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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

## ~ S

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

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

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

.

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

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

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

CHEMICAL: TRIADIMEFON

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYTRIADIMEFON FINAL

ii TABLE OF CONTENTS

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

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

II. Sources of Scientific Data and Technical Information......................................4

A. EDSP Tier 1 Screening Assays............................................................4

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..................................................15

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

IV. Committee’s Assessment of Weight of Evidence..........................................24

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

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

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

B. Estrogen Pathway...........................................................................28

C. Androgen Pathway.........................................................................29

D. Thyroid Pathway...........................................................................29

E. Conclusions.................................................................................30

V. EDSP Tier 2 Testing Recommendations....................................................30

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

LIST OF TABLES: Table 1. Tier 1 A ssay for Triadimefon................................................................8
Table 2. Estrogenic/Anti-Estroge nic Pathway for Triadimefon.................................11
Table 3. Androgenic/Anti-Androge nic Pathway for Triadimefon................................17
Table 4. Thyroid Pathway for Triadimefon.........................................................23

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................35
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................41

APPENDIX 3. References Not Utilized in the Triadimefon WoE Analysis..........................61TRIADIMEFON 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 enerationTRIADIMEFON 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 ConcentrationTRIADIMEFON 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 Tie r 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)TRIADIMEFON 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-EvidenceTRIADIMEFON FINAL

Page 1 of 61 Executive Summary

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptor, 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 triadimefon interact s with E, A or T hormone pathways, the number and type of effects induced, the magnitude and pa ttern of responses observed across studies, taxa a nd sexes were considered. Additi onally, the condi tions under which effects occur were considered, in particular , whether or not endoc rine-related responses occurred at dose(s) that also resulted in ge neral systemic toxicity or overt toxicity.

On February 26, 2014, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-of-evidence (WoE) analysis of the potential interaction of triadimefon 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).

For the estrogen pathway, the in vitro assays for triadimefon were negative for ER binding, but was positive in the ERTA and were positive for alteration of steroidogenesis (inhibition of aromatase activity and ↑ estradiol production). In the OSRI studies, which satisfied the in vivo mammalian assays, no estrogen-mediate effects were s een in the absence of systemic toxicity. In the mammalian OSRI, when estrogen-related eff ects were seen they occurred only in the presence of systemic/overt toxicity (e.g., body wei ght loss, decreases in overall body weight gain and hepatotoxicity), which significantly confounds the interpretation of these estrogenic effects
within the context of endocrine disruption. For example, in the modified one-generation reproduction toxicity study in females, the dela y in VO and disruption in estrus cyclicity was accompanied by decreased terminal body weight and increased liver weight. In the twogeneration reproduction toxicity study, decreased fertility was noted along with decreased body weights in the P and F 1 parents, increased liver weights in the P generation and decreased birth and lactation body weights and viability in the F 1 and F 2 pups. In a bobwhite quail reproduction study, at the mid and high treatments (100 and 500 mg/kg-diet), there was an increase in number of cracked eggs (but no effects on eggshell thickn ess) and effects on egg viability and offspring survival; no effects were observed in the mallard duck study.TRIADIMEFON FINAL

Page 2 of 61 For the FSTRA, there was significant decreases in fecundity at the mid (39%; 0.24 mg a.i./L) and high (46%; 0.97 mg a.i./L) concentrations, and a significant increase in GSI (49%) at the high concentration. However, there were no effects on gonadal histology or plasma VTG in females. While there were no clinical signs of toxicity or significant e ffects on survival noted in the FSTRA study for females (male body weight was cons idered to be signifi cantly decreased at the mid and high concentration), test concentrations in this test were similar to concentrations in rainbow trout and fathead minnow early life cycl e tests where significant effects on survival were reported (fathead minnow: effects at 0.57 and 1.1 mg a.i./L ; rainbow trout: effects at 0.89 mg/L). Growth effects in these two early life cycle studies were reported at concentrations of

≥ 0.1 mg/L. Therefore, while distin ct overt toxicity was not observed in the FSTRA for females, given that overt toxicity has b een observed in other fish assays at similar concentrations and male body weight in the FSTRA was considered to be significantly decrease d, overall, the fish in the FSTRA may have been experiencing some stress which may have resulted in decreased resources (e.g., energy) for input into reproductive status. In summ ary, given that the effects in the mammalian studies coincided with systemic/ove rt toxicity and there is a general lack of complementarity for the FSTRA along with c oncerns about potential stress impacting reproduction, there is a lack of convincing evidence to suggest the triadimefon is potentially interacting with the estrogen pathway.

For the androgen pathway, th e results of the Tier 1 in vitro AR binding were negative, although alterations in expression of genes for steroid homeostasis were observed. In the in vivo mammalian assays, no androgenic or anti-androgenic effects were seen in the Hershberger assay. In the OSRI studies which satisfied the male pube rtal assay, androgen-rela ted effects were seen only in the presence of overt or systemic toxicity (e.g., body wei ght loss, decreases in overall body weight gain and hepatotoxicity). For exampl e, in the modified one-generation reproduction toxicity study in males, the observed anti-a ndrogenic effects occurred in conjunction with decreased terminal body weights a nd increased liver weights. In the FSTRA for males, there was a significant increase in GSI concentrations at the mid and high concentrations along with a reported decrease in fat pad development in th e high treatment. There were no effects on male gonadal histology. There was a decrease in male body weight at both of these test concentrations
that was considered to be biol ogically significant; along with the discussion above regarding test concentrations, this confounded the ability to di scern a potential androgen-related interaction from an overtly/systemically toxic dose. Theref ore, overall, there was a lack of convincing evidence for a potential interaction with the androgen pathway.

For the thyroid pathway, in the mammalian OSRI studies which satisfied the male and female pubertal and the AMA Tier 1 test ing requirements, no thyroid-rela ted effects were seen in the absence of systemic toxicity. No changes in th yroid weights were seen in any of the studies. Decrease in the circulating levels of thyroid hormones was seen onl y in the presence of systemic toxicity or overt toxicity. For example, in the one-generation m ammalian reproduction study inTRIADIMEFON FINAL

Page 3 of 61 males, there was a decrease in T 4 levels in the presence of ove rt toxicity. In a subchronic mammalian study, decreases in T 4 and TSH were seen in the presen ce of systemic toxicity (Wolf

et al . 2006). Treatment-related histop athological lesions of the t hyroid glands were seen in multiple species only at the high dose in the pres ence of systemic toxicity included follicular cell proliferations, follicular cell hyperplasia, colloid depletion and follicular cell adenomas. For the wildlife, effects on T 3 and T 4 levels (whole body) were reported along with alterations in gene expression for thyroid in newl y-hatched larval zebrafish, how ever, these effects were only observed in the presence of system ic toxicity (bent spin es and uninflated swim bladders). In an organ morphogenesis study (Lenkowski et al. , 2010) using Xenopus laevis tadpoles, mortality
was increased at ≥ 36.5 mg/L and intestinal and muscle ma lformations were observed. However, this study is limited in its ability to inform potential thyroid interaction based on endpoints examined and age of tadpoles (thyroid pathway is still relatively inactive at this stage). Therefore it is possible that th e interaction of triadi mefon with the thyroid hormone system may occur only at high doses as a sequel to either primary effects on the liver ( i.e. , increased hepatic clearance of thyroid hormones due to induction of hepatic enzy mes PROD and UDP-GT, Wolf et al. 2006) or in the presence of systemic/overt toxicity ( i.e. , body weight/body weight gain decreases, spine deformities).

In summary, the WoE indicates th at in mammals, tria dimefon may affect circulating levels of steroid and/or thyroid hormones only in the presence of systemic toxicity. The available data
shows that the observed effects are due to increased metabolic activity of th e liver, and not direct actions on the HPG or HPT axes. In wildlife, effects were also generally observed in the presence of systemic/overt toxi city (i.e., body weight decreases, skeletal malformations). Therefore, overall, there is a lack of convinci ng evidence to suggest a po tential interaction with the E, A or T pathways.

Based on weight of evidence considerations, ma mmalian and wildlife EDSP Tier 2 testing are
not recommended since there was a lack of convincing evidence of potential interaction with the estrogen, androgen or thyroid pathways.

.TRIADIMEFON FINAL

Page 4 of 61 I. Introduction

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptor, 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.

On February 26, 2014, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-of-evidence (WoE) analysis of the potential interaction of triadimefon 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).

Triadimefon has a solubility of 64 mg/L in wate r (20°C), and a vapor pressure of 0.2 – 0.6 mPa at 20°C and 25°C, respectively (7.6 × 10 -7 mm Hg). Triadimefon is stable to hydrolysis and appears to undergo slow degradation in aerobic so ils under field conditions. It is also moderately
soluble and was detected below the soil surface in soil column leaching studies.

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

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.TRIADIMEFON FINAL

Page 5 of 61 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 Test 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 guideli ne studies). The Agency’s revi ew of the initial OSRI is provided in the Report of the Endocrine Disr uptor Review Team on O-PP (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.

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of eleven assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A or T pathway. Additionally, the consistencyTRIADIMEFON FINAL

Page 6 of 61 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 expression of responses at higher levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biol ogical organization? Fi nally, an evaluation of conditions where the responses occur including consideration of whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.

This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA Scientific Advisory Pa nel (SAP) meeting in July 2013 th at evaluated scientific issues
associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that lit tle, if any, weight should be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues should be evaluated in terms of primar y 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, ove rt toxicity was generally defined inTRIADIMEFON FINAL

Page 7 of 61 accordance with EPA’s current approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological risk assessments. Sp ecifically, in these analyses, the effects that EPA considered to be potential evidence of ove rt toxicity included, but were not limited to: mortality; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight decreases of ≥ 10% in mammals. Addi tionally, other effects including morphological ( e.g ., organ weights/histopat hology), biochemical ( e.g ., blood chemistry), and other clinical signs ( e.g ., lethargy) were also consid ered when evaluating overt toxicity, especially if the effects were ex treme. In some instances, one parameter ( i.e., death or >10% decrease in mammalian body weight) was suffi cient to consider a do se/concentration to be overtly toxic. However, in other instances, mo re 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 potentia l overt toxicity. Additionally, effects which were considered to be signs of systemic toxic ity were also captured and these effects were generally considered as less severe forms of toxicity ( e.g ., changes in organ weights or blood chemistry). The circumstances for which a dose/c oncentration was considered overtly toxic for a particular study are desc ribed in Section IV.A.

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

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

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether or not a s ubstance has the potential to interact with the E, A or T pathway. Effects that occurred in the pr esence 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.TRIADIMEFON FINAL

Page 8 of 61 A. EDSP Tier 1 Screening Assays

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

Table 1. Tier 1 Assays for Triadimefon

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48619602)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48619605)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48619603)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied by OSRI (MRID No. 48264602)

Aromatase Assay (Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied by OSRI (MRID No. 48125203)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied by OSRI (MRID Nos. 48264605; 00032541; 00155075)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied by OSRI (MRID No. 48619604)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied by OSRI (MRID Nos. 48619605; 42153901; 00032541; 00155075)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied by OSRI (MRID Nos. 48619603; 48264602; 48264607; 00032541; 00155075)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 486673901)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied by OSRI (MRID Nos. 48264601; 48264602;

48264603; 48264604; 48264605; 48264607; 00155075; 00032541; 42153901)TRIADIMEFON FINAL

Page 9 of 61 B. Effects on Hypothalamic-Pit uitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for triadimefon to interact with the estrogen pathway is summarized in Table 2

. The various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic or HPG axis effects. This
table also includes HPG-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.

Triadimefon was negative (or not interactive) in the Tier 1 in vitro ER binding assay. In contrast, in the Tier 1 ERTA, the mean RPC max was ≥ PC 10 in both runs at approximately 10 -4 M, triadimefon was considered positive for es trogen receptor transcriptional activation.

The requirement for the steroidoge nesis assay was satisfied by an in vitro and in vivo rat steroidogenesis study submitted as OSRI (Goetz et al. , 2009). In the in vitro portion of this study, H295R cells exposed to triadimefon for 48 hours had increased (approximately 180% of control) estradiol levels at 1 and 3 μM, but decreased es tradiol levels at higher concentrations. In the in

vivo portion of this study, male Wistar Han rats fed triadimefon at 126 mg/kg/day had increased ( ↑ 20%) serum estradiol levels after 30 days of treatment but was not statistically significant. The Tier 1 aromatase assay was satis fied by OSRI (aromatase assay validation study, USEPA, 2007).
Results indicate triadimefon inhibite d aromatase activity with a log IC 50 of -5.767 M.

In another study submitted as OSRI (Kojima et al. , 2004), triadimefon was negative for estrogen receptor transcriptional activation in CHO cells . In a cell proliferation/ER binding assay (Okubo

et al. , 2004), triadimefon induced cell proliferation (maximum at 100 μM) in MCF-7 cells that could be blocked by the anti-estrogen ICI 182,780, but did not bind to the ER in a non-guideline binding assay. In an ER binding/ERTA/VTG induction study (Petit et al., 1997), triadimefon was negative in a trout ER binding assay and a yeast trout ER transcriptiona l activation assay, but positive in a trout hepatocyte VTG induction assay (178% induction at 10 -4 M). Finally, in a cell proliferation/tran scriptional activa tion study (Vinggaard et al ., 1999), triadimefon caused a 2.4- fold increase in cell proliferation in an MCF-7 cell assay measuring DNA content, but was negative in a yeast estrogen screening assay.

The requirement for the uterotrophic and female pubertal assays were satisfied collectively by four studies submitted as OSRI: 1) a modified one-generation rat reproduction toxicity study (Rockett et al ., 2006); 2) a three-generation reproduc tion toxicity study (MRID 00032541); 3) a two-generation reproduction toxicity study (MRID 00155075); and 4) a combined chronic toxicity/carcinogenicity study (MRID 42153901).TRIADIMEFON FINAL

Page 10 of 61 In the modified one-generation rat reproduction toxicity study (Rockett et al. , 2006), VO was delayed, estrus cyclicity was init ially disrupted and ovarian weight was increased at termination on PND 99 in the high-dose (90 mg/kg/day) tria dimefon group. However, numerical data were not reported in this literature study. No estroge n-specific effects were observed at the lower doses (5 and 25 mg/kg/day). In a three-ge neration reproduction toxicity study (MRID 00032541), fertility was affected at the high dose (90 mg/kg/day) in all generations. Correspondingly, in the two-generation reproduc tion toxicity study (M RID 00155075), fertility was decreased in the high-dose (90/mg/kg/day) F 1 parents. These decreases appear to be related to reduce mating interest and/or ability of male rats exposed to triadi mefon as described in Section 2 below. While no estrogen-specific effect s were observed in the three-generation study at lower doses (2.5 and 15 mg/kg/day), a signific ant decrease in ovarian weight was observed at the 2.5 mg/kg/day in the two-generation study. In th e combined chronic toxi city/carcinogenicity study (MRID 42153901), no effects were noted on ovary weight or histopathology of the ovary or uterus at any dose (2 .5, 15, and 90 mg/kg/day).

