR binding, and uterine peroxidase activity, 

respectively relative to controls. Simazine did not exhibit agonist or antagonist effects on basal 

and E2-induced increases on uterine weights. E2-induced increase in uterine wet weight was not 

significantly affected by co-treatment with simazine; however, some dose-independent decreases 

in E2-induced PR binding uterine peroxidase activity were observed. In rats administered 150 

mg/kg/day simazine + E2, there was a statistically significant increase in uterine peroxidase 

activity. These data taken together indicate that simazine is not an ER agonist in this study. 

Subchronic Oral Toxicity in Rodents (Rat; OCSPP 870.3100)

In a subchronic toxicity study (MRID 00143265), groups of 10 Sprague-Dawley rats/sex were 

administered simazine (97.5% purity) in the diet at dose levels of 0, 200, 2000, or 4000 ppm 

(equivalent to 0, 14.25, 142 or 276 mg/kg/day) for 13 weeks. Organs that were weighed included 

kidneys, liver, testes, ovaries, and adrenals; histopathological examinations were conducted for 

control and high-dose animals on these tissues as well as the pituitary, thyroid, epididymis, 

prostate, seminal vesicle, urinary bladder, mammary gland, uterine cervix and horn, and vagina. 

Adrenal gland, liver, and kidney from low- and mid-dose rats were also examined 

microscopically. 

Rats at all dose levels survived the 13-week dosing period in good physical condition with no 

treatment-related clinical signs or ocular changes. There were significant dose-related reductions 

in mean food consumption during the first week of dosing in males and females, respectively, at 

4000 ppm (↓53% and ↓46%); 2000 ppm (↓37% and ↓32%) and 200 ppm (↓7% and ↓8%). Food 

consumption increased during the second week for males and females in all treated groups, but 

mean feed consumption for the 2000 and 4000 ppm rats remained significantly less than control 

rats throughout the dosing period. The 200 ppm males had significantly reduced mean food 

consumption compared to control males during Weeks 5-11. Significant weight loss occurred in 

the 2000 and 4000 ppm rats during the first week of dosing. During Weeks 2-13, significantly 

reduced mean body weights and body weight gains were observed in all treated males compared 

to controls. Mean body weights and body weight gains were significantly less than controls in 

the 2000 and 4000 ppm females during Weeks 1-13. At 13 weeks, dose-related reductions in 

mean red blood cell counts (RBC) were detected in all treated rats (both sexes) with 

accompanying decreased hematocrit (HCT) levels in females at 2000 and 4000 ppm and in the 

Page 62 of 74
SIMAZINE FINAL

 

 

Page 55 of 66 

4000 ppm males. Mean white blood cell counts (WBC) were significantly lower in all treated 

males. Neutrophil and platelet counts were significantly higher in the 2000 and 4000 ppm 

females and in all treated males (NS). Several clinical chemistry and urinalysis parameters were 

significantly different from controls in all dose groups, with most marked differences in the 2000 

and 4000 ppm groups: significantly lowered mean blood glucose levels occurred in male rats; 

high cholesterol and inorganic phosphorus levels were present in males and females; low levels 

of sodium and calcium were observed in males; low levels of BUN, LDH, SGOT and creatinine 

were observed in females; urinary ketone levels were elevated in males; and urinary protein 

levels were decreased in females. 

Decreased absolute organ weights and increased relative organ weights for brain, heart, kidney, 

liver, and testes in males and for brain and spleen in females were observed at 2000 and 4000 

ppm. Increased mean relative adrenal weights occurred in both sexes in all dose groups. 

Decreased mean absolute spleen weight in males and mean absolute ovary and heart weights 

were observed at 2000 and 4000 ppm; relative ovarian weight was decreased in the 4000 ppm 

females. There were no treatment-related gross lesions observed at necropsy. Microscopic 

examination of kidneys showed a dose-related incidence of renal calculi in treated rats, which 

reached significance in the 4000 ppm males and for males and females combined at 2000 and 

4000 ppm. The incidence of renal epithelial hyperplasia (tissue reaction to calculi) was also 

significant in the 4000 ppm males. 

The LOAEL is 200 ppm (equivalent to 12.6/15.9 mg/kg/day in males/females), based on reduced 

RBC and WBC, increased cholesterol and inorganic phosphorus, and increased renal calculi in 

males and females. A NOAEL was not determined. 

