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 EVIDEN CE ANALYSIS OF POTENTIAL
INTERACTION WITH THE ESTROG EN, ANDROGEN OR THYROID
PATHWAYS

CHEMICAL: FLUTOLANIL

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYFLUTOLANIL FINAL

ii TABLE OF CONTENTS

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

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

II. Sources of Scientific Data and Technical Information......................................5

A. EDSP Tier 1 Screening Assays............................................................5

B. Other Scientifically Rele vant Information (OSRI)......................................5

III. Weight of Evidence (WoE) Evaluation.......................................................5

A. EDSP Tier 1 Screening Assays.............................................................8

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9
1. Effects on Estrogen Pathway.....................................................9
2. Effects on Androgen Pathway..................................................13

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................17

IV. Committee’s Assessment of Weight of Evidence..........................................21

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................21

1. Tier 1 in vivo Assays.............................................................22

2. OSRI...............................................................................22

B. Estrogen Pathway...........................................................................23

C. Androgen Pathway.........................................................................24

D. Thyroid Pathway...........................................................................24

E. Conclusions.................................................................................24

V. EDSP Tier 2 Testing Recommendations....................................................24

VI. References.......................................................................................26

LIST OF TABLES: Table 1. Tier 1 Assays for Flutolanil..................................................................8
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Flutolanil ....................................10
Table 3. Androgenic/Anti-Androge nic Pathway for Flutolanil ..................................14
Table 4. Thyroid Pathway for Flutolanil.............................................................19

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................29
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................38

APPENDIX 3. References Not Utilized in the Flutolanil WoE Analysis.............................47FLUTOLANIL 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 enerationFLUTOLANIL FINAL

iv Abbreviation Terminology

F2 Second filial g eneration
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 ConcentrationFLUTOLANIL FINAL

v Abbreviation Terminology

MTD Maximum Tolerated Dose
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)FLUTOLANIL FINAL

vi Abbreviation Terminology

TP Testosterone Pro p ionate
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-EvidenceFLUTOLANIL FINAL

Page 1 of 48 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 flutolan il interacts with E, A, or T hormone pathways, the number and type of effects induced, the magnitude of respons es, and the pattern of responses observed across studies, taxa, and sexes were c onsidered. Additionally, the cond itions under which effects occur were considered, in particular, the dose(s) at which responses ha ppen and whether overt toxicity
was present.

On August 13, 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and th e Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence analysis of the potential interac tion of flutolanil with the E, A or 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 wildlife studies (40 CFR Part 158 and literature).

The conclusion of the WoE evaluation is that fl utolanil demonstrates no convincing evidence of potential interaction with the estrogen or a ndrogen pathways in mammals. However, the complementary responses observed in both sexe s in the fish short-term reproduction assay (FSTRA) suggest a potential for fl utolanil to interact with the E and A pathways in fish.

For the estrogen pathway, flutolanil was negative in the in vitro Tier 1 estrogen receptor (ER) binding, aromatase and steroi dogenesis assays. In a published ERTA study (Kojima et al. , 2004) considered as OSRI, flutolanil wa s reported to be negative for both ER α agonism and antagonism. Flutolanil was also negati ve for estrogenic activity in the in vivo Tier 1 uterotrophic assay and showed no evidence of an estrogenic res ponse in the female pubertal rat assay or in the mammalian Part 158 toxicity studies. Overall flut olanil does not appear to have a potential to interact with the estrogen pathway in ma mmals. Conversely, in the FSTRA several complementary responses were observed that sugges t that flutolanil may have the potential to interact with the estrogen pathway in fish. The FSTRA responses included decreased fecundity, decreased plasma VTG, increased female GSI and alterations in gonada l histopathology. In
addition to the decrease in fec undity in fish, a 17% decrease in eggs laid was observed in theFLUTOLANIL FINAL

Page 2 of 48 bobwhite quail reproduction (no other treatment-re lated effects observed); however, it is noted
that this effect alone is limite d in its diagnostic specificity for a potential estrogen-related effect. A 5% non-treatment responsive decrease in e ggshell thickness was observed the mallard duck reproduction study, but no other effects were obs erved in this study (and not seen in the bobwhite quail study). Therefore, while there were largely negative or li mited responses in the
Tier 1 in vitro assays, in vivo mammalian dataset, and avian dataset, for the FSTRA there were complementary responses along several levels of biological organization for the females including alterations in enzyme activity (plasma VT G) up to the apical endpoint of fecundity. As such, significant effects of the reproductive cap acity of fish were observed in the FSTRA suggests that flutolanil may potentia lly interact with the HPG axis.

For the androgen-pathway, although flutolanil show ed an equivocal response in the Tier 1 AR binding assay, in a published ARTA study (Kojima et al ., 2004) considered as OSRI, flutolanil was reported to be negative for AR transcript ional activation. Flutolanil did not alter in vitro testosterone production in the Tier 1 steroidogenesis assay. In vivo , flutolanil was negative in both the Tier 1 Hershberger and in the male pubertal assay. There were no androgen-related responses in the mammalian Part 158 toxicity studies to support an interaction with the androgen pathway in mammals. No androgen-related ef fects were observed in the Part 158 avian reproduction studies. However, in the FSTRA, median male tubercle score was decreased by 32% and multiple abnormalities in the testes were observed in the high treatment group (1.2 mg/L). These findings suggest a potential effect on the androgen pathway in fish. As with the findings in the estrogen pathway, while the in vitro and mammalian/avian in vivo assays do not suggest a potential intera ction, the results in the male fish, with complementary effects in the female fish, suggest a potential to in teract with the A pathway in fish.

There is no convincing evidence for the potential for flutolanil to interact with the T pathway in mammals or wildlife. There were no treatment-related effects on the thyroid parameters in the in

vivo male or female pubertal rat assays or in the Part 158 mammalian toxi city studies. The effects on development, growth and histopathol ogy observed in the amphibian metamorphosis assay (AMA) were not considered to be relate d to flutolanil treatme nt, but rather may be reflective of a response due to the presence of th e solvent (similar effect s observed in solvent control and treatment groups rela tive to negative control). Ther efore, the results in the AMA do not suggest a potential for interaction w ith the thyroid pathway in amphibians.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not
recommended for flutolanil since there was no convi ncing evidence of potent ial interaction with the estrogen, androgen or thyroid pathways.

Based on the complementary responses observed in the FSTRA, including effects on the apical endpoint of fecundity, EDSP Tier 2 Medaka Extended One-Generation Reproduction Test is
recommended. The results in the FSTRA indicate th at there may be a potential for interactionFLUTOLANIL FINAL

Page 3 of 48 with the estrogen and androgen pathway in fis h. Currently, the chronic fish NOAEC value is 0.233 mg a.i./L based on decreased length and wet weight in a fathead mi nnow early life-stage study. Effects on female GSI and gonadal histopathology were observed at 0.018 mg a.i./L in the FSTRA, with an increase in the number of effects observed in the males and females, including a cessation in fecundit y, at 1.2 mg a.i./L. Therefore, as the concentration of the
observed effects in the FSTRA are lower than th e current regulatory NOA EC for fish, additional
data are recommended to understand the dose-re sponse relationship for potential reproductive
effects.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for flutolanil. In both sp ecies, 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 flutolanil exposure. Relative to the EDSP, additional testing is not recommended. As there was no convincing evidence of a potential thyroid interaction in amphibi ans, no additional testing is
recommended.FLUTOLANIL FINAL

Page 4 of 48 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 flutolan il interacts with E, A, or T hormone pathways, the number and type of effects induced, the magnitude of respons es, and the pattern of responses observed across studies, taxa, and sexes were c onsidered. Additionally, the cond itions under which effects occur were considered, in particular, the dose(s) at which responses ha ppen and whether overt toxicity
was present.

On August 13, 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and th e Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) analysis of the potential inte raction of flutolanil with the E, A or T hormone pathways. The T1 WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in

vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited
OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Flutolanil, is a systemic benzanilide fungicide. Flutolanil’s water solubility is 6.53 mg/L at 20°C with a vapor pressure of 4.88 x 10

− 8 mm Hg at 25°C. In the environment, flutolanil is anticipated to be stable and pers istent. Flutolanil has a low propens ity to volatilize, is resistant to hydrolysis and is stable to photol ysis. Flutolanil is re latively resistant to aerobic and anaerobic degradation in soils and aquatic e nvironments, with half-lives of more than 200 days in soils and greater than one year in water. Because of its reported water solubility and high soil binding capacity, there is potential for flutolanil sorbed to soil to be transported into surface water via
erosion. Flutolanil has a low lik elihood to bioaccumulate in fi sh and other aquatic organisms
with an estimated bioconcentration factor of 100 with more than 95-99% depurated in 3 days.

The available information considered in determin ing the potential interact ion of flutolanil with the E, A and/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 th e Agency (USEPA, 2011), is presented in
Section IV. The EDSP Tier 2 studies recommendations are presented in Section V.FLUTOLANIL FINAL

Page 5 of 48 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 Screening assays were submitted as OSRI by the Te st Order recipients and/or the public. This included studies published in the open literatu re and/or data submitted to support pesticide registration ( e.g. , Part 158 Guideline studies). The Agen cy’s review of the initial OSRI is provided in the Report of the Endocrine Disrupt or Review Team on flutolanil (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any additional relevant info rmation. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/ studies considered but not uti lized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria, and approach used in the WoE determination on the potential of a
substance to interact with endocrine-related processes ( i.e., E, A, or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory 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.FLUTOLANIL FINAL

Page 6 of 48 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 other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a speci fic endpoint for discerni ng whether or not the chemical has interacted with the endocrine syst em, are considered. The co llective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indi cation of potential interaction as measured by the study.

The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data, as well as characterizing the extent to which the available data suppor t a hypothesis that a chemical has the potential to interact E, A, or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, coherence, strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are
meant to evaluate whether or not a chemical can interact with E, A and T consisting of different levels of biological organization from a molecular initiating ev ent, such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment and fecundity at the whole organism level. The extent of expressi on of response s 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 bi ological organization? Finally, under what conditions did responses occur including consider ation of whether the responses were observed in the presence of overt or systemic toxicity? Th e presence of overt and/ or systemic toxicityFLUTOLANIL FINAL

Page 7 of 48 introduces uncertainty in the ability to distinguis h effects specifically re lated to an endocrinerelated effect from a non-endocrine toxic response.

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 screening proce ss. 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 ( i.e ., death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g ., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addres sing the three component s 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.FLUTOLANIL FINAL

Page 8 of 48 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 biol ogically relevant in regard to a treatment-relate d effect which supports a conclusion of whether a substance has the potential to interact with the E, A, or T pathway. Effects that occurred in th e presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

A. EDSP Tier 1 Screening Assays

The Tier 1 assays submitted in response to the ag ency’s test order for flutolanil are shown below
in Table 1. Table 1. Tier 1 Screening Assays for Flutolanil

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied (MRID 48616805)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300; OECD 455 Requirement satisfied by
OSRI (Kojima et al., 2004)

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied (MRID 48616802)

Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550; OECD 456 Requirement Satisfied (MRID 48616808)

Aromatase Assay (human recombinant microsomes) OSCPP 890.1200 Requirement Satisfied (MRID 48616803)

Uterotrophic Assay (Rat) OSCPP 890.1600; OECD 440 Requirement Satisfied (MRID 48616809)

Hershberger Assay (Rat) OSCPP 890.1400; OECD 441 Requirement Satisfied (MRID 48616807)

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied (MRID 48671101)

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied (MRID 48671101)

Fish Short-term Reproduction Assay OSCPP 890.1350; OECD 229 Requirement Satisfied (MRID 48616806)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; OECD 231 Requirement Satisfied (MRID 48616801)FLUTOLANIL FINAL

Page 9 of 48 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential interactions of flutolanil w ith the estrogen pathway are summarized in Table 2. The various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for estrogen ic, 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.

