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

## ~ S

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

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

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

.

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

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

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

CHEMICAL: TRIFLURALIN

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYTRIFLURALIN FINAL

ii TABLE OF CONTENTS

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

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

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

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

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

III. Weight of Evidence (WoE) Evaluation.......................................................6

A. EDSP Tier 1 Screening Assays.............................................................9

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis.............................10

1. Effects on Estrogen Pathway......................................................10

2. Effects on Androgen Pathway....................................................16

C. Effects on Hypothalamic-Pituit ary-Thyroidal (HPT) Axis...........................21

IV. Committee’s Assessment of Weight of Evidence..........................................25

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................25

1. Tier 1 in vivo Assays..............................................................25

2. OSRI..................................................................................26

B. Estrogen Pathway...........................................................................27

C. Androgen Pathway.........................................................................27

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

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

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

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

LIST OF TABLES:

Table 1. Tier 1 A ssay for Trifluralin...................................................................9
Table 2. Estrogenic/Anti-Estroge nic Pathway for Trifluralin ....................................12
Table 3. Androgenic/Anti-Androge nic Pathway for Trifluralin ..................................17
Table 4. Thyroid Pathway for Trifluralin ..........................................................23
APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................36

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................48

APPENDIX 3. References Not Utilized in the Trifluralin WoE Analysis............................57TRIFLURALIN FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

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

CTA Comparative Thyroid Assay

CV Coefficient of Variation

CYP Cytochrome 450

DER Data Evaluation Recor d
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 r 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 enerationTRIFLURALIN FINAL

iv Abbreviation Terminology

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 Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py 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 Intera g enc y Coordinatin g 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 h
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 DoseTRIFLURALIN FINAL

v Abbreviation Terminology

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 th
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 ionateTRIFLURALIN FINAL

vi Abbreviation Terminology

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-EvidenceTRIFLURALIN FINAL

Page 1 of 58 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 thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether trifluralin interacts with E, A or T hormone path ways, the number and type of effects induced, the magnitude and pattern of responses observed acr oss studies, taxa, and
sexes were considered. Additi onally, the conditions under which effects occu r were considered, in particular, whether or not e ndocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On January 8, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of evidence (WoE) analysis of the potential interaction of trifluralin with the E, A or T hormone pathways. The T1Wo ERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

For the estrogen pathway, trifluralin was negative in the estrogen receptor (ER) binding assay which is capable of detecting an interaction of the test compound with the ER (both agonists and antagonists). Although the ER transactivation assay (ERTA) was weakly positive (14-18% of the response seen with 1 nM 17 β -estradiol in three out of four assay runs) at 10

− 4 M trifluralin, there was no evidence of ER agonism (or antagoni sm) in response to trifluralin treatment in the female pubertal assay or uterot rophic assay. Furthermore, the results of the aromatase assay
indicate that trifluralin does not inhibit aromatas e activity. An slight incr ease (1.2- to 1.4-fold) in estradiol production was observed in the steroidogenesis assay at relatively high concentrations of trifluralin (10 and 100 μM). In the fish short-term reproduction assay (FSTRA), all effects were observed in the presence of overt toxicity, ex cept for a 0.5% decrease in fertility at the low
concentration for which there were no other eff ects observed. Triflurali n did not alter estrous cyclicity or reproductive or gan histopathology in the fema le pubertal rat assay.

In the mammalian Part 158 studies, following dietary exposure to rats for 13 weeks, a decrease in pituitary weight was observed at doses >40 mg /kg/day. No treatment-related effects on the pituitary were observed in the tw o-generation rat reproduction or in the chronic toxicity studiesTRIFLURALIN FINAL

Page 2 of 58 with rats. The decrease in ovarian weight s een at 337 mg/kg/day in the rat reproduction study occurred in the presence overt toxicity. No ot her estrogen-related effect s were observed in the mammalian reproduction or developmental studies. Decreases in fecundity were observed in the Part 158 sheepshead minnow study in the absence of overt toxicity ( ≥ 1.3 μg/L), however, there is a lack of diagnostic specifici ty for this endpoint in of itself regardi ng potential endocrinerelated effects. A 4% decrease in eggshe ll thickness and 7% decrease in 14-d offspring body weight at 1000 mg/kg-diet in the mallard duck reproduction study was observed; however, no other reproductive parameters were affected in this study or the bobw hite quail reproduction study. Overall, there is a lack of convincing evidence for a potential in teraction with estrogen pathway for trifluralin.

For the androgen pathway, trifluralin was negative for androgen receptor (AR) binding and did not alter testosterone production in the steroidogenesis assay. No androgenic effects were seen
the mammalian Tier 1 in vivo assays at doses that did not cause either systemic toxicity (male pubertal assay) or overt toxicity (Hershberger assay). The observed effects in the Hershberger assay (decreases in androgen-responsive tissu es weights) at the hi gh dose (450 mg/kg/day)
occurred at a dose that was consid ered overtly toxic (10% decrease in body weight). In the male pubertal assay, decreases in androgen-responsiv e tissues weights (prostate, Levator anibulbocavernosus and epididymal weights) and decr eased serum testosterone were seen at the high dose (250 mg/kg/day). This dose caused sign ificant decreases in body weights approaching overt toxicity (9%) and increases in absolute (29%) and relative (37%) liver weights. In the FSTRA, except for GSI which was increased (p<0.05) dose-dependently at al l treatments as well as a slight decrease in fertilit y (0.5%), all other observed effect s occurred at a c oncentration that was considered overtly toxic.

In the mammalian Part 158 studies, following dietary exposure of trif luralin to rats for 13 weeks, pituitary weights were decreased at dose levels >40 mg/kg/day. No treatment-related effects on the pituitary were observed in the two-generatio n rat reproduction or in the chronic toxicity
studies with rats in the absence of overt toxicity. Therefore, as the in vitro assays were negative and the findings in the in vivo assays occurred in the presence or approaching overt toxicity, or were limited to an isolated effect, there is a lack of complementary evidence within the assays as well as a lack of redundancy of evidence across the assays. Hence, trifluralin does not appear to potentially interact with the androgen-pathway.

For the thyroid pathway for trifluralin, in the pub ertal assays, dose-dependent decreases in serum T4 levels were seen in females at the mid-(75 mg/kg/day) and high (250 mg /kg/day) dose and in males at doses of 25, 75 and 250 mg/kg/day. Incr eased serum TSH levels were observed in both males and females at the high dose only. Eff ects on thyroid histopath ology (alterations in follicular cell height and colloid area) were also observed in high dose female pubertal rats. All thyroid effects in the pubertal as says occurred at doses where incr eases in liver weights were also observed. In the amphibian metamorphosis assa y (AMA), while reduced growth and effects onTRIFLURALIN FINAL

Page 3 of 58 the development endpoint of normalized hind -limb length was observed, there were no corresponding histopathological diagnoses to sugge st the developmental is thyroid-related.

In the mammalian Part 158 studies, following dietar y exposure to rats for 13 weeks, pituitary weights were decreased at dose levels > 40 mg/k g/day. In the chronic/carcinogenicity study with rats, increased thyroid weights and an increase in the incidence of thyroid gland follicular cell tumors in males was observed only in the presen ce of overt toxicity. Additional OSRI (Saghir et
al ., 2008) investigating the mode of action for trifluralin-related thyroid perturbations showed
that two week treatment of trifluralin in male ra ts resulted in decreases in serum T3 and T4 and elevated thyroid stimulating hormone (TSH). Tr ifluralin also induced T4-specific hepatic UGT activity, and 2-fold increases in bile-flow rate and biliary elimination of T4 also were demonstrated. Furthermore, in previously submitted mechanistic studies for benfluralin (a
dinitroaniline similar to triflu ralin), induction of hepatic mi crosomal enzyme activity was
observed in both sexes in rats tr eated with benfluralin. In a 14-day study submitted to support a postulated mode of action for liver and thyroid tumors in the rat, benfluralin was shown to induce constitutive androstane receptor (CAR) and pregnane X receptor (PXR) related gene transcripts and associated pentaoxyresorufin O-dealkylation (PROD) enzyme activities and benfluralin also up-regulated T4-specific urid ine diphosphate glucuronosyltransferase (UGT) genes associated with T4 metabolism. These eff ects occurred at a dose that resulted in decreased
serum T4 and T3 levels and increased liver weight and hepatic hypertrophy.

Based on the profile of li ver and thyroid effects seen in the pubertal assays, the available mode of action data and the lack of evidence for a po tential interaction in the AMA, thyroid effects due to trifluralin exposure are likel y attributed to enhanced serum T 4 clearance due to liver enzyme (UGT) induction.

The conclusion of the WoE evaluation is that tr ifluralin demonstrates no convincing evidence of potential interaction with the estrogen or androge n pathways. The available data suggests the thyroid effects observed in the mammalian in vivo studies may result from liver enzyme induction and subsequent increased hormone clearance. In the wildlife data, while there is a lack of data to support liver enzyme induction which may result in hormone clearance, this mode of action may be conserved across other vertebrate ta xa. However, there is no convincing evidence in the wildlife studies to suggest that there is a pote ntial interaction with E, A or T, as the majority of the effects occurred in the presence of overt toxicity and lacked complementarity in the absence of overt toxicity.

In the EDSP Tier 1 in vivo male and female pubertal assays as well as in the OSRI, there was evidence for potential interaction with the thyr oid pathway in studies conducted with adult animals, but no such data exists for potenti al thyroid toxicity in the young animals.TRIFLURALIN FINAL

Page 4 of 58 For trifluralin, the point of de parture (POD) for risk assessmen t of 2.4 mg/kg/day is based on increased frequency of abnormal stool, decreas ed body weights and body weight gains and toxicity to the hematopoietic system seen at 40 mg/kg/day (LOAEL) in a chronic toxicity study in dogs. This POD is used for deriving the chroni c Reference Dose (RfD) used for dietary risk assessment and for the calculating Margins of Exposure (MOEs) used for non-dietary risk

assessments.

In general, since the POD and the toxicity endpo ints of concern are base d on data obtained from adult animals, there is a concern that the POD may not be protectiv e of potential thyroid toxicity in the young. However in the case of trifluralin, although no data exists for potential thyroid toxicity in the young, a comparativ e thyroid assay (CTA) is not r ecommended since: 1) the POD is 10-fold lower than the lowest dose (25 mg/kg/ day) that caused dose-dependent decrease in serum T4 levels in the female pubertal assay; 2) available data sugge sts the thyroid effects
observed in the mammalian in vivo studies may result from liver enzyme induction and subsequent increased hormone cl earance; 3) the CTA would be te sted at higher doses and would not be likely to yield a POD lower than the leve l currently used for risk assessment; and 4) the chronic RfD and MOEs are protectiv e of the thyroid-related findings seen in the Tier 1 pubertal

assay.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not recommended for trifluralin since additional testing is unlikely to impact the current EPA established regulatory endpoints for human risk assessments.

No fish or amphibian EDSP Tier 2 testing is re commended since there is no convincing evidence for an interaction with estrogen, androgen or thyroid pathways in fish or amphibians. Additionally, it is noted that life cycle studies which eval uate reproduction with fathead minnow and sheepshead minnow are av ailable for tr ifluralin.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard
duck are available for tr ifluralin. In the mallard duck study, effects on reproductive parameters were observed; no effects were observed in the bobwhite quail study. The t ype of data obtained from Part 158 avian reproduction studies (O CSPP 850.2300) are considered sufficient for evaluating potential reproductive eff ects to birds from trifluralin exposure. Additional testing is
not recommended.TRIFLURALIN FINAL

Page 5 of 58 I. Introduction

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 thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI) ,including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether trifluralin interacts with E, A or T hormone path ways, the number and type of effects induced, the magnitude and pattern of responses observed acr oss studies, taxa and
sexes were considered. Additi onally, the conditions under which effects occu r were considered, in particular, whether or not e ndocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On January 8, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of evidence (WoE) analysis of the potential interaction of trifluralin with the E, A or T hormone pathways. The T1Wo ERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

The water solubility of trifluralin is approximately 0.2 mg/L at pH 7 to 9 at 25°C with a octanol/water partition coeffici ent of 5.34 (log Pow). It does not hydrolyze, but does degrade rapidly in water due to photolysis. Trif luralin has a low vapor pressure (4.58 x 10 -5 mm Hg at 25°C), and therefore, volatilizati on is not expected to significan tly contribute to the dissipation
of trifluralin in the environment. Trifluralin has a whole body bioconcentr ation factor value in
fish of 1689 to 9586.

The available information considered in determin ing the potential interaction of trifluralin with the E, A, or T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxi city studies and other pu blished articles. These data are summarized in Sections III.A through I II.C. An analysis of the data submitted to the agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The EDSP Tier 2 study recomme ndations are presented in Section V.TRIFLURALIN FINAL

Page 6 of 58 II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

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

1.

B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Order recipients a nd/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration (e.g., Part 158 guideline studies) . The Agency’s review of the initial OSRI is provided in the Report of the Endocrine Disrupt or Review Team on triflurali n (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 endocrine-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 Council (S AP) (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.TRIFLURALIN FINAL

Page 7 of 58 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 eleven assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. 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 expression of responses at higher levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biol ogical organization? Fi nally, an evaluation of conditions where responses occur including cons ideration of whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemicTRIFLURALIN FINAL

Page 8 of 58 toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.

This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October, 2013 the SAP stated that, “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (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 40 CFR Part 158 studies for both human and ecological risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g ., organ weights/histopathology), biochemical ( e.g ., blood chemistry), and other clinical signs ( e.g., lethargy) were also considered when evaluating overt toxicity, es pecially if the effects were 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 weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

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

Page 9 of 58 The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the tabl e 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 trifluralin are shown
below in Table 1

.

Table 1: Tier 1 Screening Assays for Trifluralin.

Tier 1 Assays Test Guideline Test Order Status

Estrogen Receptor (ER) Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied (MRID No. 48619704)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48619705)

Androgen Receptor (AR) Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied (MRID No. 48619702)

Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48619708)

Aromatase Assay (Human recombinant microsomes) OSCPP 890.1200 Requirement Satisfied (MRID No. 48619703)

Uterotrophic Assay (Rat) OSCPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48619709)

Hershberger Assay (Rat) OSCPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48619707)

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied (MRID No. 48674001)

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied (MRID No. 48674002)

Fish Short-term Reproduction Assay OSCPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48619706)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 48619701)TRIFLURALIN FINAL

Page 10 of 58 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for trifluralin to interact with the estrogen pathway are summarized in Table 2. The various targets of the estrogen pathway across the re levant Tier 1 assays are delineated so as to facilitate determination of potential for estrogenic , anti-estrogenic or HPG axis effects. This table also includes HPG-relevant findings from the 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 table and not considered further in the WOE assessment.

