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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

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

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

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

.

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

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

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

PATHWAYS

CHEMICAL: BENFLURALIN

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYBENFLURALIN 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...................................................15

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

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

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

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

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

B. Estrogen Pathway...........................................................................24

C. Androgen Pathway.........................................................................25

D. Thyroid Pathway...........................................................................25

E. Conclusions.................................................................................26

V. EDSP Tier 2 Testing Recommendations.....................................................26

VI. References.......................................................................................28

LIST OF TABLES: Table 1. Tier 1 Assay for Benfluralin...............................................................9
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Benfluralin ...............................12
Table 3. Androgenic/Anti-Andr ogenic Pathway for Benfluralin..............................17
Table 4. Thyroid Pathway for Benfluralin........................................................21

APPENDIX 1. EDSP Tier 1 Screening Assays........................................................32
APPENDIX 2. Other Scientifically Relevant Information (OSRI)................................45BENFLURALIN 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 A R 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 CA R 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

DE R 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 )
E R 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 enerationBENFLURALIN FINAL

iv Abbreviation Terminology

Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA 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 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 DoseBENFLURALIN FINAL

v Abbreviation Terminology

N Ne g ative
NE Not examined/evaluate d
NF sta g e 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 N R 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 )
PX R 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 ionateBENFLURALIN FINAL

vi Abbreviation Terminology

T R 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-EvidenceBENFLURALIN FINAL

Page 1 of 53 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, 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 a pesticide chemical inte racts with E, A, or T hormone pathways, the number and type of effects i nduced, the magnitude and pattern of responses observed across studies, taxa, and sexes we re considered. Additi onally, the conditions un der which effects occur were considered, in part icular, whether or not endocrine-rela ted responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On November 13, 2013, the 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) an alysis of the potential interaction of benfluralin with the E, A or thyroid T hormone pathways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the
results of the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the
results of the cited OSRI for mammalian and wi ldlife studies (40 CFR Pa rt 158 and literature).

For benfluralin and the estrogen pathway, a posi tive response for estrogen receptor (ER) agonism was observed in the Tier 1 estrogen receptor transactivation assay (ERTA); however, this interaction with the estrogen pathway via the ER wa s not consistent with th e negative findings in the Tier 1 ER binding and uterotrophic assays. Estrogen-related effect s were observed at the high dose in the Tier 1 female pubertal assay in cluding decreases in pitu itary weight, ovarian weight, blotted uterine weight, delayed vaginal opening and increased age at first estrus. All estrogen-related findings in the Tier 1 in vivo assay and Part 158 mamma lian studies occurred in the presence of liver effects including increase s in absolute and rela tive liver weights, and significant increases in creatinine, GGT and choles terol. Additionally, an increase in relative ovarian weight in the Part 158 mammalian subchronic rat study occurred at the high dose along with significant increases in absolute and re lative liver weights. The observed effects are consistent with the findings in the mode of action studies which support hepatic enzyme induction resulting in enhanced hormone clearance in mammals.BENFLURALIN FINAL

Page 2 of 53 No effects on the estrogen pathway were observed in the fish short term reproduction assay (FSTRA). In a one-generation Part 158 avian reproduction study with bobwhite quail, a decrease in eggs laid, as well as effect s on offspring survivability and gr owth, were observed; however,
these effects coincided with decreases in feed consumption. In the companion mallard duck reproduction study, an increase in eggs cracked were reported, but no differences in eggshell thinning or other reproduc tive effects were observed.

For benfluralin, for the androgen pathway, th e only positive findings were seen in the Hershberger assay and occurred at the high dose in th e presence of overt t oxicity. Although the presence of overt toxicity conf ounds the interpretation of the eff ects on the endocrine parameters in the study, a follow-up study to evaluate testosterone clearance showed enhanced
metabolism/clearance of testoster one at the dose that resulted in a decreased androgen-sensitive tissue weights. This suggests that the antiandr ogenic effects observed in the Hershberger assay are likely due to enhanced testosterone clearan ce. In the male pubertal assay, decreased serum
testosterone and decreases in androgen-sensitive tissue weight s and epididymal weights were seen only at the high dose (400 mg/kg/day). The endocrine-related effect s were seen in the presence of hepatotoxicity which manifested as increases in absolute (4 0%) and relative (52%) liver weights and an increase (60%) in GGT level. Increased liver weights were also observed at lower doses where no androgen-related effects were seen. The only androgen-related effect
observed in the Part 158 mammalian studies was an increase in te sticular weight in the rat chronic/carcinogenicity study, which occurred in the presence of overt toxicity. The evidence from the mammalian toxicity stud ies suggest that the androgen-re lated effects observed are due to liver enzyme induction and in creased hormone clearance and excretion. In the FSTRA the only notable effect in the absen ce of overt toxicity was an incr ease in male GSI seen at the
middle treatment concentration. In the Pa rt 158 avian bobwhite quail study, decrease in offspring survivability was observed; however decreased feeding was seen in the parental generation.

For benfluralin, for the thyroid pa thway, the effects in the Tier 1 assays were seen in the male and female pubertal assays, but no evidence of thyroid activity was seen in the amphibian metamorphosis assay (AMA). In the pubertal as says, significant decreases in serum T4 were
seen at all doses in the male and at the mid a nd high dose in the female. TSH was significantly increased at the mid and high dose in males and hi gh dose only in females. Increases in thyroid weight and altered thyroid histopathology were seen only in males at the high dose. All effects on the thyroid parameters in the pubertal assays were accompanied by increased relative liver weights. Thyroid perturbations were observed in the subchronic rat toxicity study (increased thyroid weights) at a high dose that caused significant increases in liver weight. Thyroid follicular cell hypertrophy and hyperplasia were observed in both sexes in the chronic/ carcinogenicity study in rats. An increased in cidence of follicular ce ll adenomas/ carcinomas was observed in this study, but at a dose that was considered to be overtly toxic.BENFLURALIN FINAL

Page 3 of 53 In studies that evaluated the mode of action of benfluralin, previously submitted mechanistic
studies showed that benfluralin induces hepatic microsomal enzyme activity in both sexes in the rat. In a 14-day study submitted to support a po stulated mode of acti on for liver and thyroid tumors in the rat, benfluralin was shown to i nduce constitutive androstane receptor (CAR) and pregnane X receptor (PXR) related gene transcri pts and associated PROD enzyme activities and benfluralin also up-regulated T4-specific UGT ge nes associated with T4 metabolism. These
effects occurred at a dose that resulted in decreas ed serum T4 and T3 leve ls and increased liver weight and hepatic hypertrophy. Furthermore, benf luralin is structurally similar to another dinitroaniline (trifluralin) which has been shown to induce hepatic UGTs, resulting in decreased serum T3 and T4 levels, elevated TSH and alte red thyroid histopathology leading to increased thyroid tumors in rats (Saghir et al. , 2005).

An evaluation of the Tier 1 and OSRI data sh ows that the observed effects on the endocrinerelated endpoints for the estroge n, androgen and thyroid pathway occur only when there were
concurrent increases in liver we ight. Based on a WoE, the available data suggests that the antiestrogenic, anti-androgenic and anti-thyr oid effects observed in the mammalian in vivo Tier 1 assays may result from liver enzyme induction and subsequent increased hormone clearance. However, there is no convincing evidence for an interaction with estrogen, androgen or thyroid pathways in wildlife.

For benfluralin, the current point of departur e (POD) of 0.5 mg/kg/day is based on increased histopathological lesions of th e kidneys seen in male and in female rats at 5.4 mg/kg/day (LOAEL) in a combined chronic toxicity/carcinoge nicity study in rats. This POD is used to derive the chronic Reference Dose (RfD) used for dietary exposure risk assessments and for calculating the Margins of Exposure (MOEs) fo r non-dietary exposure risk assessments. This POD is 50-fold lower than the lowest dose (25 mg /kg/day) that caused thyr oid-related effects in the Tier 1 male pubertal assay, and 600 to 800-fold lower than the doses that caused estrogenrelated (300 mg/kg/day) or andr ogenic-related (400 mg/kg/day) effects in the pubertal assays. Consequently, the chronic RfD and MOEs derive d from current PODs are protective of the endocrine-related findings in the Tier 1 in vivo assays and OSRI. In addition the totality of the
data demonstrated that the endocrine-r elated effects seen in the mammalian in vivo Tier 1 assays may result from liver enzyme induction and s ubsequent increased hormone clearance.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is NOT
recommended for benfluralin since additional tes ting is not expected to impact EPA’s current regulatory point of departur es and endpoints for human h ealth risk assessments.

As there is no convincing evidence for a poten tial interaction with th e estrogen or androgen pathway in fish or thyroid pathways in amphi bians, no additional testing is recommended.BENFLURALIN FINAL

Page 4 of 53 For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard
duck are available for benflurali n. In both species, effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from benfluralin exposure. Relative to the EDSP, additional testing is not recommended.BENFLURALIN FINAL

Page 5 of 53 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, 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 a pesticide chemical inte racts with E, A, or T hormone pathways, the number and type of effects i nduced, the magnitude and pattern of responses observed across studies, taxa, and sexes we re considered. Additi onally, the conditions un der which effects occur were considered, in part icular, whether or not endocrine-rela ted responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On November 13, 2013, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs and the Offi ce of Science Coordination and Policy conducted a weight-of evidence analysis of the potential in teraction of benfluralin with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE eval uation in this report are presented by pathway (E, A and then T) be ginning 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).

Benfluralin has a low vapor pressure (6.57 x 10 -5 mm Hg at 25°C), and theref ore, volatilization is not expected to significantly cont ribute to the dissipation of benflu ralin in the environment. The compound has a low water solubility (0.1 ppm at pH 7.00 and 25°C) and has a high octanol/water partition coefficient (log K ow = 5.29). It does not hydrolyze but does degrade rapidly in water due to photolysis. Benfluralin has a whole body bioconcentr ation factor value in fish of 1,580. Acute toxicity studi es in various fish species have shown that benfluralin is highly toxic (LC 50 values of 0.06 to 1.4 mg a.i./L).

The available information considered in determin ing the potential interacti on of benfluralin 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.BENFLURALIN FINAL

Page 6 of 53 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 and/or the public. The agency’s review of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team (USEPA, 2010). Since then, the Ag ency has also conducted a more recent search of available scientific literature for any a dditional relevant information. Summ aries of the available OSRI are presented in Appendix 2.

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 hormonal pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

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

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays 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 otherBENFLURALIN FINAL

Page 7 of 53 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 biological organizati on. Finally, under what conditions did the responses occur including co nsideration of whether the responses were observed in the presence of overt or systemic toxicity. The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.BENFLURALIN FINAL

Page 8 of 53 This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the 2013 FIFRA SAP meeting that evaluated scientific issues associated with the WoE
evaluation of the EDSP Tier 1 sc reening 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 effects in endocrine se nsitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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 ( e.g. , 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 availa ble toxicity data into account.

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

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effectBENFLURALIN FINAL

Page 9 of 53 which supports a conclusion of whether a substance has the potential to interact with the E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

A. EDSP T1 Screening Assays

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

.

Table 1: Tier 1 Screening Assays for Benfluralin.

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48614904)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48614905)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48614902)

Steroidogenesis Assay (Human cell line 295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48614908)

Aromatase Assay (human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48614903)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48614909)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48614907)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID No. 48669301)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID No. 48669302)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48614906)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 48616704)BENFLURALIN FINAL

Page 10 of 53 B. Effects on Hypothalamic-Pit uitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for benfluralin to interact with the estrogen pathway is summarized in Table 2. The various targets of the estrogen pathway across the relevant Tier 1 a ssays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

Benfluralin was classified as not interactive (negative) in the in vitro Tier 1 estrogen receptor (ER) binding assay, and negative in the steroidogenesis and aromatase assays. A weak positive response in the Tier 1 ERTA was observed at benfluralin co ncentrations of 10

− 5 and 10

− 4 M.

Effects in the Tier 1 female pube rtal assay at the high dose (300 mg/kg/day), in the absence of overt toxicity, included statis tically significant (p<0.05) decr eases in ovarian weight [(  17% and  18% for unadjusted and adjusted (for body we ight on PND 22)], respectively) and blotted uterine weights (  21%, adjusted), delayed vaginal opening (3 days, p<0.05), and increased age at first estrus. A decrease (p<0.05) in absolute a nd relative pituitary weig hts was also observed (  12 and 19%, respectively) in this assay. Indica tions of hepatotoxicity at the high dose (300 mg/kg/day) included: increases (p<0.05) in gamma glutamyl transpeptidase (GGT, 47%); cholesterol ( ↑ 12%); and creatinine ( ↑ 44%); additionally liver we ights were significantly
increased at all doses in this assay (8-37%).

In the FSTRA, there were no changes in es trogen-related endpoints such as gonadal histopathology, gonadal-somatic i ndex (GSI), or plasma vitellogenin (VTG) concentrations. While there were no significant differences (p >0.05) in fecundity in the treatment groups compared to the negative control, decreases were observed in the low (0.302 μg a.i./L; ↓ 29%) and mid (3.14 μg a.i./L; ↓ 21%) treatment groups compared to the negative control (high treatment group terminated due to overt toxicity); how ever, a similar decrease was observed in the solvent control ( ↓ 39%) suggesting the observed decrease was not treatment-related.