In an evaluation of estrogenic/anti-estrogenic acti vity from Tier 1 testing results, flutolanil was classified as negative in the estrog en receptor (ER) binding, aromatase, ER α transcriptional activation (ERTA; Kojima et al ., 2004) and steroidoge nesis assays.

Flutolanil was also classified as negative for estrogenic activity in the in vivo uterotrophic assay, and there were no estrogen-related effects obs erved in the female pubertal rat assay.

In the FSTRA, fecundity was decreased ( ↓ 100%) at the high concentration (1.2 mg/L) and
fertilization success could not be assessed as no eggs were produced. Plasma vitellogenin (VTG) was decreased ( ↓ 72%) in high-concentration females compar ed to the negative control. Female GSI was increased ( ↑ 15-64%) relative to the ne gative control in all tr eatment groups (0.018-1.2 mg/L). There was an increase in the number and severity (mild to severe) of oocyte atresia in high-concentration females and an increase in gonad si ze in each of the treatment groups; the
latter observation was attributed to an increase in vitellogenic oocyt es. Five fish were reported as lethargic on Day 1 in the high concentration, which progressively decreased to one fish by Day 4 which suggests the fish may have been st ressed at test initiati on but did not persist throughout the duration of the study. Additionally, there were no e ffects on survival or growth
parameters.

There were no estrogen-related e ffect observed in the Part 158 mammalian toxicity studies. In a Part 158 reproduction study with bobwhite qua il, there was a slight, but not statisticallysignificant, reduction in the number of eggs laid at the highest treatment concentration by 17% compared to the negative control and was consider ed biologically significa nt (4800 mg/kg-diet). In a Part 158 reproduction study with mallard ducks, there was a st atistically significant, but nontreatment responsive, 5% reduction in eggshell thickness at 4800 mg/kg-di et; no other treatmentrelated effects were observed. In a fish early life stage study with fathead minnow, effects on survival and/or growth (length and wet weight) were observed at ≥ 0.486 mg/L.FLUTOLANILFINALPage 10 of 48Table 2: Estrogenic/Anti-Estrogenic Pathway for FlutolanilLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Flutolanil1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/ GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48616805)NERTARequirement satisfied by OSRI (Kojimaet al.,2004; MRID 48033008; see below)Aromatase(MRID 48616803)NSteroidogenesis(MRID 48616808)NUterotrophic(MRID 48616809)NNEN NFemale Pubertal Rat(MRID 48671101)NNNNNNNNFSTRA(MRID 48616806)NE↑15,30,64%(L,M, H)P(L, M,H)4NFrt: N5;Fcd:↓100%(H)↓72%(H)N(H)6OSRIERTA(Kojimaet al., 2004)MRID 48033008NTwo-generationreproduction (Rat;MRID 41850805)NENENNENENNFLUTOLANILFINALPage 11 of 48Table 2: Estrogenic/Anti-Estrogenic Pathway for FlutolanilLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Flutolanil1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/ GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity 2 Overt Toxicity Observed 3Developmentaltoxicity (Rat; MRID41850804)NENNNDevelopmentaltoxicity (Rabbit;MRID 40342924)NRNNNChronic toxicity/carcinogenicity (Rat;MRID 40342921)NENNNX(H)NCarcinogenicity(Mouse; MRID41850803)NENENNEN NChronic toxicity (Dog;MRID 40342922)NENNNX(M,H)NSubchronic Toxicity(Rat; MRID 4342919)NE N N NEX(M, H)NSubchronic Toxicity(Dog; MRID40342920)NENNNEX(H)NAvian reproduction(Quail; MRID42932301)P (H)7N NAvian reproduction(Duck; MRID42932302)P (H)8N NFLUTOLANILFINALPage 12 of 481. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑) indicate the direction of the response. A shaded cell indicatesthat is parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated.2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an Xin this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Ovaries displayed a mild to severe increase in atresia (15 of 16) at the high concentration (1.2 mg/L). Increases in gonadsize were observed in all treatmentgroups (8 to 14 of 16) concomitant with an increased presence of vitellogenic oocytes.5. Fertility could not be assessed at the high concentration asno eggs were produced. Fertility was not affected at the two lower concentrations.6. Five fish were reported as lethargic on Day 1 which progressively decreased to one fish by Day 4 which suggests the fish mayhave been stressed at testinitiation but did not persist throughout the duration of the study. Additionally, there were no effects on survival or growthparameters.7. Reduction in eggs laid at the highest treatment level by 17% compared to the negative control and was considered biologically significant.8. Egg shell thickness was decreased 5% (effect not treatment-responsive) at the high dietary concentration.P Positive findingN Negative finding (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedFLUTOLANIL FINAL

Page 13 of 48 2. Effects on Androgen Pathway

The potential interactions of flutolanil w ith the androgen pathway are summarized in Table 3. The various targets of the androgen pathway across the relevant Ti er 1 assays are delineated so as to facilitate determination of potential for a ndrogenic, anti-androgenic, or HPG axis effects.
This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

In an evaluation of androgenic/an ti-androgenic activity from Tier 1 testing results, flutolanil was classified as equivocal in the in vitro androgen receptor (AR) bindi ng assay and negative in the steroidogenesis assay. The equivocal response in the AR binding assay resulted from negative responses in two of the three assay runs, and a variable response in a single assay run that yielded decreases in specific binding between 25 and 50%. In addition, flutolanil was negative for agonist and antagonist activity up to 10 -5 M in an AR transac tivation assay conducted by Kojima et al . (2004).

Flutolanil was also classified as negative fo r androgenic and anti-androgenic activity in the in

vivo Hershberger assay and there we re no changes in the androgen -responsive endpo ints in the male pubertal rat assay.

In the FSTRA, median male tubercle score was decreased (21 vs. 27) in the high treatment group (1.2 mg/L) with a reported 36% de crease in dorsal nape pad (fatpa d). In addition, three of the eight males in the high treatment group showed multiple, often related, abnormalities in the testes including increases in the number of sp ermatogonia (mild to moderate), increases in primary spermatocytes (moderate), decreases in secondary spermatocytes (mild to moderate) and/or decreases in spermatids (mild to moderate). These microscopic alterations interfered with staging of the testes in two of these males.

There were no androgen-related e ffects seen in the Part 158 mamma lian toxicity studies. There were no androgen-specific effects noted in the Part 158 avian re production studies.FLUTOLANILFINALPage 14 of 48Table 3: Androgenic/Anti-Androgenic Pathway for FlutolanilLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Flutolanil1Study Type/Literature Citation AR Binding/ AR activation 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 (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48616802)E4Steroidogenesis(MRID 48616908)NHershberger(MRID 48616807)NENN NMale Pubertal Rat(MRID 48671101)NNNNNNNNNNFSTRA(MRID 48616806)NENP(H)5Tuberclescore:(21 v 27)(H)fat pad↓36% (H)N6NN7OSRIARTA(Kojimaet al.,2004)NTwo-GenerationReproduction (Rat;MRID 41850805)NENNENNENEN NChronic Toxicity /Carcinogenicity(Rat; MRID40342921)NNNENNNEX(H)NCarcinogenicity(Mouse; MRID41850803)NNNENNENEN NFLUTOLANILFINALPage 15 of 48Table 3: Androgenic/Anti-Androgenic Pathway for FlutolanilLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Flutolanil1Study Type/Literature Citation AR Binding/ AR activation 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 (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity 2 Overt Toxicity Observed 3Chronic toxicity(Dog; MRID40342922)NENNENNNEX(M, H)NSubchronic Toxicity(Rat; MRID40342919)NNNENNENEX(M, H)NSubchronic Toxicity(Dog; MRID40342920)NNNNNENEX(H)NAvian reproduction(Quail; MRID42932301)NN NAvian reproduction(Duck; MRID42932302)NN N1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑)indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight areabsolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate(PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP).2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in thiscolumn. 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. For details seeSection IV. A4. Mean specific-binding decreases in the presence of flutolanil were variable,and not dose specific, in Run 1 (>50% but <75%at several concentrations). Flutolanildid not reduce binding <75% in Runs 2 and 3.5. Multiple, often related, abnormalities in the testes including increases in the number of spermatogonia (mild to moderate),increases in primary spermatocytes(moderate), decreases in secondary spermatocytes (mild to moderate), and/or decreases in spermatids (mild to moderate) in the three of eight males in the hightreatment group (1.2 mg/L). These microscopic alterations interfered with staging of the testes in two of these males.6. Fertility could not be assessed at the high concentration asno eggs were produced. Fertility was not affected at the two lower concentrationsFLUTOLANILFINALPage 16 of 487. Five fish were reported as lethargic on Day 1 which progressively decreased to one fish by Day 4 which suggests the fish mayhave been stressed at test initiationbut did not persist throughout the duration of the study. Additionally, there were no effects on survival or growth parametersE Equivocal assay responseP Positive findingN Negative finding (in vitro)/No effect (in vivo)NE Not examinedFLUTOLANIL FINAL

Page 17 of 48 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it
does include three in vivo assays that provide data to de tect changes in the HPT axis, i.e ., the pubertal male and female (rat) assays, and the AMA (frog).

The potential interactions of flutolanil 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.

No effects on the thyroid pathway (thyroid weight, histopathology, or hor mone concentrations)
were observed in the in vivo male and female pubertal rat assays. In the AMA, flutolanil significantly advanced median Nieuwkoop-Faber (N F) developmental stage by 2 stages at Day
21 at all treatment concentrati ons (0.015, 0.13, and 1.1 mg/L) relati ve to the negative control, with no effect at Day 7. Incr eases in the severity and inci dence of thyroid hypertrophy and increased colloid area were appare nt in all treatment groups. Fl utolanil significantly increased Day 21 normalized hind-limb length (HLL) at all treatment concentrations in a nonconcentration-dependent manner ( i.e. , 28, 26, and 23% at the low, medium, and high treatment concentrations) relative to the negative control, with no eff ects noted at Day 7. Significant increases in snout-to-vent leng th (SVL) and body weight were observed on Days 7 and 21 in the low and medium treatment groups. Growth para meters in the high treatment group were not different compared to the negative control. With the exception of Day 7 body weight, for all endpoints in which there was a si gnificant increase from the negativ e control, there was also a significant or marginally significan t increase in response of the solv ent control over the negative control. In addition, the severi ty of thyroid hypertrophy and incr eased colloid area was increased in the solvent control relative to the negative cont rol. Therefore, these apparent solvent effects on both growth and developmental endpoints suggest that the observed changes in flutolaniltreated animals relative to the negative control were related to the presence of the solvent, and not a thyroid-related interaction.

