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

CHEMICAL: TEBUCONAZOLE

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYTEBUCONAZOLE FINAL

ii TABLE OF CONTENTS

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

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

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

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

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

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

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

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis.............................9
1. Effects on Estrogen Pathway....................................................9
2. Effects on Androgen Pathway..................................................17

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................23

IV. Committee’s Assessment of Weight of Evidence..........................................27

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................27

1. Tier 1 in vivo Assays.............................................................28

2. OSRI...............................................................................29

B. Estrogen Pathway...........................................................................35

C. Androgen Pathway.........................................................................35

D. Thyroid Pathway...........................................................................35

E. Conclusions.................................................................................36

V. EDSP Tier 2 Testing Recommendations....................................................36

VI. References.......................................................................................38

LIST OF TABLES: Table 1. Tier 1 Assays for Tebuconazole.............................................................8
Table 2. Estrogenic/Anti-Estroge nic Pathway for Tebuconazole ................................12
Table 3. Androgenic/Anti-Androgeni c Pathway for Tebuconazole ...............................19
Table 4. Thyroid Pathway for Tebuconazole ......................................................25

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................43
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................55

APPENDIX 3. References Not Utilized in the Tebuconazole WoE Analysis........................80TEBUCONAZOLE 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 enerationTEBUCONAZOLE 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 ConcentrationTEBUCONAZOLE 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)TEBUCONAZOLE 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-EvidenceTEBUCONAZOLE FINAL

Page 1 of 81 Executive Summary

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether tebuconazole interacts wi th E, A or T hormone pathways, the number and type of effects induced, the magnitude and pa ttern of responses obser ved across studies, taxa and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, the dose(s) at which responses happen and whether overt toxicity was
present.

On June 18, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) an alysis of the potential interaction of tebuconazole with the E, A or T hormone path ways. 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, although tebuconazole was negative in an estrogen receptor (ER) binding assay available in OSRI a nd in the Tier 1 ER transactivat ion assay (ERTA), it appears to alter estrogen production. In vitro studies showed tebuconazo le can potentially alter steroidogenesis and inhibits aromatase activity. In the in vivo mammalian studies, a delay in vaginal opening (VO) was observed in the female pubertal assay; this finding was corroborated by other findings in mammalian OSRI such as decreased number of offspring and gestational/delivery alterations in the absence of overt toxicity. Additionally, complementary findings were observed in the fish short-te rm reproduction assay (FSTRA) which included effects on plasma vitellogenin (VTG), gonadal we ight and histopathology. Furthermore, effects

on gonadal histopathology were reported in a fish sexual development test. Therefore, the majority of the findings suggests that tebuconazo le potentially interact s with the estrogen pathway both in vitro and in vivo . It is noted that generally, these in vivo findings co-occurred
with liver effects that included increased weight and/or changes in histopathology.TEBUCONAZOLE FINAL

Page 2 of 81 For tebuconazole, effects on testosterone biosynth esis and androgen recept or (AR) interaction were consistently noted in the Tier 1 and OSRI in vitro studies. In the mammalian in vivo studies, anti-androgen effects were observed in young but not adult an imals. Testosterone levels were decreased in the Tier 1 male pubertal assay, as well as in a reprodu ctive toxicity in the published literature; this concurred with decr eased weights of androge n-dependent tissues. While there were limited findings in male fish in the FSTRA which included a decrease in gonadal somatic index (GSI), gonadal histopathologi cal findings were repor ted in a fish sexual development test. The majority of the findings suggests that tebuconazo le has anti-androgenic activity in vitro and in vivo only in the young, and can interact with the androge n receptor and interfere with steroidogenesis. Again, it is noted that these in vivo findings co-occu rred with liver effects that included increased weight and/or changes in histopathology.

Based on the available data, there is limited evid ence for a direct effect of tebuconazole on the thyroid pathway or the HPT axis. Negative responses for the T pathway were seen for
tebuconazole in the in vivo Tier 1 male and female pubertal as says and six of the seven pertinent general toxicity studies which showed no effect from tebuconazole treatment. In the published studies, effects were seen in thyroid hormone levels in maternal animals in developmental toxicity studies, but these occurred in the pres ence of systemic toxicit y. Developmental delays (including NF stage and normalized hind-limb) al ong with thyroid hypoplasia were observed in the amphibian metamorphosis assay (AMA), however , this coincided with a significant reduction in growth (length and body weight) and liver effects. Although findings of thyroid gland hypoplasia are identified in the AMA guideline as diagnostic criteria, no effects were seen on the incidence of follicular cell hypertrophy or hyperpla sia, which are expected to be more commonly affected in the presence of a thyroid-related developmental delay. Overall, the thyroid anddevelopmental findings in the AMA with tebuconazole are inconsistent with a direct, thyroidrelated delay and may be more reflective of treatment-related growth inhibition.

Based on in vivo and in vitro results, tebuconazole showed poten tial to alter the steroidogenesis pathway which may result in endocrine-related eff ects in the estrogen and androgen pathways. In the androgen pathway, effects seem to have a greater impact on young than in adult mammals.
It is noted that the e ffects observed in mammals and fish coincided with liver effects.

Based on the available data, there was a lack of complementary and redundancy of evidence in
the available Tier 1 and OSRI da ta to suggest a potential intera ction with the thyroid pathway
either in mammals or wildlife.

Tebuconazole showed potential to alter the steroidogenesis pa thway which may result in endocrine-related effects in the estrogen and a ndrogen pathways. For tebu conazole, the point of departure (POD) for human health risk assessmen t is based on a developmental neurotoxicity endpoint observed in offspring in a developmenta l neurotoxicity study in rats. The basis for the
POD is alterations in m otor ac tivity, decreases in body weight a nd brain weight and changes inTEBUCONAZOLE FINAL

Page 3 of 81 brain morphometrics observed in the offspring at 8.8 mg/kg/day, th e lowest observed effect level (LOAEL). Since the study did not establish a no-observed-advers e-effect–level (NOAEL), the default 10X Uncertainty Factor (UF L ) was applied ( i.e ., for the use of a LOAEL) resulting in an extrapolated NOAEL of 0.88 mg/kg/day. This valu e is used as the current POD for calculating the chronic Reference Dose (RfD) for dietary risk assessment and calculating the Margins of Exposure (MOEs) for non-dietary exposure risk a ssessments. This POD is approximately 75-fold lower than the lowest doses (75 mg/kg/day) that caused androgen and estrogen-related effects in the Tier 1 pubertal assays. Consequently, the ch ronic RfD and the MOEs are protective of the
endocrine-related effects seen in the pubertal assays.

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

For the wildlife the EDSP Medaka Extended On e-Generation Reproduction Test (MEOGRT) is recommended. In the FSTRA, effects were observe d at 19.5 μg/L. Gonadal hi stological effects in the fish sexual development test (FSDT) were observed at 0.045 and 0.022 μg/L for females and males, respectively, in the absence of effects on liver. The current chronic NOAEC for fish is 12 μg/L, based on survival effects in the rainbow trout early- life cycle. It is noted that no effects on fecundity were observed in the sheepshead minnow life-cycle study at co ncentrations up to 196 μg/L; effects on survival (F0) and growth (F 1) were noted. Since the reproductive effects
observed in the FSTRA and FSDT occur at concen trations similar or below the current NOAEC, additional information on pot ential reproductive eff ects is recommended.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for tebuconazole. In both spec ies, effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from tebuconazole exposure. Relative to the EDSP, ad ditional testing is not recommended.TEBUCONAZOLE FINAL

Page 4 of 81 I. Introduction

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether tebuconazole interacts wi th E, A or T hormone pathways, the number and type of effects induced, the magnitude and pattern of respons es observed across studies, taxa and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whethe r or not endocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On June 18, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence analysis (WoE) of the potential interaction of tebuconazole with the E, A or T hormone path ways. 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).

Tebuconazole is a systemic triazo le fungicide whose mode of action is through inhibition of the C-14 demethylation step within fungal sterol biosynt hesis. Tebuconazole’s solubility in water is 36 mg/L at 20°C. The vapor pres sure of tebuconazole is 1.3 x 10

− 8 mm Hg at 20°C (HSDB, 2010) and volatilization of tebuconazole is not expected (es timated Henry's Law of 1.4  10

− 10

atm-m 3 /mole). Tebuconazole is stable to hydrolysis and is not expected to undergo photolysis. Tebuconazole is expected to have moderate to limited mobility in soil and adsorb to suspended solids and sediment. Its log K ow is 3.7.

The available information considered in determ ining the potential intera ction of tebuconazole 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.TEBUCONAZOLE FINAL

Page 5 of 81 II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

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

1. B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 Screening assays were submitted as OSRI by the Te st Order recipients and/or the public. This included studies published in the open literatu re and/or data submitted to support pesticide registration (e.g., Part 158 guideli ne studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disr uptor Review Team on tebuconazole (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any additional re levant information. Summarie s of the available OSRI are presented in Appendix 2. Additiona lly, literature/studies considered but not utilized for the WoE analysis are liste d 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 (SAP0 (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

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

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

Page 6 of 81 consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data, as well as characterizing the extent to which the available data suppor t a hypothesis that a chemical has the potential to interact E, A or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, coherence, strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are
meant to evaluate whether or not a chemical can interact with E, A and T consisting of different levels of biological organization from a molecular initiating ev ent, such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment and fecundity at the whole organism level. The extent of expression of responses at higher levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.

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

Page 7 of 81 This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

For these WoE analyses, overt toxicity was gene rally defined in accordance with EPA’s current approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g ., organ weights/histopathology), biochemical ( e.g ., blood chemistry), and other clinical signs ( e.g. , lethargy) were also considered when evaluating overt toxicity, es pecially if the effects were extreme. In some instances, one parameter ( i.e ., death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g ., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

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

Page 8 of 81 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.

A. EDSP T1 Screening Assays

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

Table 1. Tier 1 Screening Assay for Tebuconazole

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied by OSRI (USEPA, 2002)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48619403)

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied (MRID No. 48619401)

Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48619404)

Aromatase Assay (human recombinant microsomes) OSCPP 890.1200 Requirement Satisfied (MRID No. 48619402)

Uterotrophic Assay (Rat) OSCPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48673703)

Hershberger Assay (Rat) OSCPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48673702)

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied (MRID No. 48688302)

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied (MRID No. 48688302)

Fish Short-term Reproduction Assay OSCPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48688301)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 48673701)TEBUCONAZOLE FINAL

Page 9 of 81 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential interactions of tebuconazole wi th the estrogen pathway are summarized in Table 2. If an effect was observed but considered by WoE not to reflect a direct interaction, then the
effect is discussed in the text but not presented in the table. The various targets of the estrogen pathway across the relevant Tier 1 assays are de lineated so as to facilitate determination of potential for estrogenic, anti-est rogenic or HPG axis effects. This table also includes HPGrelevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reported in the t able and not considered further in
the WOE assessment.