The only estrogen-related effect s een in the Part 158 mammalian t oxicity studies was an increase in relative ovarian weight (  14%, p<0.05) in the subchronic rat toxicity study at 605 mg/kg/day.

In a Part 158 avian reproduction study with the northern bobwhite quail reductions in eggs laid, embryo viability, hatchling survival, and o ffspring body weight were observed at 990 ppm (increase in % of cracked eggs at this concentr ation but relatively large % in control as well, no effects on eggshell thinning); additionally, eggs laid and embryo viability/hatchlings was reduced at the mid concentration (295 ppm) and offspr ing survival/weight were affected at allBENFLURALIN FINAL

Page 11 of 53 concentrations tested ( ≥ 96 ppm). While there were no re ported treatment-related effects on parental survival, body weight, or behavior, food consumption prior to egg laying was significantly reduced (p<0.05) at the two lowest treatment concentrations while food consumption during egg laying wa s significantly reduced (p<0.05) at the two highest treatment
concentrations. In an avian reproduction study with the mallard duck at 975 mg/kg-diet, there was a higher incidence ( ↑ 11.5%) of cracked eggs in the hi ghest treatment concentration compared to control that wasn’t statically significant (p = 0.061), but considered to be biologically significant. Eggshell thickness for both cracked and normal eggs was not significantly affected by benflura lin exposure; no other treatment- related reproductive effects or signs of toxicity were observed. In a Part 158 rainbow trout fish early life-cycle study, larvae body length was significantly reduced at ≥ 5.0 μg a.i./L, and body weight was reduced at ≥ 14.8 μg a.i./L.BENFLURALINFINALPage 12 of 53Table 2: Estrogenic/Anti-Estrogenic Pathway for BenfluralinLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Benfluralin1Study 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/Fecundity Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48614904)NERTA(MRID 48614905)PSteroidogenesis(MRID 48614908)NAromatase(MRID 48614903)NUterotrophic(MRID 48614909)NN NFemale Pubertal Rat(MRID 48669301)↓21%(H)↓17-18%(H)N↓12-19%(H)↑2 days(firstestrus)(H)↑3 days(VO)(H)↑LW(M,H)↑LE(H)NFSTRA(MRID 48614906)NENNNNNX4(H)OSRISubchronic toxicityRat(MRID 44050001)N↑14%NNXNSubchronic toxicityDog(MRID 43072301)NNNNXNChronic toxicity/Carcinogenicity-RatNNNNXX(H)BENFLURALINFINALPage 13 of 53Table 2: Estrogenic/Anti-Estrogenic Pathway for BenfluralinLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Benfluralin1Study 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/Fecundity Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3(MRID 44050002,44545001)Chronic toxicity -Dog(MRID 43628702)NNNNXNCarcinogenicity –Mouse(MRID 41021501)NNNNXX(H)Developmentaltoxicity- Rat(MRID 000147535)NX NDevelopmentaltoxicity- Rabbit(MRID 42039101)NNXX(H)Two-generationreproduction- Rat(MRID 43628701,43628702)N N N NNXX(H)Avian ReproductionQuail(MRID 42145502)P5(L,M,H)N N6Avian ReproductionMallard(MRID 42145501)P7(H)N N1. Key to responses: L= Low treatment, M= Medium treatment, H= High treatment. Arrows (↓or↑) indicate the direction of the response. A shaded cellindicates that is parameter is not routinely evaluated or is notapplicable in this assay. LW = liver weight, LE = serum levelsof liver enzymes, LH = liverhistopathological changes2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an Xin this column. For details see Section IV. ABENFLURALINFINALPage 14 of 533. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Signs of toxicity including mortality, lethargy, loss of equilibrium and bloating were observed with≥38% of the fish affected.5. Reduction in eggs laid and embryo viability reported at≥295 ppm; offspring survival affected at all doses (≥96 ppm).6. While there were no reported treatment-related effects on parental survival, body weight, or behavior, food consumption prior to egg laying wassignificantly reduced (p<0.05) at the two lowest treatment concentrations while food consumption during egg laying was significantly reduced (p<0.05) atthe two highest treatment concentrations7. Increase in percentage of cracked eggs at 975 mg/kg-diet.P Positive findings (in vitro)/No effect (in vivo)N Negative findings (in vitro)/No effect (in vivo)BENFLURALIN FINAL

Page 15 of 53 2. Effects on Androgen Pathway

The potential for benfluralin to interact with the androgen path way are summarized in Table 3. The various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for androgenic, anti-androgenic, or HPG axis effects. . . This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reporte d in the table and not considered further in the WOE assessment.

In an evaluation of androgenic/an ti-androgenic activity from Tier 1 testing results, benfluralin was classified as negative (non-binder) in the in vitro androgen receptor (AR) binding (rat prostate cytosol) assay, and the st eroidogenesis assay.

In the Hershberger assay, benfluralin did not i nduce significant increases in accessory sex tissue (AST) weights in the androgenic po rtion of the assay. However, at the high dose (750 mg/kg/day) which was determined to be an overtly toxic dose ( ↓ 12% in body weight), an imals had significant decreases in LABC (  27%) and seminal vesicles (  33%) weight in the an ti-androgenic portion of the assay. The changes in androgen respons ive tissue weights were accompanied by a nonsignificant decrease (~ 29%) in se rum testosterone levels. There were significant treatment-related increases (p ≤ 0.5) in absolute liver weight of abou t 38% and 68% at 250 and 750 mg/kg bw/day benfluralin, respectively, in the androgenic por tion, and about 39% and 65% in the anti-androgenic portion of assay.

A follow-up study to the Hershberger assay was conducted to examine 14 C-testosterone clearance.
The results confirmed that animals administered 750 mg/kg bw/day benfluralin for ten days had similar effects on body weight, body weight gain, and liver and a ndrogen-responsive tissue weights as high-dose animals in the definitive Hershberger assay. These animals showed an approximately
3.7-fold increase in 14 C-testosterone clearance compared to the control animals, resulting in lower plasma area under the curve (AUC) va lues (65%) relative to control after ten days of benfluralin treatment. These data suggest th at testosterone in benfluralin-tre ated animals was less available to interact with androgen-responsive tissues and stimulate/maintain organ weights. The dose of 750 mg/kg/day was an overtly toxic dose and the effects of benfluralin on testoste rone clearance at lower
doses in the absence of ov ert toxicity is unclear.

In the male pubertal assay no changes in androgen -related endpoints were se en at the low or mid dose. At the high dose (400 m/kg/day), in the ab sence of overt toxicit y, significant (p<0.05) decreases in the weights of the seminal vesicles (  32%), ventral prostate (26 %), dorsolateral prostate (  21-22%), LABC (  20%) and epididymis (  13%) were seen. In addi tion to the tissue weight changes, a 52% decrease (p<0.05) in serum testos terone was also observe d at the high dose. Absolute and adjusted pituitary weights were decreased at 400 mg/kg bw/day benfluralin, but relative pituitary weights were not significantly different. No histopathol ogical lesions wereBENFLURALIN FINAL

Page 16 of 53 observed in the testis, but two animals, in the hi gh dose, were noted to have slightly decreased spermatic elements in the epididymis. The age and body weight at preputial separation were not affected at any dose level. At 100 mg/kg/day, there were significant (p<0.05) increases in absolute (23%) and relative (22%) liver weights. At 400 mg/kg/day, hepatotoxicity was characterized by significant (p <0.05) increases in absolute (40%) and relative ( 52%) liver weights as well as increases in serum creatinine ( 50%) and GGT (60%) levels.

In the FSTRA, there were no changes in androgen -related endpoints such as gonadal histopathology, VTG concentrations, or male secondary sex charact eristics. There was a st atistically significant increase (27%) in male GSI at the middle treatment con centration (3.14 μg a.i/L; high concentration terminated prior to test termination due to signs of overt toxicity).

In the mammalian Part 158 studies, the only effect on androgen-related tissue weights was a 48-50% increase in testicular weight observed in the rat chronic/car cinogenicity study at a dose (275 mg/kg/day) that showed overt toxicity. In the Part 158 bobwhite quail reproduction study, reductions in embryo viability and/or offspring survival were reported at concentrations of ≥ 96 ppm; however, it is noted that reductions in food consump tion were noted in all te st concentrations along the duration of the study.BENFLURALINFINALPage 17 of 53Table 3: Androgenic/Anti-Androgenic Pathway for BenfluralinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Benfluralin1Study Type/Literature Citation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histo p atholo gy Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility/Fecundity Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48614902)NSteroidogenesis(MRID 48614908)NHershberger(MRID 48614907)NN↑LW(M,H)↓BW(12%)(H)Male Pubertal Rat(MRID 48699302)↓52%(H)NN↓13 -18%(H)NN↓SV, GP,DP, VP,LABC(H)N↑LW(M,H),↑LE(H)NFSTRA(MRID 48614906)NEGSI:↑27%(M)NNNNX4(H)OSRISubchronic toxicityRat(MRID 44050001)NNNNNNX NChronic toxicity-Rat(MRID 44050002,44545501)↑48 -50%(H)NNNNNXX(H)Chronic toxicity -Dog(MRID 43628702)NNNX NBENFLURALINFINALPage 18 of 53Table 3: Androgenic/Anti-Androgenic Pathway for BenfluralinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Benfluralin1Study Type/Literature Citation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histo p atholo gy Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility/Fecundity Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Carcinogenicity –Mouse(MRID 41021501)N N N N N NXX(H)Developmentaltoxicity-Rat(MRID 000147535)NX NDevelopmentaltoxicity- Rabbit(MRID42039101)NX NTwo-generationreproduction- Rat(MRID 43628701,43628702)NNNNNNNXX(H)AvianReproduction- Quail(MRID 42145502)P5(L,MLH)N N6AvianReproductionMallard(MRID 42145501)NN N1. Key to responses: L= Low treatment, M=Medium treatment, H=High treatment. Arrows (↓or↑) indicate the direction of the response. A shaded cellindicates that is parameter is not routinely evaluated or is notapplicable in this assay. LW= liver weight, LE = serum liverenzymes, Fert = fertility (i.e. littersize, fertile eggs).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 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Signs of toxicity including mortality, lethargy, loss of equilibrium and bloating were observed with≥38% of the fish affected.BENFLURALINFINALPage 19 of 535. Reduction in embryo viability reported at≥295 ppm; offspring survival affected at all doses (≥96 ppm)6. No reported treatment-related effects on survival or behavior, but food consumption was reduced at two lowest treatments prior to egg laying and reducedin two highest treatments during egg production.P Positive findings (in vitro)/No effect (in vivo)N Negative findings (in vitro)/No effect (in vivo)BENFLURALIN FINAL

Page 20 of 53 C. Effects on Hypothalamic-Pitui tary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it
does include three in vivo assays that provide data that can detect changes in the HPT axis, i.e

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

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

In the amphibian metamorphosis assay (AMA), there were no treatment-related effects on developmental stage, asynchronous development, nor did it show remarkable thyroid histological effects. A significant (p<0.05) 10% increase in 21-day wet weight, relative to the negative control, was observed at the lowest 0.0027 mg a. i./L level; however, higher levels were not significantly different from th e negative control (p>0.05).

In the Tier 1 male pubertal assay, significan t (p<0.05) dose-responsiv e decreases in T4 (  12%, 29%, and 53%) were seen at 25, 100 and 400 mg/kg/day, respectively. This correlated to a corresponding increase in TSH (  53% and 105%) at 100 and 400 mg/kg/day, respectively. In the Tier 1 female pubertal assay, serum T4 levels were signifi cantly (p<0.05) decreased 13% and 44% at 75 and 300 mg/kg/day, respectively. A 109% increase in TSH was observed at 300 mg/kg/day in females. In addition to the cha nges in thyroid-related hor mones, thyroid weights (adjusted for body weight on PND 23 ) were increased 15% at the high dose in males. Increases in follicular cell height with a related decrease in colloid area were also observed at the high dose in males. The thyroid-related e ffects observed in the male and pube rtal assays occurred at doses
that also resulted in increased liver weights in both sexes.