No thyroid effects were noted in any of the Pa rt 158 mammalian chronic toxicity studies, but increased thyroid weights were observed in th e subchronic rat and dog studies. Generally nonsignificant and/or non-dose-depende nt increases in thyroid weight s were noted in the high-dose female as well as the medium- and high-dose male rats. In the subchr onic dog thyroid weighs were increased 25% at the high dose (2000 mg/kg/day) and 21 a nd 23% in females at the mid (400 mg/kg/day) and high (2000 mg/kg/day) dose, respectively. The thyroi d weight increases in the dog were not statistically significant. Thyroxine (T 4 ) and thyroid stimulating hormone (TSH)FLUTOLANIL FINAL

Page 18 of 48 concentrations were not determined in any ge neral toxicity studies. Histopathological examination of the pituitary did not rev eal any treatment-related changes in any in vivo
mammalian studies.FLUTOLANIL FINAL

Page 19 of 48 Table 4. Thyroid Pathway for Flutolanil.

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

Study Type/

Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(± or asynchronous,HLL)Growth (BW, SVL)Systemic Toxicity2Overt ToxicityObserved3

EDSP Tier 1 Assays

Male Pubertal Rat (MRID 48671101) N N N N N N N

Female Pubertal Rat (MRID 48671101) N N N N N N N

AMA (MRID 48616801) N N 4 Day 21 NF stage: ↑ 2 stages 5

Day 21
HLL: ↑ 28,26,23% (L, M, H) 6 Day 7 BW: ↑ 40, 54% Day 21 BW:

↑ 20, 35% Day 7 SVL:

↑ 15, 18% Day 21 SVL: ↑ 5, 9% (L, M) 7 N

OSRI Chronic Toxicity/ Carcinogenicity (Rat; MRID 40342921) N N N X (H) N

Carcinogenicity (Mouse; MRID 41850803) NE N NE N N
Chronic toxicity (Dog; MRID 40342922) N N N N X (M, H) Subchronic Toxicity (Rat; MRID 40342919) N N NE X (M, H) N

Subchronic Toxicity (Dog; MRID 40342920) N N NE X (H) N

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. . Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. 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. Although not statistically-significant, increases in the severity and incidence of thyroid hypertrophy and increased colloid area were apparent in all treatment groups. However, the severity of thyroid hypertrophy and increased colloid area was significan tly increased in the solvent control relative to the negative control, suggesting that the observed changes were related to the presence of the solvent. 5. Increased median developmental stage (59 vs. 57 in the negative control). Median NF stage of the solvent control was also 59, suggesting that the observed chan ges were related to the presence of the solvent.FLUTOLANIL FINAL

Page 20 of 48 6 Day 21 HLL of the solvent control was significantly increased by 47% compare to the negative control, suggesting that the observed changes were re lated to the presence of the solvent. 7 Day 7 and 21 SVL of the solvent control was increased marginally (p = 0.053) by 11 and 5%, respectively, relative to the negative control. Day 7 and 21 BW of the solvent control were increased by 28% (NS) and 22% (p<0.05), respectively, relative to the negative control. Increased body weight on Day 7 ( ↑ 40 and 54%) and Day 21 ( ↑ 20 and 35%) in the low- and medium-dose groups, respectively. This suggests that the observed changes in growth were related to the presence of the solvent. P Positive finding N Negative finding NE Not examinedFLUTOLANIL FINAL

Page 21 of 48 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of flutolanil to interact with endocri ne 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 a sequelae of system ic effects. Endocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses are mediated through an endocrine pathway, therefore nonendocrine toxic responses (incl uding but not limited to mortalit y or body weight changes) are
also considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence evaluation of the results of the Tire 1 assays stated that “In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All 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).

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. Generally, one parameter (i .e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concen tration to be considered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were generally less severe forms of toxicity (e.g., changes in orga n weights or blood chemistry).FLUTOLANIL FINAL

Page 22 of 48 1. Tier 1 in vivo Assays

There was no evidence of overt toxicity in the Tier 1 in vivo studies. Systemic effects in the
mammalian Tier 1 in vivo assays were confined to minimal ch anges in liver weights. Finally, no
treatment-related mortalities or clinical si gns were present in the FSTRA or the AMA.

2. OSRI

Systemic or overt toxicity in the OSRI in vivo studies was also minimal. The toxicology studies
conducted on flutolanil demonstrat e few biologically significant effe cts, mainly in the liver and gastrointestinal (GI) system.

In a subchronic oral toxicity study in rats, no mort ality or clinical signs were observed. Slight decreases in overall body weight gain were noted at the high dose . Absolute liver weights in males and females were increased by 9 and 19%, respectively, and relative liver weights were increased by 14 and 20%, respectively, in the hi gh-dose group. Absolute and relative liver weights in females also were increased by 11 and 8%, respectively, in the medium-dose group. In a subchronic oral toxicity study in dogs, no mo rtality or clinical signs were observed, although swollen mammary areas were noted in the medium- and high-dose females. The absolute and relative weights for the liver were increased in both sexes in the high-dose animals.

In a two-generation reproduction toxicity study in rats, no mortality or clinical signs were
observed. Absolute and re lative liver weights were increased in high-dose F 0 -generation animals
and F 1 females. There were no treatment-related effects on mortality or clinical signs in a prenatal developmental toxicity study in rats or rabbits. Reduced body weight gains during dosing were observed in all treatment groups in the rabbit developmental study, and lower overall body weight gain was observed in the high-dose group ( ↓ 12%). However, these
differences were attributed to higher control group weights ( ↑ 11-15%) relative to the treated groups prior to dosing.

In a combined chronic toxicity/carcinogenicity st udy in rats, no mortality or clinical signs were observed. Body weight gains were decreased up to 10% during Weeks 1-13 (with body weight decreases of 3-6%) in the high- dose male rats. Body weight ga in differences decreased after Week 13. Relative liver weights were incr eased by 12 and 14%, respectively, at 3 and 24 months in high-dose males. In the high-dose fema les, absolute liver weights were increased by 15-18% at 3, 12, and 24 months, and relative liver weights were increased by 11 and 10%, respectively, at 3 and 12 months. In an oncogenic ity study in mice, no mortality or clinical signs were observed. Body weight gains were decrea sed by 17 and 16%, respec tively, in the high-dose males and females during Weeks 0-24. In medium -dose females, body weight gains also were decreased by 15% during Weeks 0-24. Absolute liver weight in the high-dose females was increased by 20%, and liver-to- body weight ratio in the high-do se females was increased by 33%. No similar increases in liver weight we re observed in high-dose males. In a chronicFLUTOLANIL FINAL

Page 23 of 48 toxicity study in dogs, all dogs survived until study termination. There was a dose-related
increase in the incidence of clinic al signs (emesis, salivation, and so ft stool) in both sexes in the medium- and high-dose groups from Week 65 un til study termination. Decreased body weights were observed in high-dose males from Weeks 93-104 ( ↓ 4-8%) with decreased body weight gains ( ↓ 8-16%). Additionally, decreased body weights were observed in high-dose females from Weeks 87-104 ( ↓ 3-8%) with decreased body weight gains ( ↓ 6-17%).

In general, absolute and /or relative liver wei ght increases were considered treatment-related adaptive changes due to the lack of adverse e ffects during gross and mi croscopic evaluations.

In a fish early life stage study with fathead minnow, effects on survival and/or growth (length and wet weight) were observed at ≥ 0.486 mg/L.

No clinical signs of toxicity were obs erved in the avian reproduction studies.

B. Estrogen Pathway

Flutolanil was negative in the in vitro Tier 1 estrogen receptor (ER) binding, aromatase and steroidogenesis assays. In a published ERTA study (Kojima et al. , 2004) considered as OSRI, flutolanil was reported to be negative for both ER α agonism and antagonism. Flutolanil was also negative for estrogenic activity in the in vivo Tier 1 uterotrophic a ssay and showed no evidence of an estrogenic response in the female pubertal rat assay or in the mamm alian Part 158 toxicity
studies. Overall flutolanil does not appear to have a potential to interact with the estrogen pathway in mammals.

Conversely, in the FSTRA several complementary responses were observed that suggest that flutolanil may have the potential to interact with the estroge n pathway in fish. The FSTRA responses included decreased fecundity, decreas ed plasma VTG, increased female GSI and alterations in gonadal histopathology. In additio n to the decrease in fecundity in fish, a 17% decrease in eggs laid was observed in th e bobwhite quail reproducti on (no other treatmentrelated effects observed); however, it is noted that this effect alone is limited in its diagnostic specificity for a potential estroge n-related effect. A 5% non-tr eatment responsive decrease in eggshell thickness was observed the mallard duck reproduction study, but no other effects were observed in this study (and not seen in the bobwh ite quail study). Therefore, while there were largely negative or limited responses in the Tier 1 in vitro assays, in vivo mammalian dataset, and avian dataset, for the FSTRA there were complementary responses along several levels of biological organization for the females including a lterations in enzyme activity (plasma VTG) up to the apical endpoint of fecundit y. As such, significant effects of the reproductive capacity of fish were observed in the FSTRA suggests that flutolanil may potentially interact with the HPG
axis.FLUTOLANIL FINAL

Page 24 of 48 C. Androgen Pathway

Although flutolanil showed an equivocal response in the Tier 1 AR binding assay, in a published ARTA study (Kojima et al ., 2004) considered as OSRI, flutolanil was reported to be negative for AR transcriptional activation. Flutolanil did not alter in vitro testosterone production in the Tier 1 steroidogenesis assay. In vivo , flutolanil was negative in both the Tier 1 Hershberger and in the male pubertal assay. There were no androgen- related responses in the mammalian Part 158 studies to support an interaction with the androgen pathway in mammals. No androgen-related
effects were observed in the Part 158 avian reproduction studi es. However, in the FSTRA, median male tubercle score was decreased by 32 % and multiple abnormalities in the testes were observed in the high treatment group (1.2 mg/L). These findings suggest a potential effect on the androgen pathway in fish. As with the findings in the estrogen pathway, while the in vitro and
mammalian/avian in vivo assays do not suggest a potential in teraction, the results in the male fish, with complementary effects in the female fi sh, suggest a potential to interact with the A pathway in fish.

D. Thyroid Pathway

There were no treatment-related effect s on the thyroid parameters in the in vivo male or female pubertal rat assays or in the Pa rt 158 mammalian toxicity studies. The effects on development, growth and histopathology observed in the AMA were not considered to be related to flutolanil treatment, but rather may be re flective of a response due to th e presence of the solvent (similar
effects observed in solvent contro l and treatment groups relative to negative control). Therefore,
the results in the AMA do not s uggest a potential for interaction with the thyroid pathway in amphibians.

E. Conclusions

The conclusion of the WoE evaluation is that fl utolanil demonstrates no convincing evidence of potential interaction with the estrogen or a ndrogen pathways in mammals. However, the complementary responses observed in both sexes in the FSTRA suggest a potential for flutolanil to interact with the E and A pathways in fish. There is no convincing evidence for the potential for flutolanil to interact with the T pathway in mammals or wildlife.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not
recommended for flutolanil since there was no convi ncing evidence of potent ial interaction with the estrogen, androgen or thyroid pathways.