In the FSTRA, fecundity was reduced by 39% and 46% at the mid (0.24 mg/L) and high (0.97 mg/L) concentrations, respec tively, relative to the negati ve control. Female GSI was increased by 49% relative to the negative control at the high concentration. There were no effects on female VTG or gonadal histopathology. In a bobwhite quail reproduc tion study, at the mid and high treatments (100 and 500 mg/kg-diet), there was an increase in number of cracked eggs, decreases in the number of fertile eggs, viab le embryos, hatchlings and 14-day survival; no effects on eggshell thickness was observed. The reproduction study with the mallard duck did not report any significant effects. In three early life stage studies (one with fathead minnow, two with rainbow trout), there were si gnificant effects (p<0.05) on growth at concentrations as low as 0.116 mg a.i/L (rainbow trout study). There were no significant effects determined on percent
hatch for all three studies.TRIADIMEFONFINALPage 11 of 61Table 2: Estrogenic/Anti-Estrogenic Pathway for TriadimefonLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Triadimefon1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity 2 Age & Weight at VO 2° Sex Characteristics Fertility/Fecundity Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1ER Binding(MRID 48619602)NERTA(MRID 48619605)PAromataseRequirement satisfied by OSRI (MRID 48125203; see below)SteroidogenesisRequirement satisfied by OSRI (MRID 48264602; see below)UterotrophicRequirement satisfied by OSRI (multiple studies; see below)Female Pubertal RatRequirement satisfied by OSRI (multiple studies; see below)FSTRA(MRID 486673901)NEGSI↑49%(H)NNFcd:↓39,46%(M, H)NNTRIADIMEFONFINALPage 12 of 61Table 2: Estrogenic/Anti-Estrogenic Pathway for TriadimefonLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Triadimefon1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity 2 Age & Weight at VO 2° Sex Characteristics Fertility/Fecundity Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRIAromatase(USEPA, 2007;MRID 48125203)PSteroidogenesis(Goetzet al., 2007;(MRID 48264602)PPAR, ERα, and ERβtranscriptional activationassays(Kojimaet al., 2004;MRID 48033008)NCell proliferation, ER,and AR binding assays(Okoboet al., 2004;MRID 48074205)NPER binding, ERTA, andvitellogenin inductionassays(Petitet al., 1997)N NPCell proliferation andtranscriptional activationassays(Vinggaardet al., 1999;MRID 48041733)P PTRIADIMEFONFINALPage 13 of 61Table 2: Estrogenic/Anti-Estrogenic Pathway for TriadimefonLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Triadimefon1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity 2 Age & Weight at VO 2° Sex Characteristics Fertility/Fecundity Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Modified one-generationreproduction toxicity(Rockettet al., 2006;MRID 48264605)N↑P(H)NP(H)↑4d(H)NX(H)NThree-generationreproductive toxicity(Rat; MRID 00032541)NENENNN↓P(H)N NTwo-generationreproductive toxicity(Rat; MRID 00155075)NE↑12%(L)NENENE↓P(H)X(M.H)NCombined chronictoxicity/carcinogenicity(Rat; MRID 42153901)NENNNENEX(H)NDevelopmentalneurotoxicity(MRID 47377101)NNX(H)NDevelopmental toxicity(Rat: MRID 00089023)NNX(M,MH,H)NDevelopmental toxicity(Rat; MRID 00149336)NNN NDevelopmental toxicity(Rabbit;MRID 41446201)NNN NSubchronic toxicity(Rat; MRID 00048624)NE N N NN NTRIADIMEFONFINALPage 14 of 61Table 2: Estrogenic/Anti-Estrogenic Pathway for TriadimefonLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Triadimefon1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity 2 Age & Weight at VO 2° Sex Characteristics Fertility/Fecundity Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Subchronic toxicity(Dog; MRID 00048625)N N N N NN NCarcinogenicity(Mouse;MRID 40752101)NE NE N NEX(H)NChronic toxicity(Dog; MRID 00032539)NE N N NX(H)NAvian reproductivetoxicity(Quail; MRID 00110431)P(M, H)4N NAvian reproductivetoxicity(Duck; MRID 42342302)NN N1. Key to responses: L=Low treatment, M=Medium treatment, MH=Medium-High treatment, H=High treatment. Arrows (↓or↑) indicate thedirection of the response. A shaded cell indicates that 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 isindicated by an X in 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 thiscolumn. For details see Section IV. A4. At the mid and high concentrations (100 and 500 mg/kg-diet),there was an increase in number of cracked eggs, and decreasesin the number offertile eggs, viable embryos, hatchlings, and 14-day survival.P Positive findings (in vitro) / No effect (in vivo)N Negative findings (in vitro) / No effect (in vivo)NE Not examinedTRIADIMEFON FINAL

Page 15 of 61 2. Effects on Androgen Pathway

The potential for triadimefon to interact with the androgen path way is 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.

The Tier 1 in vitro AR binding assay was negative. Th e Tier 1 steroidoge nesis assay was satisfied by an in vitro and in vivo rat steroidogenesis study su bmitted as OSRI (Goetz et al.

, 2009). In vitro , triadimefon reduced media levels of testosterone by 40–68% in the adult and neonatal testis culture, and altered steroid production in a manner that indicated CYP17A1 inhibition at the highest concentration. Follo wing 48 hours of exposure, there was an overall

decrease in testoste rone at 10 and 30  M triadimefon in H295R cells. In vivo , triadimefon (126 mg/kg/d) increased ( ↑ 288%; p <0.01) serum testosterone at 30 days and intra-testicular testosterone ( ↑ 210%; p<0.01). Significant (p<0.0 5) decreases were seen in absolute epididymis ( ↓ 5%) and pituitary ( ↓ 10%) weights; however, no changes were seen in the relative weights of these organs. No treatment-related changes were s een in the absolute or relative weights of the testes, seminal vesicle or ventra l prostate. Liver weights, both absolute and relative, were increased (p<0.01) after 30 days of exposure ( ↑ 27% and ↑ 37%, respectively) and body weights

were decreased (8-10%, p<0.01).

In the Tier 1 Hershberger assay in the androge n agonist portion of the assay, ventral prostate weight was increased (29%, p< 0.05) at the high dose (100 mg/ kg/day) and in the androgen antagonism portion of the assay, LABC weight was decreased (12%) at the high dose. Since only one of the five androgen-sensitive tissue wei ghts were altered in the agonism and antagonism portion of the assay, triadimefon is consid ered negative in the Hershberger assay.

The requirement for the Tier 1 in vivo male pubertal assay was satisfied with five studies submitted as OSRI: 1) a modified one-gener ation rat reproduction toxicity study (Goetz et al.

, 2007); 2) the in vitro and in vivo rat steroidogenesi s study previously discussed (Goetz et al

., 2009); 3) a gene expression study (Tully et a l ., 2006); 4) a three-generati on reproduction toxicity study (MRID 00032541); and 5) a two-genera tion reproduction toxicity study (MRID 00155075). A modified one-generation male ra t reproduction toxicity study (Goetz et al ., 2007), triadimefon was administered at 0, 5, 25, and 90 mg/kg/day. No androgen -related effects were observed at 5 or 25 mg/kg/day triadimefon. Administration of a high dose of triadimefon (90 mg/kg/day) resulted in andr ogen-related effects in the F 1 males (increased AGD, delayed PPS, decreases in accessory organs weights, increased testis weights) which co-occurred with overt toxicity (body weight ↓ 17-29%).TRIADIMEFON FINAL

Page 16 of 61 In a study by Tully et al., (2006), adult rats were administered triadimefon at doses of 5, 50 or 115 mg/kg/day via gavage for 14 days. No adverse, treatment-related effects were seen on sperm morphology or motility, testis or epididymis wei ghts, or testis histopathology. Mean serum
testosterone levels were elev ated at the mid and high doses, but the increases did not reach statistical significance. Relativ e liver weights were increased by 27% at the high dose (115 mg/kg/day) with no effect on body weights. At the mid dose (50 mg /kg/day), 3/5 rats were found with mild centrilobular hepatocyte hypertrophy, while high dose animals displayed mild centrilobular to panlobular hepatocyte hypert rophy, with 1/5 having moderate mid-zonal hepatocyte vacuolation. Gene expr ession profiles of th e liver and testis were also evaluated. Triadimefon affected the expres sion of numerous genes includi ng CYP genes and genes involved
in steroid hormone metabolism.

In the three-generation reproduction toxicity study (MRID 00032541), fertility was affected at the high dose (90 mg/kg/day) in all generations . In the two-generation reproduction toxicity study, fertility was decreased in the high-dose F 1 parents. These decreases appear to be related to reduced mating interest or ability of male rats ex posed to high doses of triadimefon rather than an effect on fertility/reproduction. This was further examined in the two-generation reproduction toxicity study by cross-mating control F 1 parents with high-dose F 1 parents (MRID 00155075). Control females mated with high-dose males disp layed greatly decreased fertility compared to high-dose females mated with control males (47.4% vs. 80%). In the absence of sperm abnormalities, the decreased fertility was attributed to a decrease in interest or ability to mate in the high-dose males.

In the FSTRA, male gonado-somatic index (GSI) exhibited a significant increase of 33 and 75% relative to the negative cont rol at the mid (0.24 mg a.i. /L) and high (0.97 mg a.i./L) concentrations, respectively. At study termination, fat pads of males were reported as poorly developed within three of the four replicate chambers at the high concentration (not statistically analyzed). No effects on male gonadal hist ology were observed. Additionally, there was a
reduction of 21% and 19% in mean male body weight in the 0.27 and 0.97 mg a.i./L, respectively, relative to the ne gative control, which was not statistically significant but was considered to be biologically significant. However, effects on mortality or other clinical signs of toxicity were not observed.

In a bobwhite quail reproduction study, at the mi d and high treatments (100 and 500 mg/kg-diet), there was an increase in number of cracked eggs , decreases in the number of fertile eggs, viable embryos, hatchlings and 14-day survival. Ther e were no effects observe d in the reproduction study with the mallard duck.TRIADIMEFONFINALPage 17 of 61Table 3: Androgenic/Anti-Androgenic Pathway for TriadimefonLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Triadimefon1Study Type / LiteratureCitation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1AR Binding(MRID 48619603)NSteroidogenesisRequirement satisfied by OSRI (multiple studies; see below)Male Pubertal RatRequirement satisfied by OSRI (multiple studies; see below)Hershberger(MRID 48619604)N↑LW(H)NFSTRA(MRID 48673901)NEGSI↑33%(M),↑75%(H)NP(H)4NNN5(M, H)OSRISteroidogenesis(Goetzet al., 2007;MRID 48264602)PPN↓5%↓10%NXNTRIADIMEFONFINALPage 18 of 61Table 3: Androgenic/Anti-Androgenic Pathway for TriadimefonLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Triadimefon1Study Type / LiteratureCitation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3AR, ERα, and ERβtranscriptional activationassays(Kojimaet al., 2004;MRID 48033008)NCell proliferation, ER,and AR binding assays(Okoboet al., 2004;MRID 48074205)PGene expression study(Tullyet al., 2006;MRID 48264607)E6N N N NE NE NNX(M, H)NGene expression(Goetz and Dix, 2009aand 2009b)PModified one-generationreproductive toxicity(Goetzet al., 2007;MRID 48264603)NNNNNNNN NX(H)X(H)Three-generationreproduction toxicity(Rat; MRID 00032541)NE N N N N NE NEP(H)X(M, H)NTRIADIMEFONFINALPage 19 of 61Table 3: Androgenic/Anti-Androgenic Pathway for TriadimefonLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Triadimefon1Study Type / LiteratureCitation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Two-generationreproduction toxicity(Rat; MRID 00155075)NE N NE N NE NE NEP(H)X(H)NDevelopmentalneurotoxicity(MRID 47377101)NX(H)NSubchronic toxicity(Rat; MRID 48264601)NENENENENENEX(H)NSubchronic toxicity(Rat; MRID 00048624)NN N N NNN NSubchronic toxicity(Dog;MRID 00048625)NN N N NNN NCombined chronictoxicity / carcinogenicity(Rat; MRID 42153901)NEX(H)NCarcinogenicity (Mouse;MRID 40752101)NNNENNENEX(M, H)NChronic toxicity(Dog;MRID 00032539)NN NE N NNX(H)NAvian reproductivetoxicity(Quail; MRID 00110431)P(M, H)7N NTRIADIMEFONFINALPage 20 of 61Table 3: Androgenic/Anti-Androgenic Pathway for TriadimefonLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Triadimefon1Study Type / LiteratureCitation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Avian reproductivetoxicity(Duck; MRID 42342302)NN N1. Key to responses: L=Low treatment, M=Medium treatment, MH=Medium-High treatment, H=High treatment. Arrows (↓or↑) indicate the direction of theresponse. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unlessotherwise indicated. Abbreviations for androgen sensitive tissues:Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Levator anibulbocavernosus (LABC), Epididymides (EP).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. At study termination, fat pads of males were reported as poorly developed within three of thefour replicate chambers at thehigh concentration (notstatistically analyzed).5.There was a reduction of 21% and 19% in meanmale body weight in the 0.27 and 0.97 mg a.i./L, respectively, relative to the negative control, which wasnot statistically significant but was considered to be biologically significant. Effects on mortality or clinical signs of toxicity were not observed.6. Serum testosterone were elevated at the mid- (50 mg/kg/day) and high-dose (115 mg/kg/day), but not significantly. Data waspresented graphically.7. At the mid and high concentrations (100 and 500 mg/kg-diet), there was an increase in number of cracked eggs, and decreases in the number of fertile eggs,viable embryos, hatchlings, and 14-day survival.P Positive findings (in vitro) / No effect (in vivo)N Negative (in vitro) / No effect (in vivo)NE Not examinedE EquivocalTRIADIMEFON FINAL

Page 21 of 61 C. Effects on Hypothalamic-Pitui tary-Thyroidal (HPT) Axis

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

The potential for triadimefon to interact with thyroid regulation is 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 the potential for thyroi d 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.

The EDSP Tier 1 in vivo male and female pubertal assays and the AMA were not conducted but were satisfied with eight studies submitted as OSRI: 1) a modified one-generation rat reproduction toxicity study focusing on males (MRID 48264603); 2) a modified one-generation rat reproduction toxicity study focusi ng on females (MRID 48264605); 3) an in vitro and in vivo rat steroidogenesis study (MRID 48264602); 4) a gene expression study (MRID 48264607); 5) a three-generation rat repr oduction toxicity study (MRID 00032541); 6) a two-generation rat reproduction toxicity study (MRID 00155075); 7) a co mbined chronic toxici ty/carcinogenicity study in rats (MRID 42153901); and 8) a subchron ic toxicity study in male rats (Wolf et al

., 2006).

In the modified one-generat ion rat reproduction toxicity study with focus on males (MRID 48264603), total serum T 4 levels were decreased by PND 92 by 25% at 90 mg/kg/day; however, T 3 , TSH levels, thyroid and p ituitary weights were unaffected by treatment. In the modified one-generation rat reproduction to xicity study with focus on females (MRID 48264605), total serum T 4 and TSH levels were not measured, and thyroid pituitary weights were unaffected by treatment; however, 2/4 (50%) female s had mild diffuse thyroid follicular cell hypertrophy at 90 mg/kg/day. In the in vivo portion of the rat steroidogenesis study (MRID 48264602), pituitary weights were decreased af ter 30 days by 10% at 126 mg/kg/day of triadimefon in the diet, but thyroid weights, serum thyroi d hormone levels and thyroid histopathology were not examine d. In the three-generation rat reproduction toxicity study (MRID 00032541), the thyroids of the F 3b pups were examined microscopi cally and showed no effects of treatment at the low and mid doses (2.5 and 15 mg/kg/day); high-dose (90 mg/kg/day) pups were not examined due to reduced fertility of the previous generations. In the two-generation rat reproduction toxicity study (MRID 00155075), the thyroid and pituitary glands of the F 1 parents
and F 2 pups were unaffected by triadimefon tr eatment. In the rat combined chronic toxicity/carcinogenicity study (MRID 42153901) , treatment-related non-neoplastic and neoplastic lesions of the thyroid glands were seen at the high dos e (90 mg/kg/day). Follicular cellTRIADIMEFON FINAL

Page 22 of 61 cystic hyperplasia was noted in 4/50 (8%) high-dose females vs . 2/50 (2%) control females; however, the increase did not reach statistical sign ificance. Also at the high dose, follicular cell adenomas were observed in 3/49 (6%) treated males and 2/50 (4%) females compared to 0/50 (0%) control individuals of the re spective sexes. The incidences of adenomas were increased in males (4/49; 8%) which showed a significant tre nd and exceeded the historical control mean (5.2%). In the rat subchroni c toxicity study (Wolf et al, 2006; MRID 48264601), thyroid follicular cell hypertrophy, increase d follicular cell proliferation and colloid depletion was noted at 30 days in rats treated with a high dose (90 mg/kg/day) of tr iadimefon. There was also a dosedependent decrease in T 4 levels after 4 and 30 days for treatment, and T 3 levels were decreased at the high dose of triadimefon after 4 and 30 days. Notably, thyroid hormone levels did not differ from control values after 90 days of exposure a nd TSH levels were not in creased in any exposure

group.

In other OSRI evaluated to inform potential in teraction with the thyroid pathway, there were no treatment-related findings affecting the thyroid or pituitary.

In a study by Liu et al. (2011), zebrafish embryos were expo sed from 6 h post-fertilization to 120 hpf (hatched larvae). The zebrafish exposed to 2 and 4 μg/mL triadimefon exhibited bent spine and uninflated swim bladde r (results not presented). The T 3 content in the larvae (whole body) exposed to 2 and 4 μg/mL triadimefon was signi ficantly less than the controls. In contrast, the larvae exposed to 2 and 4 μg /mL had significantly higher T 4 content than in the controls. The beta-subunit of TSH (TSH- β ) gene transcription was si gnificantly upregulated ( ↑ 4.0- and ↑ 5.2-fold) upon exposure to 2 and 4 μ g/mL triadimefon, respectivel y; triadimefon exposure significantly downregulated dio 1 gene transcription (1.5-fold) in the 2 μg/mL exposure group.
The thrb gene transcription was si gnificantly down-regulated ( ↓ 2.6-fold) after exposure to 2 μg/mL triadimefon. Given the re ported malformations, the repor ted results appear to have coincided with systemic toxicity.TRIADIMEFON FINAL

Page 23 of 61 Table 4. Thyroid Pathway for Triadimefon.