Tebuconazole was classified as not interactive (negative) in the Tier 1 in vitro estrogen receptor (ER) binding assay and as negative in the Tier 1 in vitro ER α transcriptional activation assay (ERTA). In contrast, tebuconazole was positive in both the aromatase and steroidogenesis Tier 1 assays, and in a series of in vitro cell proliferation and steroi dogenesis assays by Kjærstad et al. (2010) considered as OSRI. In the Tier 1 aromatase study, tebuconazole inhibited aromatase activity with an IC 50 of approximately

− 5.7 log M. Tebuconazole completely inhibited estradiol production at 10

− 5 M and above in the Tier 1 steroidoge nesis assay, and inhibited estradiol production with an IC 50 of 1.2 μ M in an steroidogenesis assay by Kjærstad et al. (2010). Kjærstad et al. (2010) also found that tebuconazole in hibited proliferation of MCF-7 cells induced by estradiol (IC 50 = 49 μM) or testosterone (IC 50 = 17 μM). While the authors attribute
the inhibition of testosterone-indu ced proliferation in part to arom atase inhibition (testosterone is converted to estradiol by aromatase), th is was not demonstrated in the study.

Tebuconazole was negative in the Tier 1 uterotro phic assay. In the Tier 1 female pubertal assay, a 2-day delay in VO (p ≤ 0.05) was accompanied by a slight increase in body weight at attainment of VO (not statistic ally significant) at 150 mg/kg/da y. Also in the pubertal study, ovarian weight decreases at low (6%) and high (7%) doses and uterus we ight decreases of 15% (wet or blotted, not grouped by st age) at the high dose were not statistically significant. These effects in pubertal females occurred in the absence of overt toxicity. Liver weights were increased in low (8%) and high (15-18%) dose groups, and relative kidney weights were increased (6%) at the high dose.

In the FSTRA, there was a significant decrease of 18.5% in female GSI at the high concentration (0.0195 mg a.i./L), and significant decreases of 35 and 55% in pl asma vitellogenin at the mid (0.0113 mg a.i./L) and high treatment. Gonada l histopathology findings included increased incidence of granulomatous inflammation in the high treatment compared to the control; observations of oocyte atresia was reported in both controls and treatment groups with no indication of treatment-related response. Additionally, microscopic findings in the liver were reported including treatment-related hepa tocyte necrosis in all treatments.TEBUCONAZOLE FINAL

Page 10 of 81 In the Part 158 developmental studies, increased resorptions per dam were observed in rats and rabbits. In the developmental neurotoxicity study, delivery was delayed on average 0.5 days and 2/25 dams died as a result of pr olonged gestation. In the same study, a 1.6-day increase in age at VO was observed at the high dose (125 mg/kg/da y); however, this finding co-occurred with reduced body weights ( ↓ 5-24%; PND 5-72). In the 2-genera tion reproductive study, at the high dose (50 mg/kg/day), F1a, F2a and F2b pup weights were decreased (p ≤ 0.05) compared to
controls from birth to week 4. There were no es trogen-related effects noted in the other Part 158
mammalian studies.

In published literature considered as OSRI, post-implantation loss was increased at 50 mg/kg/day in a developmental study by Taxvig et al. (2008) while in another developmental study by the same researcher (2007) significant post-implant ation loss was observed at 100 but not at 50 mg/kg/day. Similarly, increased anogenital distance (AGD, a potenti al sign of virilization) was observed in females at 50 mg/kg/day by Hass et al. (2012), and at 100 (but not 50) mg/kg/day by Taxvig et al. (2007). Both Hass et al. and Taxvig et al. observed increased incidence of nipple retention in males (a sign of fe minization) at 50 mg/kg/day. Taxvig et al. also found that delivery was delayed an average of 0.9 day and dams had a 7-fold increase in levels of progesterone at 100 mg/kg/day tebuconazole, compared to controls (2007 study), and a 58% decrease in estradiol levels at 50 mg/kg/day (2008 study). Other sporadic findings in lite rature studies include a delay age at VO at 20 but not 60 mg/kg/day (Moser et al. 2001), and one male genital malformation at 50 mg/kg/day (Hass et al. 2012). Another literature developmental study by Jacobsen et al. (2012) found that gavage dosing of dams on GD 7 to LD 16 up to 50 mg/kg/day had no effect on gestation length or implantation loss, nor on offspring VO, organ wei ghts or histopathology, AGD, nipple retention or genital malformations. Finally, in a study to obs erve puberty onset and gene expression by Overgaard et al. (2013), tebuconazole had no e ffect on attainment of VO, however in that study pups were not direc tly dosed (dams were dosed via gavage).

In the available Part 158 avian one-generation reproducti on toxicity studies (two quail and two duck), dietary administration of tebuconazole re sulted in reproductive effects in two of the studies. In the first bobwhite quail study, no trea tment-related reproductive effects or clinical signs were noted at dietary levels up to 73.5 ppm . However, in the second bobwhite quail study, significant reproductive effects were observed at the high dose (290 ppm), including a reduction of 33% in the number of eggs laid, 11% reduction in normal hatchlings, as well as a 37% decrease in offspring survival and 7-11% decr eases in offspring body weight. Abnormal livers (5/18) in females were also report ed at this dose. In this study, significant reductions in offspring body weight (4-6%) were also reported in the low treatment group (156 ppm). In the first mallard duck study, no treatment-related reproductive effects or clinical signs were noted at dietary levels up to 76 ppm ; however, in the seco nd mallard duck study, while clinical signs were not observed, significant decreases in the number of hatchlings per hen were reported at the high dose of 611 ppm.TEBUCONAZOLE FINAL

Page 11 of 81 In a fathead minnow fish sexua l development study, there was no effect on hatch but survival was significantly reduced at ≥ 0.045 mg/L with effects on female growth at 0.022 mg/L. There was no difference in sex ratio after 54 days of exposure but effects on gonadal histopathology were reported at ≥ 0.011 mg/L in females. However, systemic toxicity was also reported at these concentrations including histopa thological changes in the liver , pancreas and intestines ( ≥ 0.011 mg/L), kidneys (0.045 mg/L) and spinal deformities ( ≥ 0.022 mg/L). No reproductive effects
were noted in a Part 158 sh eepshead minnow life cycle study, but parental survival was significantly reduced (day 58 & 177) at the highest treatment test ed (0.197 mg/L), and parental body length was significantly reduced at the mi d-high treatment (0.097 mg /L; highest treatment not analyzed due to mortality). Additionally, offspring length wa s significantly reduced at the mid and mid-high concentrations ( ≥ 0.044 mg/L). In a Part 158 rainbow trout fish early life-cycle study, there were no effects on ha tch but survival and growth was significantly reduced at ≥ 0.025 mg/L. In another Part 158 fish early life cy cle toxicity study with sheepshead minnows, there
were no effects on hatch or survival, but wet weight wa s significantly reduced at ≥ 0.048 mg/L and length was reduced at 0.099 mg/L.TEBUCONAZOLEFINALPage 12 of 81Table 2: Estrogenic/Anti-Estrogenic Pathway for TebuconazoleLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Tebuconazole1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Gene Expression Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER BindingAssay satisfied by USEPA, 2002; see below.ERTA(MRID 48619403)NAromatase(MRID 48619402)PSteroidogenesis(MRID 48619404)PUterotrophic(MRID 48673703)NNX(H)Female Pubertal Rat(MRID 48688302)NNNNN↑2.0 days(H)LW(M, H);KW(H)NFSTRA(MRID 48688301)NE↓18.5%(H)P4(H)NN↓35, 55%(M, H)P5(L, M,H)NOSRIER Binding(USEPA, 2002)NCell Proliferation andAromatase Assays(Kjærstadet al., 2010)P PTEBUCONAZOLEFINALPage 13 of 81Table 2: Estrogenic/Anti-Estrogenic Pathway for TebuconazoleLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Tebuconazole1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Gene Expression Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Puberty Onset andKiss1Expression(Overgaardet al., 2013)NNSubchronic Toxicity(Rat; MRID 40700930)NENENNENEX(L, M,H)X(H)Subchronic Toxicity(Dog; MRID40700934)NENNNENEX(L, M,H)X(H)Chronic Toxicity (Dog;MRID 40700940)NE N N NE NEX(M, H)NChronic Toxicity (Dog;MRID 42030601)NE N N NE NEX(H)NChronic toxicity/carcinogenicity (Rat;MRID 40700939)NE N N NE NEX(M, H)NEight-Week RangeFinding Study (Mouse;MRID 40700933)NE N N NE NEX(M, H)NCarcinogenicity(Mouse; MRID40700941)NENENNENEX(L, M,H)NCarcinogenicity(Mouse; MRID42175001)NENEPNENEX(L, H)NTEBUCONAZOLEFINALPage 14 of 81Table 2: Estrogenic/Anti-Estrogenic Pathway for TebuconazoleLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Tebuconazole1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Gene Expression Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3DevelopmentalToxicity (Rat; MRID40700943)NRP6X(M, H)NDevelopmentalToxicity (Rabbit;MRID 40700945)NRP7X(H)NDevelopmentalToxicity (Rabbit;MRID 43776201)NRNX(L, M,H)NDevelopmentalToxicity (Rat; Taxvigetal., 2008)PN P8NRNDevelopmentalToxicity (Rat; Hassetal., 2012)PP9(H)NN NDevelopmentalToxicity (Rat; Jacobsenet al., 2012)NNNNN NTwo-GenerationReproduction Toxicity(Rat; MRID 40700946)NENNNENX(H)NReproductive Toxicity(Rat; Taxviget al.,2007)P(H)N N NE NEP9(H)P10(H)X(H)NDevelopmentalNeurotoxicity (Rat;MRID 45074301)↑1.6days(H)P11(H)X(L, M,H)NTEBUCONAZOLEFINALPage 15 of 81Table 2: Estrogenic/Anti-Estrogenic Pathway for TebuconazoleLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Tebuconazole1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Gene Expression Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Reproductive Function(Rat; Moseret al.,2001)NNNNX(H)NAvian reproduction(Bobwhite quail; MRID40700910)NN NAvian reproduction(Bobwhite quail; MRID41624201)P12(L, H)X(H)NAvian reproduction(Duck; MRID40700909)NN NAvian reproduction(Duck; MRID41818301)P13(M,H)N NFish SexualDevelopment Toxicity(Fathead minnow,MRID 48109802)P14(MMH)NX(M-H)X(MH-H)Fish Full Life CycleToxicity (Sheepsheadminnow, MRID43009601)NX(H)1.Key to responses: L=Low treatment, ML=Medium-low treatment,M=Medium treatment, MH=Medium-high treatment, H=High treatment. LW =liver weight, KW = kidney weight. Arrows (↓or↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinelyevaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated.TEBUCONAZOLEFINALPage 16 of 812.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 anX in this column. For details see Section IV. A3.The overt toxicity in the Tier 1 assays are presented inthis column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4.In the ovary, the microscopic findings included increased granulomatous inflammation (minimal to mild).5.Microscopic findings in the liver were reported including treatment-related hepatocyte necrosis in all treatments.6. Mean number of live fetuses/dam was decreased at the high dose due to an increase in both early (embryonic) and late (fetal)resorptions (totalresorptions: 2.8/dam treated vs. 0.6/dam controls). Mean live fetuses/dam were decreased in the high dose group, due to an increase in both early andlate resorptions (mean total resorptions: 2.8/dam treated vs. 0.6/dam controls).7. There was a reduction in live fetuses/dam observed in the high dose group as compared to the controls (6.4 treated vs. 7.4 controls), due to an increasein total (both early and late) resorptions/dam (2.4 treated vi. 0.7 controls).8. At 50 mg/kg/day, tebuconazole increased (p<0.05) the frequency of post-implantation loss (11.4%) compared to controls (0%).9. Increased incidence of nipple retention in males (a sign of feminization) at 50 mg/kg/day. Increased anogenital distance (AGD, a sign of virilization)was observed in females at 50 mg/kg/day by Hasset al.(2012), and at 100 (but not 50) mg/kg/day by Taxviget al.(2007).10. Significant post-implantation loss was observed at 100 but not at 50 mg/kg/day. At 100 mg/kg/day, delivery was delayed an average of 0.9 day.11. At the high dose (1000 ppm), two P females died as a result of prolonged gestation and the number of live fetuses/dam was decreased (p<0.01); whilethe number of dead fetuses/dam was increased (p<0.01), the stillborn index was increased (p<0.01) and the number of pup deathswas increased duringPND 1-5. Additionally at high dose mean gestation length was increased (23.0 days) compared to the controls (22.5 days), and two P females died as aresult of prolonged gestation.12. At high dose (290 ppm) reduction of 33% in the number of eggs laid, 11% reduction in normal hatchlings, 37% decrease in offspring survival and 7-11% decreases in offspring body weight.Reductions in offspring body weight (4-6%) in the low treatment group (156 ppm)13. Significant decreases in the number of hatchlings per hen were reported at the high dose of 611 ppm14. No difference in sex ratio; effects on gonadal histopathology were reported at≥0.011 mg/LP Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reported.TEBUCONAZOLE FINAL