Based on the complementary responses observed in the FSTRA, including effects on the apical endpoint of fecundity, EDSP Tier 2 Medaka Extended One-Generation Reproduction TestFLUTOLANIL FINAL

Page 25 of 48 (MEGORT) is recommended. The results in the FST RA indicate that there may be a potential for interaction with the estrogen a nd androgen pathway in fish. Currently, the chronic fish NOAEC value is 0.233 mg a.i./L, based on decreased leng th and wet weight in a fathead minnow early life-stage study. Effects on female GSI and gonadal histopathology were observed at 0.018 mg a.i./L in the FSTRA, with an increase in the number of eff ects observed in the males and females, including a cessation in fecundity, at 1.2 mg a.i./L. Therefore, as the concentration of the observed effects in the FSTRA are lower than the current regulatory NOAEC for fish,
additional data are recommended to understand the dose-response relationship for potential reproductive effects.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for flutolanil. In both sp ecies, 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 flutolanil exposure. Relative to the EDSP, ad ditional testing is not recommended.

As there was no convincing evid ence of a potential thyroid interaction in amphibians, no additional testing is recommended.FLUTOLANIL FINAL

Page 26 of 48 VI. References

Amanuma, T. (2011) Hershberger assay for flutol anil technical grade in rats. Unpublished study conducted by Research Center , Nihon Nohyaku Co., Ltd., Osaka, Japan. Final Report Number: LSRC-T10-117A, April 8, 2011. (MRID 48616807)

Amanuma, T. (2012) Male and Female Pubertal Assay with Flutolan il Technical in Rats. Unpublished study conducted by Nihon Nohyaku Co., Ltd., Osaka, Japan. Laboratory Report No.: LSRC-T12-068A, June 18, 2012. (MRID 48671101)

Amanuma, T. (2011) Uterotrophic Assay for Flutol anil Technical Grade in Ovariectomized Rats. Unpublished study conducted by Nihon Nohyaku Company, Ltd., Research Center, Osaka, Japan. Final Report No.: LSRC- T11-018A, April 15, 2011. (MRID 48616809)

Atkinson, J. (1986) T21-Flutolanil : A three-month oral toxicity study of flutolanil in rats. Unpublished study conducted by Bio/dynamics, Inc. Project #85-2926. (T-3024). (MRID 40342919)

Atkinson, J. (1986) T22-Flutolanil : A three-month oral toxicity study of flutolanil in dogs. Unpublished study conducted by Bio/dynamics, Inc. Project #85-2927. (T-3025) (MRID 40342920)

Beavers, J.B., Hoxter, K.A., and Jaber, M. (1993) W64 flutolanil: Flutolanil technical: A one generation reproduction study with the bobwhite ( Colinus virginianus ). Wildlife International Ltd. Project No.: 244108 (W-3025). (MRID 42932301)

Beavers, J.B., Hoxter, K.A., and Jaber, M. (1993) W65 flutolanil: Flutolanil technical: A one generation reproduction study with the mallard (Anas platyrhynchos). Wildlife International
Ltd

. Project No.: 244109 (W-3026). ( MRID 42932302)

Cowlyn, I.E., Nicholls, I.M., Virgo, D.M., and Fowler, J.S.L. (1990) Flutolanil: Oncogenicity study by dietary administration to CD-1(1CR)BR mice for 78 weeks. Unpublished study conducted by Life Science Research, Ltd. LSR Report # 89/0043 (T-3041). (MRID 41850803)

Hamasaka, Y. (2011) Steroidogenesis assay for Flutolanil in Human cell line- H295R. Unpublished study conducted by Research Ce nter, Nihon Nohyaku Co., Ltd., Osaka, Japan. LSRCM11- 100A, November 22, 2011. (MRID 48616808)

Ikuta, J. (2011) ER Binding Assay of Flut olanil. Unpublished study conducted by Research Center, Nihon Nohyaku Co. Ltd. Osaka, Japan. Laboratory Re port No.: GA-27, 11-0063, November 24, 2011. (MRID 48616805)FLUTOLANIL FINAL

Page 27 of 48 Jäger, M. (2011) Human Androgen Receptor Bi nding Assay using Rat Prostate Cytosol (ARRPC) with Flutolanil. Unpublished study conducted by Harlan Cytotest Cell Research GmbH, Germany. Laboratory Study Number: 1423900, December 8, 2011. (MRID 48616802)

Katoh, M., Suzuki, A., Ito, M., Chida, T., and Matsuzawa, I. (1982) Twenty-four month chronic toxicity study of NNF-136 in rats. Unpublished study conducted by Mitsubishi -Kasei Institute of Toxicological and Environmental Scie nces. Report #54-039 (T-3013). (MRID 40342921)

Kojima, H., Iida, M., Katsura, E., Kanetoshi, A., Hori, Y., and Kobayashi, K. (2003) Effects of a diphenyl ether-type herbicide, chlornitrofen, and its amino de rivative on androgen and estrogen receptor activities. Environ Health Perspect. 111:497–502.

Kojima, H., Katsura, E., Takeuchi, S., Niiyama, K., and Kobayashi, K. (2004) Screening for estrogen and androgen receptor activi ties in 200 pesticides by in vitr o reporter gene assays using Chinese hamster ovary cells. Environ Health Perspect . 112(5):524-31.

Lambert, E.P. (1992). Flutolanil: Teratology stud y in the rat. Amendment to final report. Unpublished study conducted by Life Scien ce Research Ltd. LSR Report #87 INHH023/554. First Amendment #92/NHH023/0490. (T3043). Or iginal study completed 1987. (MRID 41850804)

Nagata, T

. , Sato, M., Nagata, T., Takeuchi, T., Murakami, U., and Enomoto, M. (1982) T3- Flutolanil: Chronic toxicity study for NNF-136 administered orally to beagle dogs for 104 weeks. Unpublished study conducted by Shin Ni ppon Biomedical Laboratories, Ltd. (T-3014). (MRID 40342922)

Palmer, S.J., Kendall, T.Z., and Krueger, H.O. (2011) Flutolanil: Amphibian Metamorphosis Assay for the Detection of Thyroid Active Subs tances. Unpublished st udy conducted by Wildlife International, Ltd., Easton, Maryland 21601. Laboratory project number 397A-149. Study sponsored by Nihon Nohyaku Co. Ltd., Chuo-ku, Tokyo 103-8236 Japan. (MRID 48616801)

Palmer, S.J., Zendall, T.Z., Martin, K.H., and Krue ger, H.O. (2011) Flutolanil: Fish Short-term Reproduction Assay with the Fathead Minnow (Pimephales promelas). Unpublished study conducted by Wildlife Interna tional, Ltd., Easton, Maryla nd. Lab Study No.: 397A-148. Study sponsored by Nihon Nohyaku Co. Ltd., Chuo-ku, Tokyo, Japan. Study completed December 7, 2011. (MRID 48616806)

Rhodes, H., and Muckerman, M. (1995) Early lif e-stage toxicity of flutolanil to the fathead
minnow (Pimephales promelas) under flow-through conditions. Analytical Bio-Chemistry Laboratories, Inc

. Final Report #41685. Sponsor 's Study #523AU (W-3030). (MRID 43552001)FLUTOLANIL FINAL

Page 28 of 48 Schroeder, R.E. (1991). A two-ge neration reproduction study in ra ts with flutolanil. Unpublished study conducted by Bio/dynamics, Inc. Project #89-3417. (T-3069). (MRID 41850805)

Tauchi, K., Kawanishi, H., Takeshima, T., Toyohara, S., and Imai, S. (1987) Teratogenicity study of flutolanil in the rabbit. Unpublished study conducted by Imam ichi Institute for Animal Reproduction. Study #173 (T-3042). (MRID 40342924)

Tesh, J.M., McAnulty, P.A., Lambert, E.P., and Manning, T.J. (1987) Flutolanil: Preliminary teratology study in the rat. Life Science Research. Report #87 /NHH022/336. (T-3092) (MRID 41850804)

USEPA (2010) FLUTOLANIL - Report of the OPP Endoc rine Disruptor Review Team- Test Order#: EDSP-128975-112. December 9, 2010. Office of Chemical Safety and Pollution
Prevention. U.S. Environmental Pr otection Agency, Washington, D.C.

USEPA. (2011a) Endocrine Disruptor Screeni ng Program – Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. September 14, 2011. Office of Chemical Safety and Pollution Preven tion. U.S. Environmental Protection Agency, Washington, DC.

USEPA (2011b) FLUTOLANIL - Response to Nichino Am erica, Inc. request to evaluate supplemental information provided to satisfy the Test Order requirement for the Estrogen Receptor Transcriptio nal Activation Assay

. October 13, 2011 . Office of Chemical Safety and
Pollution Prevention. U.S. Environmenta l Protection Agency, Washington, D.C.

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.

Yasunaga, R. (2011) Aromatase Study of Flutolanil. Unpublished study conducted by Research Center, Nihon Nohyaku Co., Ltd., Osaka, Japa n. Laboratory Study N o.: GA-27, 10-0122. June 27, 2011. (MRID 48616803)FLUTOLANIL FINAL

Page 29 of 48 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100)

The 21-day assay (MRID 48616801) of flutolanil (98.7% purity) on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at developmental Nieuwkoop-Faber Stage (NF) 51 (80 per contro l and treatment group) were exposed to negative and solvent controls (0.02 mL/L dimethyl formamide, DMF) and
nominal flutolanil concentrati ons of 0.015, 0.15, and 1.5 mg a.i./L. Mean-measured concentrations were <0.01 (<LOQ; control), 0.015, 0.13, and 1.1 mg a. i./L. The test system was maintained at 21.2 to 22.2°C and a pH of 7.9 to 8.3.

All effects, unless otherwise not ed, are reported based on compar ison to the negative control.
There were no treatment-related effects on survival of tadpoles during the 21-day test; 1, 3, and 1 deaths were observed in the negative control, low, and high treatment groups, respectively. Clinical signs of toxicity in cluded one tadpole in the mid treat ment group that was periodically noted as small with a misshapen head, and anothe r was noted as weak. At test termination, one tadpole in the mid treatment group was noted to have an extra front leg. All other surviving tadpoles appeared normal throughout the test. Spin al curvature was observe d in several tadpoles in the negative control (8) and solvent control (7) and low (1), and mid (1) treatment groups.

By Day 21, several late stage ta dpoles (>NF 60) were observed in the solvent control (12), low (6), mid (9), and high (5) treatment groups; none were observed in the negative control. Late stage tadpoles were excluded during an alysis of growth parameters ( e.g. , SVL, BW, and HLL).

7-day wet body weight (p ≤ 0.016; ≥ 40%) and 7-day snout-vent length (SVL; p ≤ 0.005; ≥ 15%) were significantly increased in the mean-measured low and mid treatment groups as compared to the negative control. No significan t acceleration or delay of medi an NF developmental stage or normalized hind-limb length (HLL) was observed on Day 7.