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

Study Type / Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmentalstage orasynchronous &NHLLGrowth(BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assays

Male Pubertal Rat Requirement satisfied by OSRI (see below)

Female Pubertal Rat Requirement satisfied by OSRI (see below)
AMA (Frog) Requirement satisfied by OSRI (see below)

OSRI Steroidogenesis (Goetz et al. , 2007; MRID 48264602) NE NE ↓ 10% (H) X N

Modified one-generation reproduction toxicity focusing on males (Goetz et al. , 2007; MRID 48264603) N N N N N X (H) X (H)

Modified one-generation reproduction toxicity focusing on females (Rockett et al. , 2006; MRID 48264605) N P N X (H) N

Two-generation reproduction (Rat; MRID 00155075) NE N N X (H) N
Three-generation reproduction (Rat; MRID 00032541) NE N NE X (M, H) N
Developmental neurotoxicity (MRID 47377101) NE NE NE X (H) N

Developmental toxicity (Rat: MRID 00089023) NE NE NE X (M, MH, H) N

Developmental toxicity (Rat; MRID 00149336) NE NE NE N N
Developmental toxicity (Rabbit; MRID 41446201) NE NE NE N N
Subchronic toxicity (Rat; Wolf et al. , 2006; MRID 48264601) NE P P P NE X (H) N

Subchronic toxicity (Rat; MRID 00048624) N N N N N
Subchronic toxicity (Dog; MRID 00048625) N N N N N
Chronic toxicity (Dog; MRID 00032539) N N N X (H) NTRIADIMEFON FINAL

Page 24 of 61 Table 4. Thyroid Pathway for Triadimefon.

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

Study Type / Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmentalstage orasynchronous &NHLLGrowth(BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

Combined chronic toxicity/carcinogenicity (Rat; MRID 42153901) NE P NE X (M, H) N

Carcinogenicity (Mouse; MRID 40752101) NE N NE X (H) N
Development and gene expression (Zebrafish; Liu et al ., 2011) NE NE ↑ (MH, H) 4 NE NE NE P X (MH, H) N
1. Key to responses: L=Low treatment, M=Medium treatment, MH=Medium-High treatment, H=High treatment Arrows ( ↓ or ↑ ) indicate the direction of the response. A shad ed 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. Zebrafish exposed to triadimefon at 2 and 4 μ g/mL for as embryos through hatch exhibited elevated T 4 levels, decreased T 3 levels, and altered expression of genes involved in the thyroid pathway. Larvae at these levels also reported to exhibit bent spines and uninflated swim bladders. N Negative finding P Positive finding NE Not examined

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 triadimefon to interact with endocrine related processes ( i.e., E, A or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated analysis of those lines of evidence.TRIADIMEFON FINAL

Page 25 of 61 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 ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence eval uation of the results of the Tier 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues shou ld be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l disruptions in homeostasis” (USEPA, 2013).

A. Systemic/Overt toxicity in the Ti er 1 and Assays OSRI Studies

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 and OSRI
studies are described below. In addition to the endocrine-related effects described above for the

in vivo Tier 1 assays and OSRI, ot her effects observed which may be considered overt/systemic toxicity are also describe 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 ge nerally less severe forms of toxicity (e.g., changes in orga n weights or blood chemistry).

1. Tier 1 Assays

In the androgen agonist phase of the Hershbe rger assay, body weight gains were decreased (p<0.05) by 44% in the high-dose group (100 mg/kg/da y) compared to the vehicle control. Final body weights and body weight gains in the low- dose group were comparable to controls throughout the duration of the assay.

In the anti-androgen phase of the Hershberger assay, final body weight was decreased ( ↓ 9%; p<0.05) in the high dose group ( 100 mg/kg/day) and overall body we ight gains were decreased (p<0.05) in the 50 mg/kg/day ( ↓ 29%) and 100 mg/kg/day ( ↓ 44%) groups. Final body weights and body weight gains in the 25 mg/kg/da y group were comparable to controls.TRIADIMEFON FINAL

Page 26 of 61 In the FSTRA, there were no significant effects on mortality and no apparent effects on clinical signs ( i.e. , behavioral and other sublethal effects) except for one dark fish observed at 0.057 mg a.i./L. There was a redu ction of 21% and 19% in mean male body weight in the 0.27 and 0.97 mg a.i./L dose group, respectively, relative to the negative control, but the effect was not significant (Mann Whitney U; p>0.05); however , these decreases were considered to be biologically significant.

2. OSRI

In the modified one-generati on rat reproduction toxicity stud y with focus on males (Goetz et al., 2007), overt toxicity at the high dose (90 mg/kg/ day) manifested as decreases in the body weights of F1 male offspring during PND 1–22 ( ↓ 17–27%) and weeks 4–13 ( ↓ 22–29%). Daily food consumption (g/rat/day) was decreased by 9–26% during Weeks 4–12. Relative liver weights were increased at PND 1, 50 and 92, but th is was a result of the decreased body weights at these time points. Liver histology reveal ed strong panlobular hype rtrophy in high-dose
triadimefon males.

In the modified one-generation rat reproduction toxicity study with focus on females (Rockett

et al. , 2006), there was a decr ease in body weight and increase in liver weight at the high dose (90 mg/kg/day); however, the author s did not report numerical data.

In the in vivo portion of the rat steroi dogenesis study, liver weights were increased 27% after 30 days of exposure at 126 mg/kg/day.

In the rat gene expression study, relative liver weights were increased by 27% at 115 mg/kg/day. In the same study, 3/5 (60%) rats were found w ith mild centrilobular hepatocyte hypertrophy at 50 mg/kg/day, while animals dosed at 115 mg/kg/da y displayed mild centrilobular to panlobular hepatocyte hypertrophy, with 1/ 5 (20%) having moderate mid-z onal hepatocyte vacuolation.

In the two-generation rat reproduction toxic ity study, at the high dose (90 mg/kg/day), body weights were decreased (p<0.05) in the P males during weeks 4, 5 and 6; in the P females during weeks 1–16 and 21–26; and in the F 1 parental males and females during weeks 9–40. Terminal body weights were decreased (p<0.01) by 12% in the P females. Absolute and relative (to body weight) liver weights were incr eased (p<0.01) in the P males ( ↑ 23–29%) and females ( ↑ 23-39%).

In the three-generation rat reproduction study, at 90 mg/kg/d, body weights of the P generation parents were decreased ( ↓ 15%; p<0.01) during F 1b pre-mating, mating, gest atio, and lactation. Body weight gains of the F 1 generation parental males and females were decreased (p<0.01) throughout pre-mating, gestation an d lactation for both matings.

In the rat combined chronic toxicity/carcinogeni city study, at 90 mg/kg/day females, survival was slightly decreased (NS) at weeks 78, 90, and 105 ( ↓ 9–14%). Decreases (p<0.05) in bodyTRIADIMEFON FINAL

Page 27 of 61 weights were observed from week 1 until study termination ( ↓ 5–12%). Cumulative body weight gain was decreased at weeks 0–53 ( ↓ 15%) and 0–104 ( ↓ 23%). In the 90 mg/kg/day group, food consumption was increased (p<0.05) throughout the study ( ↑ 12–34% males; ↑ 34–67% females), resulting in increased over all mean food consumption ( ↑ 19% males; ↑ 53% females). The liver was a target organ. In the 90 mg/kg/day females, increased (p<0.05) serum cholesterol was observed at Weeks 26, 53 and 78 ( ↑ 24–32%), and absolute and re lative (to body) liver weights were increased (p<0.05; ↑ 15% and ↑ 27%, respectively) at Week 52. At week 104, absolute and relative liver weights were dose- dependently increased (p<0.05) in the females at 300 ppm and above ( ↑ 11–17% and ↑ 8–32%, respectively). A dose-dependent increase in fat in the hepatocytes was observed in the males and females at 90 mg/kg/d (p<0.05; 44–60% treated vs . 6–16% controls).

In the carcinogenicity study in mice, mean body weights and ove rall body weight gains were decreased in high-dose (765 mg /kg/day) males (body weight ↓ 6–15%; body weight gain ↓ 22%). There were non-neoplastic and pren eoplastic changes in the liver as well as increases in liver weights and correlating effects on serum enzy mes. Hepatocellular hypertrophy was increased (p<0.01) in males receiving 300 or 1800 ppm a nd in females receiving 50, 300 or 1800 ppm. Hyperplastic nodules, altered cell foci, and microve sicular fatty changes were increased in both sexes (p<0.01) at 1800 ppm. Kupffer cell prolif eration, lipofuscin pigment in liver macrophages and single cell necrosis were in creased significantly (p<0.01) in males receiving 1800 ppm and females receiving 300 and 1800 ppm. At study te rmination, liver weights were increased (p<0.01) in mid- and high-dose males ( ↑ 19–64%) and in high-dose females ( ↑ 69%) when compared to controls. Kidney weights were d ecreased (p<0.01) by 16% in high-dose males. Serum alkaline phosphatase, aspartate aminotrans ferase and alanine aminotransferase were increased in both sexes receiving 1800 ppm. Ther e was an increase (p<0.05) in hepatocellular adenomas in high-dose males and females; however , there was no increase in liver carcinomas.

In a rainbow trout fish ( Oncorhynchus mykiss ) early life stage toxi city study, the 17-day LC 50 was 1.270 mg/L (growth not evaluated). In another rainbow trout fish early life stage toxicity study, sixty-day cumulative mortality was signifi cantly greater than control mortality at 0.89 mg/L. Growth in terms of final length and/or weight was significantly reduced in at 0.116, 0.30 and 0.89 ppb. In the fathead minnow fish ( Pimephales promelas ) early life stage toxicity study, survival at the two highest test concentrations (0.57 and 1.1 mg a.i./L) was significantly reduced when compared to the pooled c ontrol. At test termination, mean standard length and mean wet weight were significantly reduced at the 0.27 mg a.i./L (top two treatments excluded from growth analyses). Abnormalities in the two highest test levels were noted primarily during the first week following the completion of hatch an d were considered dose related. There were no reported signs of systemic or overt toxicity reported in the avian reproduction studies.

Overall, the liver is a target orga n in mammals which is consistent with other triazo le fungicides. In the available data, effects on the liver ranged from increases in liver weight only, up toTRIADIMEFON FINAL

Page 28 of 61 evidence of hepatotoxicity (h istopathology, increased serum alanine aminotransferase). In addition, mechanistic studies evaluating the mode of action for triadimefon showed changes in CYP expression (gene expression study, Goetz a nd Dix 2009, Goetz et al. 2007) and induction of hepatic enzymes PROD and UDP-G T (subchronic rat study, Wolf et al . 2006). Therefore the systemic effects, particularly in the liver are likely influencing endocrine-related endpoints in mammals. In addition to effects on the liver, tria dimefon is known to cause neurotoxicity and its MoA involves blocking the re-uptake of dopamine which leads to increased motor activity and hyperactivity in rodents. Triadimefon acts as an indirect dopamine agonist by binding to the dopamine transporter and increasing levels of s ynaptic dopamine. Signs of neurotoxicity seen following triadimefon exposure include hyperact ivity, increased motor activity, rearing, body temperature, habituation and spatial distribution. Additionally, it is noted that in some cases,
endocrine-related effects coincide d with signs of overt toxicity (decreased body weight) in both
mammals and wildlife.

B. Estrogen Pathway

The in vitro assays for triadimefon were negative fo r ER binding, but was positive in the ERTA and were positive for alteration of steroidoge nesis (inhibition of aromatase activity and ↑ estradiol production). In the OSRI studies, which satisfied the in vivo mammalian assays, no estrogenmediate effects were seen in the absence of sy stemic toxicity. In the mammalian OSRI, when estrogen-related effects were seen they occurred only in the presence of systemic/overt toxicity (e.g., body weight loss, decreases in overall body weight gain and hepatotoxicity), which significantly confounds the interp retation of these estrogenic e ffects within th e context of endocrine disruption. For example, in the modifi ed one-generation repro duction toxicity study in females, the delay in VO and disruption in estrus cyclicity was accompanied by decreased terminal body weight and increased liver weig ht. In the two-generati on reproduction toxicity study, decreased fertility was noted along with decreased body weights in the P and F 1 parents, increased liver weights in the P generation a nd decreased birth and lactation body weights and viability in the F 1 and F 2 pups. In a bobwhite quail repr oduction study, at the mid and high treatments (100 and 500 mg/kg-diet), there was an increase in number of cracked eggs (but no effects on eggshell thickness) and effects on egg vi ability and offspring survival; no effects were observed in the mallard duck study.

For the FSTRA, there was significant decreases in fecundity at the mid (39%; 0.24 mg a.i./L) and high (46%; 0.97 mg a.i./L) concentrations, and a significant increase in GSI (49%) at the high concentration. However, there were no effects on gonadal histology or plasma VTG in females. While there were no clinical signs of toxicity or significant e ffects on survival noted in the FSTRA study for females (male body weight was cons idered to be signifi cantly decreased at the mid and high concentration), test concentrations in this test were similar to concentrations in rainbow trout and fathead minnow early life cycl e tests where significant effects on survival were reported (fathead minnow: effects at 0.57 and 1.1 mg a.i./L ; rainbow trout: effects at 0.89TRIADIMEFON FINAL

Page 29 of 61 mg/L). Growth effects in these two early life cycle studies were reported at concentrations of

≥ 0.1 mg/L. Therefore, while distin ct overt toxicity was not observed in the FSTRA for females, given that overt toxicity has b een observed in other fish assays at similar concentrations and male body weight in the FSTRA was considered to be significantly decrease d, overall, the fish in the FSTRA may have been experiencing some stress which may have resulted in decreased resources (e.g., energy) for input into reproductive status. In summ ary, given that the effects in the mammalian studies coincided with systemic/ove rt toxicity and there is a general lack of complementarity for the FSTRA along with c oncerns about potential stress impacting reproduction, there is a lack of convincing evidence to suggest the triadimefon is potentially interacting with the estrogen pathway.

C. Androgen Pathway

For the androgen pathway, th e results of the Tier 1 in vitro AR binding were negative, although alterations in expression of genes for steroid homeostasis were observed. In the in vivo mammalian assays, no androgenic or anti-androgenic effects were seen in the Hershberger assay. In the OSRI studies which satisfied the male pube rtal assay, androgen-rela ted effects were seen only in the presence of overt or systemic toxicity (e.g., body wei ght loss, decreases in overall body weight gain, and hepatotoxicity). For exampl e, in the modified one-generation reproduction toxicity study in males, the observed anti-a ndrogenic effects occurred in conjunction with decreased terminal body weights a nd increased liver weights. In the FSTRA for males, there was a significant increase in GSI concentrations at the mid and high concentrations along with a reported decrease in fat pad development in th e high treatment. There were no effects on male gonadal histology. There was a decrease in male body weight at both of these test concentrations
that was considered to be biol ogically significant; along with the discussion above regarding test concentrations, this confounded the ability to di scern a potential androgen-related interaction from an overtly/systemically toxic dose. Theref ore, overall, there was a lack of convincing evidence for a potential interaction with the androgen pathway.

D. Thyroid Pathway

For the thyroid pathway, in the mammalian OSRI studies which satisfied the male and female pubertal and the AMA Tier 1 test ing requirements, no thyroid-rela ted effects were seen in the absence of systemic toxicity. No changes in th yroid weights were seen in any of the studies. Decrease in the circulating levels of thyroid hormones was seen onl y in the presence of systemic toxicity or overt toxicity. For example, in the one-generation mammalian reproduction study in males, there was a decrease in T 4 levels in the presence of ove rt toxicity. In a subchronic mammalian study, decreases in T 4 and TSH were seen in the presen ce of systemic toxicity (Wolf

et al . 2006). Treatment-related histop athological lesions of the t hyroid glands were seen in multiple species only at the high dose in the pres ence of systemic toxicity included follicular cell proliferations, follicular cell hyperplasia, colloid depletion and follicular cell adenomas. For theTRIADIMEFON FINAL

Page 30 of 61 wildlife, effects on T 3 and T 4 levels (whole body) were reported along with alterations in gene expression for thyroid in newl y-hatched larval zebrafish, how ever, these effects were only observed in the presence of system ic toxicity (bent spin es and uninflated swim bladders). In an organ morphogenesis study (Lenkowski et al. , 2010) using Xenopus laevis tadpoles, mortality
was increased at ≥ 36.5 mg/L and intestinal and muscle ma lformations were observed. However, this study is limited in its ability to inform potential thyroid interaction based on endpoints examined and age of tadpoles (thyroid pathway is still relatively inactive at this stage).

Therefore it is possible that th e interaction of triadi mefon with the thyroid hormone system may occur only at high doses as a sequel to either primary effects on the liver ( i.e. , increased hepatic clearance of thyroid hormones due to induction of hepatic enzy mes PROD and UDP-GT, Wolf et al. 2006) or in the presence of systemic/overt toxicity ( i.e. , body weight/body weight gain decreases, spine deformities).

E. Conclusions

In summary, the WoE indicates th at in mammals, tria dimefon may affect circulating levels of steroid and/or thyroid hormones only in the presence of systemic toxicity. The available data
shows that the observed effects are due to increased metabolic activity of th e liver, and not direct actions on the HPG or HPT axes. In wildlife, effects were also generally observed in the presence of systemic/overt toxi city (i.e., body weight decreases, skeletal malformations). Therefore, overall, there is a lack of convinci ng evidence to suggest a po tential interaction with the E, A or T pathways.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations, ma mmalian and wildlife EDSP Tier 2 testing are
NOT recommended since there was a lack of co nvincing evidence of poten tial interaction with the estrogen, androgen or thyroid pathways.TRIADIMEFON FINAL

Page 31 of 61 VI. References

Bombard, E. (1986). MEB 6447: carcinog enicity study on NMRI mice (21-month administration in the feed). Report No. 87287. Unpublished study prepared by Bayer AG. (MRID 40752101).