Page 17 of 81 2. Effects on Androgen Pathway

The potential interactions of tebuconazole with the androgen pathway are summarized in Table
3. If an effect was observed but considered by Wo E not to reflect a direct interaction, then the
effect is discussed in the text but not presented in the table. The various targets of the androgen pathway across the relevant Tier 1 assays are de lineated so as to facilitate determination of potential for androgenic, anti-androgenic or HP G axis effects. This table also includes HPGrelevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reported in the t able and not considered further in
the WOE assessment.

Tebuconazole was classified as a binder in the Tier 1 in vitro AR binding assay, as average radioligand binding was reduced to <50% at concentrations of 10 -3 M and 10 -4 M. In the Tier 1 steroidogenesis assay, tebuconazole reduced testosterone production by 32-98% at 10

− 6 M and above. Four published studies (OSRI) also examined effects of tebuconazole on AR transactivation and/or steroidogenesis. In an AR reporter gene assay using AR-transfected CHO cells, tebuconazole was an AR antagonist in th e presence of R1881 (a strong androgen agonist),
and inhibited testosterone production in a steroid ogenesis assay (Kjærstad et al. , 2010). In an AR-transcription activation assay using MDA-kb2 cells with and without dihydrotestosterone (DHT), tebuconazole was also an AR antagonist (Orton et al. , 2011). In a study by Hass et al. (2012), tebuconazole also exhibite d some antagonistic action in an AR reporter gene assay and reduced testosterone production in a steroidogenesis assay.

Tebuconazole was negative in the Tier 1 Hers hberger assay, as well as another Hershberger assay from the published literature (Taxvig et al. , 2008). Conversely, in the male pubertal assay,
tebuconazole reduced serum testosterone levels in a dose dependent manner (17% NS at low dose and 53% p ≤ 0.05 at high dose). Weights of several androgen-target organs also decreased in a dose-dependent manner (semin al vesicle 17-33%, dorsolatera l prostate 21%, LABC 11-24%, epididymis 2-14%) although the cha nge at the low dose was statisti cally significant only for the dorsolateral prostate. A 10% testis weight decrease was not sta tistically significant. Pituitary weights decreased (p ≤ 0.05) at the low (9%) and high (12%) doses. Age at initial preputial separation (PPS) was delayed 2.5 days (p ≤ 0.05), however a similar 2.4 day delay in age at complete PPS was not statistically significant. A small increase in incidence of gross atrophy was observed in testis (1/2/3 of 15 in contro l/low/high dose), seminal vesicles (2/5/7) and LABC (0/1/3). Microscopically, increased incidences of tubular degeneration in the testes (1/0/4) and oligospermia in the epididymis (1/2/4) were also observed. These androgen-related effects occurred in the absence of overt toxicity but co -occurred with decreases in serum bilirubin (75% at low and not detected at high dose) and ur ea nitrogen (10% at high dose), decreased organ weights of kidneys (7-9%) and adrenals (18-13% not dose-depe ndent) and a 10% increase in liver weight (high dose, relative only).TEBUCONAZOLE FINAL

Page 18 of 81 In the FSTRA, a 19% decrease in GSI was marg inally significant (p=0.056) at the high concentration (0.0195 mg/L). For the liver, the occasional findings of microgranulomas (minimal) and individual hepatocyte necrosis (minimal) in the livers were likely treatmentrelated (based on findings in females); there wa s also a dose-related d ecrease in hepatocyte vacuolation compared to control.

There were no androgen-related effects observed in any of the mammalian Part 158 studies.

In addition to the Part 158 toxicity studies, five published studies considered as OSRI examined effects of tebuconazole on development and reproducti ve parameters in rodents. In a couple of rat developmental toxicity studies by Taxvig et al. (2007 and 2008), tebuconazole did not affect fetal serum testosterone levels following e xposure from GD 7 through 21 at 50 mg/kg/day, but
testicular levels were decreas ed in the 2007 study at 100 mg/kg/day. Increased male pup nipple retention was observed postnatally, without changes in male AGD, in the 2007 Taxvig et al. study and another similar study by Hass et al. (2012) at ≤ 50 mg/kg/day. However, neither the Taxvig et al. nor the Hass et al. studies found treatment-relate d changes in postnatal organ weights or histopathology, and th ere were no effects on semen quali ty. In an additional rat developmental toxicity study by Jacobsen et al. (2012), male pups dosed from GD 7 through
PND 16 showed no effects of tebuconazole treatment at ≤ 50 mg/kg/day on reproductive organ weights, semen quality or age at PPS. Finall y, in a rat reproductive toxicity study (Moser et al.

, 2001) examining effects from pre- and post-natal exposure (GD 14 to PND 7) to tebuconazole at 6, 20 or 60 mg/kg/day, high-dose male pups at PND 45 had reduced epididymides weights ( ↓ 17%) that may have been related to decrease d body weight; no effects were noted on AGD, age to PPS, testes weight, or sperm counts or mobility.

In a bobwhite quail study submitted as OSRI, wh ile not specific to the androgen-pathway, a significant effect on percent normal hatchlings and subsequent offs pring survival and weight was reported at the high dose (290 ppm); there were no androgen-related endpoints affected in the other avian reproduction studies. In a fathead minnow fish sexual development study, there was
no difference in sex ratio but effects on ma le gonadal histopathology were reported at ≥ 0.022 mg/L for males. However, histopathological change s in other tissues were also reported at these concentrations including the live r, pancreas and intestines ( ≥ 0.011 mg/L), kidneys (0.045 mg/L) and spinal deformities ( ≥ 0.022 mg/L). No effects on androgen-re lated endpoints were reported in the fish life-cycle study.TEBUCONAZOLEFINALPage 19 of 81Table 3: Androgenic/Anti-Androgenic Pathway for TebuconazoleLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Tebuconazole1Study Type/Literature Citation AR Binding/ARTA 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/Fecundity Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48619401)PSteroidogenesis(MRID 48619404)PHershberger(MRID 48673702)NENN NMale Pubertal Rat(MRID 48688302)↓53%(H)NP(L, H)↓13-14%(H)P(H)↓9, 12%(L, H)P4(H)NP5(L, H)NFSTRA(MRID 48615506)NE↓19%(H)NNNN P6NOSRIARTA/Steroidogenesisassays(Kjærstadet al., 2010)PPARTA assays(Ortonet al., 2011)PHershberger (Rat;Taxviget al., 2008)NENX(H)NSubchronic Toxicity(Rat; MRID40700930)NNNENNENEX(L, M,H)X(H)Subchronic Toxicity(Dog; MRID40700934)NNNENNENEX(L, M,H)NTEBUCONAZOLEFINALPage 20 of 81Table 3: Androgenic/Anti-Androgenic Pathway for TebuconazoleLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Tebuconazole1Study Type/Literature Citation AR Binding/ARTA 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/Fecundity Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Chronic Toxicity(Dog; MRID40700940)NNNENENENEX(M,H)NChronic Toxicity(Dog; MRID42030601)NNNENNENEX(H)NChronic toxicity/carcinogenicity (Rat;MRID 40700939)NNNENNENEX(H)NEight-Week RangeFinding Study(Mouse; MRID40700933)NNNENENENEX(L, M,H)NCarcinogenicity(Mouse; MRID40700941)NNNENNENEX(M,H)NCarcinogenicity(Mouse; MRID42175001)NNNENNENEX(L, H)NDevelopmentalToxicity (Rat; Taxviget al., 2007)NNNENNP(L, H)X(H)NDevelopmentalToxicity (Rat; Taxviget al., 2008)P7NNENDevelopmentalToxicity (Rat; Hassetal., 2012)PPPNE NTEBUCONAZOLEFINALPage 21 of 81Table 3: Androgenic/Anti-Androgenic Pathway for TebuconazoleLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Tebuconazole1Study Type/Literature Citation AR Binding/ARTA 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/Fecundity Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3DevelopmentalToxicity (Rat;Jacobsenet al., 2012)NNNNNNX(H)NTwo-GenerationReproductionToxicity (Rat; MRID40700946)NNNENX(H)NDevelopmentalNeurotoxicity (Rat;MRID 45074301)NX(L, M,H)NReproductiveFunction (Rat; Moseret al., 2001)NN↓17%(H)NNNX(H)NAvian reproduction(Bobwhite quail;MRID 40700910)NN NAvian reproduction(Bobwhite quail;MRID 41624201)P8N NAvian reproduction(Duck; MRID40700909)NN NAvian reproduction(Duck; MRID41818301)NN NFish SexualDevelopment Toxicity(Fathead minnow,MRID 48109802)P9NX(MH)X(MHH)TEBUCONAZOLEFINALPage 22 of 81Table 3: Androgenic/Anti-Androgenic Pathway for TebuconazoleLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Tebuconazole1Study Type/Literature Citation AR Binding/ARTA 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/Fecundity Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Fish Full Life CycleToxicity (Sheepsheadminnow, MRID43009601)NNX(H)1.Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter isnot routinely evaluated or is not applicable in this assay.Changes in weight are absolute unless otherwise indicated. as Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate (PR), Levatorani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP), and tubercle score (TS).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 anX 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 this column. Fordetails see Section IV. A4.Weights of several androgen-target organs also decreased in a dose-dependent manner (seminal vesicle 17-33%, dorsolateral prostate 21%, LABC 11-24%, epididymis 2-14%) although the change at the low dose was statistically significant only for the dorsolateral prostate.5.These androgen-related effects occurred in the absence of overttoxicity but co-occurred with decreases in serum bilirubin (75%at low and not detectedat high dose) and urea nitrogen (10% at high dose), decreased organ weights of kidneys (7-9%) and adrenals (18-13% not dose-dependent), and a 10%increase in liver weight (high dose, relative only).6.For the liver, the occasional findings of microgranulomas (minimal) and individual hepatocyte necrosis (minimal) in the liverswere likely treatmentrelated (based on findings in females); there was also a dose-related decrease in hepatocyte vacuolation compared to control7. In GD 21 fetal testis, levels of testosteronedecreased 50% at 100 mg/kg/day, while levels of 17α-hydroxyprogesterone increased 330% at 50 mg/kg/dayand 238% at 100 mg/kg/day. Plasma testosterone levels were not changed from controls in male fetuses or dams.8. At high dose, 290 ppm, 11% reduction in normal hatchlings, 37% decrease in offspring survival and 7-11% decreases in offspring body weight.Reductions in offspring body weight (4-6%) in the low treatment group (156 ppm)9. No difference in sex ratio but effects on male gonadal histopathology were reported at≥0.022 mg/L for malesP Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not ExaminedTEBUCONAZOLE FINAL