Thyroid perturbations were also seen in Part 158 toxicity studies with benfluralin, including the 90-day oral toxicity and the chroni c/carcinogenicity studies in the ra t. Female rats in the 90-day oral toxicity study at 605 mg/ kg/day significant (p<0.05) increas es in absolute and relative thyroid gland weight ( ↑ 22 and 27%, respectively) were obs erved, but no histological changes were reported. In the chronic/carcinogenicity study, male and female rats at 2500 and 5000 ppm (136/275 and 168/331 mg/kg/day in males and females, respectively) had increased thyroid follicular cell hypertrophy and hyperp lasia, plus increased incidences of combined follicular cell
adenomas/carcinomas. Both sexes also had in creased hepatocellular hyp ertrophy at these same doses, but males also had increased incidences of hepatocellular necrosis and adenomas.BENFLURALIN FINAL

Page 21 of 53 Table 4: Thyroid Pathway for Benfluralin

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

Study Type/ Literature CitationSerum T4LevelSerum TSHLevelPituitary WeightThyroid WeightThyroid:Gross andHistopathologyDevelopmentalstage orasynchronous &HLLGrowth(BW, SVL)SystemicToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assays

Male Pubertal Rat (MRID 48669302) ↓ 12%, 29%, 53% (L,M,H) ↑ 53%, 105% (M,H) ↓ 14%
H ↑ 15%
H P 4 ↑ LW (M,H),

↑ LE (H) N

Female Pubertal
Rat (MRID 48669301) ↓ 13%, 44% (M.H) ↑ 109% (H) ↓ 12- 16%

H N N ↑ LW (M,H)

↑ LE (H) N

AMA (Frog) (MRID 48616704) N N N N

OSRI
Two-generation reproduction- Rat (MRID 43628701, 43628702) N N N X N

Subchronic toxicity-Rat (MRID 44050001) N ↑ 17

- 27% N X N

Chronic toxicity
- Rat (MRID 44050002, 44545501) N ↑ 33

- 64% P X X (H)

Chronic toxicity - Dog (MRID 43628702) N N N X N

1. Key to responses: L= Low treatment, M=Medium 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, LE = serum liver 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. Increases in follicular cell height with a related decrease in colloid area were observed at the high dose P Positive findingsBENFLURALIN FINAL

Page 22 of 53 N Negative findings

IV. Committee’s Assessment of Weight of Evidence

This section of the document will describe the weight of evidence (WoE) determination on the potential of benfluralin 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 co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substance may contribute to the WoE evaluati on in helping el ucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively sequelae of systemic effects. E ndocrine 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 evalua tion of the results of the Ti re 1 assays stated that “I n summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described below. In addition to the endocrine -related effects described above
for the in vivo Tier 1 assays and OSRI, other eff ects observed which may be considered overt/systemic toxicity are also 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., or gan weights or blood chemistry).BENFLURALIN FINAL

Page 23 of 53 1. Tier 1 in vivo Assays

In the Hershberger assays, decreases in bodywei ght and bodyweight gain were observed at the high dose (750 mg/kg/day) in bot h the androgenic and antiandroge nic phases. The decreases in body weights were 7-11% in the androgenic phas e and 10-14% in the anti-androgenic phase. The decreases in body weight gains were 49% and 57%, in the androgeni c and anti-androgenic phases, respectively. In the high dose anti-androgenic group as well as the test group to evaluate testosterone clearance, body weights were decreased by 12-20 % resulting in a decreased ( ↓ 85%) cumulative body weight gains. In addition to body weight effects, absolute liver weights were significantly increased at the mid ( ↑ 38%) and high ( ↑ 68%) dose groups in the Hershberger assay. Liver weights were similar to controls at the low dose.

In the male pubertal assays benfluralin, there was no treatment-relate effects on bodyweights or body weight gains. In the female pubertal assa y, at the high dose (300 mg/kg/day), body weights were significantly decreased (8-10%) during PND 22-35, resulting in cumulative decreases in body weight gain (15-42%) for each interval up to PND 22-35. Hepatotoxicity was seen in both the pubertal assays. In males, absolute liver we ight were increased by 22% at the mid dose (100 mg/kg/day) and by 40% at the high dose (400 mg/kg/day). Rela tive liver weights were increased by 22% and 52% at the mid and high dose groups, respectively. Also, there was an increase (60%) in GGT at the high dose only. In females, absolute liver weights were increased by 22% at the mid- and by 32% at the high dose. Relative liver weights were increased by 19% and 37% at the mid and high dose groups, respectively. Also , there were increases in GGT (47%) and cholesterol (12%) levels at the high dose. Creatin ine levels were also elevated in males (50%) and females (44%) at the high dose, but were not accompanied by histopathological findings or significant changes in we ight of the kidney.

In the FSTRA, overt toxicity (mortality, lethargy, loss of equilibrium, bloating) was observed at the high test concentration (0.0365 mg a.i./L) with 38% of the fish affected, and as such this treatment group was terminated prio r to the scheduled test termination. No overt toxicity was
observed at the lower test concentrations. No tr eatment-related mortality or clinical signs of toxicity were observed in the AMA at the concentrations tested ( ≥ 74.4 μg a.i./L).

2. OSRI

The liver and the kidneys were the target-organs in the Part 158 studies with benfluralin. In the subchronic toxicity study with rats, benfluralin induced dose-related nephritis in males and cortical tubular epithelial pigm ent deposition, increases in absolu te and relative liver and kidney weights at 20 mg/kg/day, corti cal tubular epithelial regenerati on, urinary AST increase and liver hypertrophy in males at 32 mg/kg/day. In the chr onic toxicity study with dogs, in males at the high dose (125 mg/kg/day), there were increased absolute and relative liver weights withBENFLURALIN FINAL

Page 24 of 53 corroborative increased incidence of minimal to slight centrilobul ar to diffuse hepatocellular hypertrophy. In the two-generation reproduction study, liver effects included dose-dependent
increases in the absolute and re lative weights at 68 and 342 mg/ kg/day in the parental animals (F 0 and F 1 ). Kidney effects observed at these doses included increased incidence of hyaline droplets (graded as minimal) in males and chr onic progressive nephropat hy (graded as slight and/or moderate) in males and females. In the chronic toxicity/carcinoge nicity study with rats, significant increases (p<0.05) in absolute (19-43 %) and relative (29-104%) liver weights, were observed in both sexes at the mid (136 mg /kg/day) and high (295 mg/kg/day) dose groups.Toxicity was corroborated by changes in several serum chem istry parameters (alterations in cholesterol, bilirubin, and liver enzymes) and histopathology findi ngs (non-neoplastic and neoplastic lesions). Relative thyroid weights were elevated (p<0.05) (+33 to +64% over controls) at the mid and high dose groups and corr elated with microscopi c thyroid lesions (nonneoplastic and neoplastic). Live r and thyroid tumors were s een in both sexes. In the carcinogenicity study with mice, there were li ver nodules at the mid dose (42 mg/kg/day), alterations in a number of liver enzymes (ALSP and alanine aminotransferase) levels, and multi focal hyperplasia of the liver and liver tumors in female mice.

No treatment-related mortality or clinical signs of toxicity were observed in the Part 158 mallard duck reproduction study (MRID 42145501). For th e bobwhite quail repro duction assay, while there were no treatment-related signs of toxic ity or mortality, food consumption prior to egg laying was significantly reduced (p <0.05) at the two lowest treatme nt concentrations (96 and 295 mg/kg-diet (ppm)) while food consumption during egg laying was significantly reduced (p<0.05) at the two highest treatment con centrations (295 and 990 ppm).

In a Part 158 rainbow trout fish early life- cycle study, larvae body length was significantly
reduced at ≥ 5.0 μg a.i./L, and body weight was reduced at ≥ 14.8 μg a.i./L.

B. Estrogen pathway

Although the Tier 1 ERTA was positive for ER agonism, interaction with the estrogen pathway via the ER was not consistent with the nega tive findings in the Tier 1 ER binding and uterotrophic assays. Estrogen-rela ted effects were observed at the high dose in the Tier 1 female pubertal assay including decreases in pituitary weight, ovarian weight, blotted uterine weight, delayed vaginal opening and increased age at firs t estrus. All estrogen-related findings in the
EDSP Tier 1 in vivo assays and Part 158 mamm alian studies occurred in the presence of liver effects including increases in absolute and relative liver weights, and significant increases in creatinine, GGT and cholesterol. Additionally, an increase in relative ovarian weight in the Part 158 mammalian subchronic rat study occurred at the high dose along with significant increases
in absolute and relative liver wei ghts. The observed effects are consis tent with the findings in the mode of action studies which support hepatic en zyme induction resulting in enhanced hormone
clearance in mammals.BENFLURALIN FINAL

Page 25 of 53 No effects on the estrogen pathway were observe d in the FSTRA. In a one-generation Part 158 avian reproduction study with bobwhite quail, a d ecrease in eggs laid, as well as effects on offspring survivability and growth, were obser ved; however, these effects coincided with decreases in feed consumption. In the co mpanion mallard duck reproduction study, while an increase in eggs cracked was reported, no effects on eggshell thinning or other reproductive
effects were observed.

C. Androgen pathway

For the androgen pathway, the only positive findings seen in the Hershberger assay occurred at the high dose and in the presence of overt toxicity. Although th e presence of overt toxicity confounds the interpretation of th e effects on the endocrine para meters in the study, a follow up study to evaluate testosterone clearance showed enhanced metabo lism/clearance of testosterone
at the dose that resulted in a decreased androgen-sensitive tissue weights. This suggests that the antiandrogenic effects observed in the Hershberger assay are likely attributed to enhanced
testosterone clearance. In the male pubertal assay, decreased serum testosterone and decreases in androgen-sensitive tissue weights and epididym al weights were seen only at the high dose (400 mg/kg/day). The endocrine-related effects we re seen in the presen ce of hepatotoxicity which manifested as increases in absolute (40%) and relative (52%) liver weights and an increase (60%) in GGT level. Increased liver weights were also observed at lower doses where no androgen-related effects were seen. The only a ndrogen-related effect observed in the Part 158
mammalian studies was an increase in testicular weight in the rat chr onic/carcinogenicity study which occurred in the presence of overt toxic ity. The evidence from the mammalian toxicity studies suggests that the androge n related effects observed are due to liver enzyme induction and
increased hormone clearance and ex cretion. In the FSTRA the only notable effect in the absence of overt toxicity was an increase in male GSI s een at the middle treatment concentration. In the Part 158 avian bobwhite quail study, a decrease in offspring survivability was observed; however decreased feeding was seen in the parental generation.

D. Thyroid pathway

For the thyroid pathway, the effects in the Tier 1 assays were seen in the male and female pubertal assays, but no evidence of thyroid activ ity was seen in the AMA. In the pubertal assays, significant decreases in serum T4 were seen at all doses in the male and at the mid and high dose in the female. TSH was significantly in creased at the mid and high dose in males and high dose only in females. Increases in thyroi d weight and altered thyr oid histopathology were seen only in males at the high dose. All effects on the thyroid pa rameters in the pubertal assays were accompanied by increased relative liver weight s. Thyroid perturbations were observed in the subchronic rat toxicity study (increased thyroid weights) at a high dose that caused significant increases in liver we ight. Thyroid follicular cell hypertrophy and hyperplasia wereBENFLURALIN FINAL

Page 26 of 53 observed in both sexes in the chro nic/carcinogenicity study in rats . An increased incidence of
follicular cell adenomas/carcinomas was obser ved in this study, but at a dose that was considered to be overtly toxic.

Previously submitted mechanistic studies showed that benfluralin induces hepatic microsomal enzyme activity in both sexes in the rat. In a 14-day study submitted to support a postulated
mode of action for liver and t hyroid tumors in the rat, benfluralin was shown to induce constitutive androstane receptor (CAR) and pregna ne X receptor (PXR) related gene transcripts and associated PROD enzyme activities and be nfluralin also up-regulated T4-specific UGT genes associated with T4 metabolism. Thes e effects occurred at a dose that resulted in
decreased serum T4 and T3 levels and incr eased liver weight a nd hepatic hypertrophy. Furthermore, benfluralin is structurally similar to another dinitroaniline (trifluralin) which has been shown to induce hepatic UGTs, resulting in decreased serum T3 a nd T4 levels, elevated TSH and altered thyroid histopat hology leading to increased thyr oid tumors in rats (Saghir et
al. , 2005).

E. Conclusions

An evaluation of the Tier 1 and OSRI data sh ow that the observed effects on the endocrinerelated endpoints for the estr ogen, androgen and thyroid pathwa y occur primarily when there
were concurrent increases in liver weight. A WoE evaluation of the available data suggests that the anti-estrogenic, anti-andr ogenic and anti-thyroid eff ects observed in the mammalian in vivo Tier 1 assays may result from liver enzyme induction and subseque nt increased hormone clearance. However, there is no convincing evid ence for an interaction with estrogen, androgen or thyroid pathways in wildlife.

V. EDSP Tier 2 Testing Recommendations

For benfluralin, the current point of departur e (POD) of 0.5 mg/kg/day is based on increased histopathological lesions of th e kidneys seen in male and in female rats at 5.4 mg/kg/day (LOAEL) in a combined chronic toxicity/carcinoge nicity study in rats. This POD is used to derive the chronic Reference Dose (RfD) used for dietary exposure risk assessments and for calculating the Margins of Exposure (MOEs) fo r non-dietary exposure risk assessments. This POD is 50-fold lower than the lowest dose (25 mg /kg/day) that caused thyr oid-related effects in the Tier 1 male pubertal assay, and 600 to 800-fold lower than the doses that caused estrogenrelated (300 mg/kg/day) or andr ogenic-related (400 mg/kg/day) effects in the pubertal assays. Consequently, the chronic RfD and MOEs derive d from current PODs are protective of the endocrine-related findings in the Tier 1 in vivo assays or OSRI. In additi on the totality of the data
demonstrated that the endocrine-rela ted effects seen in the mammalian in vivo Tier 1 assays may result from liver enzyme induction and s ubsequent increased hormone clearance.BENFLURALIN FINAL

Page 27 of 53 Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is NOT
recommended for benfluralin since additional tes ting is not expected to impact EPA’s current regulatory point of departur es and endpoints for human h ealth risk assessments.