Bombard, E., and Schilde, B. (1991). MEB 6447: Chronic toxicity and ca rcinogenicity studies on Wistar rats with administration in diet over a period of 105 weeks. Unpublished study prepared by Bayer AG, Department of Toxi cology, Germany. Laboratory Study/Report No.: T-3027626/101922, October 25, 1991. (MRID 42153901).

Carlisle, J. (1982). Rainbow trout embryo-larvae study w ith triadimefon (Bayleton ® ). Unpublished study prepared by Mobay Chemi cal Compaty Study N o. 81-666-01, Report #250. (MRID 00110433).

Carlisle, J. (1983). Toxicity of Triadimefon (Bayleton ® ) to rainbow trout early life stages. Unpublished study prepared by Mobay Chemi cal Corporation Study Number 83-666-02. (MRID 00131105).

Clemens, G., and Hartnagel, R. (1990). Teratology study in the rabbit with MEB 6447 (Triadimefon): Unpublished study by Mile s Inc. Report No.: MTD0149. (MRID 41446201).

Cohle, P., and Friesen, K. (1991). Ea rly life stage toxicity of Bayleton ® to fathead minnows ( Pimephales promelas ) in a flow-through system. Unpublished study prepared by Mobay Chemical Corporation Report No. 101301. (MRID 41922103).

Davis, J. (2011). The Hershberger Bi oassay (OPPTS 890.1400); Triadimefon. Unpublished study conducted by Integrated Laboratory System s, Inc., Durham, NC. Laboratory Project Study Number C197-200, December 14, 2011. (MRID 48619604).

Eiben, R. (1984). MEB 6447 (Triadimefon): two- generation study with rats. Unpublished study prepared by Bayer AG Institute of Toxicology, Supplementary Study Report No. 12712. (MRID 00155075).

Goetz, A., Ren, H., Schmid, J., et al . (2007). Disruption of testoste rone homeostasis as a mode of action for the reproductive toxi city of triazole fungicides in the male rat. Published: Toxicol.
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Goetz, A., Rockett, J., Ren, H., et al . (2009). Inhibition of rat and human steroidogenesis by triazole antifungals. Syst. Biol. Reprod. Med. 55 : 214–226. (MRID 48264602).

Goetz, A., and Dix, D. (2009a). Mode of acti on for reproductive and hepatic toxicity inferred from a genomic study of triazole antifungals. Toxicol. Sci. 110 (2) : 449–462.

Goetz, A., and Dix, D. (2009b). Toxicogenomic effects common to tri azole antifungals and conserved between rats and humans. Toxicol. Appl. Pharmacol. 238 : 80–89.TRIADIMEFON FINAL

Page 32 of 61 Hoffman, K., and Luckhaus, G. (1974). MEB 6447: subchronic toxicity study on dogs (thirteenweek feeding experiment). Unpublished study prepared by Bayer AG Report No. 5071; Report No. 44737. (MRID 00048625).

Hoffman, K., and Groning P. ( 1978). MEB 6447: long-term toxi city study on dogs (two-year feeding study). Unpublished study prepared by Bayer AG, Institute for Toxicology, WuppertalElberfeld, Germany. Laboratory Re port No.: 7882; 66656, October 24, 1978. (MRID 00032539).

Hoffman K., Groning, P., and Lorke, D. (1978). MEB 6447: Long-term toxicity study on dogs (two-year feeding study). Unpublished study prepared by Bayer AG, Institute for Toxicology, Wuppertal-Elberfeld, Germany. Laborat ory Report No.: 7882; 66656. (MRID 00126261).

Kojima, H., Katsura, E., Takeuchi, S., et al. (2004). Screening fo r estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese Hamster Ovary cells. Environ. Health Perspect

. 112 : 524–531. (MRID 48033008).

Lamb, D., and Carsel, M. (1982). Triadimefon (Bayleton ® ) reproduction study with bobwhite quail. Unpublished study prepared by Mobay Chemical Corporation, Study #81-675-03, Report #291. (MRID 00110431).

Lenkowski, J., Sanchez-Bravo, G., and McLaughlin, K. (2010). Low concentrations of atrazine, glyphosate, 2,4-dichlorophenoxyacetic acid, and tria dimefon exposures have diverse effects on Xenopus laevis organ morphogenesis. J. Environ. Sci. 22(9) : 1305–1308.

Liu, S., Chang, J., Zhao, Y., et al. (2011). Changes of thyroid hor mone levels and related gene expression in zebra fish on early lif e stage exposure to triadimefon. Environ. Toxicol. Pharmacol. 32 (3) : 472–477.

Loser, E., and Lorke, D. (1979). MEB 6447: multigeneration reproduction study on rats. Unpublished study by Bayer AG, Report No. 8297; Apr 28, 1980. (MRID 00032541).

Mohr, U. (1976). MEB 6447: subchronic toxi city study on rats (twelve week feeding experiment). Unpublished study by Bayer AG Report No. R 840a. (MRID 00048624).

Marble,B and Hall, T (2012) Weight of evidence to evaluate the potential for triadimefon to affect estrogen, androgen or t hyroid systems. Bayer CropScience LP ,Research Triangle Park, NC 27709. Report Number US0 274. MRID No. 48688501

Nagumo, K., Teraki, Y., and Chiba, I. (1981). Teratogenicity test of MEB 6447 in pregnant rats. Unpublished study by St. Marianna Univ., First Dept. of Anatomy, Laboratory of Embryology, Kawasaki. Kanagawa Prefecture. Japan. (MRID 00089023).

Okubo, T., Yokoyamo, Y., Kano, K., et al. (2004). Estimation of es trogenic and antiestrogenic activities of selected pesticides by MCF-7 cell proliferation assay. Arch. Environ. Contam. Toxicol. 46 : 445–453. (MRID 48074205).TRIADIMEFON FINAL

Page 33 of 61 Petit, E., Le Goff, P., Crevadi, J-P., et al. (1997). Two complementar y bioassays for screening the estrogenic potency of xenobiotics: recombinan t yeast for trout estrogen receptors and trout hepatocyte cultures. J. Mol. Endocrin. 19 : 321–335.

Rockett, J., Narotsky, M., Thompson, K., et al . (2006). Effect of conazole fungicides on reproductive development in the female rat. Reprod. Toxicol. 22 : 647–658. (MRID 48264605).

Sheets, L., Gilmore, R., and Hoss, H. (2008). A developmental neurotoxicity study with technical grade triadimefon in Wistar rats. Unpublished study by Bayer CropScience Project Number: 071D72/IL, 201766. (MRID 47377101).

Stafford, T. (1992). Technical Bayleton: a one generation reproduction study with Mallard ducks. Unpublished study by Miles, Inc. Study No. BL740801. (MRID 42342302).

Tully, B., Bao, W., Goetz, A., et al. (2006). Gene expression profili ng in liver and testis of rats to characterize the toxicity of triazole fungicides. MRID 48264607. Published: Toxicol. Appl. Pharmacol. 215 : 260–273.

Unger, T., Van Goethem, D. and Shellenberger , T. (1982). A Terato logical Evaluation of Bayleton in Mated Female Rats: MRI Proj ect No. 7272-B. Unpublished study prepared by Midwest Research Institute. 52 p. (MRID 00149336).

USEPA (2007). Integrated summary report on aromatase, December 11, 2007. http://www.epa.gov/endo/pubs/aromatase_isr.pdf . (MRID 48125203).

USEPA (2010). Triadimefon: report of the Endo crine Disruptor Review Team, Test Order #: EDSP 109901-15. December 20, 2010.

USEPA (2011). Endocrine Disruptor Screeni ng Program — Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.

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

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

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Willoughby, J.A. (2009). Triadimefon: estrogen r eceptor transcriptional activation (human cell line (HeLa-9903). Unpublished st udy conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9077V-100376ERTA, December 14, 2011. (MRID 48619605).TRIADIMEFON FINAL

Page 34 of 61 Willoughby, J.A. (2011a). Triadimefon: Androgen Receptor Binding (Rat Prostate Cytosol). Unpublished study conducted by CeeTox Inc., Kalamazoo, MI. Laboratory study number 90770- 100376ARB, December 14, 2011. (MRID 48619603).

Willoughby, J.A., Sr. (2011b). Triadimefon: Estr ogen receptor binding (rat uterine cytosol). Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9077V100376ERB, December 14, 2011. (MRID 48619602).

Wolf, D., Allen, J., George, M., et al . (2006). Toxicity profiles in rats treated with tumorigenic and nontumorigenic triazole conazole fungicides: propiconazole, triadimefon, and myclobutanil. Toxicol. Path. 34 : 895–902. (MRID 48264601).

York, D. (2012). Triadimefon — short-term reproduction assay with the fathead minnow ( Pimephales promelas ) following OPPTS 890.1350 and OECD 229 Guidelines. Laboratory Study No. 13798.6262. Unpublished study by Smithers Viscient, Wareham, MA. (MRID 48673901).TRIADIMEFON FINAL

Page 35 of 61 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100)

Requirement satisfied by OSRI (MRID Nos. 48264601; 48264602; 48264603; 48264604; 48264605; 48264607; 00155075; 00032541; 42153901); See Appendix 2.

Androgen Receptor (AR) Bindi ng Assay; (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48619603), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment,
which was conducted to measure the bi nding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations of triadimefon (logarithmic increase from 10

− 11 to 10

− 4 M). Dimethyl sulfoxide (DMSO) was used as the vehicle at a final assay concentration of approximately 3.2%. The assay included dexame thasone as a weak posit ive control, and R1881 as the ligand reference standard.

The saturation binding experiment was conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and wa s functioning with appropriate affinity for the radiolabeled reference ligand prior to routinel y conducting AR competitive binding experiments. Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. Separate cyto solic preparations were used for each of the three competitive binding runs , so data from three satura tion binding experiments were evaluated. For the cytosolic preparation conducted on March 30, 2011, the mean dissociation constant (K d ) for [ 3 H]-R1881 was 0.614±0.065 nM, and the estimated B max was 1.080±0.109 fmol/100 μg protein for the single batch of prostate cytosol that was prepared. For the cytosolic preparation conducted on July 23, 2011, the K d for [ 3 H]-R1881 was 1.080 ± 0.690 nM, and the
estimated B max was 1.071 fmol/100 μg protein (±0.167) for the single batch of prostate cytosol that was prepared. For the cytosolic pr eparation conducted on September 24, 2011, the dissociation constant (K d ) for [ 3 H]-R1881 was 0.613 ± 0.041 nM, and the estimated B max was 0.817 fmol/100 μg protein (±0.049) for the single batc h of prostate cytosol that was prepared.
The mean and individual K d values were generally below the range reported in the EPA validation program (0.685 to 1.57 nM), although two individual values were within the range and one value was above the range. Confidence in these numbers is generally high according to the goodness of fit (R 2 = 0.880-0.992) and the sm all variation in R 2 and K d estimates among runs (although greater variability was observed in the July 23, 2011 satu ration binding data).

In the competitive binding experiment, precipitati on of triadimefon was visually observed at the highest concentration (10

− 3 M) in the first run; therefore, the 10

− 3 M data were not included in the evaluation and the highest concentra tion in the second and third runs was 10

− 4 M triadimefon.TRIADIMEFON FINAL

Page 36 of 61 In all three runs, he specific [ 3 H]-R1881 AR binding was >75% at all soluble conc entrations of
triadimefon tested. An IC 50 and relative binding affinity (RBA) could not be calculated for
triadimefon.

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

− 9.3 and

− 4.4 M, respectively, and the mean RBA for the weak positive control was 0.0012%. Confidence in these numbers is high due to the small variati on. All performance crite ria were generally met; however, the top percent binding for triadimefon was low (77.7 – 82.9%) at 10

− 4 M in the first run, but specific binding in the presence of triadi mefon was consistently > 80% at concentrations

≤ 10

− 5 in the first run and at all concentrations in th e other two runs. As th is deviation was minor
and was not considered to have affected the resu lts of the study, all thr ee runs were considered
valid.

Based on the results from the three runs, triadi mefon is classified as a Non-Binder in the Androgen Receptor Binding Assay. Aromatase Assay; (OCSPP 890.1200)

Requirement satisfied by OSRI (USEPA 2007; MRID No. 48125203); See Appendix 2. Estrogen Receptor (ER) Bindi ng Assay; (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48619602), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiment s. A saturation binding experiment was performed to demonstrate that th e 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 conducting ER competitive binding experiments. The competitive binding experiment was conducted by measuring the bi nding of a single concentration of [ 3 H]-17 β

estradiol (1 nM) in the presence of increasing co ncentrations of triadimefon (logarithmic increase
from 10

− 10 to 10

− 3 M). DMSO 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 li gand reference material.

Raw saturation binding data were not provide d in the study report; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. Th e mean dissociation constant (K d ) for [ 3 H]-17 β -estradiol
was 0.303 ± 0.090 nM and the mean estimated B max was 0.143 ± 0.003 nM (33.59 ± 0.70 fmol/100 μg of cytosol protein) for the uterin e cytosol preparation used in this study. The K d and

B max were within the range recomm ended in the Test Guideline.

In the competitive binding experiment, triadimefon had no effect on [ 3 H]-17 β -estradiol binding
to the ER at concentrations of ≤ 10

− 4 M in all runs. Precipitation was observed at 10

− 3 M for all runs; therefore, the data from the 10

− 3 M triadimefon concentration we re not evaluated. A meanTRIADIMEFON FINAL

Page 37 of 61 log IC 50 was not calculated for triadimefon as comp etitive binding was not observed in any run.
The mean IC 50 was 1.2 × 10

− 9 M for the natural ligand, 17 β -estradiol, and 2.95 × 10 –6 M for the weak positive control, whic h also had a relative binding affinity (RBA) of 0.041%.

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

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

In an estrogen receptor transcript ional activation assay (MRID 48649605), hER α -HeLa-9903
cells cultured in vitro were exposed to triadime fon (99.20% a.i., Batch #2009020210/2010-
006597 at concentrations of 10

− 4 to 10

− 10.3 M in DMSO (0.1%). The experiments were performed using 96-well plates and each triadime fon concentration was tested in six wells per plate. Cells were exposed to the test agent for 24 ± 2 hours to induce a luci ferase reporter gene. Luciferase expression in response to activation of the estrogen receptor was measured using a proprietary method.

Performance criteria values for reference chemi cals were generally below the expected ranges, indicating increased sensitivity of the assay. Triadi mefon was tested up to the limit of solubility and cytotoxicity, 10

− 4 M. The RPC Max was 26.6% and 29.7% in runs 1, and 2, respectively; the
associated PC Max were 10 -4.3 and 10 -4 M, respectively. The res ponse was dose-dependent.
Because the RPC Max > PC 10 in two assay runs, triadimefon was considered positive for estrogen receptor transcriptional activa tion in this test system.

Fish Short-Term Reproduction Assay (FSTRA); (OCSPP 1350)

The 21-day short-term reproduction assay of triadimefon with fathead minnows ( Pimephales promelas) was conducted under flow-through conditions. Adult fish, 16 spawning groups (2 males and 4 females in each group; 5-6 months ol d) were exposed to triadimefon (99.2% purity) at nominal concentrations of 0 (control), 0.063, 0.25, and 1.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.0046), 0.057, 0.24, and 0.97 mg a.i./L. The test system was
maintained at 24 to 26 o C and a pH of 6.9 to 8.0.

There were no significant effects on mortality (p>0.05) and no appa rent effects on clinical signs ( i.e. , behavioral and other subletha l effects) except for one da rk fish observed in the low treatment group. There was a reduction of 21 and 19% in mean male body weight in the mid and high treatment groups, respectively, re lative to the negative contro l, which was not statistically significant (Mann Whitney U; p>0.05) but was consid ered to be biologica lly significant. There were no significant differences in body weight for female fish or in body length for male or
female fish.TRIADIMEFON FINAL

Page 38 of 61 Spawning frequency and fecundity in controls, re spectively, were at leas t every 4 days and 22 eggs/female/day; fertilization su ccess in controls was 97%. Th ere was a significant (JonckheereTerpstra; p<0.05) reduction in fecundity of 39% and 46% at the mid and high treatment levels, relative to the negative control. While male vitellogenin (VTG) exhibited a significant (Jonckheere-Terpstra; p<0.05) increase of 359% in the median value at the high concentration, relative to the negative control, it is noted that va riability in VTG levels appeared to be greater in the treatment groups, and the signif icant increase in the highest tr eatment appeared to be due to two male fish with higher VTG values and not an increased treatment-related response from the fish in that treatment. Male and female gona do-somatic index (GSI) exhibited a significant increase (Jonckheere-Terpstra; p< 0.05) relative to the controls with males exhibiting a 33% and 75% increase in GSI at the mid and high treatment levels and females exhibiting a 49% increase at the high treatment level. There were no significa nt effects on fertility, female VTG, or male tubercle scores (no tubercles de tected in females) when treated groups were compared to the negative control (p>0.05). Pl asma testosterone and 17 β -estradiol (sex steroids) were not measured in this study.