Trifluralin was negative (or not interactive) in the Tier 1 in vitro estrogen receptor (ER) binding and aromatase assays. In contrast, a posit ive response was observed in the Tier 1 ER α transcriptional activation assay at trifluralin concentrations of 10 − 5 and 10 − 4 M. Additionally, a positive response also was observed in the steroi dogenesis assay, with slight increases in estradiol concentration (1.2- to 1.4-fold) at the two highes t concentrations, 10 and 100 μM.

Trifluralin was negative in both the Tier 1 in vivo uterotrophic and female pubertal rat assays.

In the FSTRA, fertility was reduced (p<0.05) at the low ( ↓ 0.5%; 0.195 μg a.i./L) and high ( ↓ 4%; 21.5 μg a.i./L) treatments, and there was an 87% decrease in fecundity in the high-dose group. Plasma VTG concentrat ions and GSI in females were not affected, but increased incidences of severe oocyte atresia and mini mal subacute multifocal ovarian inflammation were present at the high dose. However, clinical signs of toxicity also were observed at the high dose. Females in the high concentration (5 out of 16 exposed) were observed as either bloated, having partial paralysis, a bent tail or all of these signs. Therefore, all responses, with the exception of the decrease in fertilization succ ess at the low dose occurred in the presence of overt toxicity.

There were no estrogen-related effects observed in the Part 158 mamma lian studies; however, decreased pituitary weights were observed at all doses in the subchr onic rat toxicity study.

In a Part 158 fathead minnow fish life cycle study, there were no treatment-related effects on reproduction; however, effects on pare ntal survival were observed at ≥ 5.1 μg/L. In a
second Part 158 fish life cycle study with sheepshead mi nnow, significant decreases in the number of eggs spawned per female was observed at all treatment concentrations ( ≥ 1.3 μg/L). In this study, parental survival was reduced at 17.7 μg/L (highest treatm ent) and parental and offspring growth and offspri ng survival were reduced at ≥ 9.6 μg/L. Decreases in growth
were observed in a Part 158 rainbow trout early life-cycle study at 4.32 μg/L.

In a Part 158 avian reproduction study with bobw hite quail, no treatment-related effects were
observed at test concentrations up to 1000 mg/kg-diet. In a se cond Part 158 avian reproductionTRIFLURALIN FINAL

Page 11 of 58 study with the mallard duck, there was a statis tically significant (p<0.05) 4% reduction in eggshell thickness at the highes t treatment concentration (1000 mg/kg-diet) in which there was also a significant decrease in 14-day survivor body weight. For this study, while there was a significant decrease in parental male body wei ght at the highest treatment, there were no apparent overt signs of toxicit y, treatment related mortalities or reductions in food consumption or female body weight. Additional Part 158 av ian reproduction studies with the bobwhite quail
and mallard duck were available with no treatment -related effects up to te st concentrations of 50 mg/kg-diet.TRIFLURALINFINALPage 12 of 58Table 2. Estrogenic/Anti-Estrogenic Pathway for TrifluralinLinesofEvidenceIndicatingPotential InteractionwiththeEstrogenic/Anti-EstrogenicPathwayfor Trifluralin1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone 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 Tier1 AssayER Binding(MRID48619704)NERTA(MRID48619705)PAromatase(MRID48619703)NSteroidogenesis(MRID48619708)PUterotrophic(MRID48619709)NFemale Pubertal Rat(MRID 48674001)N N N N N NLW(M,H)LE(H)NFSTRA(MRID 48619706)NENNNFrt:↓0.5%,(L)NX(H)4OSRISubchronic toxicity(Rat; MRIDs 40138301and 00151906)NRNR NR↓(L, M,H)X NRDevelopmental toxicity(Rat; MRID 00152419)NRNRXX(MH, H)TRIFLURALINFINALPage 13 of 58Table 2. Estrogenic/Anti-Estrogenic Pathway for TrifluralinLines of Evidence Indicating Potential Interaction with theEstrogenic/Anti-EstrogenicPathway for Trifluralin1Study Type /Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity 2 Age & Weight at VO 2° Sex Characteristics Fertility (Frt) / Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Developmental toxicity(Rabbit; MRID00152421)NRNRXX(M, H)Two-generationreproduction(Rat; MRID 00151901)N N N N NENXNTwo-generationreproduction(Rat; MRID 00162543)NE NE N NENENXNTwo-generationreproduction(Rat; MRID 40405007)NE N N NENENXX(H)Chronic toxicity(Rat; MRIDs 00044337,00162456, 00162457,00162458)XX(M, H)Chronic toxicity(Mouse; Jaeger, 1986and MRIDs 00044338,00158935, 40392313)XNRChronic toxicity(Dog; MRIDs42447001, 00151908,00159618)XX(H)TRIFLURALINFINALPage 14 of 58Table 2. Estrogenic/Anti-Estrogenic Pathway for TrifluralinLines of Evidence Indicating Potential Interaction with theEstrogenic/Anti-EstrogenicPathway for Trifluralin1Study Type /Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormon e Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and His t o p a t holo g y Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt) / Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Life-cycle toxicity(Fathead minnow;MRID 05008271)NX (M,MH, H)Life-cycle toxicity(sheepshead minnow;MRID 42449901)P(L, ML)5X(H,MH)Avian reproduction(Bobwhite Quail; MRID00131134)NNNAvian reproduction(Mallard Duck MRID00131132)NNNAvian reproduction(Bobwhite Quail; MRID40334706)NN NAvian reproduction(Mallard Duck; MRID40334704)P(H)6N N71. Key to responses: L=Low treatment, =M treatment, H=High treatment. Arrows (↓or↑) indicate the direction of the response. A shaded cell indicates that isparameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated. LE = serum liver enzymes2. 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 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. ATRIFLURALINFINALPage 15 of 584. There were five females (out of 16) in the high concentration observed as either bloated, observed having partial paralysis,observed having a bent tail, or allof these signs. Similar clinical signs were also observed in males at this concentration.5. Decreases in number of number of eggs spawned per female at all test concentrations.6. 4% decrease in eggshell thickness and decrease in 14-d offspring body weight at 1000 mg/kg-diet.7. While there was a significant decrease in parental male body weight at 1000 mg/kg-diet, there were no apparent overt signs of toxicity, treatment relatedmortalities, or reductions in food consumption or female body weightP Positive findings (in vitro) / No effect (in vivo)N Negative findings (in vitro) / No effect (in vivo)NE Not examinedNR Not reportedTRIFLURALIN FINAL

Page 16 of 58 2. Effects on Androgen Pathway

The potential for trifluralin to interact wi th the androgen pathwa y are summarized in Table 3. The various targets of th e androgen pathway across the releva nt Tier 1 assays are delineated so as to facilitate determination of potential for a ndrogenic, anti-androgenic or HPG axis effects.
This table also includes HPG-relevant fi ndings from the data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

In an evaluation of androgenic/anti-androgenic activity from Tier 1 testing results, trifluralin was negative (or a non-binder) in the in vitro AR binding assay and there was no effect on testosterone production in th e steroidogenesis assay.

In the anti-androgenic portion of Hershberger assay at the high dose (450 mg/kg/day), decreases (p<0.05) in three AST weights (i.e., seminal vesicles with co agulating glands, ventral prostate, and LABC) and decreased serum testoste rone levels (36%; p<0.0 5) were observed at the high dose (450 mg/kg/day). However, bodyweights at this dose were decreased 10% and bodyweight gains were decreased 46 % indicating overt toxicity. No statistically significant effects on AST weights were seen at the low (50 mg/kg/day) and mi d dose (150 mg/kg/day) groups. In the male pubertal assay, in the hi gh dose, serum testosterone concentration was decreased (NS) by 30% and adjusted weights of AST (DP and LABC) and epididymal weights were significantly decreased (p<0.5). No histopat hological lesions were observed in the testes or epididymides. Age and body weight at preputial separation (PPS) were also not affected at any dose level. The high dose in this study was approaching an overtly toxic dose as indicated by significant decreases (p<0.05) in body weight at post-na tal day (PND) 30 and 37 of 11 and 9%, respectively, with decreases (p<0.5) in body weight gain of 11 to 24%.

In the FSTRA, with the exception of effects on male GSI and fertility, all effects occurred at the highest treatment concentration where overt toxicity was apparent (abnormal swimming/partial paralysis in male (5/8) and fe male fish). Significant increases in GSI was observed at all treatments ( ↑ 29-39%) and was treatment-dependent. Fertility was decreased
0.5% at the mid concentration. Additionally, at the high test c oncentration (21.5 μg a.i./L), male nuptial tubercles score were decr eased (20 vs. 31 in control) and 17- β estradiol concentration in the high-dose males was increased by 78%. Furthermore at this concentration, in male gonadal development, there was a shift to the late spermatogenic stage and an increased proportion of spermatogonia.

The only effect on male reproductive parameters in the Part 158 mamm alian studies was an increase in testes weight (8%) in F1 offspr ing only in the rat two-generation reproduction study. Decreased pituitary weights were observed at all doses in the su bchronic rat toxicity study. No androgen-related effects were observed in the Part 158 fish or avian studies.TRIFLURALINFINALPage 17 of 58Table 3. Androgenic/Anti-Androgenic Pathway for TrifluralinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Trifluralin1Study Type / LiteratureCitation AR Binding Steroidogenesis Sex Steroid Hormones 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 Observed 3EDSP Tier 1 AssayAR Binding(MRID 48619702)NSteroidogenesis(MRID 48619708)NHershberger(MRID 48619707)NNLW(M, H)X(H)4Male Pubertal Rat(MRID 48674002)NNN↓8%(M),↓11 -14% (H)NN↓12%,DP;↓13%,LABC(H)NLW(M, H)LE(H)N5FSTRA(MRID 48619706)N↑29%,↑36%,(L, M)NNFrt:↓0.5%,(L)NX(H)6TRIFLURALINFINALPage 18 of 58Table 3. Androgenic/Anti-Androgenic Pathway for TrifluralinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Trifluralin1Study Type / LiteratureCitation AR Binding Steroidogenesis Sex Steroid Hormones 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 Observed 3OSRISubchronic toxicity(Rat; MRIDs40138301, 00151906)NR NR NR NR↓(L,M, H)NRX NDevelopmental toxicity(Rat;MRID 00152419)NRXX(MH,H)Developmental toxicity(Rabbit; MRID00152421)NRXX(M, H)Two-generationreproduction(Rat; MRID 00151901)↑8%(H, F1)N NE N N NENX NTwo-generationreproduction(Rat; MRID 00162543)NE N NE N NE NENX NTwo-generationreproduction(Rat; MRID 40405007)NNNENNENENXX(H)Chronic toxicity(Rat; MRIDs00044337, 00162456,00162457, 00162458)XX(M, H)Chronic toxicity(Mouse; MRIDs42447001, 00151908,00159618)X NChronic toxicity(Dog;MRIDs42447001, 00151908,00159618)NRNRNRNRNRNRXX(H)TRIFLURALINFINALPage 19 of 58Table 3. Androgenic/Anti-Androgenic Pathway for TrifluralinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Trifluralin1Study Type / LiteratureCitation AR Binding Steroidogenesis Sex Steroid Hormones 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 Observed 3Life-cycle toxicity(Fathead minnow;MRID 05008271)NX (M,MH, H)Life-cycle toxicity(sheepshead minnow;MRID 42449901)NX(H,MH)Avian reproduction(Bobwhite Quail;MRID 00131134)NNNAvian reproduction(Mallard Duck MRID00131132)NNNAvian reproduction(Bobwhite Quail;MRID 40334706)NN NAvian reproduction(Mallard Duck; MRID40334704)NN N71. Key to responses: L=Low treatment, =M treatment, H=High treatment. Arrows (↓or↑) indicate the direction of the response. A shaded cell indicates that isparameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated. Abbreviations for androgensensitive tissues: Seminal vesicles (SV), Ventral prostate (VP),Dorsal prostate (DP), Levatorani-bulbocavernosus (LABC), Epididymides (E).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 inthis column. For details see Section IV. A3. 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. Fordetails see Section IV. A4. Effects at the high dose (450 mg/kg/day) in the anti-androgenic phase of the assay occurred in the presence of overt toxicity (Body weight↓10% by Day 11).5. The high dose in this study was approaching an overtly toxicdose as indicated by significantdecreases (p<0.05) in body weight at post-natal day (PND) 30and 37 of 11 and 9%, respectively, with decreases (p<0.5) in body weight gain of 11 to 24%6. Five males (out of 8) that exhibited either a partial lossof equilibrium and partial paralysis, appearing extremely dark, having a bent tail, or appearing verythin). Similar clinical signs were also observed in females at this concentration.7. While there was a significant decrease in parental male body weight at 1000 mg/kg-diet, there were no apparent overt signs of toxicity, treatment relatedmortalities, or reductions in food consumption or female body weightTRIFLURALINFINALPage 20 of 58P Positive findings (in vitro) / No effect (in vivo)N Negative findings (in vitro) / No effect (in vivo)NR Not reportedNE Not examinedTRIFLURALIN FINAL

Page 21 of 58 C. Effects on Hypothalamic-Pituitary-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 hy pothalamic or pituitary regulatio n 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 (r at) assays, and the AMA (frog).

The potential for trifluralin to interact with thyroid regulation are summarized in Table 4. The various targets of the thyroid path way across the relevant Tier 1 a ssays 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. This tabl e also includes HPG-relevant findings from the 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 table and not considered further in the WOE

assessment.

In the male and female pubertal assays , dose-dependent decreases (25-58%) in T 4 concentrations were seen at the low (25 mg /kg/day), mid (75 mg/ kg/day) and high (250 mg/kg/day) doses. TSH concentr ations were elevated (males: ↑ 74%, NS; females: ↑ 73%) at the high dose only. There was no effect on thyroi d weight for either sex, but increases in follicular cell height and decreases in colloid area were noted in females at the high-dose.

In the AMA, there were no histological effects on the thyroid, nor any effects on developmental stage. There was an increase of 25 and 18% in normalized hind-limb length (HLL) at the low and mid concentratio n (2.64 and 26.2 μg/L, respectively) and a 20% decrease at the high concentration (232 μg/L) compared to the negati ve control. For this endpoint, a marginally significan t increase of 16% (p = 0.05) was observed in the solvent control compared to the negative control which we re of the same magnitude as effects observed in the low and mid treatment levels. Additionally, there were significan t (p<0.05) increases in the solvent control as compared to the negative in the grow th measurements including Day 7 snout-vent length (SVL) and Day 21 body weight. For both of these e ndpoints, the low and
middle treatment concentrations we re also increased over the nega tive control (not all effects significant at α =0.05), while the high treatment concentration was decreased as compared to the negative control. The results of the AMA indicate a reduc tion of growth of X. laevis tadpoles, but there were no corresponding histopathological di agnoses to suggest that this developmental delay is thyroid-related.