As there is no convincing evidence for a poten tial interaction with th e estrogen or androgen pathway in fish or thyroid pathways in amphi bians, no additional testing is recommended.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard
duck are available for benflurali n. In both species, effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from benfluralin exposure. Additional testing is not recommended.BENFLURALIN FINAL

Page 28 of 53 VI. References

Adams, E.R., and Jordan, W.H. (1996). Subc hronic toxicity studies of benefin (Compound 054521) administered orally in the diet to Fi scher 344 rats for 3 months. Unpublished study performed by Lilly Research Laboratories, Gr eenfield, IN. Laboratory Project ID: R33989, R44089, and R29990, February 29, 1996. MRID 44050001.

Coady K.K., and Sosinski L.K. (2011). Bene fin: Evaluation of Benefin in the Human Recombinant Aromatase Assay. Unpublished study performed by Toxicology & Environmental Research and Consulting, The Dow Chemical Co mpany, Midland, MI. La boratory Report No.: 101143, 04 October 2011. MRID 48614903.

Coady, K.K., Marino, T.A., Hutchinson, K.L., McClymont, E.L., and Thomas, J. (2011). Benefin: The Amphibian Metamorphosis Assay Using the African Clawed Frog, Xenopus laevis

. Unpublished study performed by Toxicology & En vironmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory report number 101097. MRID 48614901.

Cocke, P.J., Gunnoe M.D., and Koenig, G.R. (1990) . The Toxicity of Benefin to Rainbow Trout ( Salmo gairdneri ) in a 49-Day Early Life-Stage Study. Performed by Lilly Research Laboratories, Greenfield, IN. La boratory Study No.: F00690. MRID 41613805.

Dalgard, D. W. (1993). Benefin: 13-Week Oral Toxicity Study in Beagle Dogs. Unpublished study prepared by Hazleton Washington, Inc., Vi enna, VA. Lab Project No.: HWA 174-135, December 28, 1993. MRID 43072301

Koenig, G.R., Jordan, W.H., (1998) A Chronic Toxicity and Oncogenicity Study in B6C3F 1 Mice given Benefin (EL-11 0, Co mpound 54521) in the Diet for Two Years. Unpublished study performed by Lilly Research Laboratories, Gree nfield, IN. Laboratory Project ID: M02785 and M02885, December 14, 1988. MRID 41021501.

LeBaron, M.J., Schisler, M.R., and Visconti, N.R. (2011). Evaluation of Benefin in an in vitro androgen receptor binding assay. Unpublished st udy performed by Toxicology & Environmental Research and Consulting, The Dow Chemical Co mpany, Midland, MI. Laboratory Project ID: 111097, October 21, 2011. MRID 48614902.

LeBaron, M.J., Schisler, M.R., Visconti, N. R. (2011). Evaluation of Benefin in an in vitro Estrogen Receptor Binding Assay. Unpub lished study performed by Toxicology &BENFLURALIN FINAL

Page 29 of 53 Environmental Research and Consulting, Dow Chemical Company, Midland, MI. Laboratory Project ID: 111123, Oct ober 25, 2011. MRID 48614904.

LeBaron, M.J.; Kan, H.L. (2011) Evaluation of Benefin in an in vitro Estrogen Receptor Transcriptional Activation Assa y in Human Cell Line HeLa-9903. Unpublished study performed by Toxicology & Environmental Research and Consulting, Dow Chemical Company, Midland, MI. Laboratory Project ID: 101184, October 20, 2011. MRID 48614905.

Lebaron, M.J.; Kan, H.L.; Perala, A.W. (2011). Evaluation of Benefin in the In Vitro Steroidogenesis Assay. Unpublis hed study performed by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan, 48674. Laboratory Study No.: 101183, October 14, 2011. MRID 48614908.

Marino, T.A., K.K. Coady, R.R. Hukkanen, D.W. Louch, N.A. Malowinski, L.K. Sosinski. (2011). Benefin: A Fish Short-Term Re production Assay with the Fathead Minnow, Pimephales promelas . Unpublished study performed by Toxico logy and Environmental Research and Consulting, The Dow Chemical Company, Midl and, Michigan. Laboratory Study No.: 101165, September 22, 2011, revised October 17, 2011. MRID 48614906.

Marty, M.S., Marshall, V.A., Hansen, S.C., et al . (2011). Benefin: Hershberger assay in
castrated adult male Crl:CD (SD) rats. Unpublished st udy performed by Toxicology & Environmental Research and Consulting, Th e Dow Chemical Company, Midland, MI. Laboratory Project Study ID: 101114, October 17, 2011. MRID 48614907.

Marty, M.S., Brooks, K.J., and Jeong, Y.C. (2011). Benefin: uterotrophic assay in the immature female Crl:CD(SD) rat. Unpublished st udy performed by Toxicology & Environmental Research and Consulting, The Dow Chemical Co mpany, Midland, MI. Laboratory Project ID: 101116, June 23, 2011. MRID 48614909.

Marty, M.S., Zablotny, C.L., Hukkanen, R.R. ( 2012). Benefin: pubert al development and thyroid function in intact j uvenile/peripubertal female Cr l:CD(SD) rats. Unpublished study performed by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. La boratory Study No.: 111073, January 25, 2012. MRID 48669301.

Marty, M.S., Andrus, A.K., Stebbins, K.E. (2012) Benefin: pubertal development and thyroid function in intact juvenile/per ipubertal male Crl:CD(SD) rats. Unpublished study performed by Toxicology & Environmental Research and C onsulting, The Dow Chemical Company, Midland, MI. Laboratory Study No.: 111074, January 25, 2012. MRID 48669302.BENFLURALIN FINAL

Page 30 of 53 MacKenzie, K.M. (1985). Rat Teratology Study with Benefin. Unpublished study performed by Hazleton Laboratories, Greenfiel d, Indiana. Laboratory Study No.: 6180-101, June 24, 1985.

MRID 000147535.

Mercieca, M.D. (1991). Teratology Study in Rabbits with Benefin: Final Report. Unpublished study performed by Springborn Laboratories, Inc., Spencerville, OH. Laboratory Study No.: 3130.9, June 3, 1991. MRID 42039101.

Moore, M.R. (1996). Benefin: Two-Year Dietary Chronic Toxicity/Oncogenicity Study in Fischer 344 Rats. Unpublished study performe d by Corning Hazleton, Inc. (CHV), Midland, Michigan. Laborat ory Study ID.: CHV 174-133: DR-0097-3397-005, July 1, 1996. MRID

44050002.

Moore, M.R (1998). Benefin: Two-Year diet ary Chronic Toxicity/Oncogenicity Study in Fischer 344 Rats. Unpublished study performed by Covance Labs. Inc., VA. Laboratory Study No.: CHV174-133, DR-0097-3397-005, April 22, 1998. MRID 44545501.

Murray, A.G., Smith, J.R., and Grothe, D.W. (1991). The Toxicity of Benefin to Mallards in a One-Generation Reproduction Study. Performed by Toxicology Research Laboratories, Lilly Research Laboratories, Greenfield, Indiana. Laboratory Project No.: A01090. MRID

42145501.

Murray, A.G., Seacat, J.L., and Grothe, D.W. (1991). The Toxicity of Benefin to Bobwhite in a One-Generation Reproduction Study. Prepared by Toxicology Research Laboratories, Lilly Research Laboratories, Greenfield, Indiana. Laboratory Project No.: A00690. MRID

42145502.

S. Papineni, S., Marty, S.M., Kramer, V.J., Juberg, D.R., Gehen, S., and K. K. Coady. Benfluralin: Evaluation of Potential Estrogen, An drogen or Thyroid Eff ects. Prepared by Dow AgroSciences (MRID No.48698801)

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

Trotter, J. (1995). Two-Genera tion Reproduction Study in Rats w ith Benefin: Final Report:. Unpublished study performed by Hazleton Washingt on, Inc., Vienna, VA. Laboratory Project No.: HWA 174-136, February 9, 1995. MRID 43628701. USEPA (2010) Benfluralin: Report of the Endocrine Disruptor Revi ew Team – Test Order #: EDSP 084301-15 .December 16, 2010. Office of Chemical Sa fety and Pollution Prevention. U.S. Environmental Protection Ag ency, Washington, D.C.BENFLURALIN FINAL

Page 31 of 53 USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.

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

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

Walker, M .D. (1995). Benefin: One-Year Oral Chroni c Toxicity Study in Beagle Dogs: Final Report. Unpublished study performed by Corni ng Hazleton Inc., Vienna, VA. Laboratory Project No.: CHV 174-143 : DR-0097-3397-004, April 26, 1995. MRID 43628702.BENFLURALIN FINAL

Page 32 of 53 APPENDIX 1. EDSP T1 Screening Assays

Amphibian Metamorphosis Assay

The 21-day assay (MRID No. 48616704) of benf luralin (97.5% purity) on amphibian metamorphosis of the African clawed frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Fabe r (NF) stage 51 (80/co ntrol and treatment group; 20/replicate) were exposed to negative and solv ent (0.1 mL/L dimethylformamide; DMF)
controls and benfluralin at nominal concen trations of 0.004, 0.020, and 0.100 mg a.i./L. Timeweighted average (TWA), measured concentr ations were <0.00020 (<LOQ; controls), 0.0027, 0.0141, and 0.0744 mg a.i./L. The test system was ma intained at 21.8 to 22.3°C and a pH of 6.9

to 7.9.

There were no significant diffe rences (p>0.05) between the negative and solvent control responses for any endpoint. Unless otherwise in dicated, all effects are reported based on comparison to the negative control.

No mortalities or clinical signs ( i.e. , behavioral and other subletha l effects) were observed. Day 21 wet weight was significantly increased (p<0.05) by 10% in the low treatment group; the mid and high treatment groups were not significantly different (p>0.05) from the negative control.

Normalized (for snout-vent length) hind-limb length (HLL) was comparable for both controls and all treatment groups on Day 7 and Day 21. Th e median NF developmental stage was the
same for both controls and a ll treatment groups on both Day 7 (NF stage 54) and Day 21 (NF stage 57); no asynchronous development was observ ed throughout the test. No treatment-related effects on thyroid gland histopa thology were observed in any treatment group. Mild follicular cell hypertrophy was detected in both controls and all treatment groups.

Androgen Receptor (AR) Binding Assay

In an androgen receptor (AR) binding assay (MRID 48614902), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR. Prior to conducting the AR competitive binding experiments, saturation bi nding experiments were conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radiolabeled ligand. The satura tion binding experiment was conducted in duplicate and summarized in a submitted study profile (MRID 48614902).

The competitive binding assay was conducted to meas ure the binding of a single concentration of [ 3 H]-R1881 (10 nM) in the presence of increasing concentrations of benfluralin (logarithmic
increase from 10 -10 to 10 -3 M). Ethanol was used as a vehicl e at a final assay concentration of <3%. The assay included dexamethasone as a weak positive control, and R1881 as the ligandBENFLURALIN FINAL

Page 33 of 53 reference standard. Three independent runs were conducted with 3 replic ates per concentration

per run.

In the saturation binding experime nt, the maximum binding capacity (B max ) was 3.245 fmol/100 μg protein and the di ssociation constant (K d ) was 0.4641 nM. These values were below the range of values from the validation studies (7 to 16 fmol/100 μg protein and 0.685-1.57 nM, respectively). The Scatchard plot indicated a line ar response across the c oncentrations of ligand added. Nonspecific binding as a percent of total binding was less than 20% across the entire concentration range in the satu ration binding assays (range 6.2-19.8 %), with the exception of the high concentration (10 nM) in one assay, which was 24.6%.

The estimated log IC 50 s for R-1881 and the weak positive co ntrol (dexamethasone) were -8.975 and -4.509 M, respectively. The mean RBA for the positive control wa s 0.0034%. The solvent control responses indicate d no drift in the study assa y. All performance crit eria were met, with the exception of the bottom (% bindi ng) of R1881. In Run 2, the bottom was 2.1, just outside the recommended range of -2.0 to 2.0%. The range fo r the other two runs was -1.0 to -1.7, with a mean value for the three assays of -1.6%. The re viewers consider this a minor deviation and find that the performance criteria were met.

The lowest percent total [ 3 H]-R1881 binding at all concentrati ons of benfluralin was 77%, 78%, and 75% resulting in classification for the runs as non-binder, non-binder a nd equivocal in runs 1, 2, and 3, respectively. An estimated mean log IC 50 for benfluralin could not be determined since benfluralin did not displace more that 50% of the radioligand at any concentration.