By Day 21, wet weight (p ≤ 0.001; ≥ 20%) and SVL (p ≤ 0.005; ≥ 5%) were significantly increased
in the mean-measured low and mid treat ment groups. Hind-limb length (HLL; p ≤ 0.003; ≥ 23%), normalized (NHLL; p ≤ 0.05; ≥ 23%), and NF stage (p=0.05) were significantly increased at all treatment levels as compared to the negativ e control. No asynchronous development was observed throughout the test.

No treatment-related effects were observed for thyroid gland histopathol ogy, however there were significant differences between the n egative and solvent control.FLUTOLANIL FINAL

Page 30 of 48 For all variables in which there was a significant increase (p<0.05) from the negative control, there was also a significant incr ease (p<0.05) in response of the so lvent control over the negative
control. . Both the solvent and the concentrati on used are within what is recommended in the guideline for this study. These e ffects however make it difficult to ascertain whether the effects
observed were treatment related or mediated by the solvent.

Androgen Receptor (AR) Bi nding Assay (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48616802), prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a saturation binding experiment and a competitive binding experiment. The satura tion binding experiment was conducted to demonstrate that the AR in the rat prostate cy tosol was present in reasonable numbers and was functioning with appropriate affi nity for the radio-labeled refe rence androgen (R1881) prior to routinely conducting AR competitive binding e xperiments. The competitive binding assay was conducted to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (l ogarithmic increase from 10

− 9 to 10

− 4 M) of flutolanil (98.7% purity, Lot #: 9AE0011P). Ethanol was used as a vehicle at a fina l assay concentration of 2%. The assay included dexamethasone as a weak pos itive control, and R1881 as the ligand reference
standard.

In the saturation binding experime nt, the dissociation constant (K d ) for [ 3 H]-R1881 was 3.02 (±0.38) nM and the estimated B max was reported as 9567 (±537) dpm. The K d was not within the range reported in the EPA validation program ( 0.685 to 1.57 nM), but the study reports that the expected saturation kinetics were observed in the curve. Although the standard deviation for the average K d (6% of mean) and B max (13% of mean) values were provided, the individual run K d and B max values were not provided, therefore the variation among runs and confidence in the reported K d and B max numbers is uncertain. However, the data produced a linear Scatchard plot.

In the competitive binding experiment, flutolanil was evaluated at a concentration range from 10

-

9 M to 10 -4 M, not the 10 -10 to 10 -3 M (or up to the limit of solubi lity) range recommended in the test guideline. The mean specific binding of the radiolabeled ligand reduced in the presence of flutolanil was variable and not dose sp ecific in the first run: 78-98% at 10

− 4 , 10

− 5 , and 10

− 6 M;
67% at 10

− 7 M; and 73-74% at 10

− 8 and 10

− 9 M. In the second and third runs, flutolanil did not reduce the mean specific binding at any concen tration (92-105% and 81-98%, respectively). Flutolanil is classified as equi vocal as specific radioligand bind ing was greater than 50% but less
than 75% at several concentrati ons in the first run. As speci fic binding was not <50% at any flutolanil concentration, an IC 50 and RBA could not be ca lculated for flutolanil.

The estimated log IC 50 s for R1881 and the weak positive control (dexamethasone) were

− 8.8 and

− 4.5 M, respectively. The mean RBA for the w eak positive control was 0.0046%. ConfidenceFLUTOLANIL FINAL

Page 31 of 48 in these numbers is high due to the small vari ation. No precipitation of the test compound was
noted and the solvent cont rol responses indicated no drift in the study assay.

The performance criteria were not met for R1881 and dexamethasone indicating possible problems with the assay. For R1881, the top and bottom % binding was low in Run #1, and the Hill slope was high in Run #2. For dexamethasone, the top % binding was low in Runs #1 and #3, the bottom % binding was high in all Runs , and the Hill slope was low in Run #3.

Examination of the curve for the reference materi al across the runs indica ted variability in the top plateaus for R1881. The study reported that the maximal percent bi nding at the lowest concentration of R1881 was unexpectedly lowe r than 18% of the negative control but
nonetheless the IC 50 calculated were comparable to the re sults obtained for the other two runs showing the expected plateau level. Additionally , variability in the t op % binding and Hill slope curves for the weak positive control, dexame thasone, was observed and the bottom of binding curves (14-33%) could not be achieved at the expected levels.

Based on the results from the three assay runs, flutolanil is classified as Equivocal in the Androgen Receptor Binding Assay.

Aromatase Assay (OCSPP 890.1200)

In an in vitro aromatase (CYP19) assay (MRID 48616803) , flutolanil (99.5% a.i., Lot #: 8AE0010P) in ethanol ( ≤ 1%) was incubated with human recombinant aromatase and tritiated androstenedione ([1 β

3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]-ASDN) for 15 minutes at 37°C to assess the potential of flutolanil to inhibit aromat ase activity. Flutolanil wa s tested at logarithmic
concentrations from 10 –10 M to 10

− 3 M (Run 1) and from 10 –10 M to 10

− 4 M (Runs 2 and 3), due to precipitation observed at 10

− 3 M and 10 -3.5 M in Run 1.

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation period for each con centration of chemical. Tritiated water was quantified using liquid scintillation counting (L SC). Three independent runs were conducted,
and each run included a full activ ity control, a background activity control, a positive control series (10

− 10 M to 10

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

− 10 M to 10

− 3 M) with 3 repetitions per concentration.

Aromatase activity in the fu ll activity controls ranged from 0.203 to 0.253 nmol·mgprotein

− 1 ·min

− 1 for the three test runs, with a mean and standard deviation of 0.230 ± 0.017 nmol·mg-protein

− 1 ·min

− 1 . Activity in the background contro ls averaged 0.08% of the full activity. The response of the full activity controls and background controls was acceptable for
each run.FLUTOLANIL FINAL

Page 32 of 48 Results for the positive control were generally within the recommended ra nges for the top of the curve, bottom curve, Hill slope, log IC 50 , and coefficient of varia tion for replicates of each concentration within runs. Also, the coefficien ts of variations (%CVs) for replicates of each
concentration of flutolanil with in a run were within the 15% guideline. For 4-OH ASDN, the estimated log IC 50 averaged –7.2 M, and the slope was -1.0.

For flutolanil, aromatase activity averaged 0.230 ± 0.017 nmol·mg-protein

− 1 ·min

− 1 at the lowest
tested concentration of 10 –10 M and 0.242 ± 0.011 nmol·mg-protein –1 ·min

− 1 at the highest successfully tested concentration of 10

− 4 M (highest non-precipitating dose). Precipitation was
observed in Run 1 at 10

− 3 M and 10 -3.5 M. The data for flutolanil were not suitable for modeling. The lowest portion of the response curve was greater than 75% activity at all concentrations of The mean aromatase activity of 10 –4 M flutolanil was 105.56% of c ontrol activity, and the best fit mean inhibition response curve was a straight line (essentially no inhi bition was observed at other doses).

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

Estrogen Receptor (ER) Bi nding Assay (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48616805), uterine cytosol from Sprague Dawley rats was used as the source of estr ogen receptors (ER) to conduct saturation binding experiments and competitive binding experiments in this assay. The saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was functioning with appropr iate affinity for the radiolabeled reference estrogen prior to routinely conducting ER compe titive binding experiments. 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 increasi ng concentrations (logarithmic increase from 10

− 10 to
10

− 4 M) of flutolanil (99.5% a.i ., Lot#: 8AE0010P). Ethanol was used as a vehicle at a final concentration of 2%. 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.

In the saturation binding experime nts, the maximum binding capacity (B max ) was 41.3 ± 1.7 fmol/100 μg protein and th e dissociation constant (K d ) was 0.044 ± 0.004 nM. For the competitive binding experiments, perfor mance criteria were met; the log PC 50 , log PC 10, log

EC 50, and Hill slope for the reference chemicals 17 β -estradiol, 17 α -estradiol, 19-norethindrone and octyltriethoxysilane all performed within the acceptable range. The mean RBA for 19-
norethindrone was 1.06 × 10

− 4 %. Flutolanil displaced less than 25% of the radiolabeled estradiolFLUTOLANIL FINAL

Page 33 of 48 from the ER over the range of so luble concentrations tested (10 -10 to 10 -4 M) in all competitive binding runs.

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

ER α Transcriptional Activatio n (ERTA) Assay (OCSPP 890.1300)

Requirement satisfied by OSRI (Kojima et al., 2004; MRID 48033008); See Appendix 2.

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350)

The 21-day short-term reproduction assay (MRI D 4861606) of flutolanil with fathead minnows ( Pimephales promelas) was conducted under flow-through c onditions. Adult fish, 20 spawning groups (2 males and 4 females in each group: ca . 6 months), were exposed to flutolanil (98.7% purity) at nominal concentrations of 0 (negative control), 0 (solvent control), 0.020, 0.20 and 2.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.010), <LOQ (<0.010), 0.018, 0.18 and 1.2 mg a.i./L. The solvent control consiste d of dimethylformamide (DMF) at a final concentration of 0.02 mL/L. The test sy stem was maintained at 24.6 to 25.9 o C and a pH of 7.7 to

8.2.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. There were no sign ificant effects on survival in any treatment group as compared to the negative control. At study termination, all surviving fish appeared normal. Female body weight and length were signifi cantly increased (p<0.05; 22% a nd 4%, respectively of negative control) at the high treatment level; there were no significant effects on male body weight or length.

Flutolanil significantly reduced fecundity (p<0.05) 100% (rela tive to control) at the high treatment level; the fer tilization success in the high treatment group was not calculated because no eggs were produced in any replicate. Fertili zation success in the rema ining treatments was not significantly different from ne gative control (p>0.05). While not statistically significant, fecundity was reduced 17% from the negative co ntrol at the low and mid concentrations.

In females in the high treatment group, plasma vitellogenin (VTG) was significantly reduced relative to negative control (p< 0.01, 72%); while not statistically significant, female VTG at the
mid treatment level was reduced 16% from the negative control. Female gonado-somatic index (GSI) levels were significantly promoted in a concentration-dependent manner, relative to the negative control, at all treatmentlevels (p<0.05; 15-64%). There were no significant differencesFLUTOLANIL FINAL

Page 34 of 48 in male VTG or GSI compared to the negati ve control (p>0.05). Sex steroids (plasma 17 β

estradiol and testos terone) were not measured during th is study. Nuptial tubercle scores were significantly reduced (p<0.05) in male fi sh at the high treatment level (21 vs. 27); no
tubercles were observed for females.

There was a statistically significan t decrease in mean male fatp ad score at the high treatment level compared to the pooled control (p<0.05; Wilcoxon Rank-Sum test; calculated by the study author). In males at the high concentration, there was an incr eased presence of spermatogonia and primary spermatocytes with a decreased pr esences of spermatocysts containing secondary spermatocytes and spermatids (p<0.05; Jonckheere- Terpstra test); the testes could not be normally staged in 2 of 8 fish. In females, ovaries showed a mild to marked increase in atresia at the high treatment level (p<0.05; Jonckheere-Terpstra test; 15 of 16 fish) and significant changes in gonad stage and size at all treatment le vels (p<0.05; Jonckheer e-Terpstra test).

