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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

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

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

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

.

Page I of2Page 2 of 2 Attachment A. EDSP List 1 Chemicals
Chemical Name PC Code TXR Number 2,4-D 030001 0057151 Abamectin 122804 0057152 Acephate 103301 0057153 Acetone 044101 0057154 Atrazine 080803 0057155 Benfluralin 084301 0057156 Bifenthrin 128825 0057157 Captan 081301 0057158 Carbaryl 056801 0057159 Carbofuran 090601 0057160 Chlorothalonil 081901 0057161 Chlorpyrifos 059101 0057162 Cyfluthrin 128831 0057163 Cypermethrin 109702 0057164 DCPA 078701 0057165 Diazinon 057801 0057166 Dichlobenil 027401 0057167 Dimethoate 035001 0057168 EPTC 041401 0057169 Esfenvalerate 109303 0057170 Ethoprop 041101 0057171 Fenbutatin-Oxide 104601 0057172 Flutolanil 128975 0057173 Folpet 081601 0057174 Glyphosate 417300 0057175 Imidacloprid 129099 0057176 Iprodione 109801 0057177 Isophorone 847401 0057178 Linuron 035506 0057179 Malathion 057701 0057180 Metalaxyl 113501 0057181 Methomyl 090301 0057182 Metolachlor 108801 0057183 Metribuzin 101101 0057184 MGK-264 057001 0057185 Myclobutanil 128857 0057186 Norflurazon 105801 0057150 o-Phenylphenol 064103 0057146 Oxamyl 103801 0057142 PCNB 056502 0057138 Permethrin 109701 0057149 Phosmet 059201 0057145 Piperonyl Butoxide 067501 0057141 Pronamide 101701 0057137 Propargite 097601 0057148 Propiconazole 122101 0057144 Pyriproxyfen 129032 0057140 Simazine 080807 0057136 Tebuconazole 128997 0057143 Tetrachlorvinphos (TCVP) 083701 0057147 Triadimefon 109901 0057139 Trifluralin 036101 0057135

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

CHEMICAL: SIMAZINE

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS 

### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYSIMAZINE 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 Rele vant 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-Estr ogenic Pathway for Simazine.....................................13
Table 3. Androgenic/Anti-Andr ogenic 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.........................65SIMAZINE FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ACTH Adrenocorticotropic hormone

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

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

CG Cowper’s Gland

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

DEA Desethyl- s -atrazine

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

iv Abbreviation Terminology

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

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

K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

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

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

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

v Abbreviation Terminology

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

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

OPP Office of Pesticide Programs

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

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g thSIMAZINE FINAL

vi Abbreviation Terminology

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

T4 Thyroxine (tetraiodothyronine)

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

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

VP Ventral Prostate VTG Vitello g eni n WoE Wei g ht-of-EvidenceSIMAZINE FINAL

Page 1 of 66 Executive Summary

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or t hyroid (T) hormone pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (H PG) and hypothalamic-pituitary thyroid (HPT) perturbations.
In addition to the available Ti er 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/atrazi ne/atrazine_update.htm)

In determining whether simazine interacts with E, A or T hormone pa thways, the number and type of effects induced, the magnitude of respons es, and the pattern of responses observed across studies, taxa and sexes were considered. Additionally, the conditions under which effects occur were considered, in part icular, whether or not endocrine-rela ted 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 we ight-of-evidence (WoE) analysis of the potential interaction of simazine with the E, A or T signaling pa thways. 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 cy tosol assay and negative in a cell proliferation assay (using MCF-7 cells) conducted by Connor et al . (1996). Additionally, sim azine was also negative in the Tier 1 and OSRI ER transcriptional activation (ERTA) assays. Altered aromatase activity was reported in studies usi ng 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 corticos terone were increased (Laws et al ., 2009). In the in vivo assays, simazine was negative for estrogenic activity in several OSRI uterotrophi c 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 inSIMAZINE 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 mammalia n toxicity studies.

There was a marginally significant (p=0.0512) re duction in female GSI at the high treatment level (3.4 mg/L) in the FSTRA; however, no e ffects on reproduction we re observed. Female mallard ducks exposed to simazine in the diet pr esented a greater incidence of regressed ovaries and produced fewer eggs, fewer hatchling survivor s, and fewer 14-day surv ivors 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 we re observed in the quail study.

Since simazine belongs to the tr iazine CMG, evidence of estrogen -related effects with atrazine and atrazine metabolites (Laws et al ., 2000; 2003; SAP, 2000) were al so considered. In studies by Laws et al . (2000), atrazine significantly delayed VO and reduced the weights of reproductive tissues (uterine and ovarian) associated with estr ogenic responses in peripubertal rats. Similar effects were observed in peripubert al rats exposed to chlorinated metabolites of atrazine (Laws et al ., 2003) indicating a similar mode of action for th e 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 al terations in the hormonal control of pubertal development. Decreased LH secretion would le ad to insufficient stimulation of the gonads during puberty resulting in delayed VO and a lterations in estrogen-responsive tissues.

For the androgen pathway, simazine was negative in the androgen receptor (AR) binding and AR transcriptional activation (ARTA) assays. Sim azine was positive in the steriodogenesis assay by
Sanderson et al . (2001). In a single dos e 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. A ll androgen-related effects in the male pubertal assay and Part 158 mammalian toxicity st udies occurred in the presence of overt toxicity. Th ere were no androgen-related effect s in the FSTRA. Regressed or regressing testes were observed in 25% of male bobwh ite quail at the highest treatment level in an avian reproduction study; decreases in embryo vi ability 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 nu mber 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 posse ss 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 w ith androgenic responses. The e ffects are consistent with theSIMAZINE 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 contro l of pubertal development.
Decreased LH secretion would lead to insuffici ent stimulation of the gonads during puberty to produce the necessary steroids fo r 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 hypothalami c control of pubertal progression.

The available data do not provide convincing evid ence of potential interac tion 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 de parture (POD) used for human health risk
assessment for the triazine CMG is based on e ffects seen with atra zine. The POD of 2.56 mg/kg/day based on a luteinizing hormone suppressi on 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 chroni c 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 atr azine, 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 appr oximately 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 Rf D and the MOE are protective of the estrogen and androgen-related effects seen in the EDSP pubertal assays.

With respect to determining whether additional st udies 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 r ecent 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, whic h 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, Ma yer and Sanders 1975). The LOAEC valueSIMAZINE 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 a ndrogen 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 bird s, Part 158 avian reproduc tion studies with both northern bobwhite quail and mallard duck are availa ble for simazine. In both species, effects on reproductive parameters were observed. The t ype of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are consid ered 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 curren t EPA established regulatory endpoints for human health or ecological risk assessment.SIMAZINE FINAL

Page 5 of 66 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) hormone path ways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (H PG) and hypothalamic-pituitary thyroid (HPT) perturbations.
In addition to the available Ti er 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 th e determination were the FIFRA Science Advisory Panel (SAP) reports on atrazine (2009 – 2011) and the common mechan ism grouping (CMG) of simazine with other similar triazines (refer to the Introduction section for more details).

In determining whether a pesticide chemical in teracts with E, A or T hormone pathways, the number and type of effects i nduced, 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 consid ered, in particular, whether or not endocrine-related responses occurred at dose(s) that also resulted in ge neral 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 we ight-of-evidence (WoE) analysis of the potential interaction of simazine with the E, A or T signaling pa thways. 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 herbicid e, has a water solubility of 3.5 mg/L at 20 o C. Laboratory absorption data show low water/soil partitioning for simazine, or ability to be absorbed into the soil. Laboratory studies indicate th at simazine is able to persis t 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. Si mazine is also stable to ab iotic 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 He nry’s Law constant (3.2×10

− 10

atm·m 3 mol

− 1 at 25°C), losses of simazine from soil and water systems due to volatilization are expected to be insignificant co mpared to dissipation by chemical degradation and metabolism. The octanol-water partitioning coefficient is (K ow ) 122 with bioconcentration factors of 4.92-7.30
in whole fish.SIMAZINE FINAL

Page 6 of 66 The available information considered in determin ing the potential interact ion 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 stud ies 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 (U SEPA, 2011), is presented in Section IV. The
EDSP Tier 2 studies r ecommendations are presen ted in Section V.

Other information considered in this WoE a ssessment 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 propazi ne and their three chlorinated degradates should be included in a CMG and cons idered through a cumulativ e risk assessment.
EPA’s triazine cumulative risk assessment consider ed the combined effects of atrazine, simazine and propazine, closely related tria zine herbicides. These chlorotriazine herbicides, along with respective three major chlorinated metabolites [ i.e ., desethyl-s-atrazine (DEA), desisopropyl-satrazine (DIA), and diamino ch lorotriazine (DACT)], have b een determined by EPA (2002; “ The Grouping of a Series of Triazine Pesticid es 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 resu lts in both reproductive and developmental
alterations. These three chemi cals (atrazine, simazine, and pr opazine) and their three major
chloro metabolites constitute the triazine CM G. 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 d ecreases LH levels by suppres sion of gonadotrophin-releasing hormone (GnRH) from the hypothalamus. Suppression of LH is regarded as a precursor event in
the neuroendocrine mode of action of the triazines.