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

Aromatase Assay

In an in vitro aromatase (CYP 19) assay (MRID 48614903) , Benfluralin (97.5% a.i., Lot No. ACD13683, TSN100037) was incubate d with human recombinant aromatase and tritiated androstenedione (1- β [ 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) in dimethyl sulfoxide (DMSO) at concentrations of 0, 10 -10 , 10 -9 , 10 -8 , 10 -7 , 10 -6 , 10 -5.5 , 10 -4 , or 10 -4.5 M for 15 minutes to assess the potential of benflu ralin to inhibit aromatase activit y. Evidence of test material insolubility was observed at 10 -3 and 10 -4 M therefore the highest test concentration analyzed

was 10 -4.5 M.

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. Tritia ted water was quantified with liquid scintillation counting (LSC). Th ree runs were conducted, and each run included aBENFLURALIN FINAL

Page 34 of 53 full activity control, a bac kground activity control, a pos itive control series (10 -10 to 10 -5 ) with a known inhibitor (4-hydroxyandrostenedione; 4-OH AS DN), and the test chemical series (10 -10 to
10 -4.5 ) with three repetitions per concentration. The proficiency evaluation data for the investigating laboratory regard ing the aromatase assay were not included in the study.

The response of the full activity controls and background controls were acceptable for each run, although the mean background count from assay Run 3 was approximately 100-fold greater compared to the previous two assay runs.

The standard curves for 4-OH ASDN met three out of four performance criteria in Run 1, all four criteria in Run 2, and only two out of four perfor mance criteria in Run 3. These deviations from the guideline recommendations for performan ce criteria were considered minor by the investigators because acceptable Hill slope valu es and goodness of fit values were obtained for all three assay runs. In general, values in Run 3 were more variable compared to the previous two assay runs, including a highe r background count and higher %CVs for the replicate tubes.

Aromatase activity was similar across the test ed concentration range for benfluralin (10 -10 M to
10 -4.5 M). The aromatase activity curves after exposur e to benfluralin either did not fit the model (Run 1) or were not lower than 75% of full activity (Runs 2 and 3). The log IC 50 of the test
material was -8.0 M.

Based on the data from the average response curv e, and despite the larg e variability introduced by the data from assay Run 3, benfluralin is classi fied as a Non-inhibitor of aromatase activity in this assay.

Estrogen Receptor (ER) Binding Assay

In an estrogen receptor (ER) binding assa y (MRID 48614904), uterine cytosol from SpragueDawley rats was used as the s ource of estrogen receptors (ER) to evaluate the potential for benfluralin to displace the bound refe rence estrogen, radiolabeled [ 3 H]-17 β -estradiol from the ER. A saturation binding experiment was conduct ed prior to the competitive binding experiment
to demonstrate that the ER in the uterine cytosol was present in adequate numbers and functioning with the appropriate affinity for th e radiolabeled ligand. Saturation binding data were not presented in the study report; however, saturation binding data was presented in the study profile submitted separately by the test order recipient (study profile, MRID 48675404).

The competitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of increasing concentrations ofBENFLURALIN FINAL

Page 35 of 53 benfluralin (logarithmic increase from 10 -10 to 10 -3 M). Ethanol was used as a vehicle at a final concentration of <3%. The assay included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17- β -estradiol as the natural ligand reference material. Three independent runs were conducted with 3 replicates per concentration per run.

In the saturation binding experime nt, the maximum binding capacity (B max ) was 59.28 fmol/100 μg protein and the di ssociation constant (K d ) was 0.1032 nM. The B max fell within the expected range of 10 to 150 fmol/100 μg protein and the K d for the run was within the expected range of 0.03 to 1.5 nM. Nonspecific binding as a percent of total binding was 1.7%-8.6% across the entire concentration range in the saturation bind ing assay. Only a single run of the saturation binding experiment was conducted on the batch of cytosol preparation used for the competitive binding experiment.

For the competitive binding experiment, all pe rformance criteria were met for appropriate responses by 17 β -estradiol and 19-norethindrone. The log IC 50 and RBA could not be determined for benfluralin as it did not displace ≥ 50% for the radiolabeled estradiol from the ER at any concentration. The bottom of the curve for per cent of total binding at the 95% confidence interval for benfluralin was 87.7%, 70.2%, and 75.9% in Runs 1, 2, and 3, respectively.

Based on the results from the three runs, benflura lin is classified as Not Interactive in the Estrogen Receptor Binding Assay.

ER α Transcriptional Activation Assay

In an estrogen receptor transcriptiona l activation assay (MRID 48614905) conducted by Toxicology & Environmental Research and Consulting, hER α -HeLa-9903 cells cultured in vitro were exposed to benfluralin (97.5% purity Lot# ACD13683, TSN100037 at concentrations of

10

− 4 , 10

− 5 , 10

− 6 , 10

− 7 , 10

− 8 , 10

− 9 and 10

− 10 M in DMSO (final concentration 0.1%) for 24 hours. The experiments were performed using 96-well plates and each benfluralin concentration was tested in triplicate (3 wells/plate) in each experiment. Cells were exposed to the test substance for approximately 24 hours to induce reporter (lucif erase) gene products. Luciferase expression in response to activation of the estrogen recep tor was measured upon addition of a luciferase
substrate and detection with a lumi nometer with acceptable sensitivity.

Benfluralin was tested up to the limits of both cytotoxicity and solubility; 10

− 3.5 M was both cytotoxic to the cells (79.1% viability compared to controls) and precipit ated in the cell culture media at the end of the 24-hour treatment duration. The positive control and reference chemicals induced the appropriate responses, al though the assay's responsiveness to 17 α -methyltestosterone indicated increased sensitivity to weak agonists. The RPC Max (maximum level of responseBENFLURALIN FINAL

Page 36 of 53 induced by chemical expressed a percentage of the response induced by 1 nM estradiol (E2) on the same plate) was 13.3-19.1%

. The PC Max was the limit of solubility and cytotoxicity, 10

− 4 M.

Because the RPC Max > PC 10 in all three assay runs, benfluralin was considered positive for estrogen receptor transcriptional activation in this test system.

Fish Short Term Reproduction Assay

The 21-day short-term reproduction assay of benfluralin with fathead minnows ( Pimephales promelas ) was conducted under flow-through conditions. Adult fish (20 spawning groups; 2 males and 4 females in each group; 1 group/replicate and 4 replicates/control or treatment ; ca . 6 months old) were exposed to negative and solvent (0.0 5 mL/L dimethylformamide; DMF) controls and benfluralin (97.5% purity) at nominal c oncentrations of 0.00033, 0.0033, and 0.033 mg a.i./L; time-weighted average (TWA), measured conc entrations were <0.0000 505 (<LOQ; controls), 0.000302, 0.00314, and 0.0365 mg a.i./L. The test syst em was maintained at 24.3 to 25.5 o C and a pH of 7.5 to 7.8.

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

Overt toxicity ( e.g. , mortality, lethargy, loss of equilibri um, bloating) was observed among fish exposed to the high treatment level with ≥ 38% of the fish affected; therefore, this treatment group was removed from the study and excluded from any s ubsequent statistical an alyses. In the low and mid treatment levels, no effect on mortality was observed; overa ll mortality was 4.2% in the negative and solvent cont rols and low treatment group and 0% in the mid treatment group. No treatment-related effects on secondary se x characteristics or clinical signs ( i.e. , behavioral and other sublethal effects) were observed; however, one female in the mid treatment group was observed to have slight vertical banding present. Benfluralin exposure was not associated with any significant changes (p>0.05) in body weight or length in males or fe males relative to the negative
control.

With the exception of one negative control repli cate and two solvent cont rol replicates, spawning occurred every 4 days. Fecundity in the ne gative and solvent cont rols was 30.7 and 18.8 eggs/female/day/replicate, resp ectively (range for negative cont rol: 13.2-49.0; range for solvent control: 11.0-26.9). Mean fertilization success in th e negative and solvent controls was >99%. No significant differences (p>0.05) in fecundity or fertilization success were observed in the benfluralin treatment groups relative to the negative control. Plasma vitellogenin (VTG) was not significantly different (p>0.05) in males or fema les compared to the negative control. Gonadosomatic index (GSI) was significantly increased (p<0.05) by 27% in males at the mid treatmentBENFLURALIN FINAL

Page 37 of 53 level compared to the negative control; there were no significant differences (p>0.05) in GSI in females compared to the negative control. Ma le nuptial tubercle scor e was not significantly different (p>0.05) relative to the negative co ntrol; no tubercles were observed in females.

No treatment-related effects on gonadal histopathol ogy were observed in males. The incidences of grade 4 (severe) oocyte atresia (9 or more atretic oocytes in a section) were slightly higher in the solvent control group (n=6) and the low treatment group (n=7) relative to th e negative control and the mid treatment group (n=0 for both). However, the reason for this increase was not clear and
was deemed not to be treatment related. Five of the six reported incidences of severe atresia in the solvent control group occurred in the two replicates in which a decrease in fecundity was observed, and all four females in the low treatment replicat e that produced no eggs also had severe oocyte
atresia.

While not statistically significant (p>0.05), mean fecundity in the so lvent control group was reduced by 39% compared to the negative control. Therefore, there is uncertainty as to whether there were solvent effects in the study. Two of th e four replicates in the solvent control had fecundity rates of 11 eggs/female/day, and these replicates did not spawn every four days; the fecundity rates for these two replicates seven da ys prior to the exposur e period were 27.3 and 20.6 eggs/female/day, suggesting adequate spawning prior to test initiation. Fec undity in one replicate of the negative control was also reduced compared to the pre- exposure period, and the replicate did not spawn every four days (13.2 eggs/femal e/day in the 21-day exposure period versus 31.1 eggs/female/day in the 7 days prior to the expos ure period). Although the observed decline in these replicates may be due to biologi cal variation, the possib ility of a solvent in terference cannot be conclusively ruled out. One replicate in the lo w treatment group did not produce any eggs during the exposure period. While not statistically different (p>0.05) from the negative control, decreases of 29 and 21% in fecundity in the low and mid c oncentration groups, respectively, were of a similar magnitude as that for the solvent control. Ther efore, the results of the study do not suggest a significant treatment-related effect on fecundity.

Hershberger Assay

A Hershberger Assay (MRID 48614907) was performe d to investigate both androgenic and antiandrogenic effects in male rats. To screen for potential androgenic activity, benfluralin (benefin; 97.5% a.i., lot # ACD13683) in corn oil was admini stered daily via oral gavage to groups of seven 55-day old, castrated male Sprague Dawley ra ts at dose levels of 0 (vehicle), 50, 250, or 750 mg/kg/day. An androgenic positive control group of 7 castrated rats exposed to 0.4 mg/kg/day testosterone propiona te (TP) by subcutaneous in jection was also included.BENFLURALIN FINAL

Page 38 of 53 To screen for potential anti-andr ogenic activity, benfluralin was administered daily via oral gavage to groups of seven 55-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 50, 250, or 750 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by subcutaneous injection. An anti-androgenic positiv e control group of 7 castrated rats exposed to 0.4 mg/kg/day TP and 3.0 mg/kg/day flutamide (FT) was included. TP alone at 0.4 mg/kg/day was used as th e anti-androgenic negative control.

For both components of the assay, the animal s were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration to determine weights of the five androgen-dependent tissues. In the androgen antagonist por tion of the study, blood samples were taken prior to sacrifice to evaluate serum testosterone levels in animals treated with TP + 0, 50, 250 or 750 mg/kg/day benfluralin for 10 days.

In addition, a 14 C-testosterone clearance expe riment was conducted to examine if the decrease in androgen-responsive tissue weights were attr ibuted to enhanced clearance of exogenous testosterone secondary to hepa tic enzyme induction by benflurali n. In this study, two groups of seven castrated, jugular vein-cannul ated male rats were dosed w ith benfluralin daily via oral gavage to at dose levels of 0 (vehicle) or 750 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/ day by subcutaneous injection. 14 C-Testosterone was administered on Days 1 and 10 to measure plasma clearance of 14 C-testosterone radioactivity. On Day 1, all animals had blood samples collected from the cannulae at 0.08, 0.17, 0.25, 0.5, 1, 2, 3, and 6 h post-dosing. On Day 10, three c ontrols and four 750 mg/kg/day rats remained patent and had blood samples collected as on Day 1, except the 6 h sample was collected via the orbital sinus. The non-patent animals only had 6 h samples collected vi a the orbital sinus. Clinical signs, body weight and androgen res ponsive tissue weights were also evaluated.

All animals survived until scheduled termina tion. There were no treatment-related gross pathological findings.