Subchronic Oral Toxicity (Dog; OCSPP 870.3150) 

In a subchronic toxicity study (MRID 00146655), 4 beagle dogs /sex were administered 

simazine (97.5% purity) in the diet at doses of 0, 200, 2000, or 4000 ppm (equivalent to 0/0, 

6.9/8.2, 65.2/64.3, and 133.6/136.7 mg/kg/day for males/females) for at least 91 days. Organs 

that were weighed included heart, brain, liver and testes (list not provided); histopathological 

examinations were conducted on these tissues as well as the adrenals, kidney, pituitary, thyroid, 

epididymis, prostate, urinary bladder, mammary gland, ovary, uterine cervix and horn, and 

vagina. 

There were no mortalities. Tremors, observed as early as Week 9 and persisting until 

termination, were observed at 4000 ppm in 4/4 males and 3/4 females. Mean daily food 

consumption was significantly decreased in males and females in the 2000 (↓22% and ↓32%, 

respectively) and 4000 (↓25% and ↓36%, respectively) ppm groups compared to controls. The 

4000 ppm males lost weight (−0.8 kg) and the 2000 ppm males only gained a small amount of 

weight (+0.1 kg) over the dosing period; the 2000 and 4000 ppm females lost weight (−0.6 kg 

Page 63 of 74
SIMAZINE FINAL

 

 

Page 56 of 66 

and −1.4 kg, respectively) over the course of the study. Mean RBC, hemoglobin (HGB), and 

HCT values were significantly decreased in the 4000 ppm males and females during Weeks 7 

and 13. Mean percent neutrophils were significantly increased and mean percent lymphocytes 

were significantly decreased in the 4000 ppm males at Week 13. Mean platelet count was 

significantly increased in the 4000 ppm males during Weeks 7 and 13. Mean percent eosinophils 

ware significantly reduced in all treated males at Week 7 and in the 4000 ppm males at Week 13. 

Mean prothrombin time was significantly less than controls in all dose groups in females at 

Week 13. Significantly decreased mean albumin levels were observed in the 2000 and 4000 ppm 

males at Week 13 accompanied by an increase in mean globulin levels and a decrease in A/G 

ratio. There were also significant reductions of mean SGOT levels in all treated males at Week 7 

and in the 4000 ppm males at Week 13, in mean creatinine levels in the 4000 ppm males at Week 

13, in mean total bilirubin in the 4000 ppm males at Week 7, and in calcium levels in the 4000 

ppm males at Week 7 and the 2000 and 4000 ppm males at Week 13. Significant decreases were 

observed in mean alkaline phosphatase in the 2000 and 4000 ppm females at Week 7, in calcium 

levels in the 4000 ppm females at Week 13, and in sodium and SGOT levels in the 2000 and 

4000 ppm females at Week 13. Urinalysis revealed significant increases in specific gravity in the 

2000 and 4000 ppm males at Week 7, and in ketone levels in the 2000 and 4000 ppm males and 

females at Week 7 and the 4000 ppm males at Week 13. Urinary pH was significantly decreased 

in the 4000 ppm males at Week 7 and in the 4000 ppm males and females at Week 13. 

Mean absolute heart weights were significantly decreased in all treated males and in the 4000 

ppm females; mean relative heart weights were also significantly reduced in the 4000 ppm males 

and females. Significantly higher mean relative brain and liver weights were observed for the 

2000 and 4000 ppm males and the 4000 ppm females. Mean absolute testes weights were 

significantly decreased in the 2000 and 4000 ppm males; mean relative testes weights were also 

decreased in the 4000 ppm males. 

There were no treatment-related gross or microscopic lesions observed at necropsy. 

The LOAEL is 2000 ppm (equivalent to 65.2/64.3 mg/kg/day in males/females), based on 

reduced albumin levels and increased globulin levels in males; and elevated urinary specific 

gravity in males and ketone levels in males and females. The NOAEL is 200 ppm (equivalent to 

6.9/8.2 mg/kg/day in males/females). 

Subchronic Dermal Toxicity (Rabbit; OCSPP 870.3200) 

In a subchronic dermal toxicity study (MRID 00057567), groups of New Zealand rabbits/sex 

were administered simazine (97.6% purity) by dermal application at dose levels of 0, 10, 100, or 

1000 mg/kg/day, five days/week for three weeks. For half the animals in each group, the shaved 

skin was abraded once per week. After 21 days, each animal was sacrificed and subjected to 

Page 64 of 74
SIMAZINE FINAL

 

 

Page 57 of 66 

gross necropsy. Organs that were weighed included liver, kidneys, testes, ovaries, thyroid, 

adrenals, and pituitary. Histopathological examinations were conducted on these tissues. 