The grouping of the CMG triazines based on a common mechanism of ac tion 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 ev idence to support the proposed grouping for the neuroend ocrine and neuroendocrine-relate d effects. For the purpose of estimating risks, the CMG triazines are all assu med 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/reregis tration/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 tri azines with a decrease in luteinizing hormone as a precursor event to developmental and repr oductive effects, which may be mediated through the estrogen and androgen pathways.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 believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Orde r recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 Guideline studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team on 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 c onsidered but not util ized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and approach used in the WoE determination on the potential of a
substance to interact with endoc rine-related processes (i.e., E, A or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the indivi dual studies for their scientific quality and relevance in evaluating potential endocrine interaction(s). The second step is to integrate the data along different levels of biological organization while examining the extent of complementarity ( i.e ., the concordance of endpoints within an assay that measures multiple endpoints) and redundancy ( i.e ., the concordance of endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to
evaluate whether additional te sting is needed based on the evidence and conclusions described
above.

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) 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 wereSIMAZINE 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 in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach 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 speci fic endpoint for discerni ng whether or not the chemical has interacted with the endocrine syst em, are considered. The co llective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indi cation of potential interaction as measured by the study.

The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data, as well as characterizing the extent to which the available data suppor t a hypothesis that a chemical has the potential to interact E, A, or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, coherence, strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are
meant to evaluate whether or not a chemical can in teract with E, A, and T consisting of different levels of biological organization from a molecular initiating ev ent, such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment and fecundity at the whole organism level. The extent of expressi on 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 releva nt, robust and consistent evidence in terms of complementarity and redundancy, as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biological organization? Finally, under what conditions did the responses occu r including discussions regardi ng whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.SIMAZINE FINAL

Page 9 of 66 This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

For these WoE analyses, overt toxicity was gene rally defined in accordance with EPA’s current approach as used by OPP in reviewing Part 158 studies for both human and ecological risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other e ffects including morphological (e.g., organ weights/histopathology), bioche mical (e.g., blood chemistry), a nd other clinical signs (e.g, lethargy) were also considered when evaluating overt toxicity, es pecially 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/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity (e.g., changes in organ wei ghts or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

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

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

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, , orSIMAZINE 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)

ER α 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)SIMAZINE FINAL

Page 11 of 66 B. Effects on Hypothalamic-P ituitary-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 a ssays 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 reporte d in the table and not considered further in the WoE assessment

.
Simazine was positive in an OSRI estrogen r eceptor (ER) binding assa y where simazine was weakly competitive with E 2 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 inhi bitor. Blair et al . (2000) found no competitive
interaction of simazine with the ER in a rat ut erine cytosol assay. Simazine was negative in the Tier 1 ERTA assay as well as multiple ER TA 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 consid ered as OSRI, Sanderson et al . (2001) reported that simazine caused concentration-dependent increa ses in aromatase activity in JEG-3 (placental choriocarcinoma) cells. In an in vivo study evaluating ster oidogenesis, Laws et al . (2009) showed a single dose of simazine (188 mg/kg) admi nistered 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 we re decreased in rats coadministered simazine and estradiol (E 2 ); however, the decreased weight s 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 potenti al anti-estrogenic response in th e female pubertal rat assay; however, the responses were not clearly dose related and/or occu rred at overtly toxic doses (Zorrilla et al ., 2010). Two protocols were conducted in th is 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 m ean 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 de layed (p<0.05) by 2.7, 2.9, 5.4 and 5.1 days atSIMAZINE 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 prolac tin levels. In Study 1, the decreased prolactin levels were not dose-related. In Study 2, th ere was a dose-dependent linear trend (not statistically significant by one-w ay ANOVA), however, the magnitude of change was slight at the lower doses (12.5 – 50 mg/kg/day) and at th e higher doses (100 and 20 0 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 effect s in females fathead minnow, with the exception of a marginally signific ant (p=0.0512) decrease ( ↓ 23%) in GSI at the highest concentration tested (3.4 mg/L). There was a 41% decrease in fecundi ty at the middle treatment concentration (0.36 mg a.i/L) that was not statistically significant an d 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 t oxicity study, absolute ovary wei ghts were increased at the mid (142 mg/kg/day)) and high (276 mg/kg/day) dose le vels; however, systemic and overt toxicity, presenting primarily as body weight loss/de creased body weights and decreased body weight gains, were noted at these dose levels. In a ra bbit developmental toxic ity study, abortions were seen at the high dose in the pr esence of maternal t oxicity, presenting prim arily 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 st udy conducted with the mallard duck, there was a 49% decrease in the number of eggs laid at th e 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 increas e from control in the nu mber of regressing or regressed ovaries in th e 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 appa rent reduction on the number of eggs laid and a possi ble effect on the number of viab le embryos of eggs set at the highest treatment concentration ( 500 ppm) that were not statistica lly significant, but considered
to be treatment-related. There we re no clinical signs of toxicity or significant (p<0.05) effects on body weight observed during the test.SIMAZINEFINALPage 13 of 66Table 2. Estrogenic/Anti-Estrogenic Pathway for SimazineLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Simazine1Study Type/ LiteratureCitation 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) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssaysER BindingRequirement satisfied by OSRI (Tennantet al., 1994b; see below)ERTA(MRID 48619302)NAromataseRequirement satisfied by OSRI (Sandersonet al., 2001; see below)SteroidogenesisRequirement satisfied by OSRI (Sandersonet al., 2001; see below)UterotrophicRequirement satisfied by OSRI (O’Connoret al., 2000, and Tennantet al., 1994a; see below)Female Pubertal RatRequirement satisfied by OSRI (Zorrillaet al., 2010; see below)FSTRA(MRID 48673602)NEP(H) NNNNNOSRIER Binding(Blairet al., 2000)NER Binding(Tennantet al., 1994b)PER Binding/ERTA(O’Connoret al.; 2000)NNERTA(Ortonet al., 2009)NERTA(Kojimaet al., 2004)NSIMAZINEFINALPage 14 of 66Table 2. Estrogenic/Anti-Estrogenic Pathway for SimazineLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Simazine1Study Type/ LiteratureCitation 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) Systemic Toxicity Observed 2 Overt Toxicity Observed 3ERTA/Cell Proliferation(Connoret al., 1996)NAromatase/Steroidogenesis(Sandersonet al., 2001)PSteroidogenesis(Lawset al., 2009)EFemale Pubertal (Zorrillaet al., 2010)P4N N N N N↓2.7 d↓2.9 d(ML, M)X(MH,H)X(MH, H)Uterotrophic (Tennantetal., 1994a)NX(L)X(H)Uterotrophic (Connoretal., 1996)NNE NSubchronic Toxicity (Rat;MRID 00143265)NEN N NE NEX(L, M,H)X(M, H)Subchronic Toxicity (Dog;MRID 00146655)NENE N NE NEX(L, M,H)X(M, H)Developmental Toxicity(Rat; MRID 40614403)NX(M,H)X(H)Developmental Toxicity(Rabbit; MRID 00161407)NN(H)X(L, M,H)X(M, H)Two-GenerationReproduction (Rat; MRID41803601)NEN N NENX(M,H)X(H)SIMAZINEFINALPage 15 of 66Table 2. Estrogenic/Anti-Estrogenic Pathway for SimazineLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Simazine1Study Type/ LiteratureCitation 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) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Chronic Toxicity/Oncogenicity (Rat; MRID40614405)NENN NEX(M,H)X(M, H)Chronic Toxicity (Dog;MRID 40614402)NENNNX(H)NAvian Reproduction(Duck; MRID 3576901)P5(H)N N6Avian Reproduction(Quail; MRID 0163134)P7(H)N N1. 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. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated. BW= Body weight; FC= Food Consumption2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an Xin this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. 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 decreasesin the number of hatchlings(↓56%) and 14-day-old survivors (↓58%). Gross pathological observationsincluded an increase from control in the number of regressing or regressedovaries in the highest treatment concentration.6. A significant decrease (10%) from control in female body weight at the high concentration. There were no treatment relatedclinical signs of toxicityobserved 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 setat the highest treatmentconcentration (500 ppm) that were not statisticallysignificant, but considered to be treatment-related.P Positive findingN Negative finding (in vitro)/No effect (in vivo)E EquivocalNE Not examinedSIMAZINE FINAL

Page 16 of 66 2. Effects on Androgen Pathway

The potential for simazine to interact w ith the androgen pathway is summarized in Table 3. The various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for androgenic, anti-androgenicor HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e 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 ac tivation (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 a nd progesterone levels in the plasma of rats following a single dose of th e 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 progeste rone, 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 prosta te and seminal vesicle weights were decreased at 75 mg/kg and above. At 300 mg/kg, minimal to mode rate testicular degeneration we re observed in all animals, whereas testes weights were una ffected 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 wei ght losses, were observed. There were no androgenic/anti-androgenic effects note d in the other Part 158 studies.

In the FSTRA, there were no statistically signifi cant 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 st udy, decreased embryo viability and regressed testes (gross pathology 4 birds) was observed.