In a chronic oral carcinogenicity study with triflu ralin in rats, an increa se in the incidence of thyroid gland follicular cell tumors (adenomas plus carcinomas combined) in males was observed at the high dose (325 mg/kg/day) . In a literature study by Saghir et al ., (2008), trifluralin was reported to induce thyroid tumors via a UGT-related MoA. After a two-weekTRIFLURALIN FINAL

Page 22 of 58 exposure to trifluralin (441 mg/kg/da y), effects on body weight gain ( ↓ 20%; p<0.05), serum T 3 ( ↓ 17%; p<0.05), serum T 4 ( ↓ 90%; p<0.05) and TSH ( ↑ 37%; NS) were observed. Induction of

T 4 -specific hepatic UGT activity and 2-fold increase s in bile-flow rate and biliary elimination of

T 4 also were demonstrated. Decreased pituitary we ights were observed at all doses in the Part 158 subchronic rat toxicity study.TRIFLURALIN FINAL

Page 23 of 58 Table 4. Thyroid Path way for Trifluralin.

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

Study Type / Literature CitationThyroid WeightThyroid: Grossand HistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmentalstage orasynchronous &HLLGrowth(BW, SVL,)Systemic ToxicityObserved2Overt Toxicity Observed3

EDSP Tier 1 Assays

Male Pubertal Rat (MRID48674002) N N ↓ 25%, ↓ 42%, ↓ 58% (L,M,H) N N  LW (M,H)  KE (H) N

Female Pubertal Rat (MRID 48674001) N ↑ cell height, ↓
colloidal area (H) ↓ 33%, ↓ 45% (M, H) ↑ 73% (H) N  LW,  KE (H) N

AMA (Frog; MRID 48619701) N HLL: ↑ 18%, ↑ 25%, ↓ 17% (L, M, H) 4 Day 7: SVL- ↑ 9% (M), ↓ 17% (H);
BW ↓ 39% (H) 5 N

OSRI

Subchronic toxicity (Rat; MRIDs 40138301, 00151906) NR NR ↓ (L, M,

H) X NR

Developmental toxicity (Rat; MRID 00152419) NR NR NR X X (M,H, H)

Developmental toxicity (Rabbit; MRID 00152421) NR NR NR X X (M, H)

Two-generation reproduction (Rat; MRID 00151901) NE N N X N

Two-generation reproduction (Rat; MRID 00162543) NE NE NE X NTRIFLURALIN FINAL

Page 24 of 58 Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Trifluralin 1

Study Type / Literature CitationThyroid WeightThyroid: Grossand HistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmentalstage orasynchronous &HLLGrowth(BW, SVL,)Systemic ToxicityObserved2Overt ToxicityObserved3

Chronic toxicity (Rat; MRIDs 00044337, 00162456, 00162457, 00162458) NR NR NR X X (M, H)

Chronic toxicity (Mouse; MRIDs 00044338, 00158935, 40392313) NR NR NR X NR

Chronic toxicity (Dog; MRIDs 42447001 and 00151908/00159618) NR NR NR X X (H)

Mechanistic Study (Rat; Saghir et al .,2008) T 4 ↓ 90%,

T 3 ↓ 17% ↑ 37% NR X N

1. Key to responses: L=Low treatment, =M treatment, H=High treatment Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay; LW= liver weight; KE= serum kidney enzymes 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. These results reflect comparison to the negative cont rol. Solvent control HLL was significantly increased compared to the negative control and was of similar magnitude as in the low and mid treatment. When compared to the solvent control, th e low and mid treatments are not significantly different, however the highest treatment is significantly decreased. Additionally, while there were no effects on developmental stage in the treatments compared to the negative c ontrol, there was a significant decrea se in stage at the high treatment compared to the solvent control. 5. These results reflect comparison to the negative control. Day 7 SVL and Day 21 body weight solvent control was significantly increased compared to the negative cont rol and was of similar magnitude as in the low and mid treatment (except for Day 21 body weight compared to mid treatment). When compared to the solvent control, the low and mid treatments are not significantly different, however the highest treatment is significantly decreased for Day 7 body weight and SVL, Day 21 NHLL, Day 21 SVL and body weight, and Day 21 NF stage. N Negative findings NR Not reported NE Not examinedTRIFLURALIN FINAL

Page 25 of 58 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of trifluralin to interact with endocrine related processes ( i.e., E, A or T hormonal pathways) as well as recommendati ons 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 pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated an alysis of those line s of evidence.

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

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

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

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

1. Tier 1 in vivo Assays

System ic toxicity was observed in several of th e Tier 1 Assays. In the anti-androgenic phase ofTRIFLURALIN FINAL

Page 26 of 58 the Hershberger assay at the high dose (450 mg/kg/ day), overt toxicity ma nifested as decreased (p ≤ 0.05) body weights on Days 7 ( ↓ 8%) and 11 ( ↓ 10%), and body weight gains (p ≤ 0.05) at each measured interval ( ↓ 46–65%). Also at the high dose, food consumption was decreased (p ≤ 0.05) during the first 4-day interval (Days 1–4; ↓ 35%) compared to vehicle control. Liver weights were increased (p ≤ 0.05, except as noted) by trifluralin trea tment in both the androgenic and antiandrogenic portions of the study: by 16% at 150 mg/kg/day; and by 36% at 450 mg/kg/day.

In the male pubertal assay, absolute and rela tive liver weights were significantly (p<0.05) increased by 29% and 37%, respectively, and serum creatinine level wa s increased (p<0.05) by 50% at the high dose (250 mg/kg/day). In the fema le pubertal assay, absolu te and relative liver weights were significantly (p <0.05) increased by 17% and 24 %, respectively, and serum
creatinine level was increased (p<0.05) by 100% at the hi gh dose (250 mg/kg/day).

In the FSTRA, there were five females (out of 16 exposed) in the high co ncentration observed as either bloated, observed having part ial paralysis, observed having a be nt tail or all of these signs. Five males (out of 8 exposed) exhibited either a partial loss of equilibrium and partial paralysis, appeared extremely dark, were observed with a bent tail or app eared very thin.

No treatment-related effects on mortal ity or clinical behavior signs of toxicity were observed in
the AMA.

2. OSRI

In mammals, the toxicity profile for trifluralin de monstrates that the kidney and the liver are the principal target organs in rats and dogs. In subc hronic oral studies, liver effects include increased liver weights and changes in cl inical chemistry parameters. Kidney effects include decreased kidney weights, kidney and bladder tumors, in creased blood urea nitrogen (BUN), increases in total protein, aspartate aminotransferase (AST ) and lactate dehydrogenase (LDH) in the urine. Also, protein electrophoresis of urin e samples showed the presence of α 1-globulin and α 2- globulin. In the kidneys, tubular hyaline casts, minimal cortical tubular epithelial regeneration, observed microscopically, and an increased inci dence of progressive gl omerulonephritis were seen. In dogs exposed to trifluralin for 1 year, multifocal cortical tubular cytoplasmic pigment deposition was noted in the kidneys of both sexes. In the subchroni c studies, blood effects such as lower hemoglobin levels and changes in clinical chemistry were reported in rats. In male rats, trifluralin was associated with increased incidence of thyroid follicular cell combined adenoma, papillary adenoma, cystadenoma and carcinoma tumors.

In the Part 158 studies with fish, survival in the parental group was decreased at ≥ 5.1 μg/L in the fathead minnow study, and at 17.7 μg/L in the sheepshead minnow study. Parental and offspring growth and offspring survival was also affect ed at 9.6 μg/L in the sheepshead m innow study. Reduction in growth were observed at 4.23 μg/L in an early life-cycle stu dy with rainbow trout. In the avian reproduction studies, no treatment re lated effects were observed in bobwhite quail upTRIFLURALIN FINAL

Page 27 of 58 to 1000 mg/kg-diet. For the mallard duck reproduc tion study, while a decrease in parental male body weight was observed at 1000 mg/ kg-diet, there were no appare nt overt signs of toxicity,
treatment related mortalities or reductions in food consumption or female body weight.

B. Estrogen Pathway

Trifluralin was negative in the estrogen receptor (E R) binding assay which is capable of detecting
an interaction of the te st compound with the ER (both agonist s and antagonists). Although the ER transactivation assay (ERTA) was weakly positiv e (14-18% of the response seen with 1 nM 17 β

estradiol in three out of four assay runs) at 10

− 4 M trifluralin, there was no evidence of ER agonism (or antagonism) in response to trifluralin treatment in the female pubertal assay or uterotrophic assay. Furthermore, the results of th e aromatase assay indicate that trifluralin does not inhibit aromatase activity. An slight increas e (1.2- to 1.4-fold) in estradiol production was observed in the steroidogenesis a ssay at relatively high concentrat ions of trifluralin (10 and 100 μM). In the fish short-term reproduction assay (FSTRA), all effects were observed in the presence of overt toxicity, except for a 0.5% decr ease in fertility at th e low concentration for
which there were no other effects observed. Tr ifluralin did not alter estrous cyclicity or reproductive organ histopathology in th e female pubertal rat assay.

In the mammalian Part 158 studies, following dietary exposure to rats for 13 weeks, a decrease in pituitary weight was observed at doses >40 mg /kg/day. No treatment-related effects on the pituitary were observed in the tw o-generation rat reproduction or in the chronic toxicity studies with rats. The decrease in ovarian weight se en at 337 mg/kg/day in the rat reproduction study occurred in the presence overt toxicity. No ot her estrogen-related effect s were observed in the mammalian reproduction or developmental studies. Decreases in fecundity were observed in the Part 158 sheepshead minnow study in the absence of overt toxicity ( ≥ 1.3 μg/L), however, there is a lack of diagnostic spec ificity for this endpoint in of itsel f regarding potential endocrine-related
effects. A 4% decrease in eggshell thickness and 7% decreas e in 14-d offspring body weight at 1000 mg/kg-diet in the mallard duck reproduc tion study was observed; however, no other reproductive parameters were affected in this study or the bobwhite quail reproduction study. Overall, there is a lack of convincing evidence for a potential interacti on with estrogen pathway
for trifluralin.

C. Androgen Pathway

Trifluralin was negative for AR binding and di d not alter testosterone production in the steroidogenesis assay. No androgenic e ffects were seen the mammalian Tier 1 in vivo assays at doses that did not cause either systemic toxi city (male pubertal assa y) or overt toxicity (Hershberger assay). The observed effects in the Hershberger assay (decreases in androgenresponsive tissues weights) at the high dose (450 mg/kg/day) occurred at a dose that was considered overtly toxic (10% decrease in body weight). In the male pubertal, decreases inTRIFLURALIN FINAL

Page 28 of 58 androgen-responsive tissues weights (prostate, LABC and epididymal we ights) and decreased serum testosterone were seen at the high dose (250 mg/kg/day). This dose caused significant decreases in body weights approach ing overt toxicity (9%) and incr eases in absolute (29%) and relative (37%) liver wei ghts. In the FSTRA, except for GS I which was increased (p<0.05) dosedependently at all treatments as well as a slight decrease in fe rtility (0.5%), all other observed
effects occurred at a concentration th at was considered overtly toxic.

In the mammalian Part 158 studies, following diet ary exposure to rats for 13 weeks, pituitary weights were decreased at dose levels >40 mg/kg/day. No treatment-related effects on the pituitary were observed in the tw o-generation rat reproduction or in the chronic toxicity studies
with rats in the absence of ove rt toxicity. No androgen-relate d effects were observed in the wildlife Part 158 studies. Therefore, as the in vitro assays were negative and the findings in the in

vivo assays occurred in the presen ce or approaching overt toxicity, or were limited to an isolated effect, there is a lack of complementary eviden ce within the assays as well as a lack of redundancy of evidence across the assays. Hence, trifluralin does not appear to potentially interact with the androgen-pathway.

D. Thyroid Pathway

For the thyroid pathway for trifluralin, in the pub ertal assays, dose-dependent decreases in serum T4 levels were seen in females at the mid-(75 mg/kg/day) and high (250 mg /kg/day) dose and in males at doses of 25, 75 and 250 mg/kg/day. Incr eased serum TSH levels were observed in both males and females at the high dose only. Eff ects on thyroid histopath ology (alterations in follicular cell height and colloid area) were also observed in high dose female pubertal rats. All thyroid effects in the pubertal as says occurred at doses where incr eases in liver weights were also observed. In the AMA, while reduced growth and effects on the de velopment endpoint of normalized hind-limb length was observed, th ere were no correspondi ng histopathological diagnoses to suggest the devel opmental is thyroid-related.

In the mammalian Part 158 studies, following dietar y exposure to rats for 13 weeks, pituitary weights were decreased at dose levels > 40 mg/k g/day. In the chronic/carcinogenicity study with rats, increased thyroid weights and an increase in the incidence of thyroid gland follicular cell tumors in males was observed only in the presen ce of overt toxicity. Additional OSRI (Saghir et
al ., 2008) investigating the mode of action for trifluralin-related thyr oid perturbations showed that
two week treatment of trifluralin in male rats resulted in decreases in serum T3 and T4 and elevated TSH. Trifluralin also induced T4-speci fic hepatic UGT activity, and 2-fold increases in bile-flow rate and biliary elimination of T4 also were demonstrated. Furthermore, in previously
submitted mechanistic studies for be nfluralin (a dinitroaniline simila r to trifluralin), induction of hepatic microsomal enzyme activity was observed in both sexes in rats treated with benfluralin. In a 14-day study submitted to support a postulated m ode of action for liver and thyroid tumors in the rat, benfluralin was shown to induce constitutive androstane receptor (CAR) and pregnane XTRIFLURALIN FINAL

Page 29 of 58 receptor (PXR) related gene transcripts and asso ciated PROD enzyme activ ities and benfluralin also up-regulated T4-specific UGT genes associated with T4 metabolism. These effects occurred
at a dose that resulted in decreased serum T4 and T3 levels and increased liver weight and hepatic hypertrophy. Data submitted for benfluralin al so showed increased testosterone clearance accompanied decrease in AST weights in ca strated rats treated with benfluralin.

Based on the profile of li ver and thyroid effects seen in the pubertal assays, the available MoA
data and the lack of evidence for a potential interaction in the AMA, thyroid effects due to trifluralin exposure are likely a ttributed to enhanced serum T 4 clearance due to liver enzyme (UGT) induction.