Gonadal histopathology was not st atistically analyzed, and ther e were no notable treatment related findings. In males, there were findings of minimal severity of increased proportion of spermatogonia, and interstitial cell hyperplasia/ hypertrophy; and findings of minimal to mild severity of asynchronous gonad development with no consistent pattern among concentrations. In females, there were findings of minimal to mo derate severity of increases oocyte atresia and egg debris in the oviduct, and findings of minima l severity of granulomatous inflammation with no indication of treatment-related effects. At study terminat ion, fat pads of males were poorly developed within three of the four replicate chambers at the high tr eatment level (not statistically analyzed). There were no other treatment-rela ted effects on secondary sex characteristics or gonadal staging.

Hershberger Assay; (OCSPP 890.1400) In a Hershberger assay (MRID 48619604) screeni ng for androgenic activity triadimefon (99.2% a.i., lot/batch# 20009020210/2010-006597) in polyethylene glycol (P EG; molecular weight 400) was administered daily via oral gavage to gr oups of eight 59- to 60-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehic le), 50, or 100 mg/kg/day. An androgenic positive control group consisting of eight castrated rats exposed to 0.4 mg/kg/day of testosterone propionate (TP) by subcutaneous (s.c .) injection was also included.

To screen for potential anti-androgenic ac tivity, triadimefon (99.2% a.i., lot/batch# 20009020210/2010-006597) in PEG was administered daily via oral gavage to groups of eight 59- to 60-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 25, 50, or 100 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 consisting of eight castrated ratsTRIADIMEFON FINAL

Page 39 of 61 exposed to 0.4 mg/kg/day TP and 3 mg/kg/day fl utamide (FT) was also included. TP alone was used as the anti-androge nic negative control.

For both components of the assay, body weights were determined da ily. The animals were dosed for 10 consecutive days and terminated a pproximately 24 hours after the final dose administration. At necropsy, the five androgen-de pendent tissues and liver were collected and weighed.

For the androgen agonist assay, one animal in the high-dose group was observed to have hunched posture beginning on Day 7 and was moribund on Day 9; this animal was euthanized on Day 9. For the anti-androgen assay, one animal in the low-dose group died on Day 7 as a result of a dosing error. All other rats survived until scheduled sacrifice. The death of these animals does not affect the validity of this screening assay because no other animals from any dosing group showed signs of moribundity. Other than th e euthanized rat in th e androgen agonist assay, no animals exhibited any clinical signs of toxicity. There we re no treatment-related gross pathological findings.

In the androgen agonist assa y, body weight gains were decrea sed (p<0.05) by 44% in the highdose group compared to the vehicle control. Final body weights and body weight gains in the low-dose group were comparable to controls throughout the duration of the assay. Final body weights were increased (p<0.05) by 9% in th e positive control group, resulting in increased (p<0.05) overall body weight gains of 66%. At 100 mg/kg/day, ventral prostate weight was significantly increased 29% (p<0.05). There we re no treatment-related effects in seminal vesicles, levator ani/bulbocavernosus (LABC) musc le complex, Cowper’s gland, or glans penis, and multivariate analysis of all five androgen-de pendent organ weights did not show an overall statistically significant differen ce with the administration of tr iadimefon. Liver weight was significantly increased (p<0.05) in the 100 mg/kg/day group compared to the control ( ↑ 25%), and relative liver weight was significantly increased (p<0.05) in the 50 and 100 mg/kg/day groups ( ↑ 15 and ↑ 30%). Animals in the TP group had increased (p ≤ 0.05) accessory sex organ weights as follows: 1079% in seminal vesicl es; 1360% in ventral pr ostate; 223% in LABC; 573% in Cowper’s gland; and 79% in glans penis. The performance criteri a indicated that this assay was performing as expected.

In the anti-androgen assay, final body weight was decreased (p<0.05) in the 100 mg/kg/day group ( ↓ 9%) and overall body weight gains were decreased (p<0.05) in the 50 and 100 mg/kg/day groups ( ↓ 29% and ↓ 44). Final body weights and body weight gains in the 25 mg/kg/day group were comparable to controls. Body weight gains were decreased (p<0.05) by 20% in the positive control group. At 100 mg/ kg/day, LABC weight was significantly decreased 12% (p<0.05). There were no treatment-related effects in seminal vesicles, ventral prostate, Cowper’s gland, or glans penis, and multivariate analysis of all five androgen-dependent organTRIADIMEFON FINAL

Page 40 of 61 weights did not show an overall statistically significant difference with the administration of
triadimefon. Absolute and relativ e liver weights were similar to the controls in the triadimefon dose groups and the positive control group. Animal s dosed with TP + FT (positive control) had decreased (p<0.05) accessory sex organ weights as follows: 78% in seminal vesicles; 77% in ventral prostate; 54% in LABC; 65% in Cowper ’s gland; and 28% in glans penis. The performance criteria indicated that th is assay was performing as expected.

Percent coefficient of variations (CVs) for the vehicle control and the 100 mg/kg/day group were compared to the performance criteria in the gu ideline. All %CV values were less than the
maximum recommended Guideline values.

The high-dose level selected for this study (100 mg/kg/da y) was considered to be adequate based on a dose range-finding study show ing reduced body weight and body weight gain and increased relative liver weights after 10 days of dosing at 120 mg/kg/day.

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

Female Pubertal Assay; (OCSPP 890.1450)

Requirement satisfied by OSRI (MRID Nos. 48619605; 42153901; 000325 41; 00155075); see Appendix

2.

Male Pubertal A ssay; (OCSPP 890.1500)

Requirement satisfied by OSRI (MRID Nos. 48619603; 48264602; 48264607; 00032541; 00155075); see Appendix 2.

Steroidogenesis Assay; (OCSPP 890.1550)

Requirement satisfied by OSRI ( Goetz et al. , 2007; MRID 48264602); see Appendix 2. Uterotrophic Assay; (OCSPP 890.1600)

Requirement satisfied by OSRI (MRID Nos. 48264605; 00032541; 00155075); see Appendix 2.TRIADIMEFON FINAL

Page 41 of 61 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 studies consid ered relevant for dete rmining the potential
interaction of triadimefon with the endocrine system are summar ized below. Given the breath of toxicity data available on most pesticides, the Part 158 studi es discussed in this appendix are those that fulfill current guideline requirements (whe re available) and/or are directly relevant to assessing potential endocrine effects (i.e. multig eneration rodent reproductive toxicity studies, developmental toxicity studies, chronic toxicity studies, etc.).

Aromatase Assay

The purpose of Integrated Summar y Report (ISR) for Aromatase ( MRID No. 48125203 ) was to summarize the development and validation of th e microsomal aromatase assay (OPPTS Test Guideline No. 890.1200) which had been proposed as an alternative in vitro assay for the Tier-1 screening battery in the Endocrine Disruptor Screening Program (EDSP). Triadimefon was included in supplementary blind testing of th e recombinant microsomal assay. The purpose of this study was to test the protocol using a se t of 16 diverse chemicals for which evidence of aromatase effects had been reporte d; triadimefon, an azole fungici de, had been reported to be a
weak to moderate aromatase inhibitor. The test ing was conducted according to the same protocol that was used in all method development and validation studies include d in the report. The substrate used for the assays was a mixture of non-radiolabeled ASDN (purity 100%) and
radiolabeled 3 H-ASDN (radiochemical purity >97%; specific activity 25.3 Ci/mmol). Aromatase activity was measured in an assay solution containing sodium phosphate buffer, propylene glycol, microsomal protein, 3 H-ASDN, and NADPH. The following controls were included: a full enzyme activity control (assay solution + solvent vehicl e), a background ac tivity control (full enzyme activity control except NADPH), and a positive c ontrol, 4-OH ASDN (purity 99%; in assay solution), which is a know n aromatase inhibitor that binds irreversibly to aromatase. The report indicated that the vehicl e control was either ethanol or DMSO, depending on the reference chemical used; no specific information was provided by chemical.

The full aromatase control activity for the triadimefon runs was 0.393–0.431 nmol/mg protein/min; uncorrected background activity was 0.001–0.002 nmol/mg protein/min, corresponding to ~0.3–0.5% of the fu ll activity control. These result s were consistent with the respective performance criteria of ≥ 0.100 nmol/mg protein/min for th e full activity control and

≤ 1% of full activity control for the background c ontrol. The positive control results for the study
indicated that there was not si gnificant variation between tec hnicians conducting the assays; based on the concentration response curve for tr iadimefon, there was minimal variation across
the three runs for this reference chemical. Fo r the three triadimefon runs, the four model parameters for positive controls were: Bo ttom = 0.6% to 0.9%; Top = 97.7% to 103%; LogIC 50 = -7.22 to -7.24; and slope = -1.0 to -1. 2. These were within the ranges of the performance criteria for the positive contro l of -5% to 6% for Bottom, 100 ± 10% forTRIADIMEFON FINAL

Page 42 of 61 Top, -7.3 to -7.0 for LogIC 50 , and -1 ± 0.2 for slope. The authors also provided data indicating
that even the bounds of the 95% c onfidence intervals were within the performance criteria ranges
for triadimefon.

Aromatase activity results for the reference chemicals were normalized to percent of control by dividing by the full enzyme activity for the run; re sults were not reported in terms of aromatase activity per se (nmol/mg protein/min). Mean percent of control values increas ed with decreasing
concentration of the test substance from 2.02% at 10 -4 M to 100.06% at 10 -8 M (CVs 8.22 decreasing to 3.11). The percent of control data we re fit to the four- and two-parameter models using nonlinear regression methods to calculate Bottom, Top, LogIC 50 , and slope values. Results for triadimefon using the 4-parameter mode l were Bottom = -0.002% to -0.292%; Top = 97.55% to 101.8%; LogIC 50 = -5.74 to -5.79; and slope = -0.9238 to -0.9571. For the 2-parameter model (Bottom = 0; Top = 100), results were: LogIC 50 = -5.728 to -5.829 and slope = -0.8861 to -0.9801. The results for all runs using the four -parameter model were within the performance criterion of Top = 100 ± 10%.

Under the chemical classification criteria for ar omatase inhibition used in the assay, triadimefon was unequivocally classified as an aromatase inhi bitor at the highest te sted concentration of

10 -4 M (i.e., the most inhibited level lies below 50% of the full activity control). Comparison of best fit estimates for the four- and two-parameter models yielded LogIC 50 values of -5.77 for both models. Percent control values spanned the en tire range of 0–100% for all concentrations of triadimefon, confirming th e validity of the LogIC 50 calculated from the data.

AR and ER Transcriptional Activation Assays

Kojima et al . ( MRID 48033008 ) screened 200 chemicals (incl uding triadimefon) for estrogen and androgen activity by conducting transc riptional activation assays for hER α , hER β and hAR using Chinese hamster ovary (CHO) cells with a luciferase reporter. Th e authors stated that cytotoxicity was not observed in any of the test ed compounds at the selected concentration range (data not shown). It was reported that triadime fon was negative for bot h estrogen and androgen receptor transcriptional activation from 10 -8 to 10 -5 M in this test system (data not shown).

Cell Proliferation, ER, and AR Binding Assays

Okubo et al. ( MRID 48074205 ) tested twenty pesticidal chem icals (including triadimefon) for estrogenic and anti-estrogenic ac tivity using a MCF-7 cell prolif eration assay. Chemicals were also tested for AR and ER binding using the Ligand Binding Screening System, a proprietary screening system by Toyobo. Anti- estrogenic activity was assesse d by measuring the suppression of cell proliferation induced by 30 pM of 17 β -estradiol. Triadimefon was reported to not be cytotoxic in the range tested.TRIADIMEFON FINAL

Page 43 of 61 Triadimefon was tested from 1 μM to 1 mM in an MCF-7 cell proliferation assay and a proprietary ER α and AR binding assay. Triadimefon result ed in maximal cell proliferation at 100 μM. The anti-estrogen ICI 182,780 blocked cell proliferation. In addition, triadimefon was shown to be an anti-estrogen at 100 to 1000 μM when tested with 30 pM of E2. In contrast, Okubo reported that triadimefon did not bind in th e ER binding assay (data not shown). In the AR binding assay, the authors reported an IC 50 of 902 ± 143 μM for triadimefon and a relative binding affinity (RBA) of 0.005.

ER Binding, ER Transcriptional Activat ion and Vitellogenin Induction Assays

In this study, Petit et al . (1997) used a trout ER binding assay, trout hepatocyte cultures, and
yeast p- galactosidase reporter gene assays transfect ed with trout ER to study the estrogenic effects of 49 chemical compounds (including tria dimefon). The yeast cel l walls were removed, thus removing the cell wall barrier. Triadimefon wa s tested at concentra tions arranging from 10 -8

to 10 -4 M in the transcriptional activation and bi nding assays. The mean induction values at maximum response for the transcriptional activat ion and vitellogenin i nduction assays were reported with E 2 being 100%. Triadimefon was negative in the yeast transcriptional activation assay and did not promote cell prolifer ation. However, it was tested at 10

− 4 M in the vitellogenin assay and found to be positive (178% induction). Data were displayed graphically for the ER binding assay and vitellogenin assays. The results of the experiments were explained as follows: the negative results in binding and transcriptiona l activation assays, but positive in the trout hepatocyte assay indicated that either a metabolite of triadime fon is an ER binder or that vitellogenin induction was occurring through an indirect gene activation pathway.

Cell Proliferation and Transcriptional Activation Assays

In a study by Vinggaard, Breinholt, and Larsen ( MRID 48041733 ), at total of 20 pesticides, including triadimefon, were tested in an MCF-7 cell proliferati on assay and in a yeast estrogen screening (YES) assay. MCF-7 cells were incubated at 0.001, 0.1, 1.0 and 10 μM with triadimefon, and proliferation was determined by measuring DNA content. Triadimefon was toxic to yeast above 30 μM. Triadimefon caused a 2.4-fold increase in cell proliferation with a relative proliferation efficiency of 53%, indicating it was a partial estrogen agonist. Triadimefon was also run in the MCF-7 assay at 10 μM and measured as positive by the resazurin metabolic
reduction method. Triadimefon was negative in the YES screen, but no data were provided.

Steroidogenesis Study ( in vitro and in vivo) in Rats

In an in vivo and in vitro steroidogenesis study (Goetz et al. 2009; MRID 48264602),
triadimefon was tested for effects on steroid production. Hormone production in vitro was measured in testis organ cultures from unt reated adult (PND 90–100) and neonatal (PND 1) Sprague-Dawley rats following exposures to 1, 10 or 100 μM triadimefon. The media was collected at 0.5, 1.5, and 2 hours following exposur e. A rat to human comparison was examinedTRIADIMEFON FINAL

Page 44 of 61 using the H295R human adrenocortical cell line exposed to 1, 3, 10, 30, or 100 μM triadimefon. Cells were examined microscopically to evalua te cell viability and number. Cell viability was calculated using a commercially available viability kit. For the in vivo portion of the study, male Wistar Han rats (PND 60) receiv ed triadimefon at 126 mg/kg/day in the diet for 30 days. On PND 74 (14 days after initiati on of dosing), tail blood was collected for testosterone measurements. On PND 90 (30 days after initiation of dosing), rats were sacrificed and trunk
blood was collected for evaluation of serum testos terone, estradiol, lute inizing hormone (LH), and prolactin. The weights of the testes, epididym is, ventral prostate, semi nal vesicles, pituitary gland and the liver were determined.

In vitro , triadimefon reduced media levels of test osterone levels by 40–68 % in the adult and neonatal testis culture, and altered steroid production in a manner that indicated CYP17Al inhibition at the highest concentration. In the H295R cells following 48 hours of exposure, there
was an overall decrease in estrad iol, progesterone, and testoste rone. No effects on cell growth were observed cells following treatment with tr iadimefon. A decrease was seen in testosterone

concentrations at 10 and 30  M of triadimefon. An increase in estradiol production was observed at 1 and 3 μM; however, a decrease in production was seen at the highe r test concentration.

In vivo , serum testosterone levels were not increased after 14 days but were increased ( ↑ 290%; p<0.01) at 30 days as was intr a-testicular testosterone ( ↑ 210%; p<0.01). Serum levels of estradiol, LH and prolactin were somewhat elevated at 30 days, but increases were not statistically significant. Liver we ights, both absolute and relativ e, were significantly increased after 30 days of exposure ( ↑ 27% and ↑ 37%, respectively). Significan t (p<0.05) decreases were seen in the absolute epididymis ( ↓ 5%) and pituitary ( ↓ 10%) weights; however, no changes were seen in the relative weights of these organs. No treatment-related changes were seen in the absolute or relative weights of the test es, seminal vesicle or ventral prostate.