Page 23 of 81 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

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

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

There were no effects of tebuconazole noted on serum T 4 or TSH levels, thyroid weights, or thyroid histopathology in either th e male pubertal or female pubertal assays. In the male pubertal study, pituitary weights were decreased by 9-12 % (75 and 150 mg/kg/day); these decreases occurred in the absence of overt toxicity but co-occurred with 7-9% de crease (dose-dependent) in mean kidney weights and 13-18% decrease (not dose-dependent) in mean adrenal weight at the low and high doses, and 10% increase relative liv er weight at the high dose. There were no effects of treatment on pituitary weights in the female pubertal study.

In the AMA at the highest test concentrati on (0.341 mg/L), mild thyroid hypoplasia was observed in all tadpoles, and a significant 56% reduction in no rmalized hind-limb length (HLL) on Day 21. Additionally, a significant delay of median Nieuwkoop-Faber (NF) developmental stage by one stage was observed at Day 7 at all treatment groups (0.017, 0.067 & 0.341 mg/L), and significant delays at Day 21 at the mid (by one stage) and high (by 5 st ages) concentrations. Asynchronous development was not observed. La te-stage tadpoles (N F Stage >60) were observed in the study in the ne gative control (55%) and low dose group (38%), whereas latestage tadpoles accounted for 7% and 0% of survivors in the middle and high dose groups. Significant reductions in snout-v ent length (SVL) and body weight (BW) were observed at the high concentration on both Day 7 (SVL: ↓ 16%; BW: ↓ 24%) and 21 (SVL: ↓ 24%; BW: ↓ 59%) and in the low treatment on Day 21 (SVL: ↓ 6%; BW: ↓ 12%). In the high treatment, two tadpoles were reported as pale while two more tadpoles showed erratic behavior/loss of equilibrium. Additionally, histological findings in the liver were reported in the mid and high treatment levels.

A total of thirteen mammalian Part 158 guideli ne studies on tebuconazole were submitted as
OSRI. There were no HPT effects observed in six of these studies, which included a rat subchronic toxicity study, a dog subchronic toxicity study, two dog chronic toxicity studies and two mouse oncogenicity studies. In a rat chronic toxicity/carci nogenicity study, the incidence of thyroid C-cell (i.e. parafollicular cells) hyperplasia was somewhat elevated at the mid (16 mg/kg/day; p<0.05) and high (55 mg /kg/day) dose males; however th ese cell function s relate toTEBUCONAZOLE FINAL

Page 24 of 81 calcium homeostasis and are not involved in thyroid hormone produc tion or homeostasis. Systemic toxicity was also observed in female rats, along with increased activity of several hepatic metabolic enzymes at all doses tested.

Three journal articles relevant to the thyroid pathway were submitted as OSRI. In a Hershberger assay (Taxvig et al. , 2008), serum T 4 levels were measured but were unaffected by treatment. In a developmental toxicity study (Taxvig et al. , 2007), dam serum thyroid hormones were decreased at the high dose (100 mg/kg/day): T 3 levels by 11% (p ≤ 0.05); and T 4 levels by 29% (NS). However, systemic toxicity was also observed at this dose, including decreased body weight gain during gestation ( ↓ 29%) and body weight loss dur ing the lactation period (

− 4.57 g treated vs. +7.53 g controls), increased gestation length and increased post implantation loss. In an embryonic zebrafish study, serum T 3 levels were increased by 28% while whole body T 4 levels were decreased by 10% at th e highest dose tested (4 mg/L) (Yu et al. , 2013). Additionally, the following HPT axis genes a ppeared to be upregulated by tebuconazole treatment: corticotrophin-rel easing hormone, thyroid stimula ting hormone, sodium/iodide symporter, transthyretin, urid ine diphosphate glucuronosyltransf erase, thyronine deiodinase 2 and thyroid hormone receptor β

.TEBUCONAZOLE FINAL

Page 25 of 81 Table 4. Thyroid Pathway for Tebuconazole.

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

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth (BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assay

Male Pubertal Rat (MRID 48688302) N N N N ↓ 9, 12% (L, H) P 4 (L, H) N

Female Pubertal Rat (MRID 48688302) N N N N N LW (M, H);
KW (H) N

AMA (MRID 48673701) P 5 (H) Day 7: ↓ 1 NF stage (L,M,H);

Day 21: ↓ 1 N F

stage (M); ↓ 5 NF stage (H); ↓ 56% HLL (H) SVL: Day 7: ↓ 16% (H); Day 21: ↓ 6.3, 24% (L, H) BW: Day 7: ↓ 33% (H); Day 21: ↓ 12, 59% (L, H) P 6 (M,H) N

OSRI Hershberger assay (Taxvig et al. , 2008) N
Developmental Toxicity (Taxvig et al. , 2007) T 3 ↓ 11% (H) X (H) N

Subchronic Toxicity (Rat; MRID 40700930) N X (L, M, H) X (H)

Subchronic Toxicity (Dog; MRID 40700934) N N X (L, M,

H) X (H)

Chronic Toxicity (Dog; MRID 40700940) N N X (M, H) N

Chronic Toxicity (Dog; MRID 42030601) N X (H) N

Chronic toxicity/ carcinogenicity (Rat; MRID 40700939) N X (L, M, H) NTEBUCONAZOLE FINAL

Page 26 of 81 Table 4. Thyroid Pathway for Tebuconazole.

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

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth (BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

Oncogenicity (Mouse; MRID 40700941) N X (L, M, H) N

Oncogenicity (Mouse; MRID 42175001) N X (L, H) N

Thyroid Endocrine Disruption (Zebrafish; Yu et al. , 2013) Whole body levels:
T 3 ↑ 28%
T 4 ↓ 10% (H) N

1. Key to responses: L=Low treatment, ML=Mediu m-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not app licable in this assay. Changes in weight are absolute unless otherwise indicated. 2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 3. The overt toxicity in the Tier 1 assays are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A
4. These androgen-related effects occurred in the absen ce of overt toxicity but co -occurred with decreases in serum bilirubin (75% at low and not detected at high dose) and urea nitrogen (10% at high dose), decreased organ weights of kidneys (7-9%) and adrenals (18-13% not dose-dependent), and a 10% increase in liver weight (high dose, relative only).
5. At the highest test concentration (0.341 mg/L) mild thyroid hypoplasia was observed in all tadpoles
6. Histological findings in the liver were reported in the mid and high treatment. P Positive findings N Negative findingsTEBUCONAZOLE FINAL

Page 27 of 81 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 tebuconazole to interact with endocrine related processes ( i.e., E, A or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing.. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated an alysis of those line s of evidence.

The WoE evaluation includes consid erations of biological plausibility of the findings from the different lines of evidence by examining the co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substance may contribute to the WoE evaluati on in helping el ucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively 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 Tire 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. Overt/Systemic Toxicity in the in vivo Tier 1 Assays and OSRI
Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. In addition to the endocrine -related effects described above
for the in vivo Tier 1 assays and OSRI, other eff ects observed which may be considered overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. However, in other instances, mo re than one parameter was needed to determine overt toxicity. Effects which were considered to be signs of system ic toxicity were generally less severe forms of toxicity (e.g., changes in organ weights or blood chemistry).TEBUCONAZOLE FINAL

Page 28 of 81 1. Tier 1 in vivo Assays

Rats were treated at doses of 75 and 150 mg/kg/da y in all in vivo studies; in the Hershberger and uterotrophic assays a dose 35 mg/kg/day was al so administered with no effects. In the Hershberger assay, overt toxicity was not observe d in 58-days old male rats at any dose and other systemic toxicity was not evaluated. Wh ile mean overall body weight gain was decrease 20% at the high dose in the andr ogen agonist portion of the He rshberger assay, this did not translate into a net decrease in mean body weight. Overt toxicity was also absent from the rat pubertal assays (22-day old female s and 23-day old males at start of assay). A small decreased in mean body weight (8% compared to controls ) was observed in pubertal males at the high dose but not in pubertal females. Liver weights were increased 8% at the lo w and 15-18% at the high dose in females, but in males only relative liver weight was increased 10% at the high dose. The mean liver weight changes seem to correlate with mean changes in blood urea nitrogen (BUN), total bilirubin and total cholesterol that are more significant in females ( ↓ 15-17%, not detected,
and ↑ 17%, respectively) than in males (10%, 75%- not detected, no change). Kidney weights
were decreased 7% at the low a nd 9% at the high dose in pubert al males but only relative kidney weight was increased at the hi gh dose in pubertal females; cr eatinine was decreased 8% in females but not in males. Adrenal weights were significantly decreased in males at the low (15- 18%) and high (13%) doses, but not in pubertal fe males. The differences in systemic toxicity between pubertal males and females are most likel y due to the different exposure durations (30 days males vs. 20 days females).