E. Conclusions

The conclusion of the WoE evaluation is that tr ifluralin demonstrates no convincing evidence of potential interaction with the estrogen or androge n pathways. The available data suggests the thyroid effects observed in the mammalian in vivo studies may result from liver enzyme induction and subsequent increased hormone clearance. In the wildlife data, while there is a lack of data to support liver enzyme induction which may resu lt in hormone clearance, this MOA may be
conserved across other vertebrate taxa. However, there is no c onvincing evidence in the wildlife studies to suggest that there is a potential interacti on with E, A or T, as the majority of the effects occurred in the presence of overt toxicity and lacked complementarity in the absence of overt toxicity.

II. EDSP Tier 2 Testing Recommendations

In the EDSP Tier 1 in vivo male and female pubertal assays as well as in the OSRI, there was evidence for potential interaction with the thyr oid pathway in studies conducted with adult animals, but no such data exists for potenti al thyroid toxicity in the young animals.

For trifluralin, the point of de parture (POD) for risk assessmen t of 2.4 mg/kg/day is based on increased frequency of abnormal stool, decreas ed body weights and body weight gains and toxicity to the hematopoietic system seen at 40 mg/kg/day (LOAEL) in a ch ronic toxicity study in dogs. This POD is used for deriving the chronic Reference Dose (RfD) used for dietary risk assessment and for the calculating Margins of Exposure (MOEs) used for non-dietary risk

assessments.

In general, since the POD and the toxicity endpo ints of concern are base d on data obtained from adult animals, there is a concern that the POD may not be protectiv e of potential thyroid toxicity in the young. However in the case of trifluralin, although no data exists for potential thyroid toxicity in the young, a comparativ e thyroid assay (CTA) is not r ecommended since: 1) the POD is 10-fold lower than the lowest dose (25 mg/kg/ day) that caused dose-dependent decrease in serum T4 levels in the female pubertal assay; 2) available data sugge sts the thyroid effectsTRIFLURALIN FINAL

Page 30 of 58 observed in the mammalian in vivo studies may result from liver enzyme induction and subsequent increased hormone cl earance; 3) the CTA would be te sted at higher doses and would not be likely to yield a POD lower than the leve l currently used for risk assessment; and 4) the chronic RfD and MOEs are protectiv e of the thyroid-related findings seen in the Tier 1 pubertal

assay.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is NOT recommended for trifluralin since additional testing is unlikely to impact the current EPA established regulatory endpoints for human risk assessments.

No fish or amphibian EDSP Tier 2 testing is recommended since there is no convincing evidence for an interaction with estrogen, androgen or thyroid pathways in fish or amphibians. Additionally, it is noted that life cycle studies which eval uate reproduction with fathead minnow and sheepshead minnow are av ailable for triflurlin.

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard duck
are available for trifluralin. In the mallard duck study, effects on reproductive parameters were observed; no effects were observed in the bobwh ite quail study. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from trifluralin exposur e. Additional testing is not
recommended.TRIFLURALIN FINAL

Page 31 of 58 VI. References

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Adams, E., Bernhard, N., Jordon, W. (1992) A Ch ronic Toxicity Study of Trifluralin (Compound 036352) Administered Orally to Beagle Dogs for One Year (Supp.): Lab Project Number: D07190. Unpublished study prepared by Li lly Research Labs. (MRID 42447001)

Bathe, R. (1984) 12-Month Oral Toxicity (Feeding ) Study in Beagle Dogs: Trifluralin Substance Technical Grade (Code: HOE 38474 O H AT210): Project No . 008864: Report Part 1. Unpublished study prepared by Research & Consulting Co. (MRID 00151908)

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Beavers, J., Dukes, V., Jaber, M. (1987) Trifluralin Technica l: A One-generation Reproduction Study with the Bobwhite ( Colinus virginianus ): Project No. 228-101. Unpublished study prepared by Wildlife International Ltd. (MRID 40334706)

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Becker, H. (1985) Trifluralin, Substance Technica l Grade: Multiple Generation Study in the Rat:Diet Preparation, Stability, and Analysis of the Test Article: 1st Amendment to Report: Project No. 008875: Report No. A32777. Unpubl ished study prepared by Research & Consulting Co. AG. (MRID 00159619)

Byrd, R. (1984) A Teratology Study of Tr ifluralin (El-152, Compound 36352) Administered Orally to Charles River CD Ra ts: Study R08484. Unpublished study prepared by Lilly Research Labs. (MRID 00152419)

Byrd, R. (1984) A Teratology Study (II) of Trifluralin (El-152, Compound 36352) Administered Orally to Dutch Belted Rabbits: Study B01784. Unpublished study prepared by Lilly Research Labs. (MRID 00152421)

Coady, K., Sosinski, L. (2012). Trifluralin: Eval uation of Trifluralin in the Human Recombinant Aromatase Assay. Unpublished study prepared by Toxicology & Environmental Research andTRIFLURALIN FINAL

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Donaubauer (1986) Chronic Feedin g Study (24 Months) in Rats: Trifluralin: Study No. 680: Report No. 85.0302. Unpublished study prepared by Hoechst Aktiengellschaft, Pharma Forschung Toxikologie. (MRID 00162457)

Donaubauer (1986) Carcinogenicity St udy in Rats (28-month Feeding Study): Trifluralin: Study No. 680: Report No. 85.0087. Unpub lished study prepared by Hoechst Aktiengesellschaft, Pharma Forschung Toxikologie. (MRID 00162458)

Ellgehausen, H. (1984) Determination of Trif luralin Substance Tech Grade (Code: HOE 38474 O H AT210) in Rodent Feed: Pr oject No. 008875. Unpublished study prepared by Research & Consulting Co. (MRID 00151902)

Emmerson, J., Pierce, E., McGrath, J. (1980) The Chronic Toxicity of Compound 36352 (Trifluralin) Given as a Component of the Diet to Fischer 344 Rats for Tw o Years: Studies R-87 and R-97. Unpublished study received Sep 18, 1980 under 1471-35; submitted by Elanco Products Co., Div. of Eli Lilly and Co., I ndianapolis, Ind.; CDL:243289-A, 243290. (MRID 00044337)

Emmerson, J., Owen, N., McGrath, J. (1980) The Chronic Toxicity of Compound 36352 (Trifluralin) Given as a Component of the Diet to the B6C3F1 Mouse for 24 Months: Studies M9067 and M-9077. Unpublished study received Sep 18, 1980 under 1471-35; submitted by Elanco Products Co., Div. of Eli Lilly and Co., Indianapolis, Ind.; CDL:243291-A; 243292; 243293. (MRID 00044338)

Fink, R., Beavers, J., Brown, R. (1978) One- generation Reproduction Study—Mallard Duck: Compound 36352, Trifluralin: Proj ect No. 151-103. Final Rept. Unpublished study received Sep 20, 1983 under 1471-70; prepared by Wildlife International Ltd., submitted by Elanco Products Co., Div. of Eli Lilly and Co., I ndianapolis, IN; CDL:251256-A. (MRID 00131132)

Fink, R., Beavers, J., Brown, R. (1979) On e-generation Reproduction Study—Bobwhite Quail: Compound 36352, Trifluralin: Project N o. 151-102. Final report. Unpublished study received Sep 20, 1983 under 1471-70; prepared by Wildlife International Ltd., submitted by Elanco Products Co., Div. of Eli Lilly a nd Co., Indianapolis, IN; CDL:251256-C. (MRID 00131134)

Hoyt, J. (1986) A One-year Two-generation Re production Study in CD Rats Maintained on Diets Containing Trifluralin (EL-152, Co mpound 36352): Studies R06384 and R13984. Unpublished study prepared by Lilly Research Laboratories. (MRID 00162543)TRIFLURALIN FINAL

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Jungbauer, A., Beck, V. (2002) Yeast reporter system for rapi d determination of estrogenic activity. Journal of Chromatography B , 777:167–178.

Kojima, H., et al. (2004) Screen ing for Estrogen and Androgen Receptor Activities in 200 Pesticides by In Vitro Reporter Gene Assays Using Chinese Hamster Ovary Cells. Environ Health Perspect , 112:524–531

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LeBaron, M., Kan, H., Perala, A. (2011) Evaluation of trifluralin in the in vitro steroidogenesis assay. Unpublished study prepared by Toxicolo gy & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Study No.: 101187. (MRID 48619708)

LeBaron, M., Schisler, M., Visconti, N. (2012). Evaluation of trifluralin in an in vitro estrogen receptor binding assay. Unpublished study prepar ed by Toxicology & Environmental Research and Consulting, The Dow Chemical Compan y, Midland, MI. Laboratory Project ID: 111124. (MRID 48619704)

LeBaron, M., Schisler, M., Visconti, N.R. (2012). Evaluation of trifluralin in an in vitro androgen receptor binding assay. Unpublished study prepared by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project ID: 111098. (MRID 48619702)

Macek, K., Lindberg, M., Sauter, S., Buxton, K. S., Costa, P.A. (1976) Toxicity of Four Pesticides to Water Fleas and Fathead Minnows. Duluth, Minn.: U.S. Environmental Protection Agency, Environmental Research Laboratory. (EPA report no. EPA-600/3-76-099; available from: NTIS, Springfield, VA; PB-262 912). (MRID 05008271)

Marino, T., et al. (2011). Triflu ralin: A Fish Short-term Reproduction Assay with the Fathead Minnow, Pimephales promelas . Unpublished study performed by Toxicology & Environmental Research Consulting, The Dow Chemical Company, Midland, Michigan. Lab Study No.: 101098. (MRID 48619706)

Martin, M., et al. (2009) Profili ng the Reproductive Toxicity of Chemicals from Multigeneration Studies in the Toxicity Reference Database. Toxicological Sciences, 110(1):181–190.

Marty, M., Zablotny, C., Thomas, J. (2012). Tr ifluralin: Pubertal Development and Thyroid Function in Intact Juvenile/Per ipubertal Female Crl:CD(SD) Rats . Unpublished study prepared by Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Report No. 111078. (MRID 48674001)

Marty, M., Andrus, A., Thomas, J. (2012) Trif luralin: Pubertal Development and Thyroid Function in Intact Juvenile/Per ipubertal Male Crl:CD(SD) Rats . Unpublished study prepared by Toxicology and Environmental Research and Consulting, The Dow Chemical Company,TRIFLURALIN FINAL

Page 34 of 58 Midland, MI. Laboratory Report No.: 111079. (MRID 48674002)

Marty, M., Brooks, K. (2012). Trifluralin: Uterotrophic Assay in the Immature Female Crl:CD(SD) Rat. Unpublished study prepared by The Dow Chemical Company, Toxicology & Environmental Research and Consulting, Midl and, MI. Laboratory Project Study ID.: 101144. (MRID 48619709)

Marty, M., Marshall, V., Jeong, YC. (2011). Triflu ralin: Hershberger Assay in Castrated Adult Male Crl:CD(SD) Rats. Unpublishe d study prepared by Toxicology & Environmental Research and Consulting, The Dow Chemical Compan y, Midland, MI. Laboratory Study No.: 101133. Unpublished. (MRID 48619707)

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Petit, F., et al. (1997) Two co mplementary bioassays for screening the estrogenic potency of xenobiotics: recombinant for trout estrogen receptor and trout he patocyte cultures. Journal of Molecular Endocrinology, 19:321-335.

Saghir, S., Charles, G., Bartels, M., Kan, L., Dr yzga, M., Brzak, K., Calr k, A. (2008) Mechanism of trifluralin-induced thyroid tumors in rats. Toxicology Letters 180, pp. 38-45.

Schutz, Weigand, Kramer ( 1980) Repeated-dose (3 M onths) Oral Toxicity Study of the Active Substance HOE 38474 (Code: HOE 38474 O H AT204) Administered in the Feed to Rats: Report No. 618/80. Unpublished study prepared by Hoechst Aktiengesellschaft. (MRID 00151906)

Schutz, Donaubauer (1986) Comb ined Chronic Toxicity and Carcinogenicity Study in Rats (24/28-month Feeding Study): Summary and Evaluati on of the Results: Tr ifluralin: Study No. 680: Report No. 86.0092. Unpublished study prepar ed by Hoechst Aktiengesellschaft, Pharma Forschung Toxikologie. (MRID 00162456)

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Suter, P., Horst, K., Vogel, W., et al. (1987) Oncogenicity Study with Trifluralin Active Ingredient Technical (HOE 38474 OH AT210) in Mice: Supplem entary Data in Support ofTRIFLURALIN FINAL

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Page 36 of 58 APPENDIX 1. EDSP Ti er 1 Screening Assays

Amphibian Metamorphosis A ssay (AMA); (OCSPP 890.1100)

The 21-day assay (MRID 48619701) of trifluralin (97.1% pu rity) on amphibian metamorphosis of the African clawed frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80/control and treatment group; 20/replicate) were exposed to a negative c ontrol and solvent contro l (0.1 mL/L dimethyl formamide, DMF) and nominal trifluralin concentrations of 0.00300, 0.0300, and 0.300 mg a.i./L. The reviewer-calculated time-weighted averag e (TWA), measured concentrations were <0.000181 (<LOQ; controls), 0.00264, 0.0262, and 0.232 mg a.i./L. The test system was maintained at 21.3 to 22.9°C and a pH of 7.1 to 7.4.

Unless otherwise indicated in this DER, all effects are reported based on comparison to the negative control.

No treatment-related mortalities or clinical si gns were observed during this study. On Day 6, one tadpole in the high treatment group was observed rest ing at the surface. In the same vessel, one tadpole was observed with abnormal suspension and appeared to have internal bleeding on Days 12 and 13 and was observed resting on the surf ace on Day 14. These observations were not
considered to be treatment-relate d and may have been due to physical harm to a single tadpole. At test termination, spinal curvature (bent tail) was noted in both controls and in all treatment groups; the percentage of affected tadpoles ranged from 2 to 13%.

Day 7 body weight and snout-vent length (SVL) were significantly reduced (p<0.05) by 17 and 39%, respectively at the high trea tment level, while Day 7 SVL was significantly increased (p <0.05) by 9% at the mid treatment level relative to the negative control. Day 21 body weight and SVL were comparable (p>0.05) between trifluralin treatment groups and the negative control.

Trifluralin had no effect (p>0.05) on Day 7 normalized (for snout -vent length) hind-limb length (HLL). Day 21 normalized HLL was significantly in creased (p<0.05) in the solvent control and low and mid treatment levels by 16, 25, and 18% , respectively, compar ed with the negative control. Day 21 normalized HLL was significantly reduced (p<0.05) by 20% at the high treatment level compared to the negative control. There were no effects on Day 7 or Day 21 NF developmental stage. No asynchr onous development was observed.