Dermal dosing did not appear to cause any treatment-related systemic toxicity. One 1000 

mg/kg/day female died during the study; gross necropsy revealed generalized congestion in most 

visceral organs. There were no effects of treatment on organ weights. Incidental differences 

occurred in both pre-treatment and post-treatment hematology and blood chemistry analyses; no 

significant differences were observed between test groups. Miscellaneous subclinical lesions 

ware observed at necropsy in control and test groups which were unrelated to dosage. 

The LOAEL was not observed. The NOAEL is 1000 mg/kg/day. 

Developmental Toxicity (Rat; OCSPP 870.3700) 

In a developmental toxicity study (MRID 40614403), groups of 25 presumed pregnant COBS 

CD SD BR rats were administered simazine (97.5% purity) in 2% carboxymethylcellulose by 

oral gavage (10 mL/kg) at dose levels of 0, 30, 300, or 600 mg/kg/day during GD 6-15. The rats 

were sacrificed at GD 20 and ovaries removed and corpora lutea counted. Uteri were removed 

and the number of live and dead fetuses and implantation sites were counted. Approximately 

one-third of the fetuses in each litter were examined viscerally and the remaining fetuses in each 

litter were examined for skeletal alterations. 

There were no treatment-related clinical signs of toxicity observed during the study; all dams 

survived to termination. Body weights were decreased (p<0.05) in the 300 and 600 mg/kg/day 

females on GDs 10 (↓7% and ↓10%), 14 (↓11% and ↓14%), and 18 (↓6% and ↓9%); body weight 

corrected for gravid uterus weight was decreased in these groups as well (↓7% and ↓9%). Food 

consumption was decreased (p≤0.05) in mid- and high-dose animals during the dosing period 

and increased in these same animals after the dosing period (GDs 15-19). There were no doserelated effects on reproductive parameters (fetal weights, viable litter size, numbers of live and 

dead fetuses, and post-implantation loss). There were no dose-related visceral malformations or 

variations. 

Dose- and treatment-related skeletal effects occurred in the mid- and high-dose groups. The 

parameters affected on a litter basis were presphenoid unossified at the high dose, and additional 

lumbar vertebra/centra at the mid and high dose. These parameters occurred in a dose-related 

manner in fetuses, but were not reported to be statistically significant. In addition, the total 

number of variations were nominally increased and appeared to be dose-related in fetuses. The 

parameters statistically significantly affected on a fetal basis at the mid and high dose were: 

incompletely ossified head, teeth not ossified, centra/vertebrae not ossified and/or additional, 

rudimentary ribs, and sternebrae not ossified. On a fetal basis, these and 5 other parameters were 

nominally elevated at all dose levels, except for the vertebra/centra not ossified and/or additional, 

and the presence of rudimentary rib. On a litter basis, 11 parameters were nominally elevated at 

Page 65 of 74
SIMAZINE FINAL

 

 

Page 58 of 66 

all dose levels, one of which was litters with no ossified sternebrae. One of the skeletal 

parameters, unossified sternebrae, demonstrated a statistically significant dose-related increase at 

all dose levels for fetuses but not for litters. 

The maternal toxicity NOAEL is 30 mg/kg/day and the LOAEL is 300 mg/kg/day based on 

decreased maternal body weight, body weight gain, food consumption, and food efficiency. The 

developmental toxicity NOAEL is 30 mg/kg/day and the developmental toxicity LOAEL is 300 

mg/kg/day based on increased incidence of incompletely ossified head, teeth not ossified, 

centra/vertebrae unossified and/or additional, rudimentary ribs, presphenoid not ossified, and 

sternebrae not ossified. 

Developmental Toxicity (Rabbit; OCSPP 870.3700) 

In a developmental toxicity study (MRID 00161407), groups of 19 female New Zealand White 

rabbits were administered simazine (97% purity) in 3% corn starch containing 0.5% Tween 80 

by oral gavage (10 mL/kg) at doses of 0, 5, 75, or 200 mg/kg/day during GD 7-19. On GD 29, all 

surviving does were sacrificed, and subject to necropsy. Ovaries were examined and uteri, 

including their contents, were weighed. The number and position of fetuses, the number of live 

and dead fetuses, the number of metrial bodies, and the number of corpora lutea in pregnant 

animals were recorded. All fetuses were sexed, weighed, and examined for gross external 

malformations. Each fetus was examined viscerally and then stained for skeletal examination. 