.SIMAZINEFINALPage 17 of 66Table 3. Androgenic/Anti-Androgenic Pathway for SimazineLines 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) Systemic Toxicity Observed 2 Overt Toxicity Observd 3EDSP Tier 1 AssaysAR Binding(MRID 48619301)NSteroidogenesisRequirement satisfied by OSRI (Sandersonet al., 2001; see below)Hershberger(MRID 48619303)NENNNMale Pubertal RatRequirement satisfied by OSRI (Stokeret al., 2005; see below)FSTRA(MRID 48673602)NE N NNNNNOSRIARTA(Birkhøjet al., 2004)NARTA(Ortonet al., 2009)PARTA(Kojimaet al., 2004)NSteroidogenesis(Sandersonet al.,2001)PNSteroidogenesis(Lawset al., 2009)PHershberger(Birkhøjet al., 2004)NNN NSIMAZINEFINALPage 18 of 66Table 3. Androgenic/Anti-Androgenic Pathway for SimazineLines 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) Systemic Toxicity Observed 2 Overt Toxicity Observd 3Male Pubertal(Stokeret al., 2005)NNNP(ML)NN NNNX(M, MH,H)Subchronic Toxicity(Rat; MRID00143265)N N NE N NE NEX(L, M,H)X(M, H)Subchronic Toxicity(Dog; MRID00146655)N N NE N NEX(L, M,H)X(H)Two-GenerationReproduction (Rat;MRID 41803601)NNNNX(M, H)X(H)Chronic Toxicity/Oncogenicity (Rat;MRID 40614405)N NNE N NX(H)X(H)Chronic Toxicity(Dog; MRID40614402)N NN N NX(H)NAvian Reproduction(Duck; MRID43576901)P4(H)N NAvian Reproduction(Quail; MRID00163134)P5(H)P5(H)N NSIMAZINEFINALPage 19 of 661. 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. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate(DP) Lateral Prostate (LP), Prostate (PR),Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). FC= FoodConsumption2. 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 inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1 assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Decreased offspring survival5. Decreased embryo viability and regressed testesP Positive findingN Negative finding (in vitro)/No effect (in vivo)NE Not examinedSIMAZINE FINAL

Page 20 of 66 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it
does include three in vivo assays that provide data to de tect 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 th yroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. This table also includes HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the ta ble and not considered further in the WOE assessment

.
Simazine decreased serum thyroxine (T 4 ) levels in Study 1 of Zorrilla et al (2000)

. (data presented graphically) at an overt ly toxic dose; thyroid stimula ting hormone (TSH) levels were
not evaluated. No effect on T 4 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, pres enting primarily as decreased terminal body weights, were observed in these stud ies. There were no thyroid effects noted in the
Part 158 mammalian studies.

In the AMA, simazine increased (p<0.05) nor malized 7-day hind limb length (HLL) by 6% and significantly (p<0.05) decreased 21-day normali zed 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 leve l for these endpoints, respectively. There was a slight increase in the inciden ce of animals with mild-to-modera te 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 tr eatment-related in the histopathology report. The study authors noted that observati ons of thyroid gland atrophy we re generally associated with smaller-sized tadpoles regardless of treatme nt, suggesting that t hyroid gland atrophy was secondary to smaller size at the high concentration. Overall, the re sults were not consistent with known patterns of thyroid agonism or antagonism in the AMA.SIMAZINE FINAL

Page 21 of 66 Table 4. Thyroid Pathway for Simazine
Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Simazine 1

Study Type/ Literature CitationThyroid WeightThyroid: GrossandSerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmentalstage (± orasynchronous,HLL)Growth (BW, BL,SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

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) P 4 (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 re sponse. A shaded cell indicates that is parameter is not routinely evaluated or is not ap plicable 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 we re 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 examinedSIMAZINE FINAL

Page 22 of 66 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the WoE determination on the pote ntial 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 studie s). WoE analysis in the context of the EDSP follows the Agency’s guidance (USEPA 2011) a nd 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 consid erations of biological plausibility of the findings from the different lines of evidence by examining the co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substance may contribute to the WoE evalua tion in helping elucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively a sequelae of systemic effects. Endocrine m odes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

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

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

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

1. Tier 1 in vivo AssaysSIMAZINE 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 spin al curvature, which o ccurred with similar frequency in the control and low and mid-treatm ent concentrations. However, dose-dependent decreases in growth were noted, with the decrease attaining statis tical significance (p<0.05) at 1.9 mg/L. Compared to the negative control, th e 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 toxici ty at doses up to 200 mg/kg/day. Howe ver, the high dose appeared to be overtly toxic based on the 10-11% decrease in te rminal body weight in th e 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 porti ons 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 decreas e 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/da y), and by 10.3 and 11.3% at medium-high (100 mg/kg/day) and high (200 mg/kg/da y) treatments, respectively, i ndicating that the two top doses were overtly toxic. In additi on, absolute and relative adrena l weights were decreased at 100 mg/kg/day in the first study.

Limited information was available for th e unpublished male pubertal study by Stoker et al

. (2005), but no clinical signs of t oxicity 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 wei ghts were decreased by 10,
24 and 25% in ovariectomized rats follo wing 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.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 treatme nt-related clinical signs of toxicity were observed during 13 weeks of dosing in the di et 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 toxic ity consisting of tremors beginning
at Week 9 were observed in both sexes at th e high dose (4000 ppm), a nd 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 indi cate 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 weight s were decreased in a ll treated males and high dose females, and relative heart weights were decreased in high-dose males and females.

In a Part 158 Guideline developm ental toxicity study in rats, th ere were no treatment-related clinical signs of toxi city observed during the study in females dosed up to 600 mg/kg/day during GD 6-15. Body weights were signi ficantly 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 Guidelin e developmental toxicity study
in rabbits. However there were treatment-rela ted increases in tremors in the 75 and 200 mg/kg/day dose groups (21 and 100%, respectivel y, vs. 0% in control). Significant body weight losses (p<0.05) during the dosing pe riod were also noted at 75 (

− 243 g) and 200 (

− 456 g) mg/kg/day compared to a +230 g increase in controls. Body we ights 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

m id- and high-dose groups during the dosing period.

In the two-generation reproduction st udy in rats, there were no treatm ent-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 weight s were decreased (p<0.05) by 7-11% beginning on Day 14 and remain decreased throughout pre-ma ting in P males, and similar results wereSIMAZINE FINAL

Page 25 of 66 observed for the 500 ppm first generation (F 1 ) males at the first mating ( ↓ 12-13%). Body weight gains for the 500 ppm P and F 1 males were also decreased (p<0.05) during the pre-mating, mating, and post-mating phases (8-51%) and during the rest phase for F 1 males at second mating. Body weight gains for the 100 ppm P male s were decreased during Days 0-70 ( ≈↓ 13%) and during Days 7-14 ( ≈↓ 11%) for the 100 ppm F 1 males at first mating, but were increased from the rest period to term for F 1 males at second mating.

Body weights of the 500 ppm P females were de creased (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 F 1 females were decreased during the first ( ↓ 11-15%) and second pre-mating periods, and rema ined decreased through the first ( ↓ 10-14%) and second ( ↓ 11-15%) gestational periods. La ctational body weights were decreased (p<0.05) during LD 0- 14 ( ↓ 7-11%) for the second generation (F 2a ) litters and only at LD 0 ( ↓ 11%) for the F 2b litters. In the 100 ppm group, decreased body weights were obs erved during the premating phase in P and
F 1 first mating females (approximately 6%). In ge neral, body weight gain for high-dose P and F 1 females (both matings) was decreased (p ≤ 0.05) during the pre-mati ng phase and increased (p ≤ 0.05) during the lactational phase. For F 1 females at first mati ng, body weight gain was decreased and then increased during pre-ma ting phase; it was also increased during the lactational phase. No treatment-related d ecreases in body weights were observed in F 1 or F 2 offspring on PND 14 or 21. Incidental clinical observations and gross malformations in pups were observed in both generations in a ll dose groups, including the control groups.

In the chronic toxicity/carcinogenicity study in ra ts, 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 incr easing 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. B ody weights in the 1000 ppm males and females were significantly decreased (p<0.05) beginnin g 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 anim als throughout the study (overall ↓ 27% and ↓ 28% for males and females). Females also had decreased body weight s 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 thr ough 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 wereSIMAZINE FINAL

Page 26 of 66 decreased (p<0.01), and mean corpuscular hemo globin (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 c oncentration (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 increa sed (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 a nd 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 contro ls on Days 63 through 140. Food consumption in
these animals was also decrease d frequently during the first 20 weeks of the study. In the 1250 ppm males, there was a transient nominal decrea se (not statistically significant) in RBC and HGB at Days 86 and 177, while HCT was significa ntly decreased (p<0.05) at Day 86; on Day 359, these values were nominally higher or eq uivalent 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 statisti cally significant but transient decrease (p<0.05) in RBC, HGB, and HC T 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 incr eased (p<0.05) in the1250 ppm females at 359 days. No treat ment-related, consistent change s were noted in the clinical chemistry or urinalysis results.

In the avian reproduction study on mallard ducks, th ere 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 significan tly decreased (p<0.05) by 10.2% compared to controls, with food consumption being decrease d in the high-dose group only during Week 1.

No signs of systemic or overt t oxicity were noted in the follow ing 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; a nd the avian reproduction study on bobwhite quail at dietary con centrations 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.SIMAZINE FINAL

Page 27 of 66 In the in vitro Tier 1 assays, although simazine was posit ive in the ER binding assay by Tennant
et al . (1994b), it was negative for ER bindi ng in an assay conducted by Blair et al . (2000) using the rat uterine cytosol assay and negative in a cell proliferation assa y (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 steriodogeni c 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 estrogeni c activity in several uterotrophic assays. In the OSRI female pubertal assa ys, 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) re duction in female GSI at the high treatment level (3.4 mg/L) in the FSTRA; however, no e ffects on reproduction we re observed. Female mallard ducks exposed to simazine in the diet pr esented a greater incidence of regressed ovaries and produced fewer eggs, fewer hatchling survivor s, and fewer 14-day surv ivors 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 we re 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 reproducti ve tissues (uterine a nd ovarian) associated with estrogenic responses in peripub ertal rats. Similar effects were observed in peripubertal rats exposed to chlorinated meta bolites 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 pituitar y secretion of LH) and subsequent alterations in the hormonal control of pubertal de velopment. Decreased LH secretion would lead to insufficient stimulation of the gonads during puberty resulting in delayed VO and alterations in estrogen-responsive tissues.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 a nd 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 andr ogen-related effects in the male pubertal assay and Part 158 mammalian toxicity studies occurre d in the presence of overt toxicity.