There were no apparent treatment-related effects on thyroid histopathology. Follicular cell height
and area were not determined.

A total of 21 individuals were excluded from th e analysis of Day 21 growth endpoints because NF stage exceeded 60 (“late stage”): these included one tadpole fro m the negative control, four fromTRIFLURALIN FINAL

Page 37 of 58 the solvent control, seven and nine tadpoles in the low and mid treatment levels, respectively, and none in the high treatment level. While the occu rrence of late stage tadpoles was not analyzed statistically, the pattern of occurre nce is at least superficially cons istent with the observations of greater Day 21 normalized HLL in the lower two treatment levels (suggesting advanced development) but smaller Day 21 normalized HLL at the highest treatment level when compared to the negative control.

Statistically significant solvent effects were observed compared to the negative control. For Day 21 normalized HLL, a significant increase (p=0.05) was observed in the solvent control which was similar in magnitude to the increase obser ved in the low and mid treatment groups. When compared to the solvent control, there were no differences at th e low and mid treatment groups for Day 21 normalized HLL; however, Day 21 normali zed HLL at the high treatment level was significantly reduced (p<0.05). A dditionally, there were signifi cant increases (p<0.05) in the solvent control relative to the negative contro l in Day 7 SVL and Day 21 body weight. For both of these endpoints, the low and mid treatment levels were also increased re lative to the negative control (not all effects significan t at p=0.05), while the high treatment level was decreased relative to the negative control. Comparisons to the so lvent control showed signi ficant reductions (p<0.05) for Day 7 SVL and Day 7 body weight in the high treatment group. Given th e significant effect (p<0.05) at all treatment levels for Day 21 normalized HLL which cannot be explained completely by solvent interference, there is ev idence to suggest that trifluralin may affect developmental endpoints associated with reduced growth in tadpoles exposed to the treatment levels and conditions of this study.

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

In an androgen receptor (AR) binding a ssay (MRID 48619702), ventral prostate cytosol homogenate from Sprague Dawley rats was used as the source of AR to perform a competitive binding experiment, which was conducted to measur e the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentratio ns (logarithmic increase from 10

− 10

to 10

− 3 M) of trifluralin (% purity: 97.1; Lot#: DKO-B916-33, TSN100656). Ethanol was used as solvent at a final assay concentr ation of <3%. A total of 3 runs were performed, and each run included dexamethasone as a weak positive contro l, and R1881 as the ligand reference standard.

Although no data from a saturation binding expe riment were provided, a saturation binding experiment was carried out by the performing la boratory with this batch of cytosol (MRID 48614301). A summary of these results indicate th at acceptable responsiveness was determined for all parameters. In the saturation binding experiment, the maxi mum binding capacity (B max ) was 3.245 fmol/100 μg protein and the dissociation constant (K d ) was 0.4641 nM. These values were below the range of values from the validation studies (B max expected range = 7 to 16 fmol/100 μg protein and the K d expected range = 0.685 to 1.57 nM . Nonspecific binding as a percent of total binding was >20 % across the entire concentration range for the saturation bindingTRIFLURALIN FINAL

Page 38 of 58 assay. Only a single run of the saturation bindi ng experiment was conducted on the batch of cytosol preparation used in th e competitive binding experiment.

In the competitive binding experiment, the estimated mean log IC 50 s for R1881 (

− 8.996 M) and the weak positive control, dexamethasone (

− 4.522 M), were within the expected ranges. The mean RBA for the weak positive control was 0.0034% . Confidence in these numbers is high due
to the small variation. The solv ent control responses indicated no drift in the study assay. All performance criteria were generally met.

Trifluralin had no effect on specific binding of the [ 3 H]-ligand to the AR; therefore, a log IC 50 and
RBA could not be calculated.

Based on the results from the three runs, triflurali n is classified as a Non-Binder in the Androgen Receptor Binding Assay.

Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP19) assay (MRID 48619703), trifluralin (97.1% a.i. Lot # DKOB916-33, TSN100656) was incubated with human recombinant aromatase and tritiated androstenedione ([1 β

3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]-ASDN) in 1% dimethyl sulfoxide (DMSO) at log concentrations from 10

− 10 to 10

− 4.5 M for 15 minutes to assess the potential of trifluralin to inhibit aromatase activity.

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC). Thr ee runs were conducted and each run included a full activity control, a background activity c ontrol, a positive control series (10

− 10 to 10

− 5 M) using a known inhibitor (4-hydroxyandrostenedione; 4-OH AS DN), and the test chemical series (10

− 10 to
10

− 4.5 M) with three repetitions per concentratio n. At higher tested concentrations (10

− 4 , 10 –3.5

, and 10

− 3 M), trifluralin was vi sibly insoluble.

Aromatase activity in the fu ll activity controls ranged from 0.133 to 0.225 nmol·mgprotein

− 1 ·min

− 1 for the three test runs, with a m ean and standard deviation of 0.192 ± 0.043 nmol·mg-protein

− 1 ·min

− 1 . The response of each full activity control within a run was between 90 to 110% of the average full activ ity. Activity in the background controls was 0.23 to 0.31% of the full activity controls.

For the positive control substance (4-OH ASDN), aromatase activity results were within the recommended ranges for the performance criteria. The estimated log IC 50 for 4-OH ASDN averaged

− 7.27 M and the Hill slope was

− 0.93.TRIFLURALIN FINAL

Page 39 of 58 For trifluralin, aromatas e activity averaged 0.198 ± 0.030 nmol·mg-protein

− 1 ·min

− 1 at the lowest tested concentration (10

− 10 M) and 0.191 ± 0.029 nmol·mg-protein

− 1 ·min

− 1 at the highest soluble concentration tested (10

− 4.5 M). As a percent of the control, ar omatase activity for trifluralin was

≥ 96.50% at all concentrations from 10 -10 to 10 -4.5 M.

Based on the data from the average response curve, trifluralin is classified as a Non-inhibitor of aromatase in this assay.

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

In an estrogen receptor (ER) binding assa y (MRID 48619704), uterine cytosol from Sprague Dawley rats was used as the source of ER for a competitive binding experiment, which was conducted to measure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of increasing concentrations (logarithmic increase from 10

− 10 to 10

− 3 M) of trifluralin (97.1% purity; Lot#DKO-B916-33, TSN100656). Ethanol was used as the solvent vehicle at a
final concentration of <3%. A total of three runs were performed, and each run included 19- norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17- β

estradiol as the natural ligand reference material.

A saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and wa s functioning with appropriate affinity for the
radiolabeled reference estr ogen prior to routinely conduc ting ER competitive binding experiments. Data from the saturation binding ex periment were not included in the original report; however, the binding data were reported in a separate report for the batch of cytosol that was utilized in the assay. In the saturati on binding experiment, the maximum binding capacity (Bmax) was 59.28 fmol/100 μ g protein and the dissociation c onstant (Kd) was 0.1032 nM. The Bmax was within the expected range of 10 to 150 fmol/100 μ g protein, and the Kd was within the expected range of 0.03 to 1.5 nM. Nonspecific bi nding as a percent of total binding was 1.7% to
8.6% across the entire concentrati on range in the saturation bindi ng assay. Only a single run of the saturation binding experiment was conducted on the batch of cytosol preparation used for the competitive binding experiment.

In the competitive binding experiment, the estimated mean log IC 50 was

− 8.934 M for 17 β

estradiol and

− 5.442 M for the weak positive control. The mean Relative Binding Affinity (RBA) was 0.032% for the weak positive control.

Trifluralin had no effect on specific binding of the [ 3 H]-ligand to the ER; therefore, a log IC 50 and RBA could not be calculated for trifluralin. Spec ific binding was >75% at all concentrations of
trifluralin tested.

Based on the results of the three runs, trifluralin is classified as Not Interactive in the Estrogen Receptor Binding Assay.TRIFLURALIN FINAL

Page 40 of 58 ER α Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300)

In an estrogen receptor transcriptio nal activation assay (MRID (48619705), hER α -HeLa-9903
cells cultured in vitro were exposed to trifluralin ( 97.1%, Lot #s DKO-B916-33, TSN100656) at logarithmically increasing concentrations from 10 -10 to 10 -4 M in DMSO. Four independent runs were performed using 96-well plat es; each concentration was tested in 3 wells/plate. Cells were exposed to the test agent for about 24 hours to induce reporter (lucifer ase) gene products. Luciferase expression in response to activati on of the estrogen receptor was measured after
addition of luciferase substrate and detection with a luminometer.

Trifluralin was tested up to the limit of solubility, 10 -4 M. Performance criteria were generally met for all reference chemicals, except values for 17 α -methyltestosterone were consistently lower than the expected range, indicating increased sensitiv ity of the assay to weak agonists. Trifluralin

RPC Max was <0%, 18%, 16.6% and 13.7% in runs 1, 2, 3, and 4, respectively; associated PC Max was 10 -4 M. The response was dose-de pendent. Because the RPC Max > PC 10 in three out of four assay runs, trifluralin was considered positive 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 (MRI D 48619706) of trifluralin with fathead minnows ( Pimephales promelas) was conducted under flow-through conditions. Adult fish (20 spawning groups; 2 males and 4 females in each group; 6 mont hs old), were exposed to trifluralin (97.1% purity) at nominal concentrati ons of 0 (negative control), 0 (solvent control; 0.05 mL/L dimethylformamide; DMF), 0.000350, 0.00350, and 0.0350 mg a.i./L; time-weighted average (TWA), measured concentrations were <0.00003 (<LOQ, controls), 0.000195, 0.00198, and 0.0215 mg a.i./L. The test system was maintained at 24.5 to 25.6 o C and a pH of 7.4 to 7.6.

Comparisons between the negative and solvent c ontrol groups revealed no significant differences (p>0.05) for any endpoint. Unless otherwise indicated in this DER, all effects are reported based on comparison to the negative control.

There was no significant effect (p>0 .05) on fish mortality. Clinical signs of toxicity were present at the high treatment level, with five males show ing partial loss of equilibrium, very thin bodies, partial paralysis, and/or a bent spine. The same number of females appeared to be bloated and/or partially paralyzed with bent tails. These clinical effects were in c ontrast to no more than two fish from the control groups appearing extremely dark in color or aggressive. Female body weight was significantly increased (p<0.05; D unnett’s) by 10% in the high tr eatment group compared to the negative control. Male body wei ght was not significantly differe nt (p>0.05) from the negativeTRIFLURALIN FINAL

Page 41 of 58 control. Total length for males and females wa s not significantly different (p>0.05) from the negative control.

Average spawning frequency and fecundity in contro ls were at least every 4 days (except for one replicate in the solvent contro l) and 36 eggs/female/ reproductive day (range: negative control, 24.3-50.6; solvent control, 17.2-41.5 egg/female/rep roductive day), respectively; fertilization success in both controls was 99%. Fertility was significantly reduced (p<0.05; JonckheereTerpstra) by 0.5 and 4% at the lo w and high treatment levels, respec tively, relative to the negative control. Fecundity was signifi cantly decreased (p<0.05; Mann Whitney U) by 87% at the high treatment level compared to the negative control.

Male nuptial tubercle scores were significantly reduced (p<0.05; Jonckheer e-Terpstra) (p<0.05) at the high treatment level (median tubercle score: 20 ) relative to negative co ntrol (median tubercle score: 31); no tubercles were noted for fema les. There was a signifi cant monotonic decrease (p<0.05; Jonckheere-Terpstra) of the male gonado- somatic index (GSI) at all treatment levels, ranging from 29% (low treatment) to 39% (high tr eatment) relative to the negative control. Female GSI was not significantly different (p>0.05) between the negative control and treatment groups. Observations of additional secondary sexual characteristics were re ported but results were not presented.

Plasma vitellogenin (VTG) was not significantly different (p>0.05) for males or females. There was a significant increase (p<0.05; Jonc kheere-Terpstra) of 78% in male 17 β -estradiol at the high treatment level relative to the negative control. Male plasma testosterone (T) was not significantly different (p>0.05). Plasma 17 β -estradiol and testosterone we re not measured in females.

Male gonadal development was altered at the high treatment level with a greater proportion of testes categorized at the late spermatogenic st age compared to the mid-spermatogenic stage of testes in control fish and those exposed to lowe r levels of trifluralin. For females, there was increased incidence of severe increased oocyte atresia in the high treat ment group. Finally, there
was an increase in minimal subacute multifocal ovarian inflammation in females exposed to the high treatment level.

Hershberger Assay; (OCSPP 890.1400)

In a Hershberger assay (MRID 48619707) screening for androgenic activity, trifluralin (97.1%, lot no. DKO-B916-33, TSN100656) in corn oil was administ ered daily via oral gavage to groups of 55-day old castrated male Sprague Dawley rats (7 /group) at dose levels of 0 (vehicle), 50, 150, or 450 mg/kg/day for 10 consecutive days. An andr ogenic positive control group consisted of seven castrated male rats exposed to testosterone propiona te (TP) at 0.4 mg/kg/ day via subcutaneous (s.c.) injection in conjunction wi th corn oil via oral gavage.TRIFLURALIN FINAL

Page 42 of 58 To screen for possible anti-androgenic activity, additional groups of 55-day old castrated male Sprague Dawley rats (7/group) were dosed daily via oral gavage with trifluralin in corn oil at 50, 150, or 450 mg/kg/day for 10 consecutive days in conj unction with a daily s.c. injection of TP at 0.4 mg/kg/day. The same seven animals used as pos itive controls in the androgenic portion of the study served as negative controls in the anti-androgenic portion of the study, and seven castrated male rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg/day for 10 days in conjugation with a daily s.c. dose of TP (0.4 mg/ kg/day) served as positive controls.

For both components of the assa y, body weights and food consump tion were determined daily. The animals were terminated approximately 24 hours after the final dose administration. At necropsy, the five androgen-dependent tissues (sem inal vesicles with coag ulating glands, ventral prostate, levator ani-bulbocavernosus (LABC), Co wper’s glands, and glans penis), liver, adrenal glands, and kidneys were excised, examined macr oscopically, and weighed. Serum testosterone levels were measured in the anti-androgen assay.

No treatment-related effects were observed on mo rtality, clinical signs of toxicity, or gross pathology.

In the androgen agonist assay at 450 mg/kg/da y, body weights were decrea sed (not statistically significant [NS]) throughout the study ( ↓ 6–8%), compared to vehicle controls, and body weight gains were decreased (p ≤ 0.05) at each measured interval (Days 1–4, 1–7, and 1–11; ↓ 31–88%). In the anti-androgen assay at 450 mg/kg/day with TP, compared to negative controls, decreased (p ≤ 0.05) body weights were observed on Days 7 ( ↓ 8%) and 11 ( ↓ 10%), and body weight gains were decreased (p ≤ 0.05) at each measured interval ( ↓ 46–65%). Overall body weight gains (Days 1-11) were decreased (p ≤ 0.05) at 150 mg/kg/day with TP ( ↓ 22%). All other body weights and body weight gains in the triflurali n-treated groups were similar to controls in both the androgen agonist and anti-androgen assays.