Hershberger Assay (OCSPP 890.1400)

In a Hershberger Assay (MRID 48619807) screeni ng for androgenic activity, flutolanil (98.7% a.i., lot # 9AE0011P) in 0.5% (w/v ) methyl cellulose 100 (MC) was administered daily via oral gavage (10 mL/kg) to groups of six 57-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 500, or 1000 (limit dose) mg/kg/day. An androgenic positive control group
consisted of six castrated rats exposed to 0.4 mg/kg/day of te stosterone propionate (TP) by subcutaneous (s.c.) injection.

To screen for potential anti-andr ogenic activity, flutolanil (98.7% a.i., lot # 9AE0011P) in MC was also administered daily via oral gavage (10 mL/kg) to gr oups of six 58-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 500, or 1000 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The anti-androgenic positive c ontrol group consisted of six castrated rats exposed to 0.4 mg/kg/day TP and 3 mg/kg/day flutamide (FT) . TP alone was used as the an ti-androgenic negative control.

For both components of the assay, body weights were determined daily and food consumption was measured for Days 1-2, 4-5, and 8-9. The an imals were dosed for 10 consecutive days and terminated approximately 24 hours after the fina l dose administration. At necropsy, the five androgen-dependent tissues and liver were collected and weighed.

All animals survived until schedul ed termination. There were no c linical signs of toxicity and no treatment-related gross pathological findings.

In the androgen agonist assa y, body weights, overall (Days 1- 11) body weight gains, and food consumption in the flutolanil treatment groups a nd the positive controls were comparable to theFLUTOLANIL FINAL

Page 35 of 48 vehicle controls. There were no treatment-related effects on wei ghts of accessory sex organs or liver for any flutolanil treatment group. Compared to the vehicle control, animals in the TP group had increased (p ≤ 0.05) accessory sex organ weights as follows: 1090% in seminal vesicles; 1280% in ventral prostate; 818% in levator ani-bulbocavernosus (LABC); 400% in Cowper’s gland; and 91% in glans penis. Ther e were no dose-related eff ects on liver weights in the positive control group. The pe rformance criteria indicated that this assay was performing as expected.

In the anti-androgen assay, body weights, overall body weight gains, and food consumption in the flutolanil treatment groups and the positive co ntrols were comparable to the TP control.
There were no treatment-related effects in weights of accessory sex organs or liver for any flutolanil treatment group. Compared to the TP control group, animals dosed with TP + FT (positive control) had decreased (p ≤ 0.05) accessory sex organ weights as follows: 78% in seminal vesicles; 67% in ventra l prostate; 56% in LABC; 63% in Cowper’s gland; and 28% in glans penis. There were no dose -related effects on liver weights in the positive control group. The performance criteria indicated that th is assay was performi ng as expected.

Statistically significant changes were not seen in two or more of the five androgen sensitive tissue weights. Flutolanil was negative for androgenicity and anti-androgenicity in the Hershberger assay.

Female Pubertal Assay (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48671101), 15 female Sprague-Dawley (Crl:CD (SD)) rats/dose group were treated daily via oral gava ge with flutolanil (98.7% a.i., Lot # 9AE0011P) in 0.5% aqueous methylcellulose at doses of0, 500, or 1000 mg/kg/day (limit dose) from postnatal day (PND) 22 to 42. Animals were examin ed for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainme nt were recorded. Following sacrifice on PND 42, blood was collected for clinical chemistry and hormone analyses. Total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels we re determined by ELISA and radioimmunoassay (RIA), respectively. Adrenals, kidneys, liver, thyroid, pituitary, and urogenital organs were weighed and microscopic examination of th e uterus, ovaries, kidne ys, and thyroid was
conducted.

No adverse, treatment-related related effects we re observed on mortalit y, clinical signs, final body weights, overall body weight gain, age at VO, body weight at VO, organ weights, and clinical chemistry or hormone levels. There were no treatment-related hist opathological findings in the thyroid, uterus, ovaries, or kidneys.FLUTOLANIL FINAL

Page 36 of 48 Relative (to body) liver weight s were increased (p<0.05) by 6% at 500 and 1000 mg/kg/day compared to control values. Th e dose levels tested were adeq uate since the high dose was the Limit Dose (1000 mg/kg/day).

Male Pubertal Assay (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48671101), 15 ma le Crl:CD(SD) Sprague Dawley rats/dose group were treated daily via oral gavage with flutolanil (98.7% a.i., Lot# 9AE0011P) in aqueous 0.5% methylcellulose at doses of 0 (vehicle), 500, or 1000 mg/kg/day from post-natal day (PND) 23 to 53. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and age and weight at day of attainment were recorded. Following sacrifice on PND 53, total testosterone levels were determined by EIA, total T 4 levels were determined with ELISA, and
TSH levels were determined with a radioimmunoassay (RIA). Adre nals, liver, pituitary, thyroid, and urogenital organ weights were recorded a nd microscopic examinations of the testes, epididymides, thyroid, and ki dneys were performed.

No adverse, treatment-related related effects we re observed on mortalit y, clinical signs, final body weights, overall body weight gain, age at PPS, body weight at PPS, organ weights (except liver), clinical chemistry, or hormone levels (serum T 4 , TSH, or testosterone). There were no treatment-related histopathological findings in the thyroid, testes , epididymides, or kidneys.

At 1000 mg/kg/day, absolute, adjusted (for body weight on PND 23), and relative liver weights were increased by 9-10% compared to control values, and at 500 mg /kg/day, relative liver weights were increased by 6%.

The dosing levels used in the study were accepta ble, as the limit dose of 1000 mg/kg/day was used for high dose group.

Steroidogenesis Assay (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48616908), H295R cells cultured in vitro in 24-well plates
were incubated with flutolanil

, (99.5%, Lot No. 8AE0010P) at concentrations of 0.0001, 0.001, 0.01, 0.1, 0.3, 1, 3, 10, and 100 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as a vehicle, at a fi nal concentration of 0.1%.

Hormone concentrations in the assay were dete rmined using an enzyme-linked immunosorbent assay (ELISA). Three independent experiments were performed. A Quality Control (QC) plate was run concurrently with each independent run of a test chemical plate to demonstrate that the assay responded properly to positiv e control agents at two concentr ation levels; positive controlsFLUTOLANIL FINAL

Page 37 of 48 included a known inhibitor (pro chloraz) and a known inducer (forskolin) of estradiol and testosterone production.

Data at flutolanil concentr ations with % live values ≤ 80% ( ≥ 3 μM) were discarded due to cytotoxicity. Precipitation of the test compound was not reported at any concentration. Guideline acceptability recommendations and requirements were generally met, including lack of cytotoxicity, adequate producti on of testosterone and estradiol, acceptable reproducibility (low coefficients of variation, %CV), and appropriate induction and inhibiti on with positive c ontrols.

No effects on testosterone and17 β -estradiol concentrations were observed at flutolanil
concentrations of 10

− 10 M to 10

− 4 M. Based on hormone responses in three independent runs,
flutolanil treatment did not re sult in statistically significant and reproducible alterations in

testosterone or es tradiol production.

Uterotrophic Assay (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48616809) conducted to screen for potential estrogenic activity, flutolanil (98.7% purity, Lot #9AE0011P) in methyl cellulose was administered daily via oral gavage to groups of six ovariectomized Sprague-Daw ley rats at dose levels of 0 (vehicle), 500 or 1,000 (limit dose) mg/kg/day on post-natal days (PND) 67-69 for three consecutive days. A positive control group was treated with a daily dose of 17 α -ethynyl estradiol (EE) in corn oil at

0.5 μ g/kg/day by subcutaneous (s.c.) injection. Body weights and food consumption were determined daily. All animals were terminated and necropsied on PND 70 approximately 24
hours after the final dose administration to determ ine wet and blotted uter ine weights. Liver weights were also determined at necropsy.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in animals for any flutolanil treated groups. B ody weights and overall body weight gains (Days 1- 4) in the flutolanil treated groups were compar able to the vehicle cont rols throughout the study. Uterine and liver weights in the fl utolanil treated groups were compar able to the vehicle controls.

Body weights in the positive cont rol (EE) group were comparable to the vehicle control group throughout the study, but overall body weight gains of the positive control group were significantly (p<0.01) lower ( ↓ 99%) than the vehicle control gr oup. Absolute wet and blotted uterus weights for the EE group were increas ed (p<0.0001) by 426% and 195%, respectively, as expected. Liver weights were compar able to the vehicle controls.

No statistically significant changes were seen in uterine weight in this assay. Flutolanil is negative in the uterotrophic assay.FLUTOLANIL FINAL

Page 38 of 48 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

ER and AR Transcriptional Acti vation Assay (ERTA & ARTA)

In a published, non-Guideline study ( Kojima et al ., 2004) , the potential of flutolanil (>95% purity) to interact with two human ER subtypes (hER α and hER β ) and one human androgen receptor (hAR) in transactivati on assays with Chinese hamster ovary cells (CHO-K1 cells) was investigated. The human ER (hER α ) and AR (hAR) expression vectors (pcDNAER α and pZeoSV2AR, respectively) were construc ted as described previously (Kojima et al. , 2003). The
hER β expression vector was cons tructed by cloning the hER β cDNA with reverse transcriptasepolymerase chain reaction from human placental R NA. The sequence was verified and inserted into the mammalian expression ve ctor pcDNA3.1Zeo(-) creating pcDNAER β . The construction of the estrogen or androgen responsive elements containing re porter plasmid (pGL3-tkERE and pIND-ARE, respectively) was described previously (Kojima et al. , 2003). pRL-SV40 containing
the Renilla luciferase gene was used as an inte rnal control for transfection efficiency.

For detection of hER α or hER β activity, cells were tr ansfected with pcDNAER α or pcDNAER β

, pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were transfected with pZeoSV2AR, pIND-ARE, and pRL-SV40. Flutol anil was tested over a concentration range
from 10

− 8 to 10

− 5 M, and the responses were compared to the activity of 17 β -estradiol (E 2 , >97% purity) or 5 α -dihydrotestosterone (DHT, 95% purity) for th e ER or AR assays, respectively. For measurement of antagonistic activity to hER α , hER β and hAR, either 10

− 11 or 10

− 10 M E 2 or 10

− 10 M DHT was added to the cell cultures alon g with the test compound, respectively. Agonistic activities were evaluated by the relative activity ex pressed as 20% relative effective concentration (REC 20 ), the concentration of the compound de monstrating 20% of the activity of

10

− 10 M E 2 , 10

− 9 M E 2 , or 10

− 9 M DHT for ER α , ER β , and AR, respectively. Antagonistic activities were evaluated similarly by the relative activity expressed as 20 % relative inhibitory concentration (RIC 20 ), the concentration of the compound showing 20% in hibition of the activity
of E 2 or DHT against ER α , ER β , and AR, respectively.

Based on the additional information provided to the Agency (USEPA, 2011b), no evidence of cytotoxicity was observed at any of the flutolanil concentrations evaluated, and the highest test
concentration was close to the test material solubility limit (20.5 μM). The supplemental
information also included raw data from each te st concentration of the three independent experimental runs.