Conversely, overt toxicity was observed in 19-day old female rats in the uterotrophic assay at 150 mg/kg/day tebuconazole as a mean body weight lo ss of 3.0 g (compared to a gain of 8.2 g in controls), which resulted in a 24% decrease in mean terminal body weight compared to controls (p<0.01). In addition, five of six females in the high dose group had reduced motor activity on the final day of the uterotrophic assay (Day 3).

In the AMA, in the high concentration treatmen t group (0.341 mg/L), two tadpoles were reported as pale while two more tadpoles showed erra tic behavior/loss of e quilibrium. Additionally,
effects on the liver were re ported at the mid (0.067 mg/L) and high treatment [included decreased hepatocyte vacuolati on, hepatocyte anisocylosis, hepa tocyte anisokaryosis, increased multinucleate hepatocytes and individual he patocyte necrosis (0.341 mg/L only)].

In the FSTRA, no signs of overt toxicity were ob served in the treated groups. For the liver histopathology, for males, the oc casional findings of microgranulom as (minimal) and individual hepatocyte necrosis (minimal) in the livers we re likely treatment-related (based on female findings); there was also a dose-rela ted decrease in hepatocyte vac uolation compared to control. In females, dose-dependent findings in the liver included individual hepatocyte necrosis which occurred in all three treatment groups. Additi onally, in the mid and high treatments (0.011 andTEBUCONAZOLE FINAL

Page 29 of 81 0.0195 mg/L) increases in multinucleated hepatocytes, microgranulomas, hepatocyte megalocytosis and decreased hepatoce llular vacuolation were reported.

2. OSRI

Conazoles such as tebuconazole were desi gned to specifically inhibit CYP51, a sterol

14- α -demethylase which catalyzes the 14- α -demethylation of lanoste rol in the ergosterol biosynthesis pathway in fungi. Th e biosynthesis of ergosterol is critical to the formation of fungal cell walls. The lack of normal sterol pro duction slows or stops the growth of the fungus, effectively preventing further infection and i nvasion of host tissues (Ghannoum and Rice, 1999). Cyp51 is a conserved gene across many taxi incl uding fungi, protists, mammals, and plants and is required for sterol biosynthesis in all eukar yotic systems. Mammalian cholesterol synthesis is blocked by conazoles at the step of the sterol 14- α -demethylase, but a much higher concentration is required to cause the same degree of inhibition as in fungi (de Brabander et al. , 1980; Van de Bossche, 1980; Zarn et al. , 2003). Tebuconazole has been shown to result in adverse health outcomes related to the liver and the endocrine sy stem. The key events le ading to these adverse effects have not been fully elucidated. However, mechanistic information related to toxicity pathways associated with the liver and endocrine system can be used to inform the EDSP WOE
for tebuconazole for mammals.

Lipid, sterol, and steroid homeos tatic pathways are regulated by the constitutive androstane (CAR), pregnane X (PXR), peroxisome pr oliferator-activated receptor alpha (PPAR α ) and other nuclear receptors in liver. In mechanistic studi es conducted by EPA’s Office of Research and Development (ORD), relatively high doses of conazo les such as propiconazole appear to perturb fatty acid and steroid metabolism in the male rat liver predominantly through the CAR and PXR signaling pathways (Goetz and Dix; Goetz et al. , 2006). Since both CAR and PXR regulate the expression of CYP genes and the associated enzy me activity of oxidative metabolism of steroid hormones as well as xenobiotics, it may be that CAR and PXR are also components of a regulatory network that regulates circulating hormone levels.

As in the Tier 1 studies, the ta rget organ for tebuconazole toxici ty was generally the liver, with minor effects observed on the kidney and adrenal gland. dogs. In a published, multi-part study including a Hershberger assay by Taxvig et al. (2008), no signs of overt toxicity were observed; however, liver weights were increase d at the high dose (150 mg/kg/day).

In the rat subchronic toxicity study, overt toxicity was observed in the form of mortality of one high dose (1600 ppm) male and one high dose fema le. In the mid (400 ppm) dose males and high dose males and females, body weights were d ecreased, and overall body weight gains were also decreased at the high dose in both se xes. Effects on liver included increases in N

demethylase activity in high-dose males and increa ses in activity of cytochrome P-450 in midand high-dose males, and in the low-, mid-, and high-dose females. Vacuole formation in theTEBUCONAZOLE FINAL

Page 30 of 81 zona fasciculata of the adrenal glands was not ed in the high dose males and mid and high dose
females.

In the dog subchronic toxicity study, overt toxicity was noted , as one high dose (5000 ppm) female was found dead on Day 2. Body weights we re decreased in the mid and high dose males and females, with an overall decrease in body weight gain in both sexes. Lens opacity of the eyes was observed in the high-dose group, such th at all high-dose dogs were observed with this finding at Week 10. Increased plat elet counts and signs of severe anisocytosis were noted at the high dose. Serum alkaline phosphatase activ ity was increased, and serum albumin was decreased in the high dose males. Liver N -demethylase activities were increased at the low (200 ppm), mid and high dose in both sexes; cytochro me P-450 activities were increased to a lesser extent in the mid and high dose males and in the high dose females. Spleen weights were increased at the high dose in both sexes. Liver weights were slightly in creased in the mid and high dose males. Microscopic findings in the high dose group included s light degeneration of the posterior wall of the lens of the eye, cataract- like eosinophilic plaques, bilateral cataracts and slight siderosis in the liver and spleen. In the females, increase d vacuole formation was noted in the adrenals of one low dose, one mid dose and two high dose dogs.

In a dog chronic toxicity study, there were no signs of overt toxicity. Lenticular lesions (opacities, lens stars) were noted in the eyes in two animals of the mid dose (400 ppm) as well as one dog at the high dose (1000 ppm). In addition, corneal opacities (clo udy, extensive) were elevated in one dog in each of the mid dose a nd high dose groups. Alkaline phosphatase levels
were elevated over the course of treatment in the high dose males and females; liver Ndemethylase activity was significantly elevated at the high dose, as well as liver triglycerides. Absolute and relative liver weights were incr eased in the mid and high dose males, with a consistent change in the appearance of the liver in mid and high dose dogs, as well as a slight increase in the presence of siderin in mid a nd high-dose males and ORO-positive lipids in one dog in each of the mid and high-dose groups. Ot her potential target organs/tissues are the hematopoietic system, the adrenals, the kidneys and the spleen.

In a second dog chronic toxicity study, no signs of overt toxicity were observed. Microscopic pathology demonstrated the adrenal gland to be a target organ at the high dose (150 ppm) in both sexes. Hypertrophy of adrenal z ona fasciculata cells and fatty ch anges in the zona glomerulosa were observed in both sexes, and lipid hyperpla sia in the cortex was noted in the males.

In the rat chronic toxicity/carci nogenicity study, no signs of overt toxicity were noted. However, female body weights were decreased at the mi d (300 ppm) and high ( 1000 ppm) dose throughout the study. The high-dose females also had slight ly increased food consumption. In the highdose females, there were small decreases in hemoglobin, hematocrit and altered mean corpuscular concentrations and volumes which corre lated with an increased deposition of splenic
hemosiderin. Dose-related decreases in female absolute and relative adrenal weights wereTEBUCONAZOLE FINAL

Page 31 of 81 associated with a dose-related decrease in th e incidence of adrenal cortical hemorrhagic degeneration at the mid and high dose. Ther e was also a dose-rela ted increase in liver microsomal enzyme induction at all dose levels te sted, based on histological examination. In the high dose males, gross pathology showed an increas e in kidney cyst/cystic kidneys and reddened lymph nodes; histological examination of the ly mph nodes revealed an el evation of blood-filled sinuses of the mesenteric lymph nodes.

In a rat eight-week range-findi ng study,, there were no signs of overt toxicity, but body weights were consistently decreased by 1-2 g in the hi gh dose (2000 ppm) females during treatment. In the males, total bilirubin was decreased at the hi gh dose, indirect bilir ubin was decreased at the low (500 ppm) and high dose, and serum iron was increased at the high dose. Absolute liver weights were increased at the lo w and high dose in both sexes. Absolute adrenal weights were decreased in the high dose females. Gross pat hology revealed swollen or increased lobulation of the liver in the low and high dose males, and pa le livers in the both se xes at the low and high dose. In the liver, there was an increase in th e incidence and severity of cell degeneration, cell necrosis, focal necrosis, and the pr esence of vacuoles and lipid cont ent at either or both doses. At the high dose, there was an increase in th e incidence of iron-related pigment and round cell infiltrates of the kidneys. An increase in co rtical cell lipids (mid and high dose) and sinus dilation (high dose) was observe d in the male adrenals. N -demethylase activity was increased in females at the low (125 ppm, mid ( 500 ppm), and high dose (2000 ppm), and O -demethylase activity was increased in the high-dose male s and in the mid-and high-dose females. Cytochrome P-450 activity was increased in the low-, mid-, and high-dose males and females. Finally, triglycerides were increased in the lo w dose males and in the mid and high dose males
and females.