In the androgen agonist assay at 450 mg /kg/day, food consumption was decreased (p ≤ 0.05) during the first 4-day in terval (Days 1–4; ↓ 35%), compared to vehicle cont rols. In the anti-androgen assay at 450 mg/kg/day with TP, compared to negative controls, decreased food consumption was observed during each interval, Days 1–4 ( ↓ 31%; NS), 4–7 ( ↓ 12%; p ≤ 0.05), and 7–11 ( ↓ 14%;

p ≤ 0.05). At 150 mg/kg/day with TP, food consumption was decreased (p ≤ 0.05) by 8% during Days 7–11, compared to negative controls. All ot her food consumption valu es in the trifluralintreated groups were similar to controls in both the androgen a gonist and anti-androgen assays.

Liver weigh ts were increased (p ≤ 0.05, except as noted) by triflu ralin treatment in both the androgenic and anti-androgenic portions of the study, by 16% at 150 mg/kg/day (NS without TP) and by 28 and 36% at 450 mg/kg/day (w ith and without TP, respectively).

For the androgenic portion of the study, there were no treatment-rel ated effects on the weights of any of the accessory sex tissues (seminal vesicl es, ventral prostate, LABC, Cowper's glands, orTRIFLURALIN FINAL

Page 43 of 58 glans penis), adrenals, or kidney at any dose of trifluralin.

For the anti-androgenic portion of the study at 450 mg/kg/ day with TP, decreases (p ≤ 0.05) were noted in the weights of the seminal vesicles with coagulating glands ( ↓ 21%), ventral prostate ( ↓ 39%), and LABC ( ↑ 21%). Compared to negative controls , serum testosterone was decreased by 27% (NS) at 150 mg/kg/day with TP and by 36% (p ≤ 0.05) at 450 mg/kg/day with TP. Body weights were decreased ≥ 10% at the dose (450 mg/kg/day) where the effects on the androgensensitive tissue weights were obser ved, indicating overt toxicity at this dose. Furthermore,

The increase in liver weight, si gnificant decrease in serum testos terone level, and results from previously performed studies, suggest that the anti-androgenic effects of trifluralin may be secondary to enhanced testoster one metabolism and clearance rather than direct interaction with the androgen receptor.

TP administration produced increases (p ≤ 0.05) of 20–48% in body weight gains at all intervals (Days 1–4, 1–7, and 1–11) and an increase in body wei ght compared to vehicle controls at Day 11 ( ↑ 8%). Food consumption was increased (p ≤ 0.05) by 16 and 18% in TP-t reated animals relative to vehicle controls during Days 4–7 and 7–11, resp ectively. Only a minor increase (NS) in food consumption was noted in the TP-treated animals during the first interval, Days 1–4. For the TP positive control group, there were treatment-related increases (p ≤ 0.05) in the weights of the seminal vesicles with coagulating glands ( ↑ 1452%), ventral prostate ( ↑ 1328%), LABC ( ↑ 199%), Cowper’s glands ( ↑ 580%), and glans penis ( ↑ 66%), indicating that the test system was sensitive to an androgenic response. All these findings were expected androgenic re sponses of the positive
control.

There was no effect of flutamide on body weights, body weight gains, or food consumption. For the flutamide positive control group, ther e were treatment-related decreases (p ≤ 0.05) in weights of the seminal vesicles with coagulating glands ( ↓ 70%), ventral prostate ( ↓ 66%), LABC ( ↓ 44%), Cowper’s glands ( ↓ 57%), and glans penis ( ↓ 14%) compared to weights in the negative control group. These data indicate that the test system was sensitive to an anti-androgenic response.

In the anti-androgen assay, all %CV levels were less than the guide line specified maximums. In the androgen agonist assay, only th e ventral prostate in the vehicl e control group did not m eet the guideline criteria for maximum %CV (49.0% vs 45% maximum).

Trifluralin was negative for androgenic activity in the Hershberger assay as no statistically significant increases in organ weights were seen in two or more of the five androgen sensitive tissues in the androgen agonist assay. Triflu ralin was positive for anti- androgenicity in the Hershberger assay based on statistically significant decreases in organ weight s seen in three of the five androgen sensitive tissues in the androgen antagonist assay. However, the target organ weight reductions were observe d at a dose (450 mg/kg/day) th at exhibited overt toxicity [decreased body weights (10%) and body we ight gains (46%)], which confounds theTRIFLURALIN FINAL

Page 44 of 58 interpretation of the study.

Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48674001), 16 Crl:CD(SD) rats/dose group were treated daily via oral gavage with trifluralin (97.1% a.i., Lot # DKO-B916-33, TSN 100656) in corn oil at doses of 0, 25, 75 and 250 mg/kg/day from post- natal day (PND) 22 to 42. Animals were examined for vaginal opening (VO) daily begi nning on PND 22, and age and weight at day of
attainment were recorded. Af ter sacrifice on PND 42, blood was collected for clinical chemistry analyses, including analysis of total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) using immunoassays. Liver, pituitary, adrenals and ur ogenital organs were collected and weighed, and microscopic examinations were performed on the ovaries, uterus, thyroid, and kidneys.

No treatment-related effects were observed on survival, clinical si gns, age of attainment of VO, body weight at VO, final body weight, or gross or histological pathology. No treatment-related effects were noted on cycle status at necropsy, mean age at first estrus, mean cycle length, percent cycling, or percent regularly cycling.

At 250 mg/kg/day, body weights we re decreased (p<0.05) by 7% on PND 28 and 8% on PND 32. The mean terminal body weight on PND 42 was decr eased (not sta tistically significant; NS) by 5%. Body weight gains were decreased (p<0.05) over the intervals from PND 22-25 (30%), PND 22-28 (21%), and PND 22-32 (17%). After this initial decrease, the body weights of recovered slightly such that the decreas e in body weight gains over the entire dosing period (PND 22-42) was 8% (NS). This effect on body weight was transient and not c onsidered adverse; there was no evidence that the delayed growth impacted se xual development. There were no effects on body weights or body weight gains at the low or intermediate dose.

Absolute, adjusted, and relative liver weights were increased (p< 0.05) at the intermediate dose by 11-13% and at the high dose by 17-24%. There was no gross or cl inical chemistry evidence to support hepatotoxicity. Ther e were no other effects of treatment on organ weights.

Serum T 4 was decreased (p<0.05) in the mid- and high-dose groups by 33% and 45%, respectively. TSH was increased (p<0.05) by 73 % at the high dose. No effect was noted on thyroid weight. In the thyroid at the high dose, increased (p<0.05) incidences of higher follicular cells (Grade 3; 8/16 treated vs 1/16 controls) with correspondingly smaller colloid areas (Grade 3; 8/16 treated vs 1/16 controls) were observed. No differences in these parameters were observed in the low- and mid-dose groups.

The high dose was considered to be an adequa te dose based on increased serum creatinine, increased liver weights, and changes in serum T 4 and TSH levels.TRIFLURALIN FINAL

Page 45 of 58 Male Pubertal A ssay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48674002), 16 Sprague Dawley Crl:CD(SD) rats/dose group were treated daily via oral gavage with tr ifluralin (97.1% a.i., Lot # DKO-B916-33, TSN100656)
in corn oil at doses of 0, 25, 75 or 250 mg/kg/day from post-natal day (PND) 23 to 53. Animals were examined for preputial separation (PPS) da ily beginning on PND 30, and age and weight at day of attainment was recorded. Following sacr ifice on PND 53, blood was collected for analysis of clinical chemistry parameters, including total serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH), which were anal yzed using immunoassays. Liver, pituitary, adrenals, thyroid, and urogenital organ weights were recorded and microscopic examinations were performed on the testes, epid idymides, thyroid, and kidneys.

No treatment-related effects were observed on mo rtality, clinical signs, or gross pathology. Age and body weight at PPS in the treated groups were sim ilar to controls. In th e thyroid, colloid area and follicular cell height scores were similar in the treated groups to the control group. No treatment-related findings were noted in the epididymis and testis.

Body weights and body weight gains at 250 mg/kg/day were not signifi cantly (NS) different from controls on PND 53; however, body weights at this dose were decreased on PND 30 and 37 by 11 and 9%, respectively (p<0.05), and body weight ga ins were decreased (p<0.05) for the periods PND 23-30, 23-37, and 23-42 by 24%, 14%, and 11%, respectively. Although this effect on body weight was transient and not considered adve rse, the delayed growth could affect sexual development. There were no effects on body weights or body weight gains at 25 or 75 mg/kg/day.

Absolute, adjusted, and relative liver wei ghts were increased (p <0.05) by 13-16% at 75 mg/kg/day and by 29-37% at 250 mg/kg/day. At 25 mg/kg/day, relative liver weights were increased (p<0.05) by 6%. There was no gross or clinical chemistry evidence to support hepatotoxicity, and histology was not performed. The slight effect at 75 mg/kg/day and the substantial effect at 250 mg/kg/day are considered equivocal findings, because this effect could be an adaptive response.

At 250 mg/kg/day, decreased (p< 0.05) adjusted organ weights were noted in the following androgen-sensitive tissues: dorsolateral prostate (12%), levator ani-bulbocavernosus muscle complex (LABC, 13%), left epididymis (14%), and right epididymis (11%). Similar effects were noted on the unadjusted weights of these organs . Additionally, a minor decrease (8%; p<0.05) was noted in the right epididymis at 75 mg/kg/day. These effects were slight and may have been related to the delayed growth observed at 250 mg/kg/day.

Serum T 4 levels were decreased (p<0.05) dose-de pendently by 25, 42, and 58% at 25, 75 and 250 mg/kg/day, respectively. At 250 mg /kg/day there also appeared to be an effect on serum TSHTRIFLURALIN FINAL

Page 46 of 58 (increased by 74%) and testos terone (decreased by 30%); ho wever, neither difference was statistically significant, nor were there any effects observed on t hyroid weight or histopathology.

At 250 mg/kg/day, creatinine was increased (p<0 .05) by 50%, which was consistent with the probe study. Alkaline phosphatase (ALP) was s lightly decreased (p<0.0 5) by 17%; however, the toxicological significance of a s light decrease is unclear. Calcium and sodium were also increased (p<0.05) by 4% and 1%, respectively, at 250 mg/kg/day, but these minor effects were
not considered adverse.

Increased hyaline droplet formation within th e kidney proximal convoluted tubule (multifocal) was observed in the 25 (13/16, very slight severity), 75 (13/15, ve ry slight), and 250 (15/15, very slight to slight) mg/kg/day groups compared to the cont rol (1/16, very slight ). However, this finding was not associated with degenerative or cytotoxic changes in the kidneys. This was the only treatment-related histological lesion, and there was no other ev idence of kidney toxicity.
There were no treatment-related hi stopathological changes in the thyroid, testes, or epididymides.

The high dose was considered to be an adequa te dose based on increased serum creatinine, increased liver weights, and decreased serum T 4 levels.

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48619708), H295R cells cultured in vitro in 24-well plates were
incubated with trifluralin (97.1% purity; LOT # DKO-B 916-33, TSN100656) at log concentrations from 0.0001 to 100 μM for 48 hours in triplicate in three inde pendent experiments. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.1%.

Testosterone and estradiol levels were meas ured using LC-APPI-MS/MS. A Quality Control (QC) plate was run concurrently with each independe nt run of a test chemic al plate to demonstrate that the assay responded properly to positive contro l agents at two concentration levels. The positive controls included a known inhibitor (pro chloraz) and a known inducer (forskolin) of
estradiol and testos terone production.

Guideline acceptability recommendations and requir ements were generally met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptabl e reproducibility (low %CV), and appropriate induction an d inhibition with positive contro ls. The exceptions were the minimum basal hormone production level of 500 pg/mL testosterone was not met in the solvent control (SC) in the testosterone assay for 2 runs (491 and 454 pg/ mL), and cells with a passage
number of 11.5 instead of 10.5 we re used in a single run.

No effects of trifluralin were observe d on testosterone levels. Decreased (p ≤ 0.05) estradiol levels were observed at 0.0001 μM trifluralin in Runs 1 and 2 (0.8-fold the SC) and at 0.001 μM in Run 2 (0.9-fold). These changes were minimal, unrelat ed to dose, and not repr oducible across all threeTRIFLURALIN FINAL

Page 47 of 58 runs; therefore, the apparent effect on decrea sed estradiol level was not considered treatment related. However, statistically significant increases (p ≤ 0.05) of estradiol levels were noted at 10 and 100 μM in all runs (1.2–1.4-fold). Although the magnitude of increase was slight, these effects of increased estradiol levels were repr oducible and were considered treatment related.

Based on hormone responses in each of the inde pendent runs, triflurali n treatment resulted in statistically significant and reproduc ible increases in estradiol pr oduction, but triflu ralin treatment did not result in statistically significant and repr oducible alterations in testosterone production.

Uterotrophic Assay; (OCSPP 890.1600)

In an uterotrophic assay (MRID 48619709) conducted to screen for potential estrogenic activity, trifluralin (97.1% a.i., Lot # DKO-B916-33, TSN100656) in corn oil was administered daily for 3 consecutive days via oral gavage to groups of si x immature female Sprague Dawley rats at dose levels of 0 (vehicle), 25, 75, or 225 mg/kg/ day on post-natal days (PND) 19-21. A positive control group was treated with a daily dose of 17 α -ethynyl estradiol (EE) at 10 μ g/kg/day by oral gavage. Body weights were determ ined daily. All animals were examined for precocious vaginal opening, and then terminated and necropsied on PND 22 approximately 24 hours after the final dose to determine wet and blotted uterine weights.

All animals survived until scheduled termination. No clinical signs of toxicity were observed, and no precocious vaginal opening was noted in the trifluralin-treated females. At 225 mg/kg/day, overall body weight gains were decreased (p<0.0 5) by 34%. Body weights in the low- and middose groups, and the positive cont rol group were comparable to the vehicle controls throughout the duration of the assay. Uterin e weights in the trifluralin treate d groups were comparable to the
vehicle controls. Absolute wet and blotted ut erus weights for the positive control (EE) group were increased (p<0.05) by 707% an d 396%, respectively, as expected. !!

The doses tested were determined to be adequa te based on a 34% decrease in overall body weight gain in the high-dose group.