Modified One-Generation Reproduc tion Toxicity Study in Rats

In a study by Goetz et al., 2007 (MRID 48264603), triazole fungicide s associated with a range of reported male reproductive effects in experimental animals were selected to assess potential toxic modes of action. In a modified one-generation reproduction toxicity study , groups of timed pregnant female Wistar Han rats were fe d diets containing either 100, 500, or 2000 ppm myclobutanil, 100, 500, or 2500 ppm propiconazo le, or 100, 500, or 1800 ppm (5, 25 or 90 mg/kg/d) triadimefon from GD 6 through lactation. Dams were allowed to litter normally, and litters were culled on PND 8 to eight pups/dam (mal es retained preferentially ). Pups were fed the same diet as their dam beginning at weaning (PND 23) until termination. Pups were maintained on their appropriate diet up to PND 120. One ma le/dose group/litter was necropsied on PND 1, 22, 50, or 92. Measurements included anogenital distance (AGD) at PND 0, weekly body weight and food consumption, age and weight at PPS, organ weights (brain, hypothalam us, hippocampus, pituitary, thyroid, liver, testes, epididymides, ventral prostate, and seminalTRIADIMEFON FINAL

Page 45 of 61 vesicle), organ histopathology (bra in, pituitary, thyroid, liver, te stes, epididymides, and ventral prostate), serum estradiol, testosterone, T 3 , and T 4 levels, sperm morphology and motility, and fertility and fecundity via mating assays usi ng PND 78 or older treated males paired with
untreated females.

High-dose triadimefon (1800 ppm; 90 mg/kg/ day) caused overt toxicity in the F 1 males. Weekly body weights were decreased during PND 1–22 ( ↓ 17–27%) and Weeks 4–13 ( ↓ 22–29%), and mean daily food consumption (g/rat/day) was decreased by 9–26% during Weeks 4–12. AGD was increased by high-dose triadimefon, suggestiv e of hyper-virilization. Th is dose level delayed PPS (PND 47.4 treated vs . PND 42.5 controls) with a concomitant decrease in body weight at PPS ( ↓ 13%), consistent with delayed puberty. Relati ve liver weights were increased at PND 1, 50, and 92, but this was a result of the decr eased body weights at these time points. Liver histology revealed strong panl obular hypertrophy in high-dose triadimefon males. Pituitary weights were unaffected by treatment. Absolute testis weights were increased by 21% at PND 22 and by 14% at PND 50 by high-dose triadimefon treatment. At P ND 92, absolute epididymides weights were decreased by 8% and absolute se minal vesicle weights were decreased by 19%.
Serum testosterone was increa sed at PND 50 and PND 92/99 by triadimefon. At 90 mg/kg/day, triadimefon affected several reproductive parame ters including pup death in 18 of 21 litters, with nine total litter losses, resulting in a 47% survival rate, compared to 96% survival in the control litters. Untreated females mated with high-dose tr iadimefon males had an insemination index of 8% with no females becoming pregnant (fertility index of 0%). Additionally, no copulatory plugs were found, and there was a complete lack of sperm in the uterine horns after the mating period. However, there were no observed differences be tween treated males and controls in sperm morphology or motility. In addition to the repr oductive system effects, total serum T 4 levels were decreased at PND 92 by high-dose triadimefon ( ↓ 25%); T 3 and TSH levels were unaffected by treatment. These reproductive effects are cons istent with the disrupt ion of testosterone homeostasis as a key event in the mode of action for triazole-induced reproductive toxicity.

Modified One-Generation Reprod uction Toxicity Study in Rats

Three triazole fungicides were evaluated for effects on female rat reproductive development (Rockett et al., 2006; MRID 48264605). In a modified one -generation reproduction toxicity study rats were exposed via feed to 100, 500, or 2500 ppm pr opiconazole, 100, 500, or 2000 ppm myclobutanil, or 100, 500, or 1800 ppm (5, 25 or 90 mg/kg/d) triadimefon from gestation day 6 to postnatal day (PND) 98. Body weight and AGD at PND 0, ag e and BW at vaginal opening (VO), estrous cyclicity, and body and orga n weight at necropsy were measured.

BW at PND 0 was unaffected by treatment. AGD was increased by 2000 ppm myclobutanil. VO was delayed by high-dose myclobutanil and tr iadimefon (PND 36.75 treated vs. PND 32.8 controls). Estrous cyclicity was initially disrupted by 500 and 2500 pp m propiconazole and 1800 ppm triadimefon, but later normalized. At P ND 99, there was a decrease in body weight atTRIADIMEFON FINAL

Page 46 of 61 90 mg/kg/day triadimefon (numerical data not reported), an increas e in liver weight by high-dose propiconazole and triadimefon (num erical data not reported), and an increase in ovarian weight by high-dose myclobutanil and triadime fon (numerical data not repor ted). Thyroid glands of 2/4 rats exposed to 1800 ppm triadimefon showed mild diffuse follicular cell hypertrophy; no thyroid lesions were seen in the other animals. It is concluded that exposure to propiconazole, myclobutanil, and triadimefon adversely impact ed female rodent reproductive development.

Three-Generation Reproductive Toxicity Study in Rats

In a multi-generation reproduction toxicity study ( MRID 00032541 ) triadimefon, (purity not reported) was administered continuously in the diet to SPF Wistar W. 74 rats (10 males: 20 females/dose) at nominal dose levels of 0, 50, 300, or 1800 ppm (0, 2.5 15 or 90 mg/kg/day). The P animals were given test article diet formulati ons for approximately 100 da ys prior to mating to produce the F 1a litters. After weaning, F 1a animals were sacrificed, and a second mating of the P animals was conducted to produce the F 1b litters. After weaning, 10 males:20 females/dose from
the F 1b litters were selected to become the parents of the F 2 generation and were given the same
concentration test formulation as their dam fo r approximately 4 weeks prior to mating. After weaning, F 2a animals were sacrificed, and a second mating of the F 1b animals was conducted to produce the F 2b litters. This process was repeated for a total of three generations. The final generation (F 3b ) was sacrificed at the end of lactation.

At 90 mg/kg/day, fertility was affected in all generations. In the P ge neration, no differences occurred in the fertility index during the fi rst mating; however, during the second mating the fertility index was slightly decreased compared to controls (17/20 treated vs. 19/20 controls, not statistically significant). Fertilit y values were significantly (p<0.05) lower in both matings of the

F 1 generation. Only one female became pregnant in the mating to produce the F 2a litters; no females became pregnant in the mating to produce the F 2b litters. Therefore, mating of the F 1 generation resulted in no F 2 pups. The number of offspring per dam at birth was decreased (p<0.05) in the first litters of both the P generation and the F 1 generation ( ↓ 16% each). As a result of low fertility, there were no animals in the 1800 ppm group of the F 2 generation to
evaluate.

Additionally at 90 mg/kg/d, body weights of th e P generation parent s were decreased ( ↓ 15%; p<0.01) during F 1b pre-mating, mating, gestation, and lact ation. Body weight gains of the F 1 generation parental males and females were decreased (p<0.01) throughout pre-mating, gestation, and lactation for both matings.

Due to low fertility in the 1800 ppm parental generations, only one F 2 litter and no F 3 litters were produced at this dose.

In the F 1 pups, at 90 mg/kg/d, the number of offspring per dam was decreased (p<0.05) in the F 1a and F 1b litters ( ↓ 20-31%) at PND 5. At PND 28, viability was decreased (p<0.01) in the F 1aTRIADIMEFON FINAL

Page 47 of 61 (90.7% treated vs. 97.8% controls) and F 1b (56.4% treated vs. 84.2% controls) pups. Additionally, body weight gains of the F 1a pups were decreased ( ↓ 10%; p<0.05) during lactation.

At15 mg/kg/d, body weight gains of the F 2b pups were decreased ( ↓ 15%; p<0.05) during PNDs 5, 7, 14, and 21. Body weight gains were also decreased ( ↓ 10%; p<0.05) in the F 3b pups at PND
28.

Two-Generation Reproductive Toxicity Study in Rats

In a two-generation reproductive toxicity study ( MRID 00155075 ) triadimefon (Bayleton ®

, 92.6% a.i.) was administered continuously in the diet to bor:w isw (SPF/Cpb) rats (10 males : 20 females/dose) at nominal dose levels of 0, 50, or 1800 ppm (0, 2.5, or 90 mg/kg/day). The P animals were given test arti cle diet formulations from 100 days prior to mating to produce the F 1 generation. After weaning, F 1 animals were selected (10 males: 20 females/dose) to become the parents of the F 2 generation and were fed the same concentration test formulation as their dam; the P animals were sacrificed. Treatment of the F1 animals followed the same pattern as the P animals; however, only the 50 ppm F 1 parents were
sacrificed when the F 2 litters were weaned. The treatment of the 1800 ppm F 1 groups was stopped; and the 1800 ppm F 1 males were mated with the control F 1 females, while the 1800 ppm

F 1 females were mated with control F 1 males. This cross-mating procedure produced a second F 2 litter and was intended to pr ovide further information rega rding the fertility of the F 1 parents. For all matings, the day that sperm was detected in a vaginal smear was designated as gestation day (GD) 0.

No parental mortalities occurred. Sperm mo tility, sperm morphology, gest ation length, and sex ratios were unaffected by treatment.

At 1800 ppm, fertility was decreased (35% tr eated vs. 85% controls; p<0.01) in the F 1 parents. This resulted in decreases in the total number of litters (7 treated vs . 17 controls) and the total number of pups (37 treated vs . 196 controls). In the F 1 cross-mating, fertility was 47.4% from the mating of 1800 ppm males with the control females and 80% fr om the mating of the 1800 ppm
females with the control males. In the absence of sperm abnormalities, it was concluded that the observed decrease in fertility wa s due to a decreased interest of the males in copulation. Copulation was not observed in 6/10 of the 1800 ppm males during the 3-week cross-mating
with control females.

Additionally at 1800 ppm, body weights were decr eased (p<0.05) in the P males during Weeks 4, 5, and 6; in the P females during Weeks 1–16 and 21–26; and in the F 1 parental males and females during Weeks 9–40. Terminal body weight s were decreased (p<0.01) by 12% in the P females. Absolute and relative (to body weight) liver weights were incr eased (p<0.01) in the P males ( ↑ 23–29%) and females ( ↑ 23–39%).TRIADIMEFON FINAL

Page 48 of 61 At 50 ppm, body weights were d ecreased (p<0.01) in the F 1 dams on GD 1, 6, and 15 by 6–7%. Absolute and relative (to body weight) ovary we ights were increased (p<0.05) by 12% in the P dams (organ weights were not determined for the F 1 adults). The biological significance of these findings is consid ered to be equivocal.

At 1800 ppm, body weights were decreased (p<0.05) at birth and lactation by 12–13% in the F 1 and F 2 pups. Viability was decreased (p<0.05) in the F 1 pups during PND 0–5 (51.4% treated vs

. 87.1% controls) and PND 5–28 (79.2% treated vs . 93.1% controls), and in the F 2 pups during PND 0–5 (37.8% treated vs . 92.9% controls) and PND 5–28 (71.4% treated vs . 89.7% controls). Additionally, litter size was decreased (p<0.01) in the F 2 pups (5.3 treated vs . 11.5 controls).

Gene Expression Study

In a study by Tully et al. ( MRID 48264607 ) to evaluate gene expression profiles in liver and testis, groups of male Sprague-Dawley rats (15/ dose group) received tria dimefon by oral gavage at dose levels of 0, 5, 50, or 115 mg/kg/day fr om PND 60 to 74. Following exposure, trunk blood
was collected for measurements of serum testoste rone, LH, FSH, and estradiol. Livers, left and right testes, thyroids, adrenals, br ains, left and right epididymides, seminal vesicles, spleens, and ventral prostates were removed; liver, adrenals, brains, left and right epididymis, seminal vesicles, spleens, and ventral pros tates were weighed; and liver a nd testes samples were collected for histology, enzyme biochemistry, or gene expression profiling. T hyroid histopathology was also examined. Sperm morphology and motility were also examined. DNA microarrays and quantitative RT-PCR were used to measure gene expression.

No adverse, treatment-related effects were seen on sperm morphology or motility, testis or epididymis weights, or testis or thyroid histopathology. Relative liver weights were increased by 27% at 115 mg/kg/day with no e ffect on body weights. At 50 mg /kg/day, 3/5 rats were found with mild centrilobular hepatocyte hypertrophy, wh ile animals dosed at 115 mg/kg/day displayed mild centrilobular to panlobular hepatocyte hypertrophy, with 1/5 having moderate midzonal hepatocyte vacuolation. Mean seru m testosterone levels were el evated at 50 and 115 mg/kg, but did not reach statistical significance. No cha nges in LH, FSH or estradiol were observed. Triadimefon affected the expres sion of numerous genes includi ng CYP genes and genes involved
in steroid hormone metabolism.

Developmental Neurotoxicity Study (Rat)

In a developmental neurotoxicity study ( MRID 47377101 ), triadimefon (95.7% a.i.) was administered in the diet to groups of 30 mated fe male Wistar rats/dose at nominal dose levels of 0, 100, 300, or 800 ppm (equivalent to 0, 8.0, 23.9, and 71.3 mg/kg/day) from GD 6 through LD 21. Doses were adjusted dur ing lactation to achieve a mo re consistent dosage throughout exposure. Dams were allowed to deliver natu rally and were killed on lactation day (LD) 21, following weaning of their respective litters. Any females that were found to be sperm positiveTRIADIMEFON FINAL

Page 49 of 61 and/or with a vaginal plug, but did not deliver, were sacrificed on GD 24. On PND 4, litters were standardized to 8 pups/litter (4 /sex); the remaining offspring and dams were sacrificed and
discarded without further examinations. Subsequently, 1 pup/ litter/group (at least 10 pups/sex/dose when available) was allocated to subsets for FOB, motor activity, acoustic startle response, learning and memory eval uation, and neuropathological examination.

No treatment-related effects were observed on mortality, clinical si gns of toxicity, food consumption, FOB parameters, or reproductive pa rameters in the dams. At 800 ppm, treatmentrelated effects were limited to decreases in body weights on GD 13 and LD 0 (5% each, p<0.05), and body weight gain during GD 0–20 (6%, not si gnificant). No treatment-related effects on viability/survival, litter paramete rs, pre-weaning clinical signs, sexual maturation, age of onset of surface righting or pupil constriction, FOB parameters, motor activity, water maze, ophthalmoscopic lesions, gross or microscopic le sions, or brain morphometric measurements
were observed in the F 1 pups.

At 800 ppm, deviated snout was noted in 3 females and 1 male during post-weaning. Preweaning body weights (males and females combin ed) were decreased (not significant, NS) by 4-5% on PND 11–21; combined body weight ga ins were decreased (p<0.05) by 6–7% on PND 4–11 and 4–17; and overall (PND 0–21) combin ed body weight gains were decreased by 5%. Post-weaning body weights were decreased (p<0.05) by 5–7% in the males during the last 2 weeks of the study, and by 8% (NS) at termin ation. During the auditory startle reflex habituation test, a treatment- related increase (p<0.05) in total session (blocks 1–5) peak amplitude was observed in the females (54%) on PND 60. Increased (47–70%) peak amplitude was noted during all test blocks in this gr oup compared to controls; however, statistical significance (p<0.05) was only achieved during Block 5 ( ↑ 70%). During the passive avoidance test, an increase (p<0.05) in the mean number of trials to criterion (2.8 treated vs. 2.1 controls) was noted in the females during the retention phase on PND 30.

Developmental Toxicity Study (Rat)

In a developmental toxicity study ( MRID 00089023 ), groups of 19–21 presumed pregnant female Sprague-Dawley rats were administer ed triadimefon in 0.5% Cremophor EL emulsion daily by oral gavage (10 mL/ kg) from GD 6–15 at 0, 10, 25, 50, or 100 mg/kg/day. All rats were observed daily for clinical si gns of toxicity, and body wei ght and food consumption were
measured. The rats were sacrificed on GD 20 an d the uterus was removed along with the ovaries. The numbers of corpora lutea, live fetuses, resorp tion sites, dead fetuses, sex ratio of fetuses, weights of live fetuses and placentae, deformed fetuses, and the number types of external morphological abnormalities were recorded. One ha lf of the live fetuses from each litter were fixed and prepared for skeletal examination. Th e remaining half were fixed and examined for
visceral abnormalities.TRIADIMEFON FINAL

Page 50 of 61 Maternal toxicity was evident at dosage levels of ≥ 25 mg/kg/day; toxicity was manifested as increased motor activity and/or depression of maternal weight gain. The degree and duration of these increased activity episodes were dose-related. The decreases (p<0.05) in body weight gain in the 50 and 100 mg/kg/day groups during the en tire course of pregnancy did not differ significantly between any treated group and the control group . Food consumption was unaffected by treatment. No treatment-related effects were observed on the numbers of corpora lutea, implants and live fetuses, sex ratio, placental weights and fetal mortality. No external
abnormalities were observed. Dilatation of the rena l pelvis was observed in all groups at low frequencies, and did not appear to be treatment-related as it also occurred in the control group.
Two fetuses from two different l itters in the 100 mg/kg/day group we re found to have cleft palate (a total of 139 fetuses were examined). The 50 and 100 mg/kg/day groups showed significantly increased incidence of the presence of four teenth (lumbar) ribs (49% and 92% treated vs . 11% controls). The authors also stated that there was a treatment-related signif icantly higher degree of skeletal ossification (coccygeal ve rtebrae) in the 100 mg/kg/day group.