The additional information allowed the Agency to confirm that flutolanil was negative for agonist or antagonist activity against ER α and ER β , and negative for agonist or antagonist activity against AR in this transcriptional activation test system.FLUTOLANIL FINAL

Page 39 of 48 Subchronic Toxicity (Rat, OCSPP 870.3100)

In a subchronic oral toxicity study in rats ( MRID 40342919, 1986 ), flutolanil (97.5% purity) was
administered in the diet to CD (SD-derived) rats (10/sex/dose group) at concentrations of 0, 500, 4000, and 20,000 ppm for 90 days. The mean dietary concentrations were equivalent to 0, 37, 299, and 1512 mg/kg/day, respectively, for males and 0, 44, 339, and 1743 mg/kg/day, respectively, for females. Organs that were weighed included the liver, kidneys, testes, ovaries, adrenals, and thyroid/parathyroi ds. Gross and microscopic pat hological examinations were conducted on these tissues, as well as the prosta te, epididymides, seminal vesicles, uterus, and mammary and pituitary glands. The GLP status of this study is unknown.

No treatment-related effects were observed on mort ality, clinical signs, or food consumption; one
male control animal was sacrificed moribund on Day 86. Slight decreases (NS) in overall body weight gain were noted for the high-dose animals in the study. No treatment-related changes in ophthalmoscopic, hematological, clinical chemistry, gross pathol ogy, or histopathology parameters were observed in either sex. Urin alysis examinations were not conducted. No treatment-related changes were obs erved for the absolute or relative weights of the kidneys, testes, ovaries, and adrenal glands. In males, absolute and relative thyroid weights were increased (p<0.01 or 0.05) at the mid (absolute, 28% and relative 23%) and high [absolute, 16% (NS) and relative, 20%] dose groups. In high- dose females, absolute and relative thyroid weights were increased (NS) by 13 and 14%, respec tively. In the high-dos e group, absolute liver weights in males and females were increased by 9 (NS) and 19% (p<0.0 1), respectively, and relative liver weights were increased (p<0.01) by 14 and 20%, resp ectively, compared to control. In the medium-dose group, the absolute and relati ve liver weights in females were increased (p<0.01 and 0.05) by 11 and 8%, respectively. Increas ed liver weight was considered to be an adaptive response to flutolanil treatment because these effects were not supported by clinical chemistry or histopathology findings. No treatme nt-related histopathol ogical lesions were observed in any examined tissues.

The reported NOAEL is 20,000 ppm (1512 mg/kg/day; HDT), and the LOAEL is not
established.

Subchronic Toxicity (Dog; OCSPP 870.3150)

In a subchronic oral toxicity study in dogs ( MRID 40342920, 1986 ), flutolanil (97.5% purity) was administered in gelatin capsu les to Beagle dogs (4/sex/dose group) at concentrations of 0, 80, 400, and 2000 mg/kg/day for 95-98 days. Organs that were weighed included the liver, kidneys, testes, ovaries, adrenals, and thyroid/parathyroi ds. Gross and micros copic pathological examinations were conducted on these tissues, as well as the prostate, epididymides, uterus, and mammary and pituitary glands.FLUTOLANIL FINAL

Page 40 of 48 No treatment-related effects were observed on mo rtality, clinical signs, body weights, or food consumption; swollen mammary areas were note d in the medium- and high-dose females. No treatment-related changes in the ophthalmo scopic parameters were observed. Alkaline phosphatase activity was increased (significance not repor ted) at 1.5 and 3 months in low- and high-dose males; observation in the low-dose male s may be artifactual due to pretreatment elevations. Additionally in the high-dose males, cholesterol wa s slightly elevated (significance not reported) at 1.5 and 3 months , and SGOT and SGPT activities were decreased (significance not reported) at 3 months. Alkaline phosphatase activity also was increased (significance not reported) at 3 months in the hi gh-dose females. In the high-d ose group, the absolute and relative weights for the liver and thyroi d were increased (magnitude a nd significance not reported) in both sexes. No treatment-related changes were obse rved in absolute or relative weights of other organs. Gross examination noted liver enlarg ement at the high dose and increased glycogen deposition (increased incidences from minimal/m ild to moderate) were observed in the mediumand high-dose dogs. No other treatment-rela ted histopathological lesions were observed.

The systemic toxicity LOAEL is 400 mg/kg/day based on enlarged livers and increased glycogen deposition; the NOAEL is 80 mg/kg/day.

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a two-generation reproducti on toxicity study in rats ( MRID 41850805, 1991 ), flutolanil (97.6 and 98.3% purity) was admini stered daily to SD rats (30/sex/dose) in the diet at nominal doses of 0, 200, 2000, and 20,000 ppm (mean-calculated inta kes of 0, 16, 159, or 1625 mg/kg/day for males and 0, 19, 190, or 1936 mg/kg/day for females). The F 0 parental animals were treated for 70 days prior to mating, and the F 1 parental animals were treated for 98 days prior to mating. Rats were dosed during mating, ge station and lactation. The orga n weights for liver and spleen were determined (no endocrine-sensitive organs were weighed).

There were no treatment-related effects on mort ality, clinical signs, body weight/body weight gain, or food consumption in either F 0 or F 1 animals. No treatment-related effects were observed
for the F 0 , F 1 , or F 2 adults in the reproductive paramete rs of mal40342924e or female mating, fertility and gestation indices, ge station length, live birth index, lactation index, parturition, or
fetal sex ratio. No treatment-rela ted histopathological lesi ons were seen in the parental animals, and no clinical or pathological findings were noted in pups from any litter or generation. Absolute and relative liver weights were incr eased (significance not reported) in high-dose F 0 generation animals and F 1 females. These weight increases were considered treatment-related adaptive changes due to the lack of adverse e ffects during gross and mi croscopic evaluation.

The LOAEL is not established (>1000 mg/kg/day) due to the lack of parental (systemic), reproductive, and offspring toxic ity; the NOAEL is >1000 mg/kg/day.FLUTOLANIL FINAL

Page 41 of 48 Developmental Toxicity (Rat, OCSPP 870.3700)

In a prenatal developmenta l toxicity study in rats ( MRID 41850804, 1987 and 1992 ), pregnant SD CD rats (22/dose group) were administer ed flutolanil (97.5% pur ity) in 0.5% aqueous methylcellulose by oral gavage at doses of 0, 40, 200, or 1000 mg/kg/day during GDs 6 through
15. All dams were sacrificed on GD 20. All fe tuses were weighed, sexed, and examined for gross external alterations. One-half of the fetuse s in each litter were examined for visceral soft tissue alterations by the Wilson technique. The re maining fetuses in each litter were examined
for skeletal alterations with a modified Dawson’s method.

No mortalities or abortions occurred during th e study. There were no tr eatment-related effects on clinical signs, body weight, or food consumption. No treatment-related changes were seen in pregnancy rate, number of corpora lutea, mean nu mber of implantations/l itter, pre-or post-natal implantations, early or late resorptions, number of live fetuses/litter, or fetal sex ratio. No developmental abnormalities (external, visceral , or skeletal) were noted at any dose.

The maternal and developmental NOAELs are 1000 mg/kg/day (HTD). The LOAELs are not determined (>1000 mg/kg/day).

Developmental Toxicity (Rabbit, OCSPP 870.3700)

In a prenatal developmental toxicity study in rabbits ( MRID 40342924, 1987 ), pregnant New Zealand White rabbits (16/dose group) were admi nistered flutolanil ( 97.5% purity) in aqueous 2% gum arabic by oral gavage at doses of 0, 40, 200, or 1000 mg/kg/day on GDs 6 through 18. On GD 28, all surviving does were sacrificed. Each fetus was weighed, examined for gross external observations, and sacrificed. All fetuses in each litter were examined for visceral soft tissue alterations by the method of Stuckhardt. Skeletal examinations were conducted by the
method of Dawson.

No treatment-related effects on mortality or clin ical signs were observed. One low-dose dam died due to gavage error. Reduced body wei ght gains during dosing were observed in all treatment groups, and lower ove rall body weight gain was obse rved in the high-dose group ( ↓ 12%; not statistically analyzed). Control group weights were 11-15% higher than the treated groups prior to dosing, so body weight gain decreases of 15-20% during dosing may be artificially high. Food consumption was slightly less in all treatment groups during the dosing period, and lower overall food consumption wa s observed in the high-dose group. The differences observed in body weight gains and food consumption were not considered treatmentrelated. Additionally, no treatment-related effects were seen on any absolute or relative organ weights.

The number of resorptions was increased in the mid- and high-dose groups, but not considered
treatment-related based on historic al control values. No treatmen t-related changes were seen inFLUTOLANIL FINAL

Page 42 of 48 pregnancy rate, number of corpora lutea, mean nu mber of implantations/l itter, pre-or postnatal implantations, or number of live fetuses/litter at any dose. There was an apparent shift in fetal sex ratio in high-dose litters (46: 54, male:female) compared to th e control, but only because the
sex ratio in the control was skewed toward male s (58:42). Sex the ratio s in all treated groups were approximately 50/50 (low - 55:45; mid - 50:50; high – 46:54). No developmental abnormalities (external, visceral, or sk eletal) were noted at any dose.

The maternal and developmental NOAELs are 1000 mg/kg/day. The LOAELs are not determined (>1000 mg/kg/day).

Chronic Toxicity/Carcinoge nicity (Rat, OCSPP 870.4300)

In a combined chronic toxicity/carcinogenicity study in rats ( MRID 40342921, 1982 ), Crj:CD (SD) SPF rats (50/sex/dose gr oup) were fed diets containing flutolan il (97.5-99.8% purity;multiple batches) at nominal concentrations of 0, 40, 200, 2000, and 10,000 ppm (meanmeasured intakes of 0/0, 1.8/2.1, 8.7/10, 86.9/103.1, and 460.5/535.8 mg/kg/day in males/females) for 24 months. Additional rats were randomly allocated for 3-month (6 rats/sex/group) and 12-month ( 10 rats/sex/group) interim sacrif ices. For clinical chemistry examinations, additional groups of 10 rats/sex were sacrificed at the 6- and 18-month intervals. Organs that were weighed included the liv er, kidneys, testes, ovaries, pituitary, thyroid/parathyroids, and adrena ls. Gross and microscopic pat hological examinations were
conducted on these tissues as well as the prostate, seminal vesicles, uterus, and mammary gland.

No treatment-related clini cal signs or differences in mortality were obser ved during the study. In the high-dose male rats, body weight gains were decreased (p<0.01) up to 10% compared to control for Weeks 1-13 (body weight differences were decreased 3-6%). The body weight gain differences between the high dose an d control were less apparent af ter the initial 13 weeks. No specific treatment-relate d pattern was observed for occasiona l food consumption reductions in treatment groups during the study.