There were no androgen-related e ffects in the FSTRA. Regresse d 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 ch lorinated metabolites (Stoker et al . 2000; 2002). There have been a number of studies demonstrating that atrazine delays male pubert y in different strains of rats following both peripubertal and perinatal exposure (Stoker et al. 2000; Friedmann, 2002;
Trentacoste et al. 2001; Rayner et al. 2006 and Rosenberg et al. 2008; cited in the SAP reports for atrazine). Stoker et al . (2002) have also demonstrated that chlorinated metabolites of atrazine possess a similar mode of action with rega rd to androgenic-related effects. In a male pubertal protocol, atrazine and the chlorinated metabolites of atrazine significantly (p<0.05) delayed preputial separation, and decreased the weights of reproduc tive tissues associated with androgenic responses. The effects are consistent with the effect of atrazine on the central nervous system ( i.e. , suppression of GnRH/decreased pituitar y secretion of LH) and subsequent alterations in the hormonal control of pubertal de velopment. Decreased LH secretion would lead to insufficient stimulation of the gonads during puberty to produce the ne cessary steroids for androgen-dependent maturation of the sex accessory tissues in the male. Therefore, the delayed growth of the reproductive tract in the male supports the reported alterati on of the hypothalamic control of pubertal progression. For non-mamma lian 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 inte raction of simazine wi th the thyroid pathway
in mammals or wildlife.SIMAZINE FINAL

Page 29 of 66 E. Conclusions

For the estrogen pathway, while there was so me 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 th e absence of overt toxicity. Additionally, while decreased GSI was observed in the FSTRA, there were no other estrogenrelated effects observed including alterations in reproduc tion. Regressed ovaries were observed an avian study, along with decreases in egg pr oduction 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 ma mmals or were observed in the presence of toxicity. Additionally, there were no androgen-re lated effects observed in the FSTRA. In the avian reproduction studies, regr essed testes were observed.

Nevertheless, the potential for interaction with the estrogen and androgen pathway is supported by the SAP conclusion that the chlorotriazi nes (including simazine ) function through a neuroendocrine MOA that suppresses the hypothala mic release of GnRH and therefore, LH which may result in downstream effects on estr ogen and androgen signaling pathways.

V. EDSP Tier 2 Testing Recommendations

There is evidence for potential interaction wi th 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. Th e 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 fo r deriving the chronic Refe rence Dose (RfD) used for chronic dietary risk assessment and calcu lating the Margin of Exposure (MOEs) for nondietary risk assessment.

In the EDSP in vivo Tier 1 male pubertal assay with atr azine, 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 alte r 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 a nd altered estrous cyclicity) with atrazine at doses greater than 50 mg/kg/day in the female pubert al assay with atrazine.SIMAZINE FINAL

Page 30 of 66 The POD (2.56 mg/kg/day) is ap proximately 5-fold lower than the dose (12.5 mg/kg/day that caused delay in PPS and approximately 8-fold lowe r than the dose (25 mg/kg/day) that caused delay in VO. In addition, a dose of 6.25 mg/kg/da y was reported to have no effect on the day of preputial separation when compared to contro ls 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 st udies 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 r ecent 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, whic h 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, Ma yer 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 a ndrogen 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 w ith both northern bobwhite quail and mallard duck are available for simazine. In both spec ies, effects on reproducti ve parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from simazine exposure. Relative to the EDSP, ad ditional testing is not recommended.

Based on the WoE analysis, EDSP Tier 2 test ing is not recommended for simazine since additional testing will not impact current EPA established regulato ry endpoints for human health or ecological risk assessment.SIMAZINE FINAL

Page 31 of 66 VI. References

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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 - Hers hberger Bioassay in Orchidoepididymectomized Rats. WIL Research Laboratories, LLC, As hland, 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 substan ces. 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 s hort-term reproduction assay with the fathead minnow ( Pimephales promelas ). Wildlife Internati onal, 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 th e 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., Su mmit, 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-estr ogenic properties of chloro- S

triazines in rat uterus

. J. Toxicol. Environ. Health, Part A

. 43 : 183-196. (MRID 43598617)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) At razine 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 Tria zine Pesticides Based on a Common Mechanism of Toxicity. Health Effect s Division, Office of Pesticid e Programs, Washington, D.C. 20460March 2002. ID : EPA-HQ-OPP-2005-0481-0011

USEPA. (2007). Risks of Simazine Use to California Red

‐ legged Frog ( Rana aurora draytonii ). Environmental Fate and Effects Division, Office of Pesticide Pr ograms, Washington, D.C. 20460

USEPA. (2007). Risks of Simazine Use to Barton Springs Salamander ( Eurycea sosorum ). Environmental Fate and Effects Division, Office of Pesticide Pr ograms, Washington, D.C. 20460

USEPA (2010) Simazine - Report of the Endocri ne Disruptor Review Team; Test Order# EDSP080807-130; 131 and 132. December 16, 2010. Office of Chemical Safety and Pollution
Prevention. U.S. Environmental Pr otection Agency, Washington, D.C.

USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.

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

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

Willoughby, J. (2011) Simazine: androgen receptor bi nding (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: la boratory 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)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)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 fl ow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 per control a nd 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 e ffects are reported based on comparison to the negative (clean water) control. A single mortalit y was observed in the low treatment group prior to Day 7; no
additional non-incidental mortalit ies were observed throughout the re mainder of the test. Clinical signs (spinal curvature) were observed in the control (8%), lo w (6%), and mid (9%) treatment groups and were not considered to be treatment related.

Simazine significantly increased (p<0.05) normalized (for snout-vent le ngth) 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 dela y of median NF developmental stage at Day 7 or 21 in any treatment group. Further, no as ynchronous development was observed throughout the test. A single late-stage tadpol e was observed in the negative control (NF=61) and in the low and mid treatment groups (NF=62). These tadpole s were excluded from analysis of growth endpoints and normalized HLL. Con centration-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 obs erved 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. Th e study authors noted that thyroid gland atrophy was generally associated with smaller sized ta dpoles regardless of tr eatment, suggesting that thyroid gland atrophy was secondary to reductio ns in size at the high concentration.

Androgen Receptor (AR) Bindi ng 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 ofSIMAZINE 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 concen tration of ~3.2%. A total of three independent runs were performed, and each run included dexa methasone as a weak positive control, and R1881 as the ligand reference standard.

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. As separate cytosolic preparati ons were used for each of the three competitive binding runs, the data from all three saturation bi nding experiments were evaluated. For the cytosolic preparation made on March 30, 2011, the mean dissociation constant (K d ) for [ 3 H]-R1881 was 0.614 (±0.065) nM, and the estimated B max was 1.080 (±0.109) fmol/100 μg protein. For the cytosolic preparation made on July 23, 2011, mean K d for [ 3 H]-R1881 was 1.080 nM (±0.690), and the estimated B max was 1.071 (±0.167) fmol/100 μg protein. For the cytosolic preparatio n made on September 24, 2011, the mean K d for [ 3 H]-R1881 was 0.613 nM (±0.041), and the estimated B max was 0.817 fmol/100 μg protein (±0.049). The
mean and individual K d values were generally below the range reported in the EPA validation program (0.685 to 1.57 nM), although two individual values were within th e range and one value was above the range. Confidence in these numbers is generally high based on the goodness of fit (R 2 = 0.880-0.992) and the small variation in R 2 and K d estimates among runs (although greater variability was observed in the Ju ly 23, 2011 saturation binding data).

In the competitive binding experiment, precipitat ion 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, a nd the highest concentration tested in
the third run was 10

− 4 M. Specific binding of [ 3 H]-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 IC 50 and relative binding affinity (RBA) could not be calculated for simazine.

The estimated average log IC 50 s for R1881 (

− 9.3 M) and the weak positive control, dexamethasone (

− 4.4 M), were within the expected range s, and the mean RBA for the weak positive control was 0.0024%. Confidence in these numbers was moderate due to variation in
IC 50 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.SIMAZINE FINAL

Page 38 of 66 Aromatase Assay; (OCSPP 890.1200)

The requirement for a Tier 1 arom atase assay is satisfied by the in vitro assays reported by
Sanderson et al. (2001). A summary of this st udy is presented in Appendix 2.

Estrogen Receptor (ER) Bindi ng 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.