In another developmental toxicity stu dy (1982, MRID 00149336), groups of 26 presumed pregnant female Sprague-Dawley rats were ad ministered triadimefon in 0.5% Cremophor EL emulsion daily by oral gavage (10 mL/kg) from GD 6–15 at 0, 10, 30, or 90 mg/kg/day. Each dam was observed twice daily for adverse reactio ns or toxicological responses to the test substance. Dams were weighed on GD 6, 13, 15, a nd at termination. On GD 20, the dams were killed and their viscera examined grossly. The uterine contents and ovaries were examined and the number of corpora lutea were counted. A ll fetuses were counte d, numbered, weighed, and checked for viability. Live fetuses were examined for external abnormalities. One half of the
viable fetuses from each litter we re dissected and examined for visceral anomalies. The other
half of the fetuses from each litter wa re processed for skeletal examination.

All dams survived to termination, and there were no treatment-relate d clinical signs of toxicity. At necropsy, hydronephrosis involving one or both kidneys was the most commonly observed finding in all groups. Pregnancy rates were unaff ected by treatment. No differences in body weight gain were noted in a ny group prior to dosing. The 90 mg/ kg/day group showed a decrease (p<0.05) in body weight gain during the dosing period, but rebounded to approximately the same level as the controls post-dosi ng. The mean number of corpora lutea, uterine implantations, nonviable implants, late resorptions, viable fetuses, litter sizes, a nd sex ratio were comparable for all groups examined. No dead fetuses were re corded. Mean fetus weights were comparable across all groups. Fetuses treate d with 90 mg/kg/day were found to have increased (p<0.05)
incidences of rib anomalies a nd distended urinary bladder.

Developmental Toxicity Study (Rabbit)

In a developmental toxicity study ( MRID 41446201 ), groups of 20 artificially inseminated female American Dutch rabbits, were administ ered triadimefon in 0.5% carboxymethylcelluloseTRIADIMEFON FINAL

Page 51 of 61 + 0.4% Tween-80 by daily oral gavage (5 mL/k g) during GD 6–18 at dose levels of 0, 20, 50, or 120 mg/kg/day. All rabbits were ch ecked daily for mortality and clinical signs of toxicity. Individual body weights were recorded on GD 6, 10, 14, 18, 21, and 28. Food consumption was recorded on GD 1, 6, 7, 12, 15, 19, 23, and 28. All does were sacrificed on GD 28, and subjected to Caesarean section and subsequent gr oss necropsy. The following maternal examinations/observations were made: inspectio n of ovaries, recording of individual intact gravid uterine weights, gross examination of th oracic and abdominal cav ities, recording of the number of corpora lutea, the number/placement of implantations, early/late resorptions, and the number of 1ive/dead fetuses. The placentas were blotted dry and weighed. Tissues/organs with gross lesions were preserved in 10% modified Millonig’s buffered formalin for possible future histologic evaluation. All fetuses were removed from gravid uteri and were subjected to the following external observations: recording of individual weight, sex, and gross abnormalities (frontal, dorsal, and lateral view s of head; size/position of pinnae; bulging of eyes; irregular contour and visceral herniations on torso; limbs position; number of digits on fore/hind paws). This was followed by internal observations of the abdominal and thoracic viscera and of the sex. All fetuses were finally processed for skeletal examination, and the skull, vertebrae, ribs, pelvis, appendages, scapulae, clavicles, and sternum were examined.

No treatment-related mortalities or clinical signs of toxicity were observed. A marginal, transient (GD 6–10), but significant loss of body weight a nd a very transient (GD 7) but significant decrease in food consumption were observed in the 120 mg/kg/day group. The biological significance of the former effect was considered equivocal since it only oc curred during the first four days of dosing and was unaccompanied by any concomitant decrease in body weight. The
latter effect was too short las ting to have any real biological significance. No treatment-related gross pathological alterations we re observed. The pregnancy rate s, gestation indexes, average numbers of corpora lutea, implantations, per centages of pre- and pos t-implantation losses, resorptions, fetuses/litter, viable number of fe tuses/doe, male and combined fetal body weight, and percentage of male fetuses/doe were comparable between groups.

Developmental toxicity was obs erved at 50 and 120 mg/kg/day. Dose-related increases in both
the fetal and litter incidences of several skel etal alterations were observed at 50 mg/kg/day. These included incomplete ossification of the pelvic pubes and the anterior and posterior phalanges, and irregular spinous process formation. The increases in incidence of each fetal alteration was statistically significant (litter incide nce increases were not statistically analyzed). Significant decreases in placental weight and feta l female body weight, and significant increases in the incidences of many exte rnal and skeletal alterations were observed at 120 mg/kg/day. External alterations consisted of rudimentary or missing tails (2 litters; 10 fetuses). Skeletal alterations with only a fetal incidence increase included incomplete ossi fication of the skull bones and enlargement of the fontanelles. Those w ith increases in both feta l and litter incidences included extra ribs, malformations of the ar ches and centra of the caudal vertebrae, incomplete/non ossification of the 1st, 2nd, 5th sternebrae, posterior talus, and posteriorTRIADIMEFON FINAL

Page 52 of 61 phalanges. Those with dose-related increases in both fetal and litter incidences were mentioned
earlier. The increases in the incidence of each fetal alteration was significant. Except for extra ribs, the litter incidences increase s were not statistically analyzed.

Subchronic Toxicity Study (Rat)

In a subchronic toxicity study ( MRID 00048624 ), groups of 15 rats/sex were administered triadimefon in the diet at 0, 50, 200, 800, or 2000 ppm (equivalent to 0, 2.5, 10, 40 and 100 mg/kg/day) for 12 consecutive weeks. Daily physical observations were performed, and body weights and food consumption were measured weekly. Hematology, clinical chemistry and urinalysis were performed prior to dosing, at W eek 6, and at termination. At the end of the study, autopsies were performed, and tissues were saved for histopathological examinations.

No animals died due to the administration of the te st material. No clinical signs of toxicity were observed. Body weights and food consumption were decreased in the high dose group; this was considered to have resulted from palatability i ssues with the high dose dietary formulation. There
were no treatment-related effect s observed on hematology, clinical chemistry, or urinalysis. No treatment-related changes were seen in absolute or relative weights of th e testes, ovaries, thyroid, adrenal or pituitary glands. No treatment-related hi stopathological lesions we re seen in the testes, thyroid, adrenal, or pituitary glands.

In another subchronic rat toxicity study ( Wolf et al., 2006; MRID 48264601 ), male Wistar/Han rats were treated with 100, 500, 1800 ppm tr iadimefon, 100, 500, 2500 ppm propiconazole, or 100, 500, 2000 ppm myclobutanil in the feed for four, 30, or 90 days. The rats were evaluated for clinical signs, body and liver we ight, histopathology of thyroid and liver, hepatic metabolizing enzyme activity, and serum T 3 , T 4 , TSH, and cholesterol levels.

There was a dose-dependent increase in liver weig ht but not body weight for all treatments. The indication of cytochrome induction, pentoxyresorufin O -dealkylation (PROD) activity, had doserelated increases at all time points for each conazole. Uridine diphopho-glucuronosyl transferase (UDPGT), the T 4 metabolizing enzyme measured as gluc uronidation of 1-naphthol, was induced to the same extent after 30 and 90 days for a ll three conazoles. Live rs from all high dose (90 mg/kg/day) treated rats had centrilobular hepatocyte hypertrophy after four days, while only triadimefon and propiconazole tr eated rats had hepatocyte hy pertrophy after 30 days, and only triadimefon treated rats had hepatocyte hype rtrophy after 90 days. Thyroid follicular cell hypertrophy, increased follicular ce ll proliferation, and colloid de pletion were present only after 30 days in rats treated with the high dose of triadimefon. A dose-dependent decrease in T 4 was present after 4 days with a ll three compounds but only the high doses of propiconazole and triadimefon produced decreased T 4 after 30 days. T 3 was decreased after high-dose triadimefon after four days and in a dose-dependent ma nner for all compounds after 30 days. Thyroid
hormone levels did not differ from control valu es after 90 days and TSH was not increased in any exposure group. A unique pattern of toxic res ponses was not identified for each conazole andTRIADIMEFON FINAL

Page 53 of 61 the hypothesized mode of action for triadimefon- induced thyroid gland tumors was not supported by the data.

Subchronic Toxicity Study (Dog)

In a subchronic toxicity study ( MRID 00048625 ), groups of four beagle dogs/sex were
administered triadimefon in th e diet at 0, 150, 600, or 2400 ppm (equivalent to 0, 3.75, 15 and 60 mg/kg/day) for 13 weeks. Daily physical obser vations were performe d, and body weights were measured weekly. Ophthalmoscopic examinations, hematology, clinical chemistry and urinalysis were performed prior to dosing, at Week 6, and at termination. At the end of the study, autopsies were performed, and tissues were save d for histopathological examinations.

All dogs survived to scheduled termination. No c linical signs of toxicity were observed. Body weights and food consumption were decreased in the high dose group; this was considered to have resulted from palatability issues with the high dose dietary formulation. At 60 mg/kg/day, hematocrit, erythrocyte count a nd hemoglobin volume were decrea sed, and alkaline phosphatase was increased. It was stated that the necropsies were unremarkable, and that there were no histopathological findings indicative of liver damage. No trea tment-related changes were seen in absolute or relative weights of the testes, pr ostate, ovaries, thyroid, or adrenal glands. No treatment-related histopathological lesions were seen in the testes, epididymides, prostate, ovaries, uterus, thyroid, adre nal, or pituitary glands.

Combined Chronic Toxicity /Carcinogenicity in Rats

In a combined chronic/carcinogenicity study ( MRID 42153901 ) triadimefon (94.4% a.i.) was
administered in the diet to 50 Wistar Bor:WI SW rats/sex/dos e at dose levels of 0, 50, 300, or 1800 ppm (equivalent to 2.5, 15, and 90 mg/kg/day in males/females) for up to 104 weeks. In addition, an interim group of 10 rats/sex/ dose was similarly treated for 52 weeks.

In the 90 mg/kg/day females, survival was s lightly decreased (NS) at Weeks 78, 90, and 105 ( ↓ 9–14%). Decreases (p<0.05) in body weights were observed from Week 1 until study termination ( ↓ 5–12%). Cumulative body weight gain was decreased at Weeks 0–53 ( ↓ 15%) and 0–104 ( ↓ 23%). In the 90 mg/kg/day group, food cons umption was increased (p<0.05) throughout the study ( ↑ 12–34% males; ↑ 34–67% females), resulting in increased overall mean food consumption ( ↑ 19% males; ↑ 53% females).

The liver was a target organ. In the 90 mg/kg/da y females, increased (p<0.05) serum cholesterol was observed at Weeks 26, 53, and 78 ( ↑ 24–32%), and absolute and relative (to body) liver weights were increased (p<0.05; ↑ 15% and ↑ 27%, respectively) at Week 52. At Week 104, absolute and relative liver weights were dose-depe ndently increased (p<0.0 5) in the females at 300 ppm and above ( ↑ 11–17% and ↑ 8–32%, respectively). A dose-depe ndent increase in fat in the hepatocytes was observed in the m ales and females at 90 mg/kg/day (p<0.05; 44–60% treatedTRIADIMEFON FINAL

Page 54 of 61 vs . 6–16% controls). Additionally, a positive trend was found in the incidence of fat in the hepatocytes of females. In the 90 mg/kg/day ma les, alanine aminotransferase was increased (p<0.05) by 22% at Week 26 and by 37% at Week 104.

Treatment-related non-neoplastic an d neoplastic lesions of the thyr oid glands seen at the high dose were: follicular cell cystic hyperplasi a in 4/50 (8%) at the high dose females vs . 2/50 (2%) in the control females; however, the increase did not reach statistical significance. Follicular cell adenomas were observed in treated males 3/49 (6 %) compared to 0/50 (0%). The incidences in males showed a significant trend and exceed ed the historical control mean (5.2%).

Carcinogenicity Study (Mouse)

In a carcinogenicity study ( MRID 40752101 ), groups of 60 NMRI mice/sex were administered
triadimefon in the diet at dose levels of 0, 50, 300, or 1800 ppm (equivalent to 0/0, 13.5/19.6, 76.0/119.4, and 550.1/765.0 mg/kg/day in males/fema les) for up to 21 months. Mice were observed for mortality and morbid ity twice daily; detailed physical examinations were performed weekly. Body weights were recorded at study initiation, weekly during Weeks 1–12, and bimonthly from Week 15 to study termination. F ood and water consumption were recorded at the same time at the body weight measurements. He matology and clinical chemistry parameters
were measured at Weeks 53 and 90/91. All animals that died or were sacrificed on schedule were necropsied. A standard suite of organs/tissu es were collected, weighed, and examined microscopically.

There was an increase (p<0.05) in hepatocellu lar adenomas in high-dose males and females; however, there was no increase in liver carcinom as. There was no effect of dosing on survival, clinical signs, food or wate r consumption, or hematology parameters. Mean body weights and overall body weight gains were decreased in high-dose (765 mg /kg/day) males (BW ↓ 6–15%;
BWG ↓ 22%). There were nonneoplastic and preneopl astic changes in the liver as well as increases in liver weights and correlating effects on serum enzy mes. Hepatocellular hypertrophy was increased (p<0.01) in males receiving 300 or 1800 ppm and in females receiving 50, 300, or 1800 ppm. Hyperplastic nodules, altered cell fo ci, and microvesicular fatty changes were increased in both sexes (p<0.01) at 1800 ppm. Kupffer cell prolif eration, lipofuscin pigment in liver macrophages, and single cell necrosis were increased significantly (p<0.01) in males receiving 1800 ppm and females receiving 300 and 1800 ppm. At study termination, liver weights were increased (p<0.01) in mid- and high-dose males ( ↑ 19–64%) and in high-dose females ( ↑ 69%) when compared to controls. Kidney we ights were decreased (p<0.01) by 16% in high-dose males. Serum alkaline phosphatase , aspartate aminotrans ferase, and alanine
aminotransferase were increased in both se xes receiving 1800 ppm. The increases were significant compared to controls for all enzyme s at 91 weeks and for some at 53 weeks. Notreatment-related changes were seen in absolute or relative testicular weights. No treatmentTRIADIMEFON FINAL

Page 55 of 61 related histopathological lesions we re seen in the testes, epididym ides, seminal vesicle, prostate, ovaries, uterus, mammary glands, thyroid, adrenal or pituitary glands.

Chronic Toxicity Study (Dog)

In a chronic toxicity study ( MRIDs 00032539 and 00126261 ), MEB 6447 (triadimefon; 88.9– 92.7% a.i.) was administered to four beagle dogs /sex/dose in the diet for up to 24 months at nominal doses of 0, 100, or 330 ppm (e quivalent to 0, 3.03/3.49, and 11.42/11.96 in males/females). In addition, the dogs were tr eated with a high dose of 1000 ppm during Weeks 1–54 (equivalent to 34.70/33.67), that was subsequently increased to 2000 ppm (equivalent to 68.80/60.42) during Weeks 55–104.

No treatment-related effects were observed on mortality, clinical signs, ophthalmology, hematology, and histologic (including tu mors) pathology. At 2000 ppm during Weeks 54–62, body weight losses of 0.7 kg and 0.4 kg were noted in males and females, respectively, resulting in decreased overall body weight gains for both males ( ↓ 20%) and females ( ↓ 11%). At the high dose, decreased food consumption was observed in females throughout the study ( ↓ 5–18%). Increased alkaline phosphata se levels were observed at 1000 ppm in males ( ↑ 12–92%) and females ( ↑ 17–55%) and at 2000 ppm in males ( ↑ 352–491%) and females ( ↑ 121–393%) throughout the respective treatment periods. Bile duct concretions were observed in 3/4 high
dose males. Increased absolute and relative (to body) liver weights ( ↑ 16–25%) were observed in the males. Cholesterol was increased in the 1000 ( ↑ 8–27%) and 2000 ( ↑ 31–63%) ppm females.

Gene Expression Profiling Studies

In a gene expression profiling study ( Goetz and Dix, 2009a ), key biological pathways affected by triazoles in the liver and te stis following exposure from gestation through adulthood were
identified in order to better understand triazole-i nduced disruptions in te stosterone homeostasis. Using tissue samples from a previous study ( Goetz et al ., 2007; MRID 48264603 ), gene expression changes were assessed in the male rat liver and testis following exposure to myclobutanil, propiconazole, or tr iadimefon from gestational da y (GD) 6 through postnatal day (PND) 92 to investigate if triazoles disrupt test osterone homeostasis by increasing expression of genes involved in testosterone synthesis in the testis and decr ease expression of genes involved
in testosterone metabolism within the liver.