In a mouse oncogenicity study, overt toxicity was not noted. A minimal decrease in male mean body weights was noted during the fi rst third of the dosing period at the high dose (180 ppm). Bilirubin values were increased in the high dose females at Week 53 and in the low (20 ppm), mid (60 ppm) and high females at Week 92. Slight increases (NS) in absolute and relative liver weights were noted in high dose males and fe males at Weeks 53 and 92/93. Gross pathology demonstrated an increase in male liver enlarg ement and the presence of liver nodules. Nonneoplastic histopathology indicated the liver as a primary target organ at the mid and high dose in both sexes. In males, the most frequent live r lesions were vacuolation and lipid deposition; in females, hepatic vacuolation, minimal extramedul lary hematopoiesis, sinusoidal cellularity and increased lipid deposition were noted. In males, the adrenals were affected at the mid and high dose with an increased enlargement and hyperplasia of cortical cells, and an increase in minimal stomach gastritis in the high dose group. Fema les also had elevations in the pancreatic interstitial edema and stomach gastritis at the mid or high dose levels.TEBUCONAZOLE FINAL

Page 32 of 81 In the second mouse oncogenicity study, there we re no signs of overt t oxicity, but distended abdomen was observed in the high dose (1500 ppm) males and females. In males, there were decreases in body weight at the low dose (500 ppm) during the ini tial half of th e testing period, and at the high dose up to Week 84. In females, th ere were sporadic decreases in body weight at the low dose during the initial third of the study, and at the high dose, there were decreased body weights in 39 of the first 59 weeks and in 7 of the last 8 weeks. Mean food consumption was increased frequently in the mid dose males and generally throughout the study at the high dose in both sexes. At sacrifice, leucoc ytosis and changes consistent with anemia, (increased platelet counts and decreased thromboplastin times) were noted in the high dose males. In the high dose females, a reduced hematocrit and leucocytosis and an increased platelet count were noted. Plasma activities of liver enzymes (GPT, GOT, AP) were increased in the high dose females. In addition, increases were observe d at the low dose for GPT in males and for GPT and GOT in
females. Cholesterol and albumin were decreas ed in both sexes at the low and high dose. Absolute liver weights were increased in the low dose males, and at the high dose in both sexes. Absolute kidney weights were decreased in th e low and high dose males. Gross observations included: enlarged liver at the high dose in both sexes; pale liver s in both sexes at the low and high dose; areas of change in the liv er in the high dose females; irre gular surfaces in livers at the high dose in both sexes; and liver masses in the high dose females. Microscopically, there were increases in extramedullary hematopoiesis (males), focal hyperplasia of he patocytes (both sexes), oval cell proliferation (b oth sexes), periacinar hepatocyte hypertrophy (females), eosinophilic foci of hepatocyte alteration (females) and pigmen t-laden Kupfer cells (females) at the high dose, and in hepatic panacinar fine fatty vacuolat ion the low dose males and high dose males and females. At the high dose, the incidence of hepatocellular adenom as and hepatocellular carcinomas was increased in males, and the incidence of hepatocellular carcinomas was
increased in females.

In a rat prenatal developmental toxicity st udy, overt toxicity was not observed. Mean body weights were slightly decreased in the high dose dams (120 mg/kg/da y) on Day 21, and mean body weight gains were decreased during dosing. These decreases were accompanied by a reduction in food consumption duri ng dosing. Mean maternal absolu te and relative liver weights were increased at the mid (60 mg/kg/day) and hi gh dose. Mean fetal weight was reduced at the high dose.

In a rabbit prenatal developmental toxicity study, there we re no signs of overt toxicity. Maternal mean body weight gains were d ecreased during dosing in the hi gh dose (100 mg/kg/day) does. When GD 6-28 body weight gains were corrected for gravid weights, a sli ght loss was noted at the high dose. Food consumption was also decreased during dosing at the high dose.

In a second rabbit prenatal deve lopmental toxicity study, overt to xicity was not noted. Single
cell necrosis of the liver was seen in all does in the low (10 mg/kg/day), mid (30 mg/kg/day) and high (100 mg/kg/day) dose groups.TEBUCONAZOLE FINAL

Page 33 of 81 In a published, multi-part study including a developmental toxicity study by Taxvig et al. (2008), no signs of overt toxicity were observed. However, the frequency of post-implantation loss was
increased ( 11.4%) compared to controls (0%) at th e only dose administered (50 mg/kg/day).

In a published developmental toxicity study by Hass et al. (2012), overt or systemic toxicity
were not observed at the high dose (50 mg/kg/day).

In a published developmental toxicity study by Jacobsen et al. (2012), overt toxicity was not noted, however, liver weight was increase the high dose (50 mg/kg/day) males.

In a two-generation reproduction to xicity study (1987), there were no signs of overt toxicity. Clinical signs of toxicity at the high dose (1000 ppm) included loss of hair in the P and F 1b females, inflammation/changes of the eye in the P males and ill/bad condition in the F 1b males.
Loss of hair was also increased in the mid dose (300 ppm) F 1b females. Body weights were consistently decreased at the hi gh dose in both sexes in both ge nerations. Organ weight data presented for the high dose F 1b parents indicated decreases in terminal body weights and absolute liver, kidney and adrenal weights in the males, and in terminal body weights and kidney weights
in the females.

In a published reproductiv e toxicity study by Taxvig et al. (2007), no signs of overt toxicity were
noted. Treatment-related effect s observed at the high dose (100 mg/kg/day) included decreased body weight gain during gestation, increased gestation le ngth, increased post implantation loss and body weight loss during lactation.

In a developmental neurotoxicity study, there we re no signs of overt toxicity. The following findings were noted at the hi gh dose (1000 ppm); body weights were decreased in the P females during gestation and early lact ation; body weight loss was no ted during GD 6-9; body weight gains were decreased during GD 6-21 and 0-21; body weight loss was also noted during LD 1-4; body weight gains were decreased during LD 4-7. Both absolute and relative maternal food consumption were reduced beginning on GD 6, and continuing through early lactation. The
number of live fetuses/dam was decreased while the number of dead fetuses/dam was increased. At the low (100 ppm), mid (300 ppm) and hi gh dose, pup body weights and body weight gains
were decreased in both sexes. Pinna unfolding was slightly delaye d at the mid and high dose.

In a published reproductiv e function study by Moser et al. (2001), no signs of overt toxicity were noted. At the high dose (60 mg/kg/day), matern al body weights were decreased on GD 21, with body weight gains during gestation decr eased. Post-mating necropsy of F 1 males showed reduced kidney weights at the high dose whic h was likely due to a decreased terminal body weight. The number of dead pups/litter was increased at the high dose. At the PND 46 necropsy, relative (but not absolute) liver weight was increased in the high-dose females; the high-dose males had increased relative spleen and liver weights, and a decreased relative kidney weight.TEBUCONAZOLE FINAL

Page 34 of 81 In a Part 158 avian reproduction toxicity study on quail (MRID 40700910) and two avian reproductive studies on ducks (MRIDs 40700909 and 41818301), no overt or systemic signs of toxicity were noted at fed levels up to 74 ppm in quail and 611 ppm in ducks. In a second avian reproduction toxicity study on quail (MRID 41624201), th ere were no signs of overt toxicity, but treatment related lesions (liv er) were observed in the bird s at the high dose (290 ppm).

In a Part 158 rainbow trout early life stage toxi city study, overt toxicity was noted at the four highest test concentrations (0.025, 0.061, 0.120, and 0.230 mg/L) based on reduced survival of larvae at test termination. Development of larvae in the mid-high (0.120 mg/L) and high (0.230 mg/L) concentrations also appeared to be de layed by several days, and throughout the post-hatch exposure period larvae at the four highest test co ncentrations also exhibited abnormal appearance and behavior ( i.e. , dark coloration and lethargy).

In the Part 158 sheepshead minnow early life stage toxicity study, no signs of overt toxicity were noted. However, mean body length at the high concentration (0.0993 mg/L) was reduced compared to the solvent contro ls. Mean wet weight at the mid-high (0.0475 mg/L) and high concentrations was reduced compared to the solvent controls.

In a Part 158 fish full life cycle toxicity study with sheepshead minnow, ov ert toxicity was noted based on decreased parental survival on Da ys 58 and 177 at the high dose (0.197 mg/L). Parental body length was also reduced on Da y 33 at the mid-high dose (0.0967 mg/L), and growth ( i.e ., length and weight) of the F 1 generation was reduced at 28 days post-hatch at the mid-high dose.

In a fathead minnow fish sexua l development study, there was no effect on hatch but survival was significantly reduced at ≥ 0.045 mg/L with effects on female growth at 0.022 mg/L. Systemic toxicity was also reported in cluding histopathological change s in the liver, pancreas and intestines ( ≥ 0.011 mg/L), kidneys (0.045 mg/L) and spinal deformities ( ≥ 0.022 mg/L).

In a published thyroid endocri ne disruption study by Yu et al. (2013 ) on zebrafish embryos, there were no signs of overt or syst ematic toxicity up to 4 mg/L.TEBUCONAZOLE FINAL

Page 35 of 81 B. Estrogen Pathway

Although tebuconazole was negative in an OSRI ER binding assay and in the Tier 1 ERTA, it appears to alter estrogen production. In vitro studies showed tebuconazo le can potentially alter steroidogenesis and inhibits aromatase activity. In the in vivo mammalian studies, a delay in VO was observed in the female pubert al assay; this finding was corr oborated by other findings in
mammalian OSRI such as decreased number of o ffspring and gestational/d elivery alterations in
the absence of overt toxicit y. Additionally, complementary findings were observed in the FSTRA which included effects on plasma VTG, gonadal weight and histopathology. Furthermore, effects on gonadal histopathology were reported in a fish sexual development test. Therefore, the preponderance of the findings sugges ts that tebuconazole poten tially interacts with the estrogen pathway both in vitro and in vivo . It is noted that generally, these in vivo findings
co-occurred with liver ef fects that included increased weight and/or changes in histopathology.

C. Androgen Pathway

Effects on testosterone biosynthesi s and AR interaction were consis tently noted in the Tier 1 and

OSRI in vitro studies. In the mammalian in vivo studies, anti-androgen e ffects were observed in young but not adult animals. Test osterone levels were decrease d in the Tier 1 male pubertal assay, as well as in a reproductive toxicity in the published literature ; this concurred with decreased weights of androgen-depe ndent tissues. While there were limited findings in male fish
in the FSTRA which included a decrease in GS I, gonadal histopatholog ical findings were reported in a fish sexual development test. The preponderance of the findings suggests that tebuconazole has anti-androgenic activity in vitro and in vivo only in the young, and can interact with the androgen receptor and interfere with steroidogenesis. Again, it is noted that these in vivo findings co-occurred with liver effects that in cluded increased weight and/or changes in histopathology.

D. Thyroid Pathway

Based on the available data, there is limited evid ence for a direct effect of tebuconazole on the thyroid pathway or the HPT axis. Negative responses for the T pathway were seen for
tebuconazole in the in vivo Tier 1 male and female pubertal as says and six of the seven pertinent general toxicity studies which showed no effect from tebuconazole treatment. In the published studies, effects were noted on da m thyroid hormone levels in de velopmental studies, but these occurred in the presence of systemic toxicity. Developmental delays (including NF stage and normalized HLL) along with thyr oid hypoplasia were observed in the AMA study, however, this coincided with a significant reduction in grow th (length and body weight ) and liver effects. Although findings of thyroid gla nd hypoplasia are identified in th e AMA guideline as diagnostic criteria, no effects were seen on the incidence of follicular cell hypertrophy or hyperplasia, which are expected to be more commonly affected in the presence of a thyroid-related developmental delay. Overall, the thyroid and developmenta l findings in the AMA with tebuconazole areTEBUCONAZOLE FINAL

Page 36 of 81 inconsistent with a direct, thyr oid-related delay and may be more reflective of treatment-related growth inhibition.

E. Conclusions

Based on in vivo and in vitro results, tebuconazole showed poten tial to alter the steroidogenesis pathway which may result in endocrine-related eff ects in the estrogen and androgen pathways. In the androgen pathway, effects seem to have a greater impact on young than in adult mammals.
It is noted that the e ffects observed in mammals and fish coincided with liver effects.