One death in each simazine treatment group occurred during the study; however, no gross 

pathological changes were found in these animals at necropsy. Four animals aborted during the 

study: one 75 mg/kg/day dam and three 200 mg/kg/day dams. Increases (p<0.05) in the incidence 

of little, none, and/or soft stool were observed (50%, 100%, and 100%, compared to 0% 

controls). Treatment-related increases in tremors were also noted (0%, 0%, 21%, and 100% for 

the 0, 5, 75, and 200 mg/kg/day dose groups, respectively). Body weight losses (p<0.05) during 

the dosing period were noted at 75 (−243 g) and 200 (−456 g) mg/kg/day compared to a +230 g 

increase in controls. Body weights gains increased (p<0.05) during post-dosing (GD 19-29); 

however; overall (GD 0-29) body weight gains were decreased (p<0.05) at 75 (↓74%) and 200 

(↓75%) mg/kg/day. Body weight gains corrected for gravid uterine weights also displayed losses 

at 75 (−167 g) and 200 (−264 g) compared to a control weight gain of +3 g. Food consumption in 

the mid- and high-dose groups was also decreased (p<0.05) in the mid- and high-dose groups 

during the dosing period. There were no treatment-related effects on the number of corpora lutea, 

implantation sites, live fetuses per dam, or fetal sex ratio. Increases in the mean number of 

resorptions per litter (and therefore post-implantation loss) were noted in all treatment groups; 

however, there was no clear dose response, and there was no decrease in the mean number of 

live fetuses per litter. Mean fetal weight was decreased at 200 mg/kg/day in males (↓9%; NS) 

and females (↓13%; p<0.05). There were no biologically significant differences in the incidences 

of fetuses with malformations in any of the treated groups. Treatment-related increases in the 

Page 66 of 74
SIMAZINE FINAL

 

 

Page 59 of 66 

incidences of fetuses with 13th full ribs, non-ossified sternebrae, free floating ribs, and nonossified patella were observed at the mid and high dose; the increases were significant (p<0.05) 

for free floating ribs and non-ossified patella in litters of the high dose group, as well as for the 

total number of fetuses with any variation in the high dose group. 

The maternal NOAEL is 5 mg/kg/day and the LOAEL is 75 mg/kg/day, based on decreased body 

weight gain, tremors, and abortions. The developmental NOAEL is 75 mg/kg/day and the 

LOAEL is 200 mg/kg/day based on reduced mean fetal weight and increased skeletal variations. 

Simazine was not teratogenic in rabbits at doses up to 200 mg/kg/day. 

Two-Generation Reproduction in Rodents (Rat; OCSPP 870.3800) 

In a two-generation reproduction study (MRID 41803601), groups of 30 Sprague-Dawley 

rats/sex were administered simazine (96.9% purity) in the diet at nominal dose levels of 0, 10, 

100, or 500 ppm (equivalent to 0/0, 0.56/0.70, 5.6/7.0, and 29/35 mg/kg/day in males/females 

during premating) for two consecutive generations. After 10 and 13 weeks, respectively, of 

dietary treatment, the P and F1 parental animals were mated. F1 parental animals consisted of 30 

randomly selected pups/sex/dose level. F1 parental animals were allowed a 3-week rest period 

before a second mating. Sibling matings were avoided. Five F2a pups/sex/dose were selected for 

complete necropsy and histopathological examination. F2b male pups were sacrificed on PND 4 

and F2b female pups were assigned to subsequent studies. The organs weighed at sacrifice 

included the testes and ovaries. For the control and high dose groups, histopathological 

examinations were conducted on these organs as well as the seminal vesicle, prostate, 

coagulating gland, uterus, epididymides, cervix, vagina, and pituitary. 

There were no treatment-related clinical signs of toxicity in any of the treated groups, including 

adults and pups. There were no treatment-related deaths. In the 500 ppm P males, body weights 

were decreased (p<0.05) by 7-11% beginning on Day 14 and remaining decreased throughout 

pre-mating; similar results were observed for the 500 ppm F1 males at the first mating (↓12-13%; 

p<0.05). It was stated that body weight gains for the 500 ppm P and F1 males were decreased 

(p<0.05) during the pre-mating, mating, and post-mating phases (8-51%) and during the rest 

phase for F1 males at second mating. Body weight gains for the 100 ppm P males were decreased 

during Days 0-70 (≈↓13%) and during Days 7-14 (≈↓11%) for the 100 ppm F1 males at first 

mating, but were increased from rest period to term for F1 males at second mating. 