No treatment-related effects were observed for th e hematological, clinical chemistry, urinalysis, gross pathology, or histopathology parameters. Clinical chemistry results for liver-specific enzymes and compounds were variable with no clea r treatment-related responses. In the highdose males, the relative liver weights were increased (p<0.05 and 0.01, respectively) by 12 and 14%, respectively, at 3 and 24 months. In the hi gh-dose females, absolute liver weights were increased (p<0.05 or 0.01) by 15-18% compared to control at 3, 12, and 24 months and relative liver weights were increased (p<0.05) by 11 an d 10%, respectively, at 3 and 12 months. These increases in liver weights in high- dose male and female rats were considered to be an adaptive response to flutolanil because th ese increases were not supported by adverse clinical chemistry or histopathology findings. No treatment-related changes were seen in absolute or relative weights of the other examined tissues, and no tr eatment-related histopat hological lesions wereFLUTOLANIL FINAL

Page 43 of 48 observed in the testes, seminal ve sicle, prostate, ovaries, uterus , or mammary, thyroid, adrenal, and pituitary glands.

The NOAEL is 460 mg/kg/day (10,000 ppm), and the LOAEL is not established (>10,000 ppm).

Carcinogenicity (Mouse, OCSPP 870.4200)

In a carcinogenicity study in mice ( MRID 41850803, 1990 ), CD-1(ICR)BR mice (52/sex/dose group) were fed diets co ntaining flutolanil (97.6% purity) at nominal concentrations of 0, 300, 1500, 7000, or 30,000 ppm (mean-measured inta kes of 0/0, 32/34, 162/168, 735/839, and 3333/3676 mg/kg/day in males/females) for 78 weeks. Organs that were weighed included the liver, kidneys, and testes. The tissues subjecte d to histological examination included these organs, as well as epididymides , prostate, seminal vesicles, ovaries, uterus, and mammary, pituitary, and thyroid/pa rathyroids glands.

No treatment-related effects were observed on mortality, clinical signs, food consumption, hematology, gross pathology, or hi stopathology. Urinalysis and c linical chemistry evaluations were not conducted. Body weight gains were decreased (p<0.01) by 17 and 16%, respectively, in the high-dose males and females during Weeks 0-24. In medium-dose females, body weight gains also were decreased (p<0.05) by 15% during Weeks 0-24. Body weights during the study were not affected by treatment.

No treatment-related changes were observed in orga n weights, including the absolute or relative
testicular weights. Absolute liver weight in the high-dose females was increased (NS) compared to control ( ↑ 20%), and liver-to-body weight ratio in the high-dose females was increased (p<0.0l) by 33%. No similar increa se in relative liver weight wa s observed in high-dose males. The increase in liver weights may be attributable to fatty vacuolation, wh ich is not considered
adverse. No treatment-related hist opathological lesions were observed in the testes, epididymides, seminal vesicle, prostate, ovari es, uterus, or mammary, thyroid, adrenal, and pituitary glands. Flutolanil was not considered oncoge nic under the conditi ons of this study.

Based on decreased body weight gains, the LOAEL is 3333 mg/kg/day for males (30,000 ppm) and 839 mg/kg/day (7000 ppm) for females. The NOAEL is 735 mg/kg/day (7000 ppm) for males and 168 mg/kg/day (1500 ppm) for females.FLUTOLANIL FINAL

Page 44 of 48 Chronic Toxicity (Dog, OCSPP 870.4100)

In a chronic toxicity study in dogs ( MRID 40342922, 1982 ), Beagle dogs (6/sex/dose group) were administered flutolanil (97.5-99.8% purity; mu ltiple batches) in gelatin capsules at doses of 0, 50, 250, or 1250 mg/kg/day for two years. Orga ns that were weighed included the liver, kidneys, ovaries, and pituitary, adre nals, and thyroid glands. Gro ss and microscopic pathological
examinations were conducted on these tissues as well as the testes, pr ostate, and uterus.

All dogs survived until study termination. There wa s a dose-related increase in the incidence of clinical signs (emesis, saliva tion, and soft stool) in both sexe s in the medium- and high-dose groups from Week 65 until study termination. Decreased (p<0.01 or 0.001) body weights were observed in high-dose males from Weeks 93-104 ( ↓ 4-8%; unknown significant decrease at Week 90) with decreased body weight gains (NS, ↓ 8-16%). Additionally, decreased (p<0.05 or 0.001) body weights were observed in high-dose females from Weeks 87-104 ( ↓ 3-8%) with decreased body weight gains (NS, ↓ 6-17%). Decreased food consumption (from Week 79 in males and from Week 74 in females; sign ificance not reported) also was observed. No treatment-related changes in ophthalmoscopic, hematological, or clinical chemistry parameters were observed. No treatment-related changes were observed in organ weights or hi stopathological lesions at any
dose.

The systemic toxicity LOAEL is 250 mg/kg/day based on increased incidence of clinical signs (emesis, salivation, and soft st ool). The NOAEL is 50 mg/kg/day.

Avian Reproduction (OCSPP 850.2300)

In an avian reproduction study ( MRID 42932301, 1993 ), Bobwhite quail ( Colinus virginianus; 19-weeks old) were administered flutolanil (98.9% purity) in the feed at nominal concentrations of 0 (0.2% acetone/0.08% corn oil), 400, 960, 1920, or 4800 ppm (not adjusted for purity) for 20 weeks (10 weeks prior to the start of egg production and during 10 weeks of egg production). Each treatment group was comprised of sixteen mating pairs, with one pair per pen. A progressive photo period was employed in this study: 8 hours/day through Week 7 and 17 hours/day until the end of the study. Mean temperature was maintained at 19.6±1.2°C throughout the study and mean humidity was ma intained at 33±13%. Individual adult body weights were recorded at study initiation, biw eekly through Week 8, and at study termination. Food consumption in each replicate was measur ed biweekly throughout the pre-treatment and treatment periods of the adult pha se. Individual chick body weight s were recorded after hatching and on Day 14 after hatching; e gg shell thickness was measured weekly during egg production.
Adult birds and chicks were observed rout inely for mortality and clinical signs.

There were no overt signs of t oxicity or treatment-related re ductions in food consumption or body weight at any test concentration compared to control. Two incident al deaths occurred in the low-medium concentration group. Alt hough there were no significant effects on anyFLUTOLANIL FINAL

Page 45 of 48 reproductive parameter, there was a slight reducti on in the number of e ggs laid at the high concentration compared to contro l. This reduction was related to an increase in the number of hens that laid a smaller number of eggs ( ≤ 20) during the study. This reduction in egg production
was also reflected in the numb er of hatchlings and 14-day su rvivors as percentages of the maximum number of eggs set. However, the pro portion of eggs set/eggs laid indicated that fertility was not necessarily a ffected at the high dose. The LOAEC is 4800 ppm based on the reduction in egg production, a nd the NOAEC is 1920 ppm.

In another avian reproduction study ( MRID 42932302, 1993 ), Mallard ducks ( Anas platyrhynchos ; 21-weeks old) were administ ered flutolanil (98.9% purity) in the feed at nominal concentrations of 0 (0.2% acetone/0.08% corn oil), 400, 960, 1920, or 4800 ppm (not adjusted for purity) for 20 weeks (10 weeks prior to the start of egg production and during 10 weeks of egg production). Each treatment group was comprised of sixteen mating pairs, with one pair per pen. A progressive photo period was employed in this study: ≤ 8 hours/day through Week 7 and 17 hours/day until the end of the study. Mean temperature was maintained at 17.8±1.8°C throughout the study and mean humidity was ma intained at 47±15%. Individual adult body weights were recorded at study initiation, biw eekly through Week 8, and at study termination. Food consumption in each replicate was measur ed biweekly throughout the pre-treatment and treatment periods of the adult pha se. Individual chick body weight s were recorded after hatching and on Day 14 after hatching; e gg shell thickness was measured weekly during egg production.
Adult birds and chicks were observed rout inely for mortality and clinical signs.

There were no overt signs of toxi city or treatment-related morta lities at any test concentration, and there were no reductions in adult body we ight or feed consumption. At the high concentration, there was a 5% decrease (p<0.05) in egg shell thickness compared to control.However, the mean shell thickness was not treatment-responsive and was comparable to the lowconcentration group mean value; the mid-low and mid-high mean eggshell thickness values were comparable to control. The reduction in e gg shell thickness at the high concentration was considered to biologically (toxicologically) re levant. There were no other treatment-related effects on any study parameters determined at a ny test concentrations. The LOAEC is 4800 ppm based on decreased egg shell thickn ess, and the NOAEC is 1920 ppm.

Fish Early Life-Cycle Toxicity (Fathead minnow, OCSPP 850.1400)

In a fish early life-cy cle toxicity study ( MRID 43552001, 1995 ), groups of Fathead minnow ( Pimephales promelas ) embryos (30 embryos/cup; 4 replicat es/treatment concentration) were exposed to flutolanil (98.9% purit y) at nominal concentrations of 0 [negative and solvent (DMF; 0.025 mL/L) controls], 0.13, 0.25, 0.50, 1.0, and 2.0 mg/L. Mean-measured concentrations were 0.116, 0.233, 0.486, 0.933, and 1.94 mg/L (89-97% of nomi nal). After hatching, >30 fry per treatment group were impartially selected to begin the 35-day exposure period. Fry wereFLUTOLANIL FINAL

Page 46 of 48 counted on Days 4, 5, 6, 7, 8, 11, 17, 21, and 35 pos t-hatch. At Day 35 post-hatch, percent survival, mean total length, and aver age wet weight were determined.

There were no effects on the pe rcentage hatch of eggs, but Day 35 survival was decreased (p<0.05) by 96-100% at the high con centration. Mean total length and wet weight of fry were decreased (significance not repor ted) at the medium and medium-high concentrations (not determined for the high-concentration group due to ≥ 96% mortality). The LOAEC is 0.486 mg/L due to decreased length and we t weight, and the NOAEC is 0.233 mg/L.FLUTOLANIL FINAL

Page 47 of 48 APPENDIX 3: References Not Utilized in the Flutolanil WoE Analysis

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

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

The following published and unpublished references were considered for use in the WoE analysis for Flutolanil but were not utilized due to one or more of the following reasons1) the article was not available in English; 2) the com pound of interest was not us ed in the study; 3) the test material was not adequately described; 4) a formulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only data on invertebrates.

Auletta, C.S. (1990) A 21-day dermal toxicity stu dy in rats with flutolanil. Bio/dynamics, Inc. Project#89-3497 (T -3070). (MRID 41841205)

Martin, M.T., Mendez, E., Corum, D.G., Judson, R. S., Kavlock, R. J., Rotroff, D.M., and Dix, D. J. (2009) Profiling the reproductive toxicity of chemicals from multigeneration studies in the toxicity reference database. Toxicological Sci. 110(1), 181–190.

Matsumoto, K., Hosokawa, M., Kuroda, K., et al . (2009) Toxicity of Agrichemicals in Daphnia

magna

. Osaka City Med. J. : 55 : 89-97.FLUTOLANIL FINAL

Page 48 of 48 Rhodes, J.E and Gormley, M. (1991) Early life-sta ge toxicity of flutol anil technical to the fathead minnow ( Pimephales promelas ) under flow-through conditions. Analytical BioChemistry Laboratories, Inc. Fi nal Report #38436. NOR-AM Study #E642.06.89 (W-3012). (MRID 41841221)

Tsujimura, S., Igarashi, N., Kawanishi, H ., and Takeshima, T. (1982) Three generation reproduction and teratogenicity study on flutolanil in the rat. Imamichi Institute for Animal Reproduction. Study #144 (T-3015). (MRID 40342923)