Based on the available data, there was a lack of complementary and redundancy of evidence in
the available Tier 1 and OSRI da ta to suggest a potential intera ction with the thyroid pathway
either in mammals or wildlife.

V. EDSP Tier 2 Testing Recommendations

Tebuconazole showed potential to alter the steroidogenesis pa thway which may result in
endocrine-related effects in the estrogen and androgen pathways

For tebuconazole, the point of de parture (POD) for risk assessment is based on developmental neurotoxicity endpoint observed in offspring in a developmental neurotoxicity study in rats. The basis for the POD is alterations in motor activity , decreases in body weight and brain weight, and changes in brain morphometrics observed in the offspring at 8.8 mg/kg/da y, the lowest observed effect level (LOAEL). Since the study did not establish a no-observed-adverse-effect–level (NOAEL), the default 10X Uncertainty Factor (UF L ) was applied ( i.e ., for the use of a LOAEL) resulting in an extrapolated NOAEL of 0.88 mg/kg/day. This valu e is used as the current POD for calculating the chronic Reference Dose (RfD) for dietary risk assessment and calculating the Margins of Exposure (MOEs) for non-dietary exposure risk assessments. This POD is approximately 75-fold lower than the lowest dos es (75 mg/kg/day) that caused estrogen and androgen-related effects in the Tier 1 pubertal assays. Conseque ntly, the chronic RfD and the MOEs are protective of the endocrine-relate d effects seen in the pubertal assays.

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

For the wildlife the EDSP Medaka Extende d One-Generation Reproduction Test is recommended. In the FSTRA, effects were obser ved at 19.5 μg/L. Gonadal histological effects in the fish sexual developmen t test (FSDT) were observed at 0.045 and 0.022 μg/L for females and males, respectively, in the absence of effects on liver. The current chronic NOAEC for fish is 12 μg/L based on survival effects in the rainbo w trout early-life cycle. It is noted that no effects on fecundity were observed in the sheep shead minnow life-cycle st udy at concentrationsTEBUCONAZOLE FINAL

Page 37 of 81 up to 196 μg/L; effects on survival (F0) and gr owth (F1) were noted. Since the reproductive
effects observed in the FSTRA and FSDT occur at concentrations similar or below the current NOAEC, additional information on potential reproductive effects is recommended.

As there were no thyroid-rela ted effects observed in the AM A or convincing evidence of a potential thyroid-related interaction for tebuc onazole, no additional data are requested for amphibians.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for tebuconazole. In both spec ies, effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from tebuconazole exposure. Relative to the EDSP, ad ditional testing is not recommended.TEBUCONAZOLE FINAL

Page 38 of 81 VI. References

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Banman, C, Matlock, D, and Lam, C (2012) Short-term reproduction assay with fathead minnow ( Pimephales promelas ) exposed to tebuconazole technical under flow-through conditions. Bayer CropScience, Stilwell, KS, a nd Experimental Patholo gy Laboratories, Inc., Sterling, VA. Laboratory Report No. EBHWP002, July 2, 2012. MRID 48688301. Unpublished.

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Bomhard, E, and Ramm, W (1988) HWG 1608: study for chronic to xicity and cancerogenicity in Wistar rats. Bayer AG, Institute of Toxi cology, Wuppertal, Federal Republic of Germany. Laboratory Project ID 96711, January 25, 1988. MRID 40700939. Unpublished.

Bomhard, E, and Ramm, W ( 1988) HWG 1608: study for cancerogenicity in NMRI mice (administration in diet for up to twenty-one months). Bayer AG, Institute of Toxicology, Wuppertal, Federal Republic of Germany. S ponsored by Mobay Corporation, Stilwell, KS. Laboratory Study No. 96709, January 25, 1988. MRID 40700941. Unpublished.

Eiben, R (1987) HWG 1608: Two-generation study in rats. Bayer AG, Institute of Toxicology, Wuppertal, Federal Republic of Germany, a nd PATCO AG, Itingen, Sw itzerland. Laboratory Project ID 91064, November 12, 1987. Sponsored by Mobay Corporation, Kansas City, MO. MRID 40700946. Unpublished.

Fletcher, D, and Pedersen, C (1990) Folicur T echnical: toxicity a nd reproduction study in
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Hass, U, Boberg, J, Christiansen, S, et al. (2012) Adverse effects on sexual development in rat offspring after low dose exposure to a mixt ure of endocrine disrupting pesticides. Reprod.
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Jacobsen, P, Axelstad, M, Boberg, J, et al. (2012) Persistent devel opmental toxicity in rat offspring after low dose exposure to a mixt ure of endocrine disrupting pesticides. Reprod.
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Kjærstad, M, Taxvig, C, Nellemann, C, et al. (2010) Endocrine disrupting effects in vitro of conazole antifungals used as pesticides and pharmaceuticals. Reprod. Toxicol. 30 : 573-582.TEBUCONAZOLE FINAL

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Laws, S, Yavanhxay, S, Cooper, R, et al. (2006) Nature of the binding interaction for 50 structurally diverse chemicals with rat estrogen receptors. Toxicol. Sci. 94 (1) : 46-56.

Laws, S, Kariya, J, Wolf, M, et al. (2009) DSSTox EPA Estrogen Receptor Ki Binding Study (Laws et al. ) Database - (KIERBL): SDF file and documentation, Launch version: KIERBL_v1a_278_17Feb2009, http://www.epa.gov/ncct/dsstox/sdf_kierbl.html

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Moser, V, Barone, S, Smialowicz, R, et al. (2001) The effects of peri natal tebuconazole exposure on adult neurological, immunological, and repr oductive function in rats. MRID 45486901.
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800.

Overgaard, A, Holst, K, Mandrup, K, et al. (2013) The effect of peri natal exposure to ethinyl
oestradiol or a mixture of e ndocrine disrupting pesticides on kisspeptin neurons in the rat hypothalamus. NeuroToxicology 37 : 154-162.

Parker, R (2000) Developmental neurotoxic ity study of technical grade tebuconazole administered orally via diet to Crl:CD®BR VAF/Plus® presumed pregnant rats. Primedica Argus Research Laboratories, Inc., Horsham, PA. Labor atory Study No. 1702-004, March 1, 2000. MRID 45074301. Unpublished.

Porter, M, Jasty, V, Troup, C. M, et al. (1989) Safety evaluation of HWG 1608: chronic (1-year) feeding study in dogs. Miles Inc., Toxicology Department, Elkhart, IL . Laboratory Study No. 99673, June 28, 1989. MRID 42030601. Unpublished.

Ramm, W (1986) Range-finding toxi cological study with NMRI mice to establish dosage for a chronic study (feeding for eight weeks) and for de terminations of enzyme induction in the liver (feeding for five days). Bayer AG, Institute of Toxicology, Wuppertal, Federal Republic of Germany. Sponsored by Mobay Corporation, St ilwell, KS. Laboratory Project ID No. 94211, July 7, 1986. MRID 40700933. Unpublished.

Rouquie, D (2011) Evaluation of tebuconazole in the androgen receptor binding assay. Bayer SAS, Sophia Antipolis Cedex, France. La boratory Study No. SA 11212, December 8, 2011. MRID 48619401. Unpublished.TEBUCONAZOLE FINAL

Page 41 of 81 Scott Ward, G. (1991) Toxicity to embryos and larvae of the Sheepshead minnow ( Cyprinodon variegatus ) under flow-through test conditi ons. Toxikon Environmental Sciences, Jupiter, FL. Laboratory Study No. J9101002b, completion date not provided. Sponsored by Mobay Corporation, Agricultural Chemicals Di vision, Kansas City, MO. MRID 42038202. Unpublished.

Surprenant, D (1988) The toxicity of HWG 1608 Technical to rainbow trout ( Salmo gairdneri ) embryos and larvae. Laboratory Report N o. 87-11-2545, completion da te not provided. Springborn Life Sciences, Inc., Wareham, MA. Sponsored by Mobay Corporation, Stilwell, KS. MRID 40700914. Unpublished.

Toll, P.A. (1988) Effects of HWG 1608 (FOL ICUR) on Mallard Duck Reproduction. Study No. 87-675-01. Prepared by Mobay Corp., Biochemi stry/Wildlife Effects Group. Research and Development Dept. Stilwell, KA. Submitted by MOBAY Corp., Agricultural Chemicals Division, Kansas City, MO. MRID 40700909. Unpublished.

Taxvig, C, Hass, U, Axelstad, M, et al. (2007) Endocrine-dis rupting activities in vivo of the fungicides tebuconazole and epoxicon azole. MRID 48277401. Published: Toxicol. Sci. 100 (2) : 464-473.

Taxvig, C, Vinggaard, A, Hass, U, et al. (2008) Endocrine-disrupting properties in vivo of widely used azole fungicides. MRID 48264606. Published: Int. J. Androl

. 31 : 170-177.

Tinwell, H (2011) Evaluation of tebuconazole in the aromatase assay. Bayer S.A.S. (Bayer CropScience, Sophia Antipolis Cedex, France. Laboratory Study ID SA 11064, Dec 9, 2011. MRID 48619402. Unpublished.

Tinwell H (2011) Evaluation of tebuconazole in the H295R steroidogenesis assay. Bayer S.A.S., Bayer CropScience, Sophia Antipolis Cede x, France. Laboratory Study No. SA 11072, November 10, 2011. MRID 48619404. Unpublished.

Toll, P (1988) Effects of HWG 1608 (FOL ICURTM) on bobwhite quail reproduction. Mobay Corp., Biochemistry/Wildlife Effects Group, Res earch and Development Dept., Stilwell, KS. Laboratory Study No. 87-675-02, completion date not provided. MRID 40700910. Unpublished.

Toll, P (1990) Technical HWG 1608 (FOLICUR): a one generation reproduction study with bobwhite quail. Mobay Corporation, Kansas City, MO. Laboratory Project ID not provided, completion date not provided. MIRD 41624201. Unpublished.

USEPA. (2002) Development of estrogen binding data for approximately 300 chemicals: final data package submittal. Battelle, EPA C ontract No. 68-W-99-033, Work Assignment 3-04. Unpublished.TEBUCONAZOLE FINAL

Page 42 of 81 USEPA (2010) Tebuconazole: Report of the Endo crine Disruptor Review Team; Test Order#: EDSP-128997-142; -143; -144; -145; -147; -148; -149; -151; -153; -154; and -155. December 20, 2010. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

USEPA (2011) Endocrine Disrupt or Screening Program – Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention Science Coor dination and Policy. U.S. Environmental Protection Agency, Washington, DC.

von Keutz, E (1987) HWG 1608: subchronic study of toxicity to dogs with oral administration (thirteen-week feeding study). Bayer AG, Institu te for Toxicology, Wuppertal, Federal Republic of Germany. Sponsored by Mobay Corporat ion, Kansas City, MO. Laboratory Study No. 94984, May 6, 1987. MRID 40700934. Unpublished.

von Keutz, E (1987) HWG 1608: study of chronic toxicity to dogs after oral administration (twelve-month feeding study). Bayer AG, Institute for Toxicology, Wuppertal, Federal Republic of Germany. Sponsored by Mobay Corporati on, Kansas City, MO. Laboratory Report No. 956090, November 11, 1987. MRID 40700940. Unpublished.