In the androgen agonist assay, 3/ 7 rats were noted to have decr eased feces and 2/7 rats were observed with clear perioral so iling at 750 mg/kg/day. Additiona lly at this dose, body weights were decreased (p ≤ 0.05, except not significan t [NS] on Day 11) on Days 4, 7, and 11 by 7-11%, resulting in a 15.0 g cumu lative body weight loss (p ≤ 0.05) for Days 1-4 and decreased (p ≤ 0.05) cumulative body weight gains for Days 1-7 ( ↓ 76%) and Days 1-11 ( ↓ 49%). Cumulative body weight gains were also decreased (p ≤ 0.05) by 61% for Days 1-4 in the 250 mg/kg/day group; however, body weights in this group were similar to controls, as were cumulative body weight gains for Days 1-7 and 1-11. Animals dosed with 0.4 mg/kg/day TP had 6-7% increases (p ≤ 0.05) in body weights on Days 7 and 11, resulting in increased (p ≤ 0.05) body weight gains for Days 1-4 ( ↑ 55%), 1-7 ( ↑ 60%) and 1-11 ( ↑ 44%). During Days 1-4, food consumption was decreased (p ≤ 0.05) by 12-44% at 250 mg/kg/ day and above. There were no significant increases in any of the five target accessory sex organs of animals dosed with the test compound. AbsoluteBENFLURALIN FINAL

Page 39 of 53 liver weights were increased (p ≤ 0.05) by 38-68% in the 250 mg/kg/day and above groups. Animals in the 0.4 mg/kg/day TP group had accesso ry sex organ weight in creases in all five accessory sex organs indicating that the assay was responding properly. The %CVs for the vehicle controls, 0.4 mg/kg/day TP group, and th e 750 mg/kg/day group were compared to the performance criteria values as stated in the Guid eline. The %CV for the ventral prostate in the
vehicle controls exceeded the criteria value (70% vs. 45% maximum recommended); however, is unlikely that these data would have affected th e interpretation of th e assay results since no
increases were observed in the other tissue we ights. All other %CVs were less than the
maximum recommended values.

In the anti-androgen assay, 3/7 rats at 750 mg/kg/day were observed to have decreased feces. Cumulative body weight gains were decreased (p ≤ 0.05, except NS on Day 11) on Days 1-4 ( ↓ 55%), 1-7 ( ↓ 37%) and 1-11 ( ↓ 23%) at 250 mg/kg/day; however , body weights in this group were similar to controls. At 750 mg /kg/day, body weights were decreased (p ≤ 0.05) on Days 4, 7, and 11 by 10-14%, resulting in a 1 5.1 g cumulative body weight loss (p ≤ 0.05) for Days 1-4 and decreased (p ≤ 0.05) cumulative body weight gains for Days 1-7 ( ↓ 69%) and Days 1-11 ( ↓ 57%). Animals dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p ≤ 0.05) cumulative body weight gains on Days 1-4 ( ↓ 22%) and 1-7 ( ↓ 19%); however body weights and cumulative body weight gains on Days 1-11 were si milar to TP controls. During Days 1-4, food consumption was decreased (p ≤ 0.05) by 23-50% at 250 mg/ kg/day and above, and was decreased at 750 mg/kg/day during Days 4-7 and 7-11 ( ↓ 13% both periods).

In the 750 mg/kg/day group, accessory sex organ we ight were significantly decreased in two of the five target tissues: levator ani-bulbocaver nosus (LABC; 33%) and seminal vesicles (27%). Non-significant decreases were obs erved in the remaining three target tissues. Absolute liver weights were increased (p ≤ 0.05) by 39-65% in the 250 mg/kg/da y and above groups. Animals dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p ≤ 0.05) accessory sex organ weights in all five access ory tissues to indicate that the test system was responding properly to detect androgen antagonists. In rats trea ted with 750 mg/kg/day + 0.4 mg/kg/day TP, serum
testosterone levels were decrea sed (NS) by 29% compared to TP controls. The %CVs for the vehicle controls, 0.4 mg/kg/day TP group, and th e 750 mg/kg/day group were compared to the performance criteria values according to the Guide line. The %CV for the ventral prostate in the
vehicle controls exceeded the criteria valu e (70% vs. 45% maximu m recommended), but was unlikely to have affected the interpretation of th e assay results. All ot her %CV were less than
the maximum recommended values.

In the 14 C-testosterone clearance experiment, 6/7 rats dosed with 750 mg/kg/day had urine perineal staining, and 2/7 rats had red peri nasal staining. In the 750 mg/kg/day group, body weights were decreased (p ≤ 0.05) by 12-20% on Days 4, 7, and 10; resulting in body weight losses of 18.7 g and 1.0 g for Days 1-4 and 1-7, and decreased (p ≤ 0.05) cumulative body weight gains for Days 1-10 ( ↓ 85%). Animals dosed with 750 mg/ kg/day of the test compound had nonBENFLURALIN FINAL

Page 40 of 53 significant decreases in accessory sex organ weig hts: 17% in Cowper’s gland; 25% in glans penis; 37% in LABC; 53% in ventral prostate; and 48% in seminal vesicles. Absolute liver weights were increased (NS) by 38% in this grou p. Rats treated for 10 days with 750 mg/kg/day of the test compound cleared 14 C-radioactivity 3.7-fold faster (p ≤ 0.05) than the TP-only controls.
This was a result of the elimin ation half-life present in the 750 mg/kg/day group. Additionally, the 750 mg/kg/day animals had an AUC that was lower (p ≤ 0.05) than the TP-only controls (0.014 μ g×h×g -1 treated versus 0.039 μ g×h×g -1 control).

Benfluralin 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. Howeve r, benfluralin was positive for anti-androgenicity in the Hershberger assay as statistically significan t decreases in organ weights were seen in two of the five androgen sensitive tissues in the androgen antagonist assay. At 750 mg/kg/day, a significant decrease in body we ight (12%; p<0.05) and body wei ght gain (57%; p<0.05) were seen after 10 days of treatmen t indicating overt toxici ty at the dose that showed decreases in androgen-sensitive tissue weights. Additional data provided on testosterone clearance in benfluralin-treated rats in conjunction with th e observed increase in liver weights, provide evidence that the reduced accessory sex orga n weights may be secondary to increased
testosterone metabolism and clearance.

Female Pubertal Assay

In a Female Pubertal Assay (MRID 48669301), groups of 16 Crl:CD(SD) rats were treated daily via oral gavage with benfluralin (97.5% a.i.; Lo t# ACD13683) in corn oil at doses of 0, 25, 75 or 300 mg/kg/day from post-natal day (PND) 22 to 42. Vaginal opening (VO) was evaluated daily, and age and weight at attainme nt of VO were recorded. Once VO was completed, vaginal lavage samples were collected daily (including the day of necropsy) to evaluate estrus cycle length and pattern. After sacrifice on PND 42, blood was co llected and a gross necropsy was conducted. Clinical chemistry parameters were evaluated, including serum thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels using a ra dioimmunoassay assay (TSH) or chemiluminescent immunoassay (T 4 ). The following organs were weighed: liver, kidneys, adrenals (weighed as pairs), ovaries (without oviducts, weighed as pairs), imbibed and blotted uterus (with oviducts and cervix), thyroid, and pituitar y. Microscopic evaluations were performed on thyroid, kidneys, uterus, and typically the right ovary.

All animals survived until sche duled termination. One 75 mg/kg/ day female was removed from the study after being dosed twice on one day. All of the 300 mg/kg/ day animals had dark yellow urine, a typical finding following dinitroaniline herbicide exposure. There were no significant
treatment-related effects on mean estrous length, percent cycling, percent regularly cycling orBENFLURALIN FINAL

Page 41 of 53 body weight at VO. There were no gross finding s at necropsy and no histopathological lesions in the uterus, ovaries, kidneys or thyroid.

Animals treated at 300 mg/kg/day ha d decreased (p<0.05) body weights ( ↓ 8-10%) and cumulative body weight gains ( ↓ 15-42%) through PND 35; te rminal body weights largely recovered by necropsy at PND 42 ( ↓ 4%; NS) with a 6% decrease (p<0.05) in overall body weight gain (PND 22-42). Both unadjusted and adjusted mean ages at VO were increased (p<0.05) in the 300 mg/kg/day group (PND 37.2-37.3 treated vs. PND 34.4 controls) but not at lower dose levels. Age at first estrus was also increased (p<0.05) at 300 mg/kg/day (PND 37.2 treated vs. PND 35.4) due to the delay in VO.

Serum T 4 levels were decreased (p<0.05) by 13% at 75 mg/kg/day and 44% at 300 mg/kg/day, and TSH levels were increased (p<0.05) by 109% at 300 mg/kg/day; however, there were no associated changes observed in thyroid weight or histopathol ogy. Other clinical chemistry changes in the 300 mg/kg/day animals include d: decreased (p<0.05) alkaline phosphatase ( ↓ 16%) and increased (p<0.05) gamma glutamyl transpeptidase (GGT, 47%), cholesterol ( ↑ 12%), and creatinine ( ↑ 44%). Unadjusted, adjusted, and re lative liver weights were increased (p<0.05) by 19-23% at 75 mg/kg/ day and 32-37% at 300 mg/kg/day, and relative liver weight was increased (p<0.05) by 8% at 25 mg/kg/da y. At 300 mg/kg/day, de creases (p<0.05) were also observed in pituitary weight (unadjusted, adjusted, and relative; ↓ 12-19%), ovary weights (unadjusted and adjusted; ↓ 17-18%) and blotted uterus weight (adjusted; ↓ 21%).

The high dose of 300 mg/kg/day is considered adequate based on decreased body weight gains over the course of the treatment period and incr eased liver weights with concomitant significant decreases in alkaline phosphatase, and increases in GGT and cholesterol.

Male Pubertal Assay

In a male pubertal assay (MRID 48669302), groups of 16 Crl:CD(SD) rats were treated daily via oral gavage with benfluralin (97.5% a.i.; Lot #ACD13683) in corn oil at doses of 0, 25, 100 or 400 mg/kg/day from post-natal day (PND) 23 to 53. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and the age and weight of each animal on the day of attainment was recorded. Following sacrifi ce on PND 53, total serum te stosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) le vels were analyzed using radioimmunoassay (RIA) or chemiluminescent immunoassay. The follo wing organs were wei ghed: liver, kidneys and adrenals (weighed as pairs), testes and ep ididymides (weighed indi vidually), thyroid and pituitary (weighed after fixation) , ventral prostate, dorsolateral prostate, seminal vesicle with coagulating glands, and the levator ani plus bulbocavernosus muscles (LABC). Microscopic evaluations were performed on testes, epididymides, thyroid, and kidneys.BENFLURALIN FINAL

Page 42 of 53 All rats survived to scheduled termination. There were no treatment related effects on mean age and weight at completion of PPS, mean weights of adrenals, ki dneys, and testes, and gross and microscopic examinations of the testes, epididymides, and kidneys.

Systemic findings were observed at 400 mg/ kg/day. Dark yellow/orange urine, a common finding following dinitroaniline he rbicide treatment, was observed in all rats. Unadjusted and adjusted final body weights were decreased (not significant [NS]) by 8% , resulting in a 9% decrease (p<0.05) in overall body weight gains (PND 23-53). Unadjusted, adjusted, and relative (to body) liver weights were incr eased (p<0.05) by 40-52%, unadjusted and adjusted pituitary weights were decreased (p<0.05) by 13-14%, and unadjusted and ad justed thyroid weights were increased (p<0.05, except unadjusted NS) by 15%. Serum alkaline phosphatase levels were decreased (p<0.05) by 40%, and serum total protei n, creatinine, and gamma glutamyl transferase were increased (p<0.05) by 7%, 50%, and 60%, respectively. Globulin was increased (not statistically analyzed) by 22%. Phosphorus, ch loride, and potassium levels were decreased (p<0.05) by 9%, 2%, an d 6%, respectively.

At 100 mg/kg/day, unadjusted, adjusted and rela tive (to body) liver weights were increased (p<0.05) by 22-23%. Serum alkaline phosphatase levels were decreased (p<0.05) by 17%. Phosphorus was decreased (p<0.05) by 9%.

At 25 mg/kg/day, only relati ve liver weight was in creased (p<0.05), by 7 %.

There was a dose-dependent de crease (p<0.05) in serum T 4 levels of 12%, 29% and 53% at 25, 100 and 400 mg/kg/day, respectively, and a dose- dependent increase (p <0.05) in serum TSH levels of 53% and 105% at 100 and 400 mg/kg/day, respectively. Serum testosterone was also decreased (NS) by 52% at 400 mg/kg/day. When one animal with a testosterone level well above the group average was excluded from the an alysis, the decrease in testosterone at 400 mg/kg/day was 69% (p<0.05). At 400 mg/kg/day, decreased (p<0.05) unadjusted and adjusted accessory sex tissue weights were observed as foll ows: (i) seminal vesicle + coagulating gland with fluid by 32%; (ii) seminal vesicle + coagul ating gland without flui d by 25-26%; (iii) ventral prostate by 26%; (iv) dorsolater al prostate by 21-22%; (v) LABC by 20%; and (vi) epididymis by 13-18%. At 100 mg/kg/day, only adjusted right epididymis weight was decreased (p<0.05), by 9%. Animals treated with 400 mg/kg/day had incr eases in thyroid follicul ar cell height with concomitant decreases in colloid area. A slight decrease in spermatic elements in the right epididymis was observed in two anim als (#6976 & #6982) at 400 mg/kg/day.

The dose levels used in this study were considered to be adequate based on markedly increased liver weights and significant changes in thyroid hormone levels and histopathology, as well as a significant decrease in body we ight gain, at 400 mg/kg/day.BENFLURALIN FINAL

Page 43 of 53 Steroidogenesis Assay

In a steroidogenesis assay (M RID 48614908), H295R cells cultured in vitro in 24-well plates
were incubated with benfluralin (97.5% purity, Lot # ACD13683, TSN100037) at logarithmic
concentrations from 10 -10 to 10 -4 M in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.1%.