ER α Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48619302), hER α -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 we lls/plate in each run. Cells were exposed to simazine to induce reporter (luciferase) gene pr oducts. 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 RPC Max for simazine was 3.5%, with an associated PC Max of 10

− 7 M in the first run; the mean RPC Max was <0% for the second run at concentrations up to 10

− 4 M. Performance criteria valu es were generally below the validated ranges, indicating that the current assa y was more sensitive th an during the validation experiments. Therefore the perf ormance criteria deviations do not affect then interpretation of the study. Because the RPC Max < PC 10 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 (M RID 48673602) of EPTC with fathead minnows ( Pimephales promelas ) was conducted under flow-through c onditions. 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 concentrati ons 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.SIMAZINE FINAL

Page 39 of 66 Mean survival was 100, 100, 75, and 13% for male s and 94, 100, 38 and 6% for females in the control, low, mid, and high trea tment groups, respectively; overal l survival (both sexes) was 96, 100, 50, and 9% respectively. Due to the high mort ality at the high concentration, these data
were not used for statistical an alysis. Mortality was significantly different in the mid and high treatment groups compared to the negative contro l (p<0.05; Fisher’s Exact Test). There were no notable observations with regards to behavi or or changes in a ppearance, 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 characterist ics or clinical signs for any treatment group compared to the co ntrols. Male and female growth parameters (wet weight and total length) were not significan tly 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 (rang e 18-25 E/F/D); fertilization su ccess 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 differe nces between controls and the low and mid treatment groups for male or female gonado-somatic i ndex (GSI). Male tubercle scores was marginally significantly (p=0.057; Mann-Whitney) reduced compared to the control (26 vs 41 in co ntrol) at the 0.69 mg a.i./L treatment level and was approaching signif icance (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 hi stopathologic observations in male s. In female fish, there was
an increase in incidence of mi nimal to moderate oocyte atresia at the mid treatment level compared to the control; however, the smaller sa mple 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 unusua lly 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 st eroids were not meas ured in this study.

Hershberger Assay; (OCSPP 890.1400)

In a Hershberger Assay (MRID 48619303) screeni ng 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 ol d (approximate), castrated male Sprague Dawley rats at dose levels of 0 (vehic le), 65, or 200 mg/kg/day. An androgenic positive control group consisted of six cas trated rats exposed to 0.2 mg/kg/ day of testosterone propionate (TP) by subcutaneous (s.c.) in jection plus vehicle (0.5% CMC) by oral gavage (5 mL/kg).SIMAZINE FINAL

Page 40 of 66 To screen for potential anti-andr ogenic 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 le vels 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 c ontrol 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 an ti-androgenic negative control.
All animals survived until scheduled termination. No animals exhibited an y dose-related clinical signs of toxicology and there were no treatmen t-related gross patholog ical findings. Animals were sacrificed approximately 24 hours after the final dose admini stration. At necropsy, the five androgen-dependent tissues and liver were collected and weighed.

In the androgen agonist a ssay, body weights for the 200 mg/k g/day group were decreased (p<0.05) by 8-10% on Days 7, 8 and 11 compared to controls, resulti ng in a 50% decrease (p<0.05) in overall (Days 1-11) body weight gain. Overall body we ight 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 treatmen t-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 contro l 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 sim azine treatment groups were compared to the performance criteria in the Guidelines. A ll %CV values were less than the maximum recommended values, with the exception of Cowp er’s glands in the control group (CV = 68.0%; maximum recommended CV = 55%).

In the anti-androgen assay, overa ll body weight gain was decreas ed (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. Th ere 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 decreas ed (p<0.01) accessory sex organ weights as follows: 69% in seminal vesicles; 75% in ventra l prostate; 47% in LABC; 74% in Cowper’s gland; and 20% in glans penis. Liver weights in the positive contro l group were comparable to the vehicle control. The performance criteria indicated that this as say was performing as expected. There were no significant weight decreases in androgen-dependen t tissues under the conditions of this assay.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 as say is satisfied by the study reported by Zorrilla
et al. (2010). A summary of this stu dy is presented in Appendix 2.

Male Pubertal; (OCSPP 890.1500)

The requirement for a Tier 1 male pubertal assa y 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 steroi dogenesis assay is satisfied by the in vitro assays reported by
Sanderson et al. (2001). A summary of this st udy is presented in Appendix 2.

Uterotrophic; (OCSPP 890.1600)

The requirement for a Tier 1 uterotrophic assay is satisfied by the st udies reported by O’Connor
et al. (2000) and Tennant et al. (1994a). A summary of these studi es is presented in Appendix 2.SIMAZINE FINAL

Page 42 of 66 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 ge neral toxicity studies considered relevant for determining the potential interaction of simazine with the endoc rine 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 us ing an AR reporter gene assay in vitro . Simazine, deltamethrin, methiocarb, prochloraz, tribenuron -methyl and a mixture of the fi ve 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 disso lved 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 lucife rase reporter plasmid MMTV-LUC. Twelve concentrations (0.025-50 μM ) of simazine with or without R1881 (0.1 nM) were tested in quadruplicate. The mixt ure experiment combined 10 μM of each of the five pesticides (including sim azine) to make a 50 μM stock solution. OHF was included in every experiment as a positive control. Cells and te st compounds were incubated, the dosing medium was removed, and the cells were lysed; substrat e containing 1 mM lucife rin and 1 mM ATP in lysis buffer were added, and chemiluminescence wa s measured. Cytotoxicity measurements were determined by replacing pSV AR0 in the proced ure 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 ac tivation 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 re ceptor 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,SIMAZINE FINAL

Page 43 of 66 cultured in medium, and cut into 10 oocyte fragments. The ooc ytes were incubated with 6.25, 12.5, 25, and 50 IU of human chorionic gonadotropi n (hCG) or control media for 20 h. The media was sampled and hormone analysis wa s performed by RIA. After incubation, a submaximal concentration of hCG (~60% of ma ximum ovulatory response) was chosen for coincubation with simazine at conc entrations of 6.25 and 62.5 μM. Pes ticides that had no effect or only an effect at 62.5 μM, were retested at the same concentrations. In addition, the 3 β -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 agonist ic in the estrogen or androgen screen. However, simazine was found to be an ti-androgenic (125 μM, p<0.03, with an increase of approximately 17% above the control) in th e anti-androgen assay, although it did not have a stimulatory effect on the androgen recepto r in the absence of testosterone.

In the ovulation assay, testoster one 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 (hER α and hER β ) and one human androgen receptor (hAR) in transactivati on assays using Chinese hamster ovary cells (CHO-K1 cells) in a luciferase reporter assay. Fo r measurement of antag onistic activity to hER α , hER β and hAR, either 10

− 11 or 10

− 10 M 17 β -estradiol (E 2 ) or 10

− 10 M 5 α -dihydrotestosterone (DHT) was added to the cell cultures along w ith the test compound, respectively. Agonistic activities were evaluated by relative activity expr essed as 20% relative e ffective concentration (REC 20 ), the concentration of the compound showing 20% of the activity of 10

− 10 M E 2 , 10

− 9 M
E 2 , or 10

− 9 M DHT for ER α , ER β , and AR, respectively. Antagonis tic activities we re evaluated by relative activity expressed as 20% re lative inhibitory concentration (RIC 20 ), the concentration of the compound showing 20% inhibition of the activity of 10

− 10 M E 2 , 10

− 9 M E 2 , or 10

− 9 M
DHT for ER α , ER β , and AR, respectively. Simazine did not have agonist or antagonist activity
for ER α and ER β , 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 fo r 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 IC 50 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 andSIMAZINE FINAL

Page 44 of 66 antagonism in the cell prolifer ation 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 a ssay with the chimeric receptor are considered to be inferior test systems to transcriptional act ivation assays using natural full length receptors.

Simazine failed to induce any luciferase activ ity in the ER-CALUX assay, indicating it has no in
vitro estrogenic properties dete ctable 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 recep tor competition binding assays and a yeast-expressed human estrogen receptor tr anscription system. Binding of simazine to the human ER was examined using a yeast reporter ge ne assay. Yeast was incubated with simazine
and β -galactosidase induction was measured. For competitive inhibition studies, an approximate
EC 50 concentration of 17 β -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 a ssays, simazine concentrations of 10

− 7 M, 10

− 5 M and
10

− 3 M were incubated with the biogenic amine a nd estrogen receptors alpha 1 adrenergic, alpha 2 adrenergic, beta, dopamine 1, dopamine 2, sero tonin 1 and serotonin 2 and the appropriate radioligand. Percent bindi ng inhibition of the 17 β -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, 17 β -estradiol and finasteride, induced a dose-dependent increase and decrease in activation, respectively.

In another publishe d, 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 fo r 30 min. prior to exposu re 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. U nder disequilibrium conditions in a time-course experiment, uterine cytosolic extr acts were first incubated with simazine or DACT at 100 μM for 30 min. prior to exposure to radi olabeled estradiol fo r 5-150 minutes. With time, radiolabeled estradiol displaced simazine and DACT that had bound during the initial exposure. In a doseresponse experiment under disequilibrium conditi ons, uterine cytosolic extracts were first
incubated with simazine or DACT at concentrations of 10

− 9 to 10

− 3 M or unlabeled estradiol atSIMAZINE FINAL

Page 45 of 66 10

− 11 to 10

− 7 M prior to exposure to radi olabeled estradiol. The IC 50 for unlabeled estradiol was approximately 10

− 9 M whereas the IC 50 was 100 μM for simazine and DACT.

An additional experiment under disequilibrium c onditions was performed to mimic a Scatchard type analysis using a constant molar excess (100 X estradiol or 10,000X sima zine) 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 ra ts 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 experime nts. The binding of radiolabeled estradiol was reduced by an average of 39% and 24% fo r 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 signi ficant) by 21 and 13%.

Overall the results indicate that simazine a nd 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: H 295R (adrenocortical carcinoma), JEG-3 (placental choriocarcinoma) and MCF-7 (breast cancer). Receptor-mediated induction of vitellogenin was tested in primary hepatocyte cultur es of adult male carp. For the aromatase experiments, cells were plated at ca. 1-2 × 10 5 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. Ne gative controls included treatment with DMSO and no treatment; all plates contained all controls and all treatments were performed in quadruplicate. CYP19 expression wa s detected by RT-PCR, and aromatase activity was detected using a method based on estrone production of tr itiated water. Cells e xposed to the positive control 8Br-cAMP produced a sign ificant increase in aromatase activity and levels of CYP19 mRNA compared to controls.