No statistically significant changes were seen in the uterine weight in this assay. Trifluralin was negative in the uterotrophic assayTRIFLURALIN FINAL

Page 48 of 58 APPENDIX 2. Other Scientifica lly Relevant Information (OSRI)

90-Day (Subchronic) Oral T oxicity in Rodents (Rat)

A subchronic oral toxicity study ( MRID 40138301 ) was conducted in Fi scher 344 rats with trifluralin (purity and Lot/Batch not repo rted) concentrations of 0.005, 0.02, 0.08, 0.32 or
0.64% in the diet. The NOAE L was 0.005% (50 ppm or 2.5 mg/kg/day). The LOAEL was 0.02% (10 mg/kg/day) based on increased hyaline dr oplet formation in cortical cells, increased total urinary protein excretion, and changes in ur ine color and clarity. Many of the effects were reversible within a six-week recovery period.

A second subchronic oral toxicity study ( MRID 00151906 ) in Wistar rats a nd trifluralin (purity and Lot/Batch not reported) do ses of 0, 800, 2000, or 5000 ppm indicated that the NOAEL was less than 800 ppm (40 mg/kg/day) due to reductions in relative liver and pituitary gland weights
at all doses tested.

Prenatal Developmen t in Rodents (Rat)

In a prenatal developm ent study in the rat ( MRID 00152419 ), Charles River rats were administered gavage doses of 0, 100, 225, 475 or 1000 mg/kg/day of trifluralin (purity and Lot/Batch not reported) on gestation days 6-15. The maternal toxicity LOAEL was 475 mg/kg/day due to reduced weight gain and food consumption; the maternal toxicity NOAEL was 225 mg/kg/day. The developmental toxicity LOAEL was 1000 mg/kg/day due to reduced mean fetal body weight; the developmental toxicity NO AEL was 475 mg/kg. No teratogenic effects
occurred in the rat.

Prenatal Development in Non-Rodents (Rabbit)

In a prenatal developmen t study in the rabbit ( MRID 00152421 ), Dutch Belted rabbits were given oral doses of 0, 100, 225 or 500 mg/kg/da y of trifluralin (purity and Lot/Batch not reported) on gestation days 6- 28. The maternal toxicity LO AEL was 225 mg/kg/day due to anorexia, cachexia and resulting abortion; the maternal toxicity NOAEL was 100 mg/kg/day. The developmental toxicity LOAEL was 500 mg/kg/ day based on depressed fetal weight and an
increased number of fetal runt s; the developmental toxicity NOAEL was 225 mg/kg/day. No teratogenic effects occurred in the rabbit.

Two-Generation Reproduc tion in Rodents (Rat)

In a multi-generation reproduction toxicity study ( MRIDs 00151901, 00151902, 00151903, and

00159619 ), trifluralin (>99% purity; Lot/Batch #AT 210) was administered continuously in the diet to outbred Wistar KFM-Han SPF quality rats at nominal doses of 0, 200, 650, or 2000 ppm (equivalent to approximately 0, 10, 32.5 and 100 mg /kg/day; calculated by reviewers using 1 ppm = 0.05 mg/kg). The P animals (30/se x/dose) were given test article diet formulations for 80 daysTRIFLURALIN FINAL

Page 49 of 58 prior to mating to produce the F1a litters. Ten days after weaning of the F1a litters, a second mating of the P generation (with different pairs) was conducted to produce the F1b litters. After weaning, 26 rats/sex/dose from the F1b litters were selected to be F1 parents, and were given the
same diet concentration as their dams for at least 100 days prior to mating. F1 parents were bred (siblings not paired) to produce F2a and F2b litters by using the same procedures as described for their parents.

Parental mortalities included one mid-dose P genera tion male and one F1 ge neration female each from the control, mid-, and high -dose groups. The death for the mid-dose female was attributed to acute renal failure; no cause of death was stated for the other mo rtalities. Yellow discoloration of the urine was reportedly dose- dependently increased; however, no data were provided. Yellow discoloration of the adipose tissue , characteristic of dinitroaniline treatment, also was noted in the mid-dose females and high-dose males and fema les of both generations. Body weight was decreased (p ≤ 0.05) in the F1 females during pre-mating (6 -15%), gestation (7-9%), and lactation (4-9%) for both litters. Terminal body weight in F1 males and females was decreased (p ≤ 0.05) by 6%. Decreases in food consumption were se en occasionally in parental animals during acclimation and in females during gestation, but were more preval ent in dams during lactation. Relative (to body) liver weights were increased (p ≤ 0.01) in the mid-dose P males, and F1 males and females (6 to 10%), and high-dose P and F1 males and fe males (16-27%). In the parental males, relative kidney weights were increased (p ≤ 0.01) 6 to 14% at all doses in the P generation,
and increased 10% at the mid- a nd high-dose in the F1 generation . Relative testes weights were increased 8-13% (p ≤ 0.05) at the mid-dose (F1 generation ) and at the high-dose in both generations.

The only finding at 200 pp m was an increase (p ≤ 0.01) of 6% in relative kidney weight in the P generation males. The LOAEL fo r parental toxicity is 650 ppm (nominally equivalent to 32.5 mg/kg/day) based on increased lesions of the re nal proximal tubules in the F1 females and increased relative (to body) weight of the liv er and kidney (males). The NOAEL is 200 ppm (nominally equivalent to 10 mg/kg/day).

There was no adverse effect of treatment on pup mortality. Pup weights were decreased (p ≤ 0.05) 5 to 12% in the mid- and high- dose F1 litters; (ii) decreased (p ≤ 0.05) 3 to 8% in the high-dose F2a litter; and decreased (p ≤ 0.05) 2 to 7% in the mid- and high-d ose F2b litters. Litter size was decreased (p ≤ 0.05) by 13-16% in the high-dose F2a litt er on PND 0, and in the high- dose F2b
litter on PND 0 and 21. Relative liver weights were increased (p ≤ 0.05) in the mid-dose F2b females, and in the high-dose F2a animals and F2b females (6-13%). Relative kidney weights also were increased (p ≤ 0.01) 5% in the hi gh-dose F2b females, and relative testes weights were
increased 8% in males in both F1 litters. There were no effect s of treatment in the low-dose pups. The LOAEL for offspring toxicity is 650 ppm (nomin ally equivalent to 3 2.5 mg/kg/day) based on decreased pup weights in both gene rations and increased relative to body liver weight in the F2b females. The NOAEL is 200 ppm (equivalent to 10 mg/kg/day).TRIFLURALIN FINAL

Page 50 of 58 There were no effects of treatment on precoital interval, gestation length, behavior of dams during parturition and lactation, pup mortality, or the proportion of parent s that mated, became pregnant, delivered, or reared litters to weanin g. A LOAEL for reproductive toxicity was not observed; the NOAEL for reprod uctive toxicity is 2000 ppm (nominally equivalent to 100 mg/kg/day).

Two-Generation Reproduc tion in Rodents (Rat)

In a multi-generation reproduction toxicity study ( MRIDs 00162543 and 44135107 ), trifluralin (96.4% purity; Lot/Batch #554AP2) was administ ered continuously in the diet to outbred Crl:CD (SD) rats at nominal doses of 0, 200, 630, or 2000 ppm (0, 15, 47 and 148 mg/kg/day). The P animals (25/sex/dose) were gi ven test article diet formulati ons for 70 days prior to mating to produce the F1a litters. Females kept progeny (Fla) for 21 days postpartum; after weaning, 25/sex/group of F1a animals were se lected to become F1 parents. One weanling/sex/litter or the remaining pups was given a gross necropsy. The P parental animals we re again mated at 25 weeks of age and allowed to deliver and rear the F1b generation for 21 days postpartum. All Flb pups were sacrificed and given a gross necrop sy at weaning. At 36 weeks of age, all P parents were killed and given a gross necrops y, and reproductive tissu es and kidneys were collected for histopathological evaluation.

All animals survived the study, except for the de ath of a single P male in the low-dose group due to urolithiasis on Study Day 126. No treatment-related clinical effe cts were present, except for a reported dose-dependent, yellow di scoloration of the urine. Decreased body weights and body weight gains were seen in the high-dose P males; F1 males also had decreased body weights and body weight gains during the gr owth period in the mid- and high-dose groups, but only highdose male were affected during the reproduct ive period. Decreased body weights and body weight gains were also seen in the high-dose P and F1 females. Statistical significance was not reported, and body weight/body weig ht gain data were not availa ble for review. A significant (p<0.05) decrease in food consumption was noted for high-dose P males and females.

No treatment-related effects on fertility, matin g performance, number of pregnant females, gestation length/survival, litter size, pup survival , or sex ratio were obser ved. An apparent, dosedependent decrease in pup body size and weight was noted. A LOAEL for reproductive toxicity was not observed; the NOAEL for reproductive toxicity is 2000 ppm (equivalent to 148 mg/kg/day). The LOAEL for offspring toxicity is 630 ppm (equivalent to 47 mg/kg/day) based on small pup size in 3 litters. The NOAE L is 200 ppm (equivalent to 15 mg/kg/day).

Histopathological examinations were conducted on selected tissues ( i.e., vagina, uterus, ovaries, mammary glands, testes, epididymis, seminal vesi cles, prostate, kidneys, and all gross lesions) from control and high-dose animals. No treat ment-related histopathological effects were observed. The LOAEL for parental toxicity is 630 ppm (equivalent to 47 mg/kg/day) based on decreased body weight and food consumpt ion. The NOAEL is 200 ppm (equivalent to 15 mg/kg/day).TRIFLURALIN FINAL

Page 51 of 58 Two-Generation Reproduc tion in Rodents (Rat)

In a multi-generation reproduction toxicity study ( MRID 40405007 ), trifluralin (97.3% purity; Lot/Batch #5320) was administered continuously in the diet to CD(CRL) rats (25/sex/dose) at nominal doses of 0, 50, 450, or 4000 ppm (e quivalent to 3.9/4.7, 35/42, 295/337 mg/kg/day, M/F). The P animals were given test article di et formulations for 10 w eeks prior to mating to produce the F1a litters. In a sub-study to evalua te dominant lethal eff ects, a second mating was
conducted with treated males a nd untreated females of the P generation to produce the F1b litters. After weaning, 25 rats/sex/dose from the F1a litters were randomly-selected (one/sex/litter, when possible) to become the parents of the F2 generation, and were given the
same concentration test formulat ion as their dams for at least 10 weeks prior to mating. There were no effects of treatment on parental su rvival, clinical sign s, or pup sex ratio.

Body weights were decreased (p ≤ 0.05) during pre-mating at the high-dose in the P generation males and females ( ↓ 6 to 15%) and F1 males and females ( ↓ 15 to 25%), and during gestation and lactation in the P ( ↓ 7 to 19%) and F1 ( ↓ 18 to 24%) dams. Body weig ht gain was consistently decreased (p ≤ 0.05) during pre-mating in the P males, dur ing Week 1 in the P females and F1 males, and during Weeks 1, 5, and 10 in the F1 females. Food consumption was decreased (p ≤ 0.05) in the P generation males and females ( ↓ 8 to 20%) and F1 males and females during pre-mating ( ↓ 13 to 30%) and during gestati on and lactation in the P ( ↓ 13 to 28%) and F1 ( ↓ 21 to 32%) dams. Food efficiency du ring pre-mating was decreased (p ≤ 0.05) consistently in the P males, and occasionally in the P and F1 females. Absolute ( ↓ 20%) and relative to body ( ↓ 10 to 11%) ovary weights were decreased (p ≤ 0.05) in both generations. Yellow discolored adipose tissue, characteristic of dinitroaniline treatmen t, was observed in the P and F1 males and females (23-24/25 treated versus 0/25 cont rol). Findings of denser st roma of the endometrium were observed in the P and F1 females (14-15/25 trea ted versus 0/25 control), indicating uterine atrophy.

Only minor and/or transient decreases in bod y weight and food consumption in mid-dose F1
males were observed. Yellow-c olored adipose tissue was obser ved in mid-dose P females (2/25 treated versus 0/25 control), and is characteristi c of dinitroaniline tr eatment. There were no
treatment-related effects at the lowest dose.

The LOAEL for parental toxicity is 4000 ppm (e quivalent to 295/337 mg/kg/day, M/F) based on decreased body weight, body weight gain, food consumption, and food effi ciency in males and females of both generations; and uterine atrophy and decreased ovar y weight in both generations.TRIFLURALIN FINAL

Page 52 of 58 Overt toxicity was apparent at this dose as demonstrated by the body weight decreases of ≥ 10%. The NOAEL is 450 ppm (equivalent to 35/42 mg/kg/day, M/F).

In the high-dose group, fetal viabil ity (86% treated versus 94% c ontrol), neonatal viability index (85% treated versus 97% control) and litter viability (80% treated versus 100% control) were decreased (p ≤ 0.05) in the P generation (F1a litters). Lact ation index also was decreased (NS) in
these animals. The number of im plantation sites were decreased (p ≤ 0.05) in the F1a ( ↓ 9%) and F2 ( ↓ 18%) litters, resulting in reductions (p ≤ 0.01) in the number of newborn pups ( ↓ 17 to 22%) and litter size on post-natal days (PND) 1 and 4 ( ↓ 17 to 25%). Litter size remained decreased (NS) throughout the remainder of lactation in both generations. Pr e-implantation loss was increased (15.5% treated versus 9.8% control; p ≤ 0.05) in the F1b litters (mating of treated males with untreated females), yielding a decreased ( ↓ 11%; p ≤ 0.05) number of live fetuses. The LOAEL for reproductive toxicity is 4000 ppm (equivalent to 2 95/337 mg/kg/day, M/F) based on decreased ovary weights in both generations; decreased lactation index in the F1a pups; and decreased number of implantati on sites, newborn pups, litter size, and pup weights in both generations. The NOAEL is 450 ppm (e quivalent to 35/42 mg/kg/day, M/F).

Pup weights in the high-dose group were decreased (p ≤ 0.05) throughout lactation in the F1a litters ( ↓ 15%) and on PND 14 and 21 in the F1a litters ( ↓ 74 to 81%). There were no offspring
effects observed in the low- a nd mid-dose animals. The LOAEL for offspring toxicity is 4000 ppm (295/337 mg/kg/day M/F), based on decreased feta l, neonatal, and litter viability; decreased pup weights in F1a litters; and decreased number of newborn pups, litter size, and pup weights in both generations; and decreased number of implanta tion sites. The NOAEL for offspring toxicity is 450 ppm (35/42 mg/kg/day, M/F).