Testosterone and estradiol leve ls were measured with LC/A PPI-MS/MS. Three independent experiments were performed. A Quality Contro l (QC) plate was run concurrently with each independent run of the test chem ical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels ; positive controls included the known inducer (forskolin) and inhi bitor (prochloraz) of estrad iol and testosterone production.

Benfluralin was tested at concentrations up to 10 -4 M with no evidence of precipitation or cytotoxicity. Although the final assay run wa s conducted with H295R cells from passage 11.5 (one passage higher than the recommended maximu m of 10), the guidelin e requirements for CV

≤ 30% between replicate wells with in a plate and between plates were met for all three assay runs. Estradiol production was st atistically increased 1.2-fold in two of three independent assays

at 10 -4 M and by 1.3-fold in one assay at 10 -5 M, but no dose-response was observed. Slight
decreases in testosterone and es tradiol production in a single run were also not considered of biological relevance as st atistically significant differences at concentrations that do not follow a dose-response curve may be due to random effects.

Based on the hormone responses in three independent runs, benfluralin treat ment did not result in statistically significant and repr oducible alterations in testoste rone or estradiol production.

Uterotrophic Assay

In an uterotrophic assay (MRID 48614909) conducted to screen for potential estrogenic activity, Benefin (97.5% a.i., lot # ACD13683) in corn oil was administered daily via oral gavage to groups of six immature female Sprague-Dawley rats at dose levels of 0 (vehicle), 0 (untreated), 25, 75, or 200 mg/kg/day on post-natal day (PND) 19-21. A positive control group was treated
with 17 α -ethynyl estradiol (EE) by daily oral gavage at a dose level of 10 μg/kg/day. All animals were terminated and necropsied appr oximately 24 hours after the final dose on PND 22 to determine wet and blotted uterine weights.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in animals for any dose groups. No precocious vagi nal opening was observed in the test animals.BENFLURALIN FINAL

Page 44 of 53 Body weights in the dose groups were comparable to the controls throug hout the duration of the assay. At a dose of 200 mg/kg/da y, body weight gains were decrea sed by 30% compared to the controls. .Uterine weights in the dose groups were comparable to the controls. Absolute wet and blotted uterus weights for the 10 μg EE group were increa sed (p<0.05) by 811% and 466%, respectively.

No statistically sign ificant changes were seen in uterine weight in this study. Benfluralin was negative in the uterotrophic assay.BENFLURALIN FINAL

Page 45 of 53 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

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

In three separate subchr onic toxicity studies ( MRID 44050001 ), benfluralin (~95.6% purity/a.i., Lot/Batch #231EF4) was administered to rats in the diet at concentrations ranging from 0.005 to 0.75% for 3 months. In Study R33989, 15 rats/sex/dos e were fed benfluralin at nominal dietary concentrations of 0, 0.025, 0.11 or 0.5% (0, 17, 74 or 341 mg/kg/day for males and 0, 20, 94 or 395 mg/kg/day for females) for 3 months. In Study R44089, 15 rats/sex /dose were fed diets containing nominal concentrations of 0 or 0.75% benfluralin (0 or 522 mg/kg/day for males, and 0 or 605 mg/kg/day for females) for 3 months. In Study R29990, 15 males/dose were fed diets containing benfluralin at no minal concentrations of 0, 0.005, 0.05 or 0.5% (0, 3, 32 or 322 mg/kg/day) for 3 months.

In Study R33989, males at 341 mg/kg/day had incr eased absolute adrena l (10%) and thyroid (18%) gland weights and both sexes had increas ed relative adrenal ( 10-12%) and thyroid (17- 22%) gland weights. In study R44089, males (522 mg/kg/day) and females (605 mg/kg/day)
exhibited increased relative ad renal gland weights (males, 11%, and females 9%). Treated females also had significant (p<0.05) increase in absolute and relative t hyroid weights (22% and 27%, respectively) and relative ovary (14%) weights when compar ed to controls. No treatmentrelated histopathological findings were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, uterus, cervix, va gina, or mammary, adrenals, pitu itary, and thyroid glands in either sex in any study.

Concentration-dependent hepatic changes obser ved at approximately 0.025% included increased absolute and relative liver weights and minima l to slight hepatocellular hypertrophy. The absolute and relative liver weights in both sexes were increased at ≥ 0.025% (0.025 to 0.75%)
treatment doses. Livers of rats in the ≥ 0.025% treatment groups exhibited mild diffuse centrilobular hypertrophy. Enlarged livers were noted in the 0.11% group males, and in both sexes treated at 0.5 and 0.75%. Hepatic microsom al enzyme activities we re increased in both
sexes treated at ≥ 0.11%. Concentration-dependent nephrot oxic effects were observed in all treatment groups. Body weights were significant ly (p<0.05) decreased at 0.75% (<10%), and food consumption was significantly (p<0.05) decreased at ≥ 0.5%. Under the conditions of these studies, the subchronic LOAEL for these studies is 3.23 mg/kg/day (0.005% benfluralin), based on hyaline droplet formation in the kidneys of males at this dose level. A NOAEL was not
established.BENFLURALIN FINAL

Page 46 of 53 90-Day (Subchronic) Oral Toxi city in Non-Rodents (Dog)

Male and female beagle dogs were orally dosed (capsules) with benfluralin (95.8% purity/a.i., Lot/Batch #ACD13683) at 0, 5, 25 or 125 mg/kg/day for 13 weeks ( MRID 43072301 ). Weights were obtained for testes, ovaries, pituitary and thyroid; histopathologi cal examinations were conducted on the testes, epididymides, prostate , ovaries, uterus, vagina, mammary gland, and pituitary and thyroid glands. No significant, treatment-related effects were observed on body weight throughout the study. Significant findings consisted of in creased absolute and relative liver weights in high-dose males. This observa tion correlated with the increased incidence of minimal to slight centrilobular to diffuse he patocellular hypertrophy. Since clinical pathology findings did not indicate any he patotoxicity, the observed ch anges in liver weight and histopathological findings are suggestive of an adaptive, rather than toxic, effect. Other histopathological findings includ ed increased splenic pigment of mid- and high-dose animals. Based on the results of this study, the L OAEL was 25 mg/kg/day and the NOAEL was 5 mg/kg/day.

Prenatal Development in Rodents (Rat)

In a developmental toxicity study ( MRID 000147535 ), benfluralin (95.3% purity/a.i., Lot/Batch #219EF4) was administered to 25 Crl:CD (S D) rats/group by gavage at 0, 50, 225, 475 or 1000 mg/kg/day in 10% acacia from gestation day (GD) 6 through 15. Body weights were determined on GD 0, 6, 11, 16, and 20 (prior to sacr ifice); food consumption was also measured.
Uterine contents were evaluate d at sacrifice (GD 20). Matern al body weights were significantly (p>0.05) decreased on GD 11 (96% of c ontrol) and 16 (96% of control) at ≥ 475 mg/kg/day and body weight gain was significantly decreased on GD 6 to 11 (48% of control) at the same dose levels. Food consumption was also significantly depressed on GD 6 to 11 (81-85% of control) and on GD 11 to 16 (93% of control) at the same doses. At necropsy, dilated renal pelvises and enlarged hepatic lobes were noted at all doses. No consistent, treatment-related differences in the intrauterine parameters be tween the treated and control gr oups were observed. The number of litters examined in the dose and control groups were 24 or 25. At 225 mg/kg/day, a statistically significant increased number of male fetuses with unossified sternebrae were found affecting 14, 16, 15, 15 and 21 litters at 0, 50, 225, 475, and 1000 mg/kg/day, respectively. In addition, at 225 mg/kg/day, a statis tically significant increased numbe r of fetuses were seen with
dark brown-red areas on the live r (0, 0, 5, 1, 4 1itters at the ab ove respective doses). The maternal NOAEL is 225 mg/kg/day and the ma ternal LOAEL is 475 mg/kg/day based on body weight decreases on GDs 11 and 16, and the body weight gain decrement between GD 6 to 11. The developmental NOAEL is 1000 mg/kg/da y and a developmental LOAEL was not
determined.BENFLURALIN FINAL

Page 47 of 53 Prenatal Development in Non-Rodents (Rabbit)

In a developmental toxicity study ( MRID 42039101 ), benfluralin (95.6% purity/a.i., Lot/Batch #231EF4) was administered to 20 New Zeala nd White rabbits/group at 0, 25, 50, 100 or 225 mg/kg/day by gavage from gestation da y (GD) 6 through 18. Body weights were determined on GD 0, 6, 9, 12, 15, 19, 24, and 29 (prior to sacrifice); food consumption was also measured. On Day 29, uterine contents were evaluated. Maternal body weight gain was significantly decreased (p<0.01; 6% of control) between GD 6- 19 in the 225 mg/kg/day does, and was nominally reduced (68% of control) during the same period in the 100 mg/kg/day
animals. One maternal death and 3 abortions oc curred at 225 mg/kg/day. In addition, incidences of few (decreased) feces were s een in the two top dose groups (6 and 6, respectively, versus 0 control). Food consumption was reduced be tween GD 6-19 in the two highest dose groups (p<0.01; 70% of control at 225 mg/kg/day), and may have been related to the body weight gain depression, as well as the few feces. No cons istent, treatment-related differences in the intrauterine parameters between the treated a nd control groups were observed. The maternal NOAEL was determined to be 100 mg/kg/day and the maternal LOAEL was 225 mg/kg/day based on dose-related nominal body weight gain decrement, few feces, and reduced food consumption. The developmental NOAEL is 22 5 mg/kg/day and a developmental LOAEL was
not determined.

Two-Generation Reproduction in Rodents (Rat)

In a two-generation reproduction study ( MRID 43628701 and 43628702 ), Sprague·Dawley CAL: CD BR rats (30/dose/sex) were fed test diets containing benflu ralin (95.8% purity/a.i., Lot/Batch #ACD13683) at nominal concentratio ns of 0 (basal diet), 100, 1000 or 5000 ppm [males (premating), F0: 0, 7.2, 68.1, or 342 mg /kg/day, F1: 0, 7.3, 74.4, or 401 mg/kg/day; females (premating), F0: 0, 8.8, 80.0, 392 mg/kg/ day, F1: 0, 8.6, 87.3, or 437 mg/kg/day] during premating, gestation and lactation periods. No treatment-related d eaths or clinical signs were observed during the study. At 5000 ppm, decreases in mean body weights were observed during the premating periods for F0 ( ↓ 10%, males; ↓ 10.4%, females) and F1 ( ↓ 20.5%, males; ↓ 15.9%, females) animals, gestation periods for F0 ( ↓ 16.7%) and F1 ( ↓ 18.5%) females, and lactation periods for F0 ( ↓ 13.1 %) and F1 ( ↓ 12.1 %) females. Mean body wei ght gains were decreased at 5000 ppm during the premating periods for F0 ( ↓ 17.2%, males; ↓ 27.7%, females) and F1 ( ↓ 14.6%, males; ↓ 2.0%, females) animals and gestation periods for F0 ( ↓ 28.4%) and F1 ( ↓ 21.3%) females; during lactation increases were noted in m ean body weight gains for F0 ( ↑ 18.5%) and F1 ( ↑ 4.94-fold) fe males. The decreases in mean body weights were accompanied by significant decreases in food consumption during the premating, lactation, and gestation periods. Mean litter sizes of F0 and F1 an imals in the 5000 ppm group were significantly decreased and lower than the mean (range) hist orical control value of 14.37 (13.46 to 15.67). All reproductive parameters of treated animals were generally compar able to control.BENFLURALIN FINAL

Page 48 of 53 At necropsy, treatment-related liver and kidney effects were noted. Liver effects included dosedependent increases in the absolute and relativ e weights of livers of 1000 and 5000 ppm parental animals (F0 and F1), and hepatocellular enla rgement in 1000 ppm (F1) and 5000 ppm (F0 and F1) animals. Treatment-related litter effects included deceases in mean litter size of high-dose, F0 and F1 maternal animals, and the weaning in dex of F1 pups. An increased incidence of dead/missing pups was also observed in mid- a nd high-dose F0 litters an d high-dose F1 litters. All other litter parameters of treated animals were comparable to control values. For both the F1 and F2 generations, 1000 and 5000 ppm male and fe male pups had signific ant (p<0.05 or 0.01 at 1000 ppm; p<0.01 at 5000 ppm) decreases in mean body weights throughout the lactation period. The gross and necropsy examinations of F1 pups di d not reveal any treatment-related effects. Based on the findings (increased absolute a nd relative liver weight s, liver and kidney histopathology, and decreased mean pup body wei ght), a LOAEL for parental/systemic and offspring toxicity was established at 1000 ppm [Premating: 68.1 mg/kg/day (males) and 80.0 mg/kg/day (females)] and a NOAEL at 100 ppm [Premating: 7.2 mg/kg/day (males) and 8.8 mg/kg/day (females)]. There were no treatmen t-related effects on fert ility or organs of reproduction.