Male carp hepatocytes were isolat ed and cells were seeded in 96-we ll plates at a density of 1 ×
10 6 cells/mL. For the estrogenicity study, cells were exposed to 17 β -estradiol (0.06-6 μM) or simazine at the same concentrations as above; fo r the anti-estrogenicity study, cells were exposed to the same simazine conditions but in the presence of 100 nM 17 β -estradiol (approximate EC 50 ).SIMAZINE FINAL

Page 46 of 66 Positive controls added to every plate were either 100 nM 17 β -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 pr oduction using indirect competitive ELISA. Cell viability was assessed and found to be acceptable. Vitellogenin concentrations in unexposed or DMSO-exposed male carp hepatocytes were un detectable. The lowest-observed effect
concentration of 17 β -estradiol of ca. 2 nM produced a bout 100-400 ng/ml cellular protein and
the EC 50 concentration of 17 β -estradiol produced ca. 4000-6000 ng/mg protein. Co-exposure of hepatocytes to 100 nM 17 β -estradiol and 0.1, 1 or 10 μM tamoxifen inhibited 17 β -estradiolinduced vitellogenin synthesis by 54%, 89%, and 91% respectively.

Simazine and its metabolites induced aromatas e activity concentration-dependently, producing statistically significant increases in activity above control at con centrations 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 activ ity and CYP19 mRNA levels in H295R cells.
Simazine did not induce or inhi bit the induction of vitellogeni n 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% purit y) using a Hershberger assay in vivo . Simazine, deltamethrin, methiocarb, prochloraz, tribenuron-m ethyl and a mixture of the five pesticides were tested. Fourweek old male Wistar rats were castrated a nd shipped to the laborat ory after one week. On arrival, rats were weighed and randomized by body weight into ni ne experimental groups having similar weights among groups (n=6). After one w eek 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 testoster one propionate (TP). Group 2 was administered TP only and was used for comparison to treated gr oups. Group 3, the positive control, received 20 mg/kg/day flutamide (2 mL/kg) subcutan eously. 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 anaesthetiz ed 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 gl and, levator ani/bulbocavernosus (LABC) muscle, bulbourethral glands, liver, thyroid gland, pa ired 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 us ing an immunofluorimetri c assay. For ventral prostates, total RNA was isolated and c DNA was produced using reverse transcriptaseSIMAZINE FINAL

Page 47 of 66 polymerase chain reaction (RT-PCR). Real-tim e 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 ha d significantly increased adrenal weights and reduced LABC weights. The pos itive control, flutamide, had significantly reduced reproductive organ weights and LH hormone levels. Prostate bind ing protein subunit C3
mRNA and ornithine decar boxylase mRNA levels were signifi cantly reduced for the simazine, flutamide, and mixture groups. Testosterone-r epressed prostatic messa ge 2 (TRPM-2) mRNA levels were decreased in the simazine group compar ed to the testosterone control. The flutamide TRPM-2 mRNA levels were significantly increase d 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 independentof 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 (vehic le control) and the doses were selected based
on earlier studies with atrazine in weanling rats. Doses were admi nistered by oral gavage at a volume of 0.5 mL/100 g bw to groups of 10 fe males beginning on PND 22. Two studies were conducted, the first followed the EDSP protocol and the second wa s 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 St udy 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 do sing period, and the age of complete VO was recorded. On the day of VO, daily vaginal smear s were collected and observed for the presence of leukocytes (diestrus), nucleated epithelial cells (p roestrus) or cornified ep ithelial cells (estrus). In the first study, vaginal cytology (VC) was cont inued daily until scheduled sacrifice. In the second study, VC was also conducte d daily and sacrifice was on the first day of estrus after PND
62.SIMAZINE FINAL

Page 48 of 66 Two hours after the final dose, animals were sacrificed by decap itation, trunk blood was collected, and liver, kidney, adrenals, ovaries, ut erus 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 glandul ar epithelial height, and stromal thickness. Sections of ovary were evaluated for corpora lutea count (all stag es), 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 coll ect serum and stored at

− 80oC for subsequent hormone assays for T 4 estradiol (E 2 ), progesterone (P4, Study 2 only) luteiniz ing hormone (LH) and prolactin (PRL). Anterior pituitaries were frozen on dry ice and stored at -80  ̊ C for PRL and LH assays. Serum E 2 , P4 and T 4 were measured by radioimmunoassay. LH and PRL RIAs were performed using materials supplied by the Nati onal Hormone and Pituitary Ag ency (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 signi ficantly 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 differe nce was noted at 12.5 or 50 mg/kg/day simazine. Mean VO occurred on PND 32.1 ± 0.4 in the control gr oup and PND 34.4 ± 0.7 and 36.1 ± 0.4 in the 25 and 100 mg/kg groups, respectively; delayed VO co incided 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, re spectively. 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 we re significantly lower in the 100 mg/kg/day group; absolute but no t relative adrenal weights were also lower at 50 mg/kg/day. Relative (but not absolute) liver weights were hi gher at 100 mg/kg/day. In Study 2 absolute (but not relative) pituitary weights were significantly lower at 100 a nd 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 diff erent from controls. Relative ovarian weights were significantly greater in the 200 mg/kg/day group.

In Study 1, decreased serum T 4 levels were observed at 100 mg/kg/day; however, in Study 2,
serum T 4 levels were unaffected at any dose. Simazine did not alter serum E 2 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 si gnificant only at 50 mg/kg/da y. In Study 2, there was a dose dependent linear trend in decreased se rum PRL by linear trend analysis (p ≤ 0.01) that was notSIMAZINE FINAL

Page 49 of 66 significant by ANOVA. No changes in baseline se rum LG, pituitary LG or pituitary PRL were observed in either study.

In Study 1, the mean number of days until the firs t estrus was significantly delayed at 100 mg/kg relative to controls (PND 42.6 ± 0.6 vs 37.9 ± 0.4, resp ectively). The number of days in estrus was also significantly decreased at 100 mg/kg, but the number of da ys in proestrus and diestrus were not changed; however as a result of the short time be tween VO and sacrifice only 1.0 ± 0.15 estrus cycles were observed in the c ontrol animals, and fewer in the treated.

In Study 2, dosing was extended 20 da ys longer than in the EDSP prot ocol to evaluate the effect of simazine on estrus cyclicity for a longer time period. There wa s a significant delay in the number of days until first estrus in th e 100 and 200 mg/kg dose groups (PND 44.4 ± 1.1 and 43.7 ± 0.5 days, respectively) compared with cont rol (PND 39.5 ± 0.73). The number of days in proestrus was significantly decreased only in the 50 mg/kg group. Although th ere 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. A dditionally, no apparent differen ces 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 ag e 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-da y exposure study, and by 2.7, 2.9, 5.4 and 5.1 days (25, 50, 100 and 200 mg/kg/day, resp ectively) in the 41- day exposure study. Although body weight changes are known to possibl y affect onset of pube rty, the delays in VO observed at 25 mg/kg/day in this study do not appear to be affect ed by the reduced weight gain following simazine exposure. The study demonstr ates 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 adopte d by the EDSP as OPPTS 890.1500. Groups of intact 23 day old weanling male Wi star 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 animal s were necropsied on PND 53. The pituitary, testes, ventral prostate, lateral prostate, epidi dymides, and seminal vesicles with coagulating gland were removed and weighed. Tissue samples were examined by histology. Blood was analyzed for TSH, T 4 , T 3 , LH, PRL, androgen and estrogens . Additionally, LH and PRL were determined in the anterior pituitaries.SIMAZINE FINAL

Page 50 of 66 A non-monotonic dose response was found for the ons et 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 decr eases were also observed in liver ( ↓ 5-16%) and paired kidney ( ↓ 10-18%). However, these decreases corr esponded 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 leve ls in an manner inverse to
dose. Alterations in serum T 3 , T 4 , 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 ot her treated groups. Mild to modera tely 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 sa me 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, i ndicating that the testicular degeneration was responsible for the changes in th e 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 chlorotr iazine herbicides, including sim azine (98% purity), and its chlorinated metabolites, DIA (91% purity) a nd 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 co mpounds 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, ra nked by body weight, and assigned to treatment groups so that the mean body weights for all gro ups were similar. Rats received a single oral dose by gavage of simazine at 188 mg/kg; DI A 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 chem ical at 0900 h and sacrificed by decapitation at designated times: 5, 15, 30, 60, or 180 minutes afte r dosing. At sacrifice, blood was processed for serum and plasma, and aliquots were stor ed for determination of adrenocorticotropic hormone (ACTH), corticosterone (CORT), pr ogesterone (P4), and prolactin levels bySIMAZINE 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 09 00 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 modificat ions of previously described gas chromatography/mass selective detection methods.

Following a single dose of the me tabolite DACT, ACTH levels we re 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 si gnificantly 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 obs erved in the 80 mg/kg at 15 (3.9 fold) and 30 (3.7 fold) minutes. The CORT response was signi ficantly increased in rat plasma with DIA dosing at 10, 40, and 80 mg/kg at 15 minutes, a nd 40 and 80 mg/kg at 30 minutes, compared to
controls.

Compared to controls, a single dose of simazine at 188 mg/kg elicited si gnificant increases in ACTH and CORT, but not P4, at 15 minutes.