Body weights of the 500 ppm P females were decreased (p<0.05) by 10-13%, starting on Day 7 

and remaining decreased (p<0.05) through the gestational (↓11-14%) and lactational (↓7-12%) 

periods. Body weights for the 500 ppm F1 females were decreased during the first (↓11-15%) and 

second pre-mating periods, and remained decreased through the first (↓10-14%) and second 

(↓11-15%) gestational periods. Lactational body weights were decreased (p<0.05) during LD 0-

14 (↓7-11%) for the F2a litters and only at LD 0 (↓11%) for the F2b litters. In the 100 ppm group, 

Page 67 of 74
SIMAZINE FINAL

 

 

Page 60 of 66 

decreased body weights were observed during the premating phase in P and F1 first mating 

females (approximately 6%). In general, body weight gain for high-dose P and F1 females (both 

matings) was decreased (p≤0.05) during the pre-mating phase and increased (p≤0.05) during the 

lactational phase. For F1 females at first mating, body weight gain was decreased and then 

increased during pre-mating phase; it was also increased during the lactational phase. 

Food consumption was generally decreased (p≤0.05) in high-dose males and females of the P 

and F1 generations (both matings) during the premating phase (approximately 9-18%). Some 

decreases were observed in males and females in the mid-dose group, generally during the premating phase. 

Relative (to body weight) testicular weights were increased (p<0.05) by 11% in the 500 ppm P 

males and by 20% in the 500 ppm F1 males, and relative ovary weights were increased by 50% 

in the 500 ppm F1 females. Microscopic examination of tissues from animals in both generations 

revealed no compound-related differences and the absolute weights were not affected by 

treatment. Therefore, these decreases were attributed to the decreased body weights of these 

groups. No treatment-related histopathological lesions were seen in any generation at any dose 

level. No treatment-related effects were observed on reproductive parameters (number of 

matings, number of pregnancies, female fertility index, gestation index, gestation length, 

implantation sites/litter, total number of live pups, mean number of live pups/litter, live birth 

index, viability index, lactation index, fetal sex ratio, and mean pup body weight/litter) at any 

dose level. In the F1 offspring, male pup body weights were increased (p≤0.05) on Days 14 and 

21 at the low dose; and in the F2 offspring, male pup body weights were increased on Day 14 at 

the mid dose. Incidental clinical observations and gross malformations in pups were observed in 

both generations in all dose groups, including the control groups. 

The parental NOAEL is 10 ppm (0.56 and 0.70 mg/kg/day in males and females, respectively). 

The parental LOAEL is 100 ppm (5.61 and 7.04 mg/kg/day in males and females, respectively) 

based on decreased body weight and body weight gain. The NOAEL for reproductive toxicity 

was 500 ppm (28.89 and 34.96 mg/kg/day for males and females, respectively); the LOAEL was 

not determined. 

Chronic Toxicity/Carcinogenicity (Rat; OCSPP 870.4300) 

In a chronic toxicity/carcinogenicity study (MRID 40614405), groups of 70 Sprague-Dawley 

[Crl:VAF/Plus CD(SD)Br] rats/sex were administered simazine (96.9% purity) in the diet at dose 

levels of 0, 10, 100, or 1000 ppm (equivalent to 0/0, 0.41/0.52, 4.17/5.34, and 45.77/63.10 

mg/kg/day in males/females) for up to two years. An interim-sacrifice group of ten 

animals/sex/group were sacrificed at twelve months. In addition, an additional 10 rats/sex were 

dosed at 0 or 1000 ppm for 52 weeks and then maintained on untreated (control) diet for 52 

weeks. At study termination, surviving rats in all groups were sacrificed for gross and 

Page 68 of 74
SIMAZINE FINAL

 

 

Page 61 of 66 

histopathological evaluation. The organs weighed at sacrifice included the adrenals, liver, 

kidneys, testes, epididymides, and ovaries. Gross and microscopic pathological examinations 

were conducted on these organs as well as the thyroid, pituitary, urinary bladder, prostate, 

seminal vesicles, mammary gland, and uterus. 