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat)

In a chronic toxicity/oncogenicity study ( MRID 44050002 and 44545501 ), benfluralin (95.8% purity/a.i., Lot/Batch #ACD13683) was administered to Fischer 344 rats (60/se x/dose) in the diet
at nominal dose concentrations of 0, 10, 100, 2500, and 5000 ppm (0, 0.5, 5.4, 136.3, and 274.8 mg/kg body weight/day for males; 0, 0.7, 6.8, 167.9, and 331.3 mg/kg body weight/day for females) for up to 2 years. Of these rats, 10/ sex/dose were sacrificed at 12 months. Organ weights were obtained for test es, ovaries, and thyroid (at a minimum); histopathology was conducted on the testes, ovaries, uterus, and t hyroids. Survival was significantly reduced (p<0.05) in males at 100, 2500, and 5000 ppm to 62, 66, and 64%, respectively, at study termination. However, the mortality rate did not reach statistical significance in males at any dose until the final week of the study. The only treatment-related c linical sign of toxicity was a yellowing of the skin in 97 and 100% of 2500 and 5000 ppm females, respectively, and yelloworange coloration of the eyes of both sexes at these doses. At study termination, body weights were significantly (p<0.05) lower than control in males ( ↓ 8%) and females ( ↓ 18%) at 2500 ppm and in males ( ↓ 17%) and females ( ↓ 28%) at 5000 ppm. Body weight gains were reduced (p<0.05) at 2500 and 5000 ppm throughout the second year of the study.

Alterations in cholesterol, bili rubin, and liver enzymes at the tw o highest doses correlated with liver enlargement and an increased incidence of liver lesions. Significan t increases (p<0.05) in absolute ( ↑ 19-43%) and relative ( ↑ up to 2-fold) liver weights ve rsus controls were observed in both sexes at 2500 and 5000 ppm, and hepatic toxi city was corroborated by serum chemistry andBENFLURALIN FINAL

Page 49 of 53 histopathology findings. Relative thyroid weights were elevated (p<0.05; ↑ 33 to 64% over control) at 2500 and 5000 ppm, and correlated with microscopic thyroid abnormalities. At terminal sacrifice, gross pathol ogy findings included enlarged testes and uterine cysts in males and females dosed at 5000 ppm. At 12 months (interim sacrifice), incr eases in non-neoplastic lesions included thyroid follic ular cell hypertrophy in the 5000 ppm males and females and hepatocellular hypertrophy in 5000 ppm males and females, and in 2500 ppm males. At the terminal sacrifice, treatment -related non-neoplastic lesions incl uded the same thyroid and liver
lesions observed in the interim sacrifice with increasing freque ncy and severity. In addition, hepatic necrosis and thyroid follicular cell hyperplasia were increased in males and females at 5000 ppm. Thyroid and liver abnormalities correlate d with increased tumor incidences at the high dose.

The NOAEL for chronic toxicity was 10 ppm fo r males and females (0.5 mg/kg/day and 0.7 mg/kg/day, respectively), based upon an increased in cidence of histological lesions of the kidney at 100 ppm. The LOAEL was 100 ppm (5.4 mg/kg/day for ma les and 6.8 mg/kg/day for females). The two highest doses, 2500 and 5000 ppm exceeded the MTD as demonstrated by decreased weight gain throughout the second year of the study. Under the conditions of this study, benfluralin was carcinogenic in both sexes. The test s ubstance induced hepatocellular carcinoma/adenoma in male rats, and thyroid follicular adenoma/carcinoma in males and females, at 2500 and 5000 ppm.

Carcinogenicity in Rodents (Mouse)

In a mouse oncogenicity study ( MRID 41021501 ), benfluralin (95.25% purity/a.i., Lot/Batch #231EF4) was administered in the diet to B6C3F/ Crl mice (60/sex/group) for up to two years at 0, 0.005, 0.03, or 0.15% (equivalent to 0/0, 6.0/6.9, 36.4/41.8, and 184.7/223.5 mg/kg/day [M/F], respectively). This mouse study consists of two replicate studies run concurrently in which 30 mice/sex/group in each study were dosed as stated above. Histopathological examinations were conducted on the testes, epididymides, prostate, seminal vesicle, ovaries, uterus, pituitary, and thyroids; it is not clear from the DER if any of these tissues were weighed prior to pathology
examinations.

Mortality, clinical signs, and food consumption for both sexes at all doses were unaffected by
treatment with benfluralin. No treatment-related findings we re observed in the 0.005% dose group. Body weights and weight gain were equivo cally decreased in males and females of all dose groups, except for female body weight ( ↓ 8%) and body weight gain ( ↓ 11%; p ≤ 0.05) that was considered treatment-related, and potentially toxicologically significant, in the highest dose group. Male body weight and body we ight gain values in all dos e groups were generally less than control at all time periods ( ↓ ≤ 8%).BENFLURALIN FINAL

Page 50 of 53 No significant, treatment-relate d effects on organ weights were observed, except for increased absolute and relative liver weights in all high-dose animals. An indication of hepatotoxicity in males was demonstrated by a nominal increase in multifocal hepatocellular hyperplasia (7/60 versus 1/60 in control) at the high dose. Apart from nominally depressed body weight, nominally increased incidence of obstructive ur ological syndrome, and mu ltifocal hepatocellular hyperplasia in males at the high dose, no other indicati ons of toxicity in male mice were seen. In females in the 0.03% dose group, the incidence of liver nodules was slightly elevated (12/60 treated versus 7/60 in control) . At 0.15%, toxicity was observed in the liver of females as follows: at termination, an incr eased incidence of liver nodules (25/59 treated versus 7/60 in control); an increased incidence of minimal to moderate focal hyperplasia (20/59 treated versus 6/60 in control); and an increase in slight to m oderate multifocal hyperplasia (6/59 treated versus 1/60 in control). The incidences of hepatocellula r adenoma and carcinoma were not significantly different from control at any treatment level, but showed a tr end to increase in the high-dose animals. The gross and microscopic findings, such as incr eased liver nodules and hypertrophy that were observed, demonstrated that benfluralin exposur e appears to affect the morphology and growth of hepatocytes.

The LOAEL is 41.8 mg/kg/day based on microsc opic and macroscopic liver changes. The NOAEL for females is 6.9 mg/kg/day. The LOAEL for males is184.7 mg/kg/day based on the slight toxicity signals observed in male mi ce. The NOAEL for males is 36.4 mg/kg/day.

Chronic Oral Toxicity in Non-Rodents (Dog)

In a chronic toxicity study ( MRID 43628702 ), male and female beagle dogs (4/sex/group) were dosed by capsule with benfluralin (95.8% purit y/a.i., Lot/Batch #ACD13683) at 0, 5, 25, or 125 mg/kg body weight/day for one year. Weights were obtained for testes, ovaries, pituitary, and thyroid; histopathological exam inations were conducted on the testes, epididymides, prostate, ovaries, uterus, vagina, mammary gland, pituitary, and thyroids.

With the exception of one high-dose female, wh ich was sacrificed in moribund condition, all
animals survived to terminal sacrifice without any adverse c linical signs. The mean body weight, food consumption, gross pathology, and organ weights of treated animals were comparable to control values. Histopathology ob servations at terminal sacrifice revealed increased incidences of sinusoidal cell pigment in the livers of mid- and high-dose females, and high-dose males. Based on the findings of liver histopathology, a LOAEL of 125 mg/kg/day was established in males and 25 mg/ kg/day in females. A NOAEL of 25 mg/kg/day was established in males and 5 mg/kg/day in females.BENFLURALIN FINAL

Page 51 of 53 Mechanism of Action Study (Rat)

The current study (Saghir et al., 2008) was desi gned to determine the mechanism of thyroid hyperactivity after trifluralin 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 sacrificed, 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 and total UGT enzyme activity. The bile ducts from the remaining rats from each group were cannulated at the en d 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%, respectively (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 expression of selected hepatic UGT isozymes (UGT1A1, 1.4-fold; UGT1A6, 6.4-fold; UGT2B1, 3.7-fold). The total amount of T 4 in bile was increased >2-fold in trifluralin-treate d rats. Trifluralin treatme nt also increased 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 peripheral metabolism (increased conjugation by (UGTs) and increased bile flow resulting in compensatory increases in TSH synthesis and secretion. Continued, and increa sed, concentrations of TSH afte r chronic exposure would cause stimulation of the thyroid gland, resulting in cellular hypertrophy, proliferation, and predisposition to the development of follicular cell tumors.

Chronic Fish Early Life Stage Toxicity Test

In a 49-day chronic fish early life stage (ELS) toxicity study w ith rainbow trout ( MRID
41613805 ), fish were exposed to mean measured benf luralin concentrations of negative control, solvent control, 0.8, 1.9, 5.0, 14.8, and 45.5 μg a.i/L. Ti me to hatch was similar (5 to 8 days) in
the controls and the treatment concentrations . Percent hatch ranged from 95-100% on the
controls and all treatment concen trations. All larvae in the highe st treatment concentration (45.5 μg a.i/L) died by day 33 post-hatch and so were subsequently excluded fr om further statisticalBENFLURALIN FINAL

Page 52 of 53 analysis. Clinical signs of t oxicity included hyperactivity and pr ostrate position was observed in the two highest treatment con centrations (14.8 and 45.5 μg a. i/L) and no treatment related deformities were observed. At test termination, mean length was significantly (p<0.05) reduced at the 5.0 and 14.8 μg a.i/L treatment levels, a nd blotted wet weight wa s significantly (p<0.05) reduced at the 14.8 μg a.i/L treatment concentra tion when compared to the pooled controls.

Based on effects on mean length and mean we t weight, the NOAEC and LOAEC for this study were determined to be 1.9 and 5.0 μg a.i/L, respectively.

Chronic Avian Reproduction Studies (Ma llard duck and Northern Bobwhite quail)

In a chronic avian reproduction study with the mallard duck ( MRID 42145501 ), benefin (benfluralin) was exposed to birds in the diet at mean measured concentrations of control, 97, 288, and 975 ppm. One incidental mortality occurred in each of the control and two lowest
treatment concentrations. There were no clinical signs of toxicity observed in the control or any
of the treatment concentrations. Necropsy of all mortalities a nd sacrificed birds revealed no compound-related gross or microscopic lesions. There were no significa nt (p>0.05) treatment related effects on body weight yet there were significant (p<0.05) decreases in mean food consumption at the two lowest treatment concentra tions when compared to the negative control. These decreases in mean food consumption we re not accompanied by a significant decrease at the highest treatment concentrati on and therefore the study author di d not attribute this effect to benfluralin exposure.

When compared to the negative control, there we re no statistically significant differences in all measured reproductive parameters. There was a high incidence of cracked eggs in the highest treatment concentration (11.5%) but this was not shown to be significantly different from the control (p = 0.061). The study reviewer determined this effect to be biologically significant. Eggshell thickness for both cracked and norma l eggs was not significantly affected by benfluralin exposure. Similarly, offspring body we ight was not significan tly different in the treatment groups when compared to the negative control.

The NOAEC and LOAEC for this study were determined to be 288 and 975 ppm, respectively,
due to an increase in incidence of cracked eggs at the highest treatment concentration (study reviewer-determined).

In another chronic avian reproduction st udy with the northern bobwhite quail ( MRID
42145502 ), birds were exposed to benfluralin in the diet at mean measured concentrations of control, 96, 295, and 990 mg/kg-diet (ppm). There we re no clinical signs of toxicity observed in the study in the control or any of the treatment concentrations. Nine bobw hite quail died during the course of the study yet these deaths were not attributed to treatment. One female in the middle treatment group was sacrificed due to pen -related injuries. Necropsy of all deaths and
sacrificed birds revealed no fi ndings of compound related gross or histopathological lesions.BENFLURALIN FINAL

Page 53 of 53 Based on the study-reviewer’s result s (based in part on Table A in the Data Evaluation Record), when compared to the control group, there was a significant reduction in the highest treatment concentration (990 ppm) for eggs laid, viable embr yos of eggs set, normal hatchlings of live 3- week embryos and eggs set, and 14-day old survi vors. Additionally, the percentage of cracked eggs was significantly higher (p<0.05) at the highest treatment concen tration (16.2%) when compared to the control group (8.5%). The study authors provided no explanation for the high percentage of cracked eggs in the control group.

Additionally, there appears to be a significant reduction in eggs laid, viable embryos and hatchlings of egg set, and reductions in 14-d o ffspring survival at the mid concentration (295 ppm). There were no significant (p>0.05) differences in eggshe ll thickness in the treatment concentrations when compared to the control. The study author reported that mean body weight and mean body weight gain were significantl y reduced (p<0.05) at the highest treatment concentration when compared to the control prio r to egg laying; however, the reviewer analysis did not detect any differences in body wei ghts in the overall study duration. During the preproduction period, there were si gnificant but slight reduction s in food consumption between
the control and all trea tment levels. A significant decrease in food consumption during the production period was noted at the two higher test concentrations. Analysis of two-week old survivors per eggs set and 14-day survivor weight showed a significant (p <0.05) reduction at all
treatment concentrations when co mpared to the negative control ( ≥ 96 ppm). Consequently, a NOAEC could not be determined based on these effects occurring at all treatment
concentrations.