Metabolite concentrations in the plasma of ra ts following a single dose of DIA or DACT were measured; uptake of DIA was rapid with the me tabolite undergoing dealky lation yielding similar
concentrations of DACT. Plasma concentrations of DACT in rats foll owing 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 increa ses in plasma ACTH we re observed within 15 minutes following exposure to simazine or the me tabolite DIA. Dose-dependent elevations in
CORT and P4 were also observed at 15 a nd 30 minutes post-dosing with these compounds, indicating an activation of adrena l 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 mi nimal 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 pr imary inducer of ACTH release. Thus, the rapid release of ACTH and su bsequent activation of adrenal steroidogenesis following a single exposure to sim azine or DIA may reflect chlo rotriazine-induced changes at the level of the brain and/or pituitary.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 simila rities among groups (five grou ps, n=20), and maintained under standard laboratory conditions. The biochemical/hormonal measurem ents used groups of 14 rats, and the remaining 6 rats per group were desi gnated 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 anesthetiz ed 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 so lution; processed tissues were imbedded in paraffin, and stained for immuno-histochemical anal ysis 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 m easuring hematoxylin and eosin stained 5 μM sections using an image analyzer. Estrous cycl ing was determined by examining daily vaginal
washes in the 14 rats used for biochemical/hor monal evaluations. Blood was collected from all animals to determine serum prolact in levels by radio-immunoassay.

Final body weights were signifi cantly 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 wa s not affected by treatm ent. Uterine epithelial cell heights were significantly increase d 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 ch emicals, 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 fo r the uterine weight a nd progesterone receptor studies; for the thymidine incorporation study, se xually 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 fo r 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 (E 2 ) on Day 2 and 3, to investigate the inhi bition of estrogen-stimulated uterineSIMAZINE FINAL

Page 53 of 66 weight gain. Body weights were recorded on the firs t day of treatment and ju st prior to sacrifice. Uteri were dissected, tr immed, and weighed fresh.

For the progesterone receptor (PgR) study, ovariect omized 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 E 2 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). E 2 was administered two hours after each simazine treatment. Body weights were record ed on the first day of treatment and just prior
to sacrifice. Uteri were dissected at sacrifice.

In the thymidine incorporation study, rats were do sed by oral gavage for 2 consecutive days with simazine at 0, 1, 10, 50, 100, or 300 mg/kg/day. E 2 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 E 2 treatment. Animals were weighed prior to Day 1 of treatment and prio r 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 significa nt increase in uterine weights were observed compared to the
vehicle controls. For ovariecto mized rats injected with E 2 , uterine weights were significantly decreased in the 300 mg/kg group compared to the vehicle controls receiving E 2 . Day 3 body weights were significantly lowe r in the 100 and 300 mg/kg groups ( ↓ 8 and 11%) in the uterine weight stimulation study (no E 2 ). 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 s uppressed for the 300 mg/kg but not 50 mg/kg treatments plus E 2 . For treatments without E 2 injections, PgR was significantly suppressed at 300 mg/kg compared to the vehicle control. The stud y 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 radiol abeled thymidine was significantly inhibited (p ≤ 0.01) in the 50, 100 and 300 mg/kg groups with co-exposure to E 2 , but not at 1 or 10 mg/kg, compared to the E 2 control. The 300 mg/kg treatments without E 2 showed significantly less (p ≤ 0.05) thymidine incorporation
than the control without E 2 and the 300 mg/kg treatment with E 2

.

The results of the three studies indicate no estrogen-l ike 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 th ree estrogen-induced
in vivo responses, including increases in uterine we t weight, progesterone receptor (PR) binding activity, and uterine peroxidase activity. Groups of 4 immature SD female rats received 50, 150SIMAZINE 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 beginni ng at 21 days of age. Other groups of 4 rats that received simazine at the same doses also received 10 μ g/kg/day of 17 β

estradiol (E 2 ) in corn oil by intraperitoneal injecti ons on the same treatment days (21-23). Control groups consisted of: 10 μg/kg/day E 2 alone; the vehicle; or corn oil.

Simazine alone did not increase uterine wet weight at any do se, but did cause significant (p<0.05) decreases in PR binding levels and uterine peroxidase activity. E 2 alone caused 6.2-, 8.9-, and 9.7-fold increases in ut erine wet weight, PR binding, a nd uterine peroxidase activity, respectively relative to controls. Simazine did not exhibit agonist or antagonist effects on basal
and E 2 -induced increases on uterine weights. E 2 -induced increase in uterine wet weight was not significantly affected by co-treatment with sim azine; however, some dose-independent decreases
in E 2 -induced PR binding uterine per oxidase activity were observed. In rats administered 150 mg/kg/day simazine + E 2 , there was a statistically significan t 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, a nd adrenals; histopathological ex aminations were conducted for control and high-dose animals on these tissues as well as the pituitary, thyroid, epididymis, prostate, seminal vesicle, urinary bladder, ma mmary 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-w eek dosing period in good phys ical condition with no treatment-related clinical signs or ocular cha nges. There were significan t 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 fo r males and females in all treated groups, but mean feed consumption for the 2000 and 4000 ppm ra ts 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 ga ins were significantly le ss 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 theSIMAZINE FINAL

Page 55 of 66 4000 ppm males. Mean white blood cell counts (WBC) were signifi cantly 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 clin ical chemistry and urinalysis parameters were significantly different from contro ls in all dose groups, with most marked differences in the 2000 and 4000 ppm groups: significantly lowered mean blood glucose levels occu rred in male rats; high cholesterol and inorganic phosp horus 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 male s; 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 weight s occurred in both sexes in all dose groups. Decreased mean absolute spleen weight in ma les and mean absolute ovary and heart weights were observed at 2000 and 4000 ppm; relative ovar ian weight was decrea sed in the 4000 ppm females. There were no treatment-related gro ss lesions observed at necropsy. Microscopic examination of kidneys showed a dose-related inci dence 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 inorga nic phosphorus, and incr eased renal calculi in
males and females. A NOAEL was not determined.

Subchronic Oral Toxici ty (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 dos es 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/fe males) for at least 91 days. Organs that were weighed included heart, brain, liver and testes (list not provi ded); histopathological
examinations were conducted on these tissues as well as the adrenals, kidney, pituitary, thyroid, epididymis, prostate, urinary bladder, mammar y gland, ovary, uterine cervix and horn, and vagina.

There were no mortalities. Tremors, observe d 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 onl y gained a small amount of weight (+0.1 kg) over the dosing period; th e 2000 and 4000 ppm females lost weight (

− 0.6 kgSIMAZINE FINAL

Page 56 of 66 and

− 1.4 kg, respectively) over the course of th e 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 a nd 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 re ductions of mean SGOT levels in all treated males at Week 7 and in the 4000 ppm males at Week 13, in mean cr eatinine levels in the 4000 ppm males at Week 13, in mean total bilirubin in the 4000 ppm males at Week 7, a nd in calcium levels in the 4000 ppm males at Week 7 and the 2000 and 4000 ppm ma les 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. Urinal ysis revealed signifi cant increases in specific gravity in the 2000 and 4000 ppm males at Week 7, and in ket one levels in the 2000 and 4000 ppm males and females at Week 7 and the 4000 ppm males at W eek 13. Urinary pH was significantly decreased in the 4000 ppm males at Week 7 and in th e 4000 ppm males and females at Week 13.

Mean absolute heart weights we re significantly decreased in a ll 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 relativ e testes weights were also decreased in the 4000 ppm males.

There were no treatment-related gross or microscopic lesions obs erved 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 le vels 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 anim als in each group, the shaved skin was abraded once per week. After 21 days, each animal was sacrificed and subjected toSIMAZINE FINAL

Page 57 of 66 gross necropsy. Organs that were weighed included liver, kidneys, te stes, ovaries, thyroid, adrenals, and pituitary. Histopathological exam inations 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 ne cropsy revealed generalized congestion in most visceral organs. There were no effects of treatment on organ we ights. Incidental differences occurred in both pre-treatment and post-treatmen t hematology and blood chemistry analyses; no significant differences were observed between te st groups. Miscellaneous subclinical lesions ware observed at necropsy in control and te st 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 administ ered 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 a nd corpora lutea counted. Uteri were removed
and the number of live and dead fetuses and im plantation sites were counted. Approximately
one-third of the fetuses in each l itter 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 dur ing the study; all dams survived to termination. Body weights were de creased (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 th e 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 o ccurred in the mid- and high-dose groups. The parameters affected on a litter basis were presphe noid 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 a nd 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, th ese and 5 other parameters were nominally elevated at all dose leve ls, except for the vertebra/centra not ossified and/ or additional, and the presence of rudimentary ri b. On a litter basis, 11 paramete rs were nominally elevated atSIMAZINE FINAL

Page 58 of 66 all dose levels, one of which wa s litters with no ossified ster nebrae. One of the skeletal parameters, unossified sternebrae, demonstrated a statistically signi ficant 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 ga in, 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 inco mpletely 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% purit y) 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 sacrifice d, and subject to necropsy. Ovarie s were examined and uteri, including their contents, were weighed. The numbe r 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 sexe d, 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 o ccurred during the study; however, no gross pathological changes were found in these animal s 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 obs erved (50%, 100%, and 100%, compared to 0% controls). Treatment-related incr eases in tremors were also no ted (0%, 0%, 21%, and 100% for the 0, 5, 75, and 200 mg/kg/day dose groups, respec tively). Body weight losses (p<0.05) during the dosing period were noted at 75 (