The mortality rate was very low during the first year of the study; however, a high rate of 

mortality was observed during the second year. For the 1000 ppm males, the mortality rate was 

decreased (p<0.01) compared to the controls (40% treated vs. 61% controls). For females, the 

mortality rate in the 100 (75%) and 1000 (80%) ppm groups was greater (p<0.01) than controls 

(66%), with a significant increasing trend. There was an increase in palpable masses and alopecia 

in 1000 ppm females and swollen appendages in the 1000 ppm males. Body weights in the 1000 

ppm males and females were decreased (p<0.05) beginning on Day 7 and continuing through 

termination (↓22% and ↓23% at termination for males and females, respectively); body weight 

gains were also decreased (p<0.05) in these animals throughout the study (↓27% and ↓28% 

overall for males and females, respectively). The 100 ppm females also had decreased body 

weights at different intervals; however, overall body weight gain was not significantly decreased 

at study termination. Food consumption was decreased (p<0.05) in the 1000 ppm males from 

Day 7 through 700 (4 weeks prior to sacrifice); for the 1000 ppm females, food consumption was 

decreased (p<0.05) from Day 7 through 560 but not during the final 6 months of the study. 

In the 1000 ppm female rats, RBC, HGB, HCT, and lymphocytes were decreased (p<0.01), and 

mean corpuscular hemoglobin (MCH), WBC, and neutrophils were increased (p<0.05) at most 

time points of sampling. In the 1000 ppm males, mean corpuscular hemoglobin concentration 

(MCHC) was increased (Day 361) and WBC was decreased (Day 537). The only changes to 

clinical chemistry parameters possibly related to treatment were dose-related decreased glucose 

in female rats at all sampling time points. In the recovery group (dosed at 1000 ppm for one year 

then fed basal diet for one year), decreased glucose was seen at almost all time points; increased 

alkaline phosphatase was observed at all time points in this group. 

Organ weight effects included decreased (p<0.05) absolute liver weights in the 52-week sacrifice 

1000 ppm females; however, relative (to body weight) liver weights were increased (p<0.01) in 

these same animals, and relative liver weights were increased (p<0.01) at terminal sacrifice. The 

following relative organ weights were increased (p<0.05) at 52-week sacrifice: liver and testes in 

the 1000 ppm males; adrenals in the 1000 ppm females; kidneys in the 100 and 1000 ppm 

females; and ovaries in the 1000 ppm females. At terminal sacrifice, relative liver and testes 

weights were increased (p<0.05) in the 1000 ppm males and relative kidney weights were 

increased in the 1000 ppm females. As there were no corroborating gross or microscopic 

findings, these relative organ weight increases were considered to be a reflection of the 

decreased body weights. 

Page 69 of 74
SIMAZINE FINAL

 

 

Page 62 of 66 

Gross pathology, which was conducted on all animals that died during the study including those 

that died or were sacrificed prior to scheduled sacrifice, indicated that the incidence of 

macroscopic lesions in treated rats was not significantly different from that of the control groups 

for either sex. In male rats, the combined incidence of thyroid C-cell adenomas/carcinomas was 

higher at 10 (8%), 100 (12%) and 1000 (16%) ppm as compared to controls (8%), however, the 

differences did not reach statistical significance. In addition, the incidence of hyperplastic 

changes was comparable to the incidence of tumors for each group. In liver in males, the 

combined incidence of adenomas/carcinomas was higher (p<0.05) in the 1000 ppm group (8%) 

compared to controls (1%). 

In females, the incidence of cystic glandular hyperplasia of the mammary glands was increased 

(p<0.001) at 1000 ppm (93%) compared to controls (73%). This finding corroborated with the 

presence of mammary gland tumors; carcinomas were increased (p<0.001) at 100 (27%) and 

1000 (50%) ppm compared to controls (20%) and fibroadenomas were increased (p<0.01) at 

1000 ppm (57%) compared to controls (31 %). The incidence of liver hematopoisis and spleen 

hematopoiesis was increased (p<0.05) in the 1000 ppm females (7% and 14% respectively) 

compared to controls (0% and 4%, respectively). In the 1000 ppm females, pituitary gland 

carcinomas were increased (11 %) compared to controls (1%) and the combined 

adenomas/carcinomas were increased (p<0.0005) at 100 (82%) and 1000 (85%) ppm compared 

to controls (83%). Although the incidences in all groups (treated and controls) were numerically 

same, examination of the survival curve showed that the onset of these tumors was 4 to 15 weeks 

earlier in rats in the 100 and 1000 ppm groups as compared to the control and 10 ppm groups 

(i.e., treatment decreased the latency period). 

The LOAEL for chronic toxicity is 100 ppm (5.3 mg/kg/day) based on decreased body weight 

gains and decreased values for hematology parameters in female rats. The NOAEL was found to 

be 10 ppm (0.5 mg/kg/day). Simazine was found to oncogenic in female rats, inducing mammary 

tumors at dose levels of 100 ppm (5.3 mg/kg/day) and 1000 ppm (63.1 mg/kg/day). In male rats, 

simazine appears to induce the formation of liver tumors (hepatocellular adenomas/carcinomas) 

at the dose level of 1000 ppm (45.8 mg/kg/day). 