Gene expression profiles indicate d that all three triazoles signif icantly perturbed the fatty acid, steroid, and xenobiotic metabolism pathways in the male rat liver. In addition, triadimefon modulated expression of genes in the liver from the sterol biosynthesis pathway. Although individual gene expression was affected, there were no common pathways modulated by all three triazoles in the testis. The pathways identified in the liver included numerous genes involved in phase I–III metabolism (Aldh1a1, Cyp1a1, C yp2b2, Cyp3a1, Cyp3a2, Slco1a4, Udpgtr2), fatty acid metabolism (Cyp4a10, Pcx, Ppap2b), and steroid metabolism (Ugt1a1, Ugt2a1), for whichTRIADIMEFON FINAL

Page 56 of 61 expression was altered by the triazoles. These di fferentially expressed genes form part of a network involving lipid, sterol, and steroid homeostatic pathways regulated by the constitutive androstane receptor (CAR), pregnane X receptor (P XR), peroxisome proliferator-activated alpha, and other nuclear receptors in li ver. The relatively high dose and l ong-term exposures to triazole antifungals appeared to pertur b fatty acid and steroid metabolism in the male rat liver predominantly through the CAR and PXR signaling pathways.

In a second gene expression profiling study (Goetz and Di x, 2009b), biological pathways consistently modulated across multiple time poi nts and various study designs were identified. Gene expression profiling was conducted on rat liver s from three separate studies with triazole treatment groups ranging from 6 hours after a singl e oral gavage exposure, to prenatal to adult exposures via feed. To explore conservation of responses across species, gene expression from the rat liver studies were compared to in vitro data from rat and human primary hepatocytes exposed to the triazoles. Toxicogenomic data on triazoles from 33 different treatment groups and 135 samples (microarrays) identified thousands of probe sets and dozens of pathways differentially expressed across time, dose, and sp ecies. Many of these were common to all three triazoles, or conserved between rodents and hum ans. Common and conserved pathways included androgen and estrogen metabolism, xenobiotic metabolism signaling through CAR and PXR, and CYP related metabolism. Differentially expr essed genes included the Phase I xenobiotic, fatty acid, sterol and steroid metabolism ge nes Cyp2b2 and CYP286, Cyp3al and CYP3A4, and Cyp4a22 and CYP4A11; Phase II conjugation en zyme genes Ugtlal and UGT1Al; and Phase III ABC transporter genes Abcbl and ABCBI. Gene e xpression changes caused by all three triazoles in liver and hepatocytes were c oncentrated in biological pathwa ys regulating lipid, sterol and steroid homeostasis, identifying a potential common mode of ac tion conserved between rodents
and humans.

Avian Toxicity and Reproduction (OCSPP 850.2200/850.2300)

In an avian reproduction study ( MRID 00110431 ), bobwhite quail were fed triadimefon in the diet at nominal concentrations of 20, 100 and 500 ppm, along with a control. The birds were 34 weeks old and weighed 160–200 g at the beginning of the study. Each test level had 24 cages with one female and one male per cage. Fresh diet was prepared each week and stored frozen until used. Body weights were recorded at initia tion, at Weeks 2, 4, 6, 8, and at the end of the study. Food consumption was measured weekly. Eggs were collected for incubation twice daily during Weeks 10–25. On incubation Day 21, the eggs were placed in a hatcher. On Day 26, hatchlings were removed from the hatcher, weig hed and then housed accord ing to parental test group. Observation of quail continued for 14 days post-hatching. Egg shell thickness was measured from eggs taken from each laying female during Weeks 1, 3, 5, 7, and 9 of egg laying. Gross postmortem examinations were performe d on birds that died during the study and on 20 randomly selected pairs from each test group.TRIADIMEFON FINAL

Page 57 of 61 Five birds died during the study, these were not consid ered to be compound-related. At 100 and 500 ppm, there was an increase in number of cracked eggs, and decreases in the number of fertile eggs, viable embryos, hatchl ings, and 14-day survival.

In another avian reprodu ction toxicity study ( MRID 42342302 ), groups of pen-reared, unmated mallard ducks were housed in 15 pens (1 duck/sex/pe n) and exposed to triadi mefon in the diet at nominal concentrations of 50, 200, or 800 ppm from test initiation until terminal sacrifice. Adult birds were observed at least once daily throughou t the study for signs of toxicity, injuries, or illness. Mortalities occurring prior to terminal adult sacrifice were recorded and necropsied. Adult body weights were measured at study initia tion, weekly through Week 8, and at terminal sacrifice (Week 20). Feed consumption was meas ured by cage weekly throughout the adult phase of the study. Eggs were collected twice daily, labeled ac cording to pen of origin, and washed to prevent pathogen contamination. The eggs were removed from the egg cooler weekly and candled for shell cracks. Cracked eggs were reco rded and discarded. Eggs not cracked or used for eggshell thickness measurements were placed in an incubato r. Eggs were candled again on incubation Day 14 to determine fertility and embryo viability, and on Day 21 to determine embryo surviva1. Eggs were collected every ot her week for eggshell strength and thickness measurements. Eggs were then opened, the conten ts removed, the shell washed, and allowed to air dry. The average thickness of th e dried shell plus membrane was determined. Hatchlings were removed from the hatcher on Days 27 and 28 of incubation, banded, weighed, and housed according to week of hatch and parental trea tment group. Hatchlings were identified by numbered wing bands and placed in batteries of brooding pens from the time of hatch through Day 14 post-hatch. Hatchlings we re fed untreated diet. Hatch ling body weights were measured and recorded at hatch and on Day 14. Feed cons umption was not monitored. Hatchlings were observed twice daily throughout the 14- day period for signs of t oxicity, injuries or illness. Mortalities occurring prior to the end of th e 14-day period were recorded and discarded. Hatchlings surviving to Day 14 were sacrificed, weighed, and discarded.

Mean measured concentrations were 75%, 96%, a nd 86% of nominal concentrations for the 50-, 200-, and 800-ppm diets, respectively, after storage for 7 days at room temperature. Only one mortality occurred during the study, and it was not treatment related. No treatment-related adult mortality occurred in any test group during the course of the st udy. Occurrences of feather loss and lacerations were observed, but were attrib uted to normal laboratory pen housing. No overt signs of intoxication were observed during the st udy in any test group. Post-mortem observations revealed anomalies however, they occurred in a small number of birds and no dose-response was indicated. There were no apparent treatment-relate d effects upon adult body weight at any of the concentrations tested. There was a significant de crease in feed consumption during Weeks 5 and 14 in the 185 ppm group, but these findings were incidental. When compared to the control group, there were no statistically significant di fferences in reproductive parameters at any
concentration tested. There were no significant differences in th e overall mean eggshell thickness and strengths of control eggs co mpared to treatment eggs. The overall mean hatch weight and theTRIADIMEFON FINAL

Page 58 of 61 overall 14-day survivor weights we re not significantly lower for birds in treatment groups when compared to controls. Mean hatch weight for hatchlings in the 185 ppm group was significantly higher than controls, but this di fference was not considered treatme nt-related due to the lack of a distinct dose-response relationship. There were no appa rent effects on adult ducks fed
triadimefon-treated diets. The L OAEC for mallard ducks is greater than 780 ppm of triadimefon.

Fish Early Life Stage Toxicity Study (Rainbow Trout) (OPPTS 850.1400)

In a fish early life st age toxicity study ( MRID 00110433 ), rainbow trout embryos (230 to 243) were exposed to triadimefon at nominal concentrations of 0, 50, 150, 450, 1350, and 4050 ppb under flow-through conditions. The embryos at each level were divided into two groups and tested in separate 5 gallon containers. The c ontainers were aerated. Temperature was measured twice daily. The flow rate was 75 L/vessel per day. The concentrations were verified by chemical analysis periodically. The photoperiod was 16 hours light/8 hours dark. The embryos were exposed for 3 to 9 days before they hatched.

Although the test did not follow the accepted protoc ol for fish embryo-larvae study, it indicated that triadimefon is highly t oxic to trout in the early life stages, with a 17-day LC 50 of 1270 ppb.

In another fish early lif e stage toxicity study ( MRID 00131105 ), rainbow trout embryos were exposed to triadimefon at nominal con centrations of 0, 10, 30, 90, 270 and 810 ppb. The concentrations were verified by chemical anal ysis at intervals thr oughout the test. The mean analytical concentrations were 23, 41, 116, 300, and 890 ppb. For each concentration 100 eggs were used, divided between four test chambers. The exposure period was 60 days.

Although there was no concentration-related eff ect on hatching success, incubation time was decreased by approximately one-half day in th e highest concentration. There was no effect on time to swim up. Sixty-day cumulative mortality wa s significantly greater than control mortality only for the 890 ppb group. Median lethal c oncentration was 717 ppb. On Days 19 and 20, mortality was unusually high (62% for the 2 days) in the 810 ppb group. Dissolved oxygen
reached its lowest concentration on those two days (65%) but remain ed at levels not ordinarily lethal to juvenile trout. Clin ical signs observed included incr eased or decreased pigmentation, abnormal swimming, spinal curvature, tremors, edema, loss of equilibrium fin erosion, partial agnathism and. general weakness. Of the 60-da y survivors, those in the 890, 300 and 116 ppb groups were significantly lighter than controls, and the magn itude of the difference was concentration- related. Only the two highest concentrations produced significantly shorter
survivors. Triadimefon had no disc ernable effect on hatching succe ss. Growth in terms of final length and/or weight was signi ficantly reduced in at 116, 300 and 890 ppb of triadimefon. The lowest lethal concentration was 890 ppb. Th e observed LOAEC was 0.116 mg/L based on reduced growth, and the NOAEC was 0.041 mg/L.TRIADIMEFON FINAL

Page 59 of 61 Fish Early Life Stage Toxicity Study (Fathead Minnow) (OPPTS 850.1400)

In a fish early life stage toxicity study ( MRID 41922103 ), fathead minnow embryos were exposed to triadimefon at nominal concentr ations of 0.075, 0.15, 0.3, 0.6, or 1.2 mg a.i./L, and
the mean measured concentrations were 0.077, 0.17, 0.27, 0.57, and 1.1 mg a.i./L, respectively (90–113% of the nominal exposure concentrations ). A dilution water control and a solvent control (0.012 mL of DMF/ L, equal to the highest concentrati on of solvent at the highest test level) were also included. Twen ty embryos (approximately 24 hours post-spawn) were impartially distributed in groups of two to each incubation cup in each rep licate chamber (i.e., 80 embryos/concentration). Egg mortality in each cu p was recorded and dead eggs removed daily. Once hatching was complete (Day 7), the fry we re released into the test chambers. Test chambers were inspected daily and observati ons of abnormal physical changes, abnormal behavior, and mortality were r ecorded. Dead fry were discar ded. At termination (Day 35), surviving fish were sacrificed, and blotted wet we ight and standard length for individual fish was recorded. Hatchability a nd total survival data were collec ted along with growth measurements. Water quality parameters were measured on Days 0, 1, 7, 14, 21, and 35. Analytical concentrations were determined for on Days 0, 1, 7, 14, 21, 28, and 35.

The egg hatch began on Day 3 and was complete by Day 7, and was not affected by treatment. No significant reduction in hatch at any test level was indicated when compared to the pooled control. At test termination, su rvival ranged from 65 to 95% in the control and 80 to 95% in the solvent control. No significant difference was det ected between the two controls. Survival at the two highest test concentrati ons (0.57 and 1.1 mg a.i./L) wa s significantly reduced when compared to the pooled control. At test terminat ion, mean standard length and mean wet weight were significantly reduced only at the 0.27 mg a.i./L test level wh en compared to those of the pooled control. Growth data for the two highest test concentrations (0.57 and 1.1 mg a.i./L) were excluded from the statistical grow th analysis, since survival wa s significantly reduced at these concentrations and no data were collected from the highest test concentration since mortality was 100%. In general, morphological and behavioral abnormalities were sporadically observed in the control, vehicle blank and th e three lowest test levels and involved only a few fish. Abnormalities in the two highest test levels were noted primarily during the first week following the completion of hatch and were considered dose related. This week was al so a period of heavy fry mortality for these two levels. Based on thes e results for survival and growth, the NOEAC was 0.17 mg/L and the LOEAC was the 0.27 mg/L.

Thyroid Hormone Levels and Gene Expression (Zebrafish)

In this study, Liu et al. (2011) exposed embryo-larval wild-type (AB stain) zebrafish, raised under standard laboratory conditions, to varying concentrations of triadimefon. Embryos at a stage of 6 hours post-fertilization) were transf erred into glass beaker s containing 500 mL of triadimefon solution (0, 0.5, 1, 2, or 4 μg/mL) until 120 hours post-fertilization. There were 3 replications for each exposure concentration.TRIADIMEFON FINAL

Page 60 of 61 The zebrafish exposed to 2 and 4 μg/mL triadime fon exhibited bent spin e and uninflated swim bladder from (results not presented). The T 3 content in the larvae (whole body) exposed to 2 μg/mL triadimefon was 1.22 ± 0.06 ng/g, which was significantly less than the controls (1.55 ± 0.02 ng/g). In Figure 2, T 3 whole body content in larvae at 4 μg/mL triadimefon was also significantly less than co ntrols. In contrast, the embryos exposed to increasing triadimefon concentrations had significantly higher T 4 content at 2 and 4 μg/mL than in the controls at 120 hpf (whole body values; graphically shown in Figure 2). The expression of genes in the HPT
axis of the zebrafish larvae we re determined at 120 hours post-fe rtilization. The beta-subunit of TSH (TSH- β ) gene transcription was si gnificantly upregulated ( ↑ 4.0- and ↑ 5.2-fold) upon exposure to 2 and 4 μ g/mL triadimefon, respectively. Two is oforms of deiodinases (dio1 and dio2) were identified. Triadimefon e xposure significantl y downregulated dio 1 gene transcription (1.5-fold) in the 2 μg/mL exposur e group. However, the levels of dio 2 gene transcription were not altered in any other triadime fon-treated groups. The mRNA e xpressions of the genes for the two isoforms of the thyroid hormone nuclear receptors ( thraa and thrb ) were evaluated. There were no significant changes in the mRNA expression of thraa . However, the thrb gene transcription was significantly down-regulated ( ↓ 2.6-fold) after exposure to 2 μg/mL
triadimefon.

Organ Morphogenesis Study (Frog)

In an organ morphogenesis study ( Lenkowski et al. , 2010 ), Xenopus laevis tadpoles (NF stage 41) were cultured in modified Marc’s Modified Ringer’s solu tion (MMR) at a density of 13–18 tadpoles in 10 mL of MMR maintained at 18°C. Groups of tadpoles were e xposed in triplicate to triadimefon at working concentrations of 7.29, 21.88, 36.47, 51.05 mg/L for 48 hours, with static renewal at 24 hours. Tadpoles we re also exposed to atrazine, 2-4, D, and glyphosate, but these
chemicals will not be discussed in this summar y. Tadpoles were humanely euthanized, fixed, and examined for intestinal malformations, ed emas, and skeletal mu scle degeneration.

Mortality was 0% in the 7.29 and 21.88 mg/L tria dimefon exposures, but significantly increased at 36.47 and 51.05 mg/L (8.5%, p<0.005; 25.4%, p< 0.0001; respectively). Mortality occurred between 24 and 48 hours. Triadimefon exposure cau sed a significant incr ease in intestinal malformations and edemas at 21.88, 36.47, and 51.05 mg/L. Parallel malformations are further illustrated by the disruption of differentiated skeletal muscle in exposed animals. Cranial muscles in triadimefon exposed tadpoles were reduced as compared to non-treated controls. Additionally, the net-like structure of the hypa xial muscle normally encases the intestine and was disrupted in exposed tadpoles. These anomalies varied in severity depending on the degree of malformation in an individual tadpole. Therefore, higher conc entrations of chemical exposure exhibited more dramatic disruption of these muscles.TRIADIMEFON FINAL

Page 61 of 61 APPENDIX 3: References Not Utilized in the Triadimefon 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 triadimefon but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the com pound of interest was not us ed in the study; 3) the test material was not adequately described; 4) a formulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only da ta on invertebrates.

Hassold, E. and T. Backhaus (2009). Chronic Toxicity of Five Structurally Diverse Demethylase-Inhibiting Fungi cides to the Crustacean Daphnia magna : A Comparative

Assessment. Environmental Toxicology and Chemistry : 28, 1218-1226.

Iyer, V.V., Ovacik, M.A., Androulakis, I.P., Roth, C.M., and Ierapetritou, M.G. (2010) Transcriptional and metabolic flux profiling of triadimefon effects on cultured hepatocytes. Toxicol Appl Pharmacol . 248(3):165-177