Chronic Oral Toxicity/Carcin ogenicity in Rodents (Rat)

A chronic oral carcinogenicity study ( MRID 00044337 ) was conducted with trifluralin (purity and Lot/Batch not reported) in Fischer 344 rats with dietar y doses of 0, 813, 3250 or 6500 ppm for two years. The high dose (325 mg/kg/day) re sulted in significant increases of combined malignant and benign urinary bladder tumors in females. An increase in the incidence of carcinomas of the renal pelvis was seen in all dose groups of males. In addition, an increase in the incidence of thyroid gland follicular cell tu mors (adenomas plus carcinomas combined) in males was observed at 6500 ppm.

Additional rat bioassays included: a two-year st udy in SD rats at trif luralin doses of 0, 200, 1000 or 2000 ppm; a 78-week study in Osborne-Mendel rats at trifluralin doses of 0, 3250 or 6500 ppm (conducted by the National Cancer Institute); and a two-year study in Wistar rats at trifluralin doses of 0, 200, 800 or 3200 ppm ( MRID 00162456, 00162457, 00162458 ). In the last study, the systemic LOAEL is 800 ppm (40 mg/kg/day) based on body weight changes; the NOAEL is 200 ppm.TRIFLURALIN FINAL

Page 53 of 58 Mechanism of Action Study (Rat)

A significant increase in thyroid follicular cell tumors was observed in male rats after oral administration of trifluralin in a 2-year chroni c toxicity/carcinogenicity study with Fischer 344 rats ( MRID 00044337 ). The current study ( Saghir et al., 2008 ) was designed to determine the mechanism of thyroid hyperactivity after triflu ralin exposure. Groups of 15 male Fischer 344 rats were exposed to trifluralin (0 or 6500 ppm) in the diet for two weeks; a positive control group was administered phenobarbital (PB; 75 mg/kg/day) by oral gavage. The time-weighted average daily intake of trifluralin was 441 ± 77 mg/kg/day in the treatment group. After dosing for two weeks, ten rats/group were sa crificed, sera was collected for T 3 , T 4 , and TSH concentration analyses by radioimmunoassay, an d livers and thyroids were harvested and weighed. The livers were al so analyzed for selected T 4 -specific uridinediphosphate glucuronosyltransferase (UGT) gene expression a nd total UGT enzyme activity. The bile ducts from the remaining rats from each group were ca nnulated at the end of the dosing period, and
bile was collected on ice for two hours. Bile samples were analyzed for T 3 , T 4 , and reverse-T 3 by an HPLC/MS/MS method.

Body weight gain was decreased by 20% (p ≤ 0.05) in the trifluralin-treated rats compared to negative control and PB-treated rats. Liver a nd thyroid weights also were increased in the trifluralin- and PB-treated animals; all increases were significant (p ≤ 0.05) except for the thyroid after trifluralin exposure. In the trifluralin-treated rats, serum T 3 and T 4 concentrations were decreased by 17% and 90%, resp ectively (compared to negative control), and TSH concentration was increased by 37%. PB treatment decreased T 4 (23%) and increased TSH (53%) concentrations, but had no effect on T 3 concentration. Trifluralin treatment induced total hepatic UGT enzymes by 2.4-fold, as well as mRNA ex pression of selected hepatic UGT isozymes (UGT1A1, 1.4-fold; UGT1A6, 6.4-fold; UGT2 B1, 3.7-fold). The total amount of T 4 in bile was
increased >2-fold in trifluralin-treated rats. Trif luralin treatment also incr eased bile flow, with a 3.2-fold increase in the biliary elimination of conjugated T 4 and a 63% increase in conjugated

T 3 . Based on these data, decr eases in total serum T 3 and T 4 concentrations in trifluralin-treated rats were due to enhanced pe ripheral metabolism (increased conjugation by (UGTs) and increased bile flow resulting in compensatory increases in TSH synthesis and secretion. Continued, and increased, concentrations of TS H after chronic exposure would cause stimulation of the thyroid gland, resulting in cellular hypert rophy, proliferation, and predisposition to the development of follicular cell tumors.

Carcinogenicity in Rodents (Mouse)

A 78-week carcinogenicity study ( Jaeger, 1986 ) was conducted by the National Cancer
Institute with B6C3F 1 mice at trifluralin (purity and Lot/Ba tch not reported) doses of 0, 2375 or 5000 ppm. The incidence of live r carcinomas, alveolar-bronchi olar adenomas, and squamouscell carcinomas of the forestomach in the female mice was suspected to be due to contamination of the test compound with N-nitroso-di-n-propylamine.TRIFLURALIN FINAL

Page 54 of 58 A two-year study conducted in B6C3F 1 mice ( MRID 00044338 ) at trifluralin (purity and Lot/Batch not reported) doses of 0, 563, 2250 or 4500 ppm, found no tumors due to the test compound.

A two-year study in NMRI mice ( MRIDs 00158935 and 40392313 ) used purified trifluralin (purity and Lot/Batch not reported) at doses of 0, 50, 200 or 800 ppm. In creased liver weights
were noted in males at the mid- and high dose (30 and 120 mg/kg/day) and in females at the high dose. The systemic LOAEL was 200 ppm (30 mg/kg/day) in male mice and 800 ppm (120 mg/kg/day) in females. The systemic NOAEL was 50 ppm (7.5 mg/kg/day) in male mice and 200 ppm (30 mg/kg/day) in females. No treatment-related tumors were observed.

Chronic Oral Toxicity in Non-Rodents (Dog)

. In a chronic oral carcinogenicity study in the beagle dog ( MRID 42447001 ), animals were fed trifluralin (purity and Lot/Batch not reported) by capsule at dose s of 0, 0.75, 2.4 or 40 mg/kg/day for one year. The NOAEL was 2.4 mg/kg/day and the LOAEL was 40 mg/kg/day based upon reduced body weight; decreased red cells and hemoglobin concentrations; increased thrombocyte, methemoglobin, cholesterol and trig lyceride concentrations; and increased liver weight.

In a second chronic oral toxicity study in the beagle dog ( MRIDs 00151908 and 00159618 ), trifluralin (purity and Lot/Batch not reported) doses of 0, 30, 150 or 750 ppm we re fed in the diet for one year. The NOAEL was 30 ppm (0.75 mg/kg/day). The LOAEL was 150 ppm (3.75 mg/kg/day) based upon increased liver weight an d methemoglobin concentr ation. Observations at the high dose included: decreased weight gai n, decreased RBC concentration, and increased concentrations of methemoglobin, serum lip ids, triglycerides, and cholesterol.

Fish Early-Life Stage Toxicity (OPPTS.850.1400)

Rainbow trout [ Salmo gairdneri (sic); Oncorhynchus mykiss ] were exposed to trifluralin (99.86% purity; Batch/Lot not repo rted) at mean measured concentr ations of negative control, solvent control, 0.59, 1.14, 2.18, 4.32, and 8.81 μg a.i/Lin a 48-day early life stage test ( MRID
41386202 ). The time to hatch, hatchability of the eggs, and larval survival were not significantly different (p>0.05) from the negative control and there were no treatment-related
behavioral effects observed in la rvae in all concentrations othe r than the highest tested. The NOAEC (μg/L), and the LOAEC, based on significan t (p<0.05) reductions in total length and body weight, were 2.18 and 4.32 μg a.i/L, respectively.

Fish Life-Cycle Toxicity (OPPTS 850.1500)

In a fish full life cycle study conducted with fathead minnows ( Pimephales promelas ), fish were exposed to mean measured concen trations of trifluralin (97% pur ity; Batch/Lot not reported) of negative control, 1.5, 1.9, 5.1, 8.2, and 16.5 μg a.i/L ( MRID 05008271 ). After 30 days, survivalTRIFLURALIN FINAL

Page 55 of 58 in the highest treatment concentration was 67% th at dropped to 0% after 60 days. While mean survival ranged from 89-91% in 30-day and 60-d ay time points, after 61 weeks of exposure, mean survival was 0% in the 8.2 μg a.i/L tr eatment concentration. Similarly, the middle treatment concentration of 5.1 μg a.i/L had mean survival of 30% by test termination at 61 weeks. Total length and weight measurements were therefore discarded for the 60-day and 61 week time points for the highest treatment conc entration and for the 61-w eek time point for the second highest treatment concentration. The me an number of spawning individuals increased from control up to the 1.9 μg a.i/L but then dipped at the 5.1 μg a.i/L. There were no spawning females at the two highest treat ment concentrations. The mean number of eggs spawned per female in the lowest treatment group was 20 as compared to 142 for the control and 84 and 329 for the 1.9 and 5.1 μg a.i/L treatment concentrations , respectively. Survival in fish in the 1.9 and 5,1 μg a.i/L treatment concentrations averaged 56% and 72%, respective ly for 30 days and 56 and 61%, respectively for 60 days. The NOAEC a nd LOAEC for this study based on effects to parental survival was 1.9 and 5.1 μg a.i/L, respectively.

Sheepshead minnows ( Cyprinodon variegatus ) were exposed to trifluralin (99% purity; Batch/Lot not reported) at mean measured conc entrations of control, solvent control, 1.3, 4.8, 9.6, 17.7, and 34.1 μg a.i/L in a 166-day fish full life stage test ( MRID 42449901 ). There was 100% mortality observed in the highest tr eatment concentration (34.1 μg a.i/L), and
therefore this treatment concentration was remove d from further statistical analysis. Parental fish at the two highest treatment concentrati on exhibited loss of equi librium after 14 days post hatch and many were found laying on the botto m of the aquaria. Parental fish at the 4.8 and 9.6 μg a.i/L mean measured concentratio ns has darkened coloration in the caudal peduncle area. Juveniles in the F 1 generation at 12 days post hatch in the second highest treatment concentration exhibited a loss of equi librium and appeared sluggish. This study was determined to be scientifically sound but there were no raw data provided for the reviewer to conduct an independe nt statistical analysis and therefore is classified as supplemental. The only parameter that could be statistically verified by the reviewer was the number of eggs spawned per day per female wh ich showed significant reductions (p<0.05) at
all treatment concentrations and therefore a NOAEC could not be determined and the LOAEC was 1.3 μg a.i/L.

Avian Reproduction Te st (Quail) (OCSPP 850.2300)

In a one-generation avian reproducti on test with bobwhite quail ( MRID 00131134 ), trifluralin (99.6% purity; Batch/Lot not repo rted) was administered at nominal dietary concentrations of 5 and 50 ppm (mean measured concentrations). NOAEC and LOAEC values were not determined; it was reported that trifluralin exposur es <50 ppm would not significantly affect reproductive success. Wh ile the study is classifi ed as acceptable, the percentage of cracked eggs in the negative co ntrol was 9.7% as a percentage of eggs laid. EPA guidance for the bobwhite quail states th at typically only 0.6 – 2.0% of eggs laid are cracked for this species.TRIFLURALIN FINAL

Page 56 of 58 In another one-generation avian reproduction test with bobwhite quail ( MRID 40334706 ), trifluralin (96% purity; Batch/Lot not reported) wa s administered at nominal concentrations in the diet of control, 250, 500 and 1000 ppm (mean measured concentrations, (mg/kg/day), were not reported). No treatment-related effects were observed (DP barcode 417055).

Avian Reproduction Te st (Duck) (OCSPP 850.2300)

In a one-generation avian reproduc tion test with mallard ducks ( MRID 00131132 ), trifluralin (99.6% purity; Batch/Lot not repo rted) was administered at nomi nal dietary concentrations and 5 and 50 ppm (mean measured concentrations ). NOAEC and LOAEC values were not determined; however, an increase in cracked eggs (2.4%) was obser ved at the highest trifluralin treatment concentration (50 pp m) and was not determined to be significant (p>0.05). A reevaluation of this study in 2010 stated that subsequent avian repro studies ( MRIDs 40334704
and 40334706 ) did not show the cracked eggs effect in concentrations up to 1000 mg a.i./kgdiet and that the increas e in cracked eggs for MRID 00131132 for this study was not biologically significant and the NOAEC shoul d be established at 50 mg a.i./kg-diet.

In another one-generation avian repr oduction test with mallard ducks ( MRID 40334704 ), trifluralin (96% purity; Batch/Lot not reported) wa s administered at nomina l concentrations in the diet of control, 250, 500 a nd 1000 ppm (mean measured concen trations). The NOAEC was determined to be 500 ppm, and a LOAEC wa s 1000 ppm based on reductions in eggshell thickness, 14-day survivor body weight, at the highest treatment concentration. The percent reductions from the negative control in eggshell thickness and 14-day survi vor body weight at the highest treatment concentration were 4 and 7%, re spectively. There were no apparent overt signs of toxicity, treatment related mortalities, or reductions in f ood consumption or female body weight.TRIFLURALIN FINAL

Page 57 of 58 APPENDIX 3: References Not Utilized in the Trifluralin 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 availa ble in a Federal Register Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted as “other scientifically relevant information (OSR I) 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 in cluded Part 158 studies previously submitted to the agency for regist ration/reregistration, publis hed literature articles and/or Tier 1 assays. The agency also conducted a more recent search (2009 to 2014) of available scientific literature for any additional relevant information for their weight of evidence (WoE)
evaluations. These articles were evaluated in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/science/efed/policy_guidance/team_a uthors/endangered_species_r eregistration_workgroup/PDF_rot/e sa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature Toxi city Studies to Support Human Health Risk Assessment ( http://www.epa.gov/pesticides /science/lit- studies.pdf).

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

Cecconi, S., Rossi, G., Carta, G., Di, L.G., Ce llini, V., Canipari, R., and Buccione, R. (2013) Effects of trifluralin on the mouse ovary. Environ Toxicol . 28(4):201-206.

Orton, F., Lutz, I., Kloas, W., Routledge, E. ( 2009) Endocrine Disrupting Effects of Herbicides and Pentachlorophenol: In Vitro and in Vivo Evidence. Environ. Sci. Technol., 43:2144–2150.

Nishihara, T., et al. (2000) Estrogenic Activi ties of 517 Chemicals by Y east Two-Hybrid Assay. Journal of Health Science , 46(4): 282–298.

Soto, A.M., Sonnenschein, C., Chung, K.L., Ferna ndez, M.F., Olea, N., and Serrano, P.O. (1995) The ESCREEN assay as a tool to identify estr ogens: An update on estrogenic environmental pollutants. Environmental Health Perspectives 103 (Suppl. 7):113-122.TRIFLURALIN FINAL

Page 58 of 58 Weir, S.M., Yu, S., and Salice, C. J. (2012) Acute toxicity of herb icide formulations and chronic toxicity of technical-grade trifluralin to larval green frogs (Lithobates clamitans). Environ Toxicol Chem . 31(9):2029-2034.

Worth, H.M., et al. (1966) Eff ect of Trifluralin Treatment on Reproduction in Rats and Dogs. Unpublished study prepared by Lilly Re search Laboratories. (MRID 00083646)