Chronic Oral Toxicity (Dog; OCSPP 870.4100) 

In a chronic toxicity study (MRID 40614402), groups of 4 beagle dogs/sex were administered 

simazine (97.5% purity) in the diet at dose levels of 0, 20, 100, or 1250 ppm (equivalent to 0/0, 

0.68/0.76, 3.41/3.64, or 42.9/44.9 mg/kg/day for males/females) for one year. Organ weights 

determined included adrenals, kidneys, liver, ovaries, testes, epididymides, pituitary, and thyroid; 

histopathological examinations were conducted on these tissues as well as the urinary bladder, 

prostate, mammary gland, uterus, and vagina. 

Page 70 of 74
SIMAZINE FINAL

 

 

Page 63 of 66 

There were no mortalities during the dosing period. Cachexia was observed at 1250 ppm in 1 

male and 1 female from Week 14 through 20 (male) or 22 (female). Other observations with an 

apparent dose relationship were fecal changes and infrequent emesis. Body weight gain was 

decreased (p<0.05) in the 1250 ppm males on Days 7 and 14, and in the 1250 ppm females on 

Days 7-224 compared to controls. Body weights of the 1250 ppm females were decreased 

compared to controls on Days 63 through 140. Food consumption in these animals was also 

decreased frequently during the first 20 weeks of the study. In the 1250 ppm males, there was a 

transient nominal decrease (NS) in RBC and HGB at Days 86 and 177, while HCT was 

decreased (p<0.05) at Day 86; on Day 359, these values were nominally higher or equivalent to 

control values. Platelets were increased (p<0.05) in the 1250 ppm males after 86 (142%), 177 

(170%), and 359 (155%) days of dosing. In the 1250 ppm females, there was a transient decrease 

(p<0.05) in RBC, HGB, and HCT at 86 and 177 days; these values were only nominally 

decreased at 359 days compared to controls, but not when compared to pre-dose values in highdose females. MCHB was slightly increased (p<0.05) in the1250 ppm females at 359 days. No 

treatment-related, consistent changes were noted in the clinical chemistry or urinalysis results. 

No treatment-related changes were seen in absolute or relative weights of the testes, 

epididymides, ovaries, or pituitary glands at any dose level. In males at the high dose, absolute 

thyroid/parathyroid weight was decreased (↓60%; p<0.001) and in females at this dose, the 

absolute thyroid/parathyroid weights were increased (↑114%). An increase (p<0.05) in relative 

(to brain) adrenal weight was seen in males at the high dose. Absolute and relative adrenal 

weights were increased (p<0.05) in females at the high dose. There were no treatment-related 

effects on gross or microscopic pathology. 

The LOAEL is 100 ppm (equivalent to 3.41/3.64 mg/kg/day in males/females), based on 

decreased body weight gain, decreases in RBC, HGB, HCT, and an increase in platelet counts in 

females. The NOAEL is 20 ppm (equivalent to 0.68/0.76 mg/kg/day in males/females).

Avian Reproduction (OCSPP 850.2300) 

In an avian reproduction study (MRID 43576901), mature mallard ducks were administered 

simazine (96.9% purity) at nominal dietary concentrations of 0, 50, 150, or 450 ppm for 

20 weeks (nine weeks prior to the start of egg production and eight weeks of egg production). 

Each treatment group was comprised of 16 pens, with one male and one female per pen. 

Hatchability was recorded on Day 27. 

There was no treatment-related mortality during the study, and no clinical signs of toxicity were 

observed. Mean body weight of hens in the 450 ppm group at study termination was significantly 

decreased by 10.2% compared to controls. Food consumption was decreased in the high-dose 

group but only during Week 1. At necropsy, a slight increase in the number of hens with 

regressed or inactive ovary was found in the high-dose group. There was a decrease in the 

number of eggs laid (↓49%), number of hatchlings (↓56%), and number of 14-day-old survivors 

Page 71 of 74
SIMAZINE FINAL

 

 

Page 64 of 66 

(↓58%) at 450 ppm. There was no effect of treatment on eggshell thickness or offspring body 

weight. The LOAEC is 450 ppm based on decreases in the number of eggs laid and female body 

weight. The NOAEC is 150 ppm. 