− 243 g) and 200 (

− 456 g) mg/kg/day comp ared 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 cont rol weight gain of +3 g. Food consumption in the mid- and high-dose groups was also decrea sed (p<0.05) in the mid- and high-dose groups during the dosing period. There were no treatment-re lated 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 ther efore post-implantation loss) were noted in all treatment groups; however, there was no clear dose response, and th ere 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 sign ificant differences in the incidences of fetuses with malformations in any of the treated groups. Treatment-re lated increases in theSIMAZINE FINAL

Page 59 of 66 incidences of fetuses with 13 th full ribs, non-ossified sterne brae, free floating ribs, and nonossified patella were observed at the mid and hi gh dose; the increases we re significant (p<0.05) for free floating ribs and non-ossified patella in litters of th e 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 L OAEL is 75 mg/kg/day, based on decreased body weight gain, tremors, and abortions. The de velopmental NOAEL is 75 mg/kg/day and the LOAEL is 200 mg/kg/day based on reduced mean fe tal weight and increased skeletal variations. Simazine was not teratogenic in ra bbits 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% purit y) 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 genera tions. After 10 and 13 weeks, respectively, of dietary treatment, the P and F 1 parental animals were mated. F 1 parental animals consisted of 30 randomly selected pups/sex/dose level. F 1 parental animals were allowed a 3-week rest period before a second mating. Sibling matings were avoided. Five F 2a pups/sex/dose were selected for complete necropsy and histopa thological examination. F 2b male pups were sacrificed on PND 4
and F 2b female pups were assigned to subsequent studies. The or gans weighed at sacrifice
included the testes and ovaries. For the c ontrol and high dose groups, histopathological examinations were conducted on these organs as well as the semina l vesicle, prostate, coagulating gland, uterus, epididymid es, cervix, vagina, and pituitary.

There were no treatment-related c linical signs of toxicity in an y 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 we re observed for the 500 ppm F 1 males at the first mating ( ↓ 12-13%; p<0.05). It was stated that body wei ght gains for the 500 ppm P and F 1 males were decreased (p<0.05) during the pre-mating, mating, and post- mating phases (8-51%) and during the rest phase for F 1 males at second mating. Body weight gain s for the 100 ppm P ma les were decreased during Days 0-70 ( ≈↓ 13%) and during Days 7-14 ( ≈↓ 11%) for the 100 ppm F 1 males at first mating, but were increased from rest period to term for F 1 males at second mating.

Body weights of the 500 ppm P females were de creased (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 F 1 females were decreased during the first ( ↓ 11-15%) and second pre-mating periods, and rema ined decreased through the first ( ↓ 10-14%) and second ( ↓ 11-15%) gestational periods. La ctational body weights were decreased (p<0.05) during LD 0- 14 ( ↓ 7-11%) for the F 2a litters and only at LD 0 ( ↓ 11%) for the F 2b litters. In the 100 ppm group,SIMAZINE FINAL

Page 60 of 66 decreased body weights were observed dur ing the premating phase in P and F 1 first mating females (approximately 6%). In genera l, body weight gain for high-dose P and F 1 females (both matings) was decreased (p ≤ 0.05) during the pre-mating phase and increased (p ≤ 0.05) during the lactational phase. For F 1 females at first mating, body wei ght 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 F 1 generations (both matings) during the prem ating 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) testicul ar weights were increased (p <0.05) by 11% in the 500 ppm P males and by 20% in the 500 ppm F 1 males, and relative ovary weights were increased by 50% in the 500 ppm F 1 females. Microscopic examination of tissues from animals in both generations revealed no compound-related differences and th e absolute weights we re not affected by treatment. Therefore, these decreases were attr ibuted to the decreased body weights of these groups. No treatment-related histopa thological 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 fertil ity index, gestation i ndex, gestation length, implantation sites/litter, total number of live pups , mean number of live pups/litter, live birth index, viability index, lactati on index, fetal sex ratio, and m ean pup body weight/litter) at any
dose level. In the F 1 offspring, male pup body weights were increased (p ≤ 0.05) on Days 14 and 21 at the low dose; and in the F 2 offspring, male pup body weights were increased on Day 14 at
the mid dose. Incidental clinical observations and gross malforma tions in pups were observed in both generations in all dose gr oups, 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 a nd females, respectively); the LOAEL was
not determined.

Chronic Toxicity/Carcinoge nicity (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 administer ed simazine (96.9% purity ) in the diet at dose levels of 0, 10, 100, or 1000 ppm (equiva lent 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 mont hs. In addition, an additional 10 rats/sex were dosed at 0 or 1000 ppm for 52 weeks and then ma intained on untreated (control) diet for 52 weeks. At study termination, su rviving rats in all groups we re sacrificed for gross andSIMAZINE FINAL

Page 61 of 66 histopathological evalua tion. The organs weighed at sacrif ice included the adrenals, liver, kidneys, testes, epididymides, and ovaries. Gro ss 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 firs t year of the study; how ever, a high rate of mortality was observed during the second year. Fo r the 1000 ppm males, the mortality rate was decreased (p<0.01) compared to th e 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 increa sing 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 fe males, respectively); body weight gains were also decreased (p<0.05) in these animals th roughout 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 consum ption 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, a nd lymphocytes were decreased (p<0.01), and mean corpuscular hemoglobin (MCH), WBC, and neutrophils were increas ed (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 wa s 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 glucos e was seen at almost all time points; increased alkaline phosphatase was observed at all time points in this group.

Organ weight effects included decr eased (p<0.05) absolute liver weights in the 52-week sacrifice 1000 ppm females; however, relati ve (to body weight) liver weight s 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 we re 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, re lative 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 we ight increases were considered to be a reflection of the decreased body weights.SIMAZINE FINAL

Page 62 of 66 Gross pathology, which was conducted on all animal s that died during the study including those
that died or were sacrificed prior to scheduled sacrifice, i ndicated that the incidence of macroscopic lesions in treated rats was not signif icantly different from that of the control groups for either sex. In male rats, the combined inci dence 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 statis tical 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 in creased (p<0.01) at 1000 ppm (57%) compared to controls (31 %). Th e incidence of liver hematopoisis and spleen hematopoiesis was increased (p<0.05) in th e 1000 ppm females (7% and 14% respectively) compared to controls (0% and 4%, respectivel y). 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 th at 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 para meters 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/da y) and 1000 ppm (63.1 mg/kg/ day). In male rats, simazine appears to induce the formation of liv er 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 le vels 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, ovarie s, testes, epididymides, pituitary, and thyroid; histopathological examinations we re conducted on these tissues as well as the urinary bladder, prostate, mammary gland, uterus, and vagina.SIMAZINE FINAL

Page 63 of 66 There were no mortalities duri ng 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 an d infrequent emesis. Body weight gain was decreased (p<0.05) in the 1250 ppm males on Da ys 7 and 14, and in the 1250 ppm females on Days 7-224 compared to controls. Body wei ghts of the 1250 ppm females were decreased compared to controls on Days 63 through 140. F ood 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 valu es were nominally highe r 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 compar ed to pre-dose values in highdose females. MCHB was sligh tly 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 ab solute or relative we ights of the testes, epididymides, ovaries, or pituitary glands at any dose level. In males at the high dose, absolute thyroid/parathyroid we ight was decreased ( ↓ 60%; p<0.001) and in females at this dose, the absolute thyroid/parathyroi d 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, decrea ses in RBC, HGB, HCT, and an in crease 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 dietar y concentrations of 0, 50, 150, or 450 ppm for 20 weeks (nine weeks prior to the start of e gg production and eight w eeks 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 th e study, and no clinical si gns of toxicity were observed. Mean body weight of hens in the 450 ppm group at study termin ation 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 sl ight 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 survivorsSIMAZINE FINAL

Page 64 of 66 ( ↓ 58%) at 450 ppm. There was no effect of tr eatment on eggshell thic kness or offspring body weight. The LOAEC is 450 ppm base d 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 nomin al dietary concentra tions 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. A ll 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 aggr ession by pen mate. No overt signs of toxicity were observed during the study. Duri ng gross necropsy of birds sacrif iced at the end of the study, one male in the 500 ppm group was found to have slig ht 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 su rvivors as a percen t 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 reduc tions 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.SIMAZINE FINAL

Page 65 of 66 APPENDIX 3: References Not Utilized in the Simazine WoE Analysis

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

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies previously submitted to the agency for registra tion/reregistration, published literature articles and/or Tier 1 assays. The agency also c onducted a more recent search (2009 to 2014) of
available scientific literature fo r any additional relevant informa tion for their weight of evidence (WoE) evaluations. These articles were evalua ted in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/s cience/efed/policy_guidance/team _authors/endangered_species _reregistration_workgroup/PDF_ro t/esa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature To xicity Studies to Suppor t Human Health Risk Assessment ( http://www.epa.gov/pesticides /science/lit-studies.pdf).

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

Balaguer, P., Joyeux, A., Denison, M., et al . (1996) Assessing the es trogenic and dioxin-like
activities of chemicals a nd 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 hea lth risk assessment? J. Environ. Sci. Health C Environ. Carcinog.
Ecotoxicol. Rev

. 29(2) : 91-144.SIMAZINE FINAL

Page 66 of 66 Fang, H., Tong, W., Branham, W., et al . (2003) Study of 202 natu ral, 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 steroidogenesi s 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 mi crosomes 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 es trogenic 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 Scien ce+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 depe ndent 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.