In another avian reproduction study (MRID 00163134), mature bobwhite quail were 

administered simazine (97.0% purity) at nominal dietary concentrations of 0, 20, 100, or 

500 ppm for 19 weeks (11 weeks prior to the start of egg production and 8 weeks of egg 

production). Each treatment group was comprised of 16 pens, with one male and one female per 

pen. On Day 21 of incubation, eggs were placed in a hatcher. All hatchlings, unhatched eggs, and 

shells were removed from the incubator on Day 25 or 26 of incubation. Hatchlings were housed 

according to the appropriate parental grouping and maintained on a control diet until 14 days of 

age. 

There were two adult mortalities during the study: one female each in the 20 and 100 ppm 

groups; both deaths were considered due to aggression by pen mate. No overt signs of toxicity 

were observed during the study. During gross necropsy of birds sacrificed at the end of the study, 

one male in the 500 ppm group was found to have slight regressing testes and three males in this 

group were found to have regressing testes. There was no effect of treatment on the body weights 

or food consumption at any of the doses tested. At 500 ppm, there was an apparent effect on the 

number of eggs laid (NS) and a possible effect (NS) on the number of viable embryos of eggs 

set; these effects were reflected in the number of 14-day-old survivors as a percent of eggs set, 

and hatchlings and 14-day-old survivors as a percent of maximum set. There were no other 

effects on reproductive parameters (eggs cracked of eggs laid; live 3-weeks embryos of viable 

embryos; hatchlings as a percent of 3-week embryos; 14-day-old survivors as a percent of 

hatchlings). There were no treatment-related reductions in egg shell thickness or differences in 

body weight of hatchlings or 14-day-old survivors. 

The NOAEC is 100 ppm based on an effect on reproductive performance at 500 ppm. 

Page 72 of 74
SIMAZINE FINAL

 

 

Page 65 of 66 

APPENDIX 3: References Not Utilized in the Simazine 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 Simazine 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. 

Balaguer, P., Joyeux, A., Denison, M., et al. (1996) Assessing the estrogenic and dioxin-like 

activities of chemicals and complex mixtures using in vitro recombinant receptor- reporter gene 

assays. Can. J. Physiol. Pharmacal. 74: 216-222. 

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. 

Page 73 of 74
SIMAZINE FINAL

 

 

Page 66 of 66 

Fang, H., Tong, W., Branham, W., et al. (2003) Study of 202 natural, synthetic, and 

environmental chemicals for binding to the androgen receptor. Chem. Res. Toxicol. 16: 1338-

1358. (MRID 48074110). 

Forgacs, A., D'Souza., M, Huhtaniemi, I., et al. (2013) Triazine herbicides and their 

chlorometabolites alter steroidogenesis in BLTK1 murine Leydig cells. Toxicol. Sci. 134(1): 155-

167. 

Hinfray, N., Porcher, J., and Brion, F. (2006) Inhibition of rainbow trout (Oncorhynchus mykiss) 

P450 aromatase activities in brain and ovarian microsomes by various environmental substances. 

Comparative Biochemistry and Physiology, Part C, 144: 252-262. (MRID 48203005) 

Legler, J., Dennekamp, M., Vethaak, A., et al. (2002) Detection of estrogenic activity in 

sediment-associated compounds using in vitro reporter gene assays. Sci. Total Environ. 293: 69-

83. (MRID 48203003) 

Rich, J., Gabriel, S., and Schultz-Norton, J. (2012) In vitro effects of herbicides and insecticides 

on human breast cells. ISRN Toxicol. 2012: 232461. 

Schulte-Oehlmann, U., Oehlmann, J., and Keil, F. (2011) Before the curtain falls: endocrineactive pesticides – a German contamination legacy. In: Reviews of Environmental Contamination 

and Toxicology, D.M. Whitacre (ed.), Springer Science+Business Media, LLC, New York, NY. 

p. 137-159. 

Sun, L., Xu, W., He, J., et al. (2010) In vivo alternative assessment of the chemicals that interfere 

with anterior pituitary POMC expression and interrenal steroidogenesis in POMC: EGFP 

transgenic zebrafish. Toxicol. Appl. Pharmacol. 248: 217-225. 

Tran, D., Kow, K., McLachlan, J., et al. (1996) The inhibition of estrogen receptor mediated 

responses by chloro-S-triazine-derived compounds is dependent on estradiol concentration in 

yeast. Biochem. Biophys. Res. Commun. 227: 140-146. 

Wu, W., Niles, E., and LoVerde, P. (2007) Thyroid hormone receptor orthologues from 

invertebrate species with emphasis on Schistosoma mansoni. BMC Evolutionary Biology 7: 150-

166. 

Page 74 of 74