Wheat, J (1993) HWG 1608 (tebuconazole): life cycle chronic toxicity to the Sheepshead
minnow (Cyprinodon variegatus) under flow-through conditions. Toxikon Environmental Sciences, Jupiter, FL. I.aboratory Study No. J9104001, completion date not provided. Sponsored by Miles Incorporated, Kansas City, MO. MRID 43009601. Unpublished.

Yu, L, Chen, M, Liu, Y, et al. (2013) Thyroid endocrine disrupti on in zebrafish larvae following exposure to hexaconazole and tebuconazole. Aquat. Toxicol. 138 : 35-42.TEBUCONAZOLE FINAL

Page 43 of 81 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100)

The 21-day assay (MRID 48673701) of Tebucon azole (97.5% purity) on amphibian metamorphosis of South African clawed frogs ( Xenopus laevis ) was conducted under flowthrough conditions. Amphibian larvae at Nieuwkoop- Faber (NF) stage 51 (80/group, 20 in each of four reps) were exposed to nominal concentrations of 0 (negativ e control), 0.0152, 0.076, and 0.380 mg a.i./L; mean-measured concentratio ns were <0.0015 (<LOQ; control), 0.0170, 0.067, and 0.341 mg a.i./L. The test system was mainta ined at 21.4 to 22.7°C and a pH of 7.9 to 8.4.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. The Day 21 survival averaged 10 0% in the control and low treatment group and 98.3% in the mid and high treatment groups. Tad poles with bent tails were observed in the control (16/80 tadpoles) and low (14) and mid (5 ) treatment groups. In the high treatment, two tadpoles were reported as pale while two mo re tadpoles showed erratic behavior/loss of equilibrium.

Late-stage tadpoles made up more than 20% of the surviving tadpoles on Day 21 in the negative control (55%) and low tr eatment group (38%), whereas late-s tage tadpoles accounted for 7 and
0% of survivors in the mid and high treatment groups. All gr owth analyses were conducted excluding late-stage tadpoles and separate analyses were not conduc ted on the late-stage tadpoles because none were present in the highest dose. Tebuconazole had no effect on 7-day normalized hind-limb length (HLL), whereas the highest dos e exhibited a significan t 56% reduction relative to the control on Day 21. Significant delay of me dian NF developmental stage by one stage was observed at 7 days in all treatment groups and at 21 days in the mid and high treatment groups (by one stage at the mid and 5 stages at the high). No asynchronous development was observed in any treatment group. Given the large differen ces in NF stage between the control and high treatment group, additional control tadpoles were reared with the goal of selecting tadpoles at developmental stages similar to the high tr eatment (NF stage 56 and 57). Mild thyroid hypoplasia was observed at the high treatment gr oup in all 20 tadpoles, whereas this finding was not observed in the concurrent control, low or mid treatment group but was observed in 11/20 tadpoles in the stage-matched control. Mild follicular cell hypertr ophy was observed in the concurrent control (6/20 tadpoles), staged-match ed controls (2/20 tadpo les) and low and mid treatment groups (3/20 and 6/20 tadpoles respec tively). The procedure of rearing additional control frogs to allow for stage-matching histopat hological evaluation is not part of the guideline, and there is uncertainty in the ability to compar e these frogs to the treatment groups given that they were not from the same batch of eggs and were not reared at the same time which may have resulted in slight differences in environmental conditions.

Significant reductions in snout-v ent length (SVL) and body weight (BW) were observed at the high concentration on both Day 7 (SVL: ⇘ 16%; BW: ⇘ 24%) and 21 (SVL: ⇘ 24%; BW: ⇘TEBUCONAZOLE FINAL

Page 44 of 81 59%) and in the low treatment on Day 21 (SVL: ⇘ 6%; BW: ⇘ 12%). In the high treatment, two tadpoles were reported as pale while two mo re tadpoles showed erratic behavior/loss of equilibrium. Histological findings in the liver occurred in the mid and high treatment groups, and included decreased hepatocyte vacuolation, hepato cyte anisocylosis, hepatocyte anisokaryosis and increased multinucleate hepatocytes. An a dditional treatment-rela ted finding in the high treatment group was individual hepatocyte necrosis.

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

In an androgen receptor (AR) binding assay (MRID 48619401), ventral prostate cytosol from male Sprague Dawley rats was used as the source of AR to conduct a saturation binding experiment and a competitive binding experime nt. The saturation binding experiment was
conducted to demonstrate that the AR in the ra t prostate cytosol was present in reasonable numbers and was functioning with appropriate affi nity for the radiolabeled reference androgen (R1881) prior to routinely conducting AR compe titive binding experiments. The competitive binding assay was conducted to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentratio ns of tebuconazole (loga rithmic increase from

10

− 10 to 10

− 3 M). DMSO was used as the solven t at a final assay concentration of ≤ 3.3%. The assay included dexamethasone as a weak posi tive control, and R1881 as the ligand reference
standard.

In the saturation binding experiment , the mean maximum binding capacity (B max ) was 14 fmol/100 μg protein and the m ean dissociation constant (K d ) was 1.01 nM. The K d obtained from Saturation Assay #2 was low (0.59 nM), but the K d s from the other two runs were within the recommended range (0.685 to 1.57 nM) and th e Scatchard plots for the three saturation assays were linear. The speci fic binding reached a plateau, a nd non-specific binding (NSB) was less than 20% of total binding ( 4.6-12.6%). The saturation binding experiment demonstrated that AR was present in a reasonable c oncentration and functioning with appropriate affinity for the R1881 ligand.

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

− 4.3 M for tebuconazole compared to that of R1881 of

− 9.1 M and the weak positive control of

− 4.4 M. The mean RBA was 0.0014% for tebuconazole compared to 0.0021% for the weak positive control. No precipitation of the test compound was visually observed at any concentration. All performance criteria were met, with the exception that liga nd depletion and NSB were not reported, but the

IC 50 s for R1881 and the weak positiv e control were within the e xpected ranges demonstrating that the ligand was available at an appropriate level. These de viations were considered minor and performance criteria were found to be met.TEBUCONAZOLE FINAL

Page 45 of 81 Tebuconazole reduced the mean specific binding of the radiolabeled liga nd in a concentration dependent manner in all three runs. The ma ximum binding reduction oc curred at the highest concentration tested (10 − 3 M), with specific binding being reduced to 1.5-6.9%. Specific binding
was also reduced at 10 − 4 M (33-37%), but not at concentrations ≤ 10 − 5 M (82-111% binding). Specific [ 3 H]-R1881 AR binding was reduced to <50% at the highest conc entrations tested in all
three runs.

Based on the results from the thr ee runs, tebuconazole is classifi ed as a Binder in the Androgen Receptor Binding Assay.

Aromatase Assay (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48619402) , tebuconazole (97.5% a.i., batch# AE F069623-01-18) was incubated with human recombin ant aromatase and tritiated androstenedione (1- β [ 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) in dimethyl sulfoxi de (DMSO) at increasing logarithmic concentrations from 10 –10 M to 10

− 3 M for 15 minutes to assess the potential of tebuconazole to inhibit aromatase activity.

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

− 10 to
10

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

− 10 to 10

− 3 ) with three repetitions per concentration.

Aromatase activity in the full act ivity controls ranged from 0.250 to 0.365 nmol/mg-protein/min for the three test runs, with a mean and sta ndard deviation of 0.306±0.038 nmol/mg-protein/min. Activity in the background controls ranged from 3.3 to 4.4% of the full c ontrol activity with an average of 3.6%. The response of the full act ivity controls and b ackground controls was acceptable.

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.2925±0.0202 nmol/mg-protein/min at the lowest tested concentration (10

− 10 M) and 0.0028±0.0005 nmol/mgprotein/min at the highest tested concentration (10

− 5 M). These results were generally within the recommended ranges for the top of the curve, bottom curve, Hill slope, and log IC 50

.

For tebuconazole, aromatase activity averag ed 0.2706 ± 0.0069 nmol/mg-protein/min at the lowest tested concentration (10

− 10 M) and 0.0004 ± 0.0009 nmol/mg-pr otein/min at the highest tested concentration (10

− 3 M). The lowest portion of the response curve for tebuconazole acrossTEBUCONAZOLE FINAL

Page 46 of 81 runs was less than 50% activity. Tebuconazo le reduced aromatase activity to 90% at 10

− 7 M,
62% at 10

− 6 M, 19% at 10

− 5 M, 2.3% at 10

− 4 M, and 0.1% at 10

− 3 M.

The estimated log IC 50 ranged from

− 5.8 to

− 5.7 M for the test material and averaged

− 5.73 M compared to the positive control 4-OH ASDN, which averaged

− 7.20 M. The average slope of the concentration response curve was

− 0.99 for tebuconazole and

− 0.88 for 4-OH ASDN.

Based on the data form the average response curve, tebuconazole is classified as an Inhibitor of aromatase activity in this assay.

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

The requirements for a Tier 1 ER receptor bindi ng assay were satisfied by the study by USEPA (2002). EPA tested tebuconazole in a rat uterin e cytosol ER competitive binding assay as one of
the chemicals used to evaluate a set of QSAR models in an u npublished report by Battelle (Work Assignment 3-04, EPA Contract number 68-W- 99-033). Tebuconazole was negative for ER binding. The procedures were described in Laws et al. (2006); however, this paper was a followup for the positive chemicals. A synopsis is presented in Appendix 2.

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

In an estrogen receptor transcript ional activation assay (MRID 48619403), hER α -HeLa-9903
cells cultured in vitro were exposed to tebuconazole (97.5% a.i., Batch # K689052) at logarithmically increasing concentrations from 10

− 10 to 10

− 4 M in DMSO (0.1%). The experiments were performed using 96-well plates and each concentration was tested in triplicate (3 wells/plate). Cells were exposed to the test agent for 23 ±1 hour to induce reporter (luciferase) gene products. Lu ciferase expression in response to activation of the estrogen receptor was measured upon addition of a lucifera se substrate and detection with a luminometer with acceptable sensitivity.

Tebuconazole was tested up to the limit of solubility, 10

− 4 M. Performance criteria were met for all reference chemicals. The log PC 10 for 17 α -estradiol and 17 α -methyltestosterone, and Hill Slope for 17 α -estradiol were lower than the expected range in the first run, indicating increased sensitivity of the assay to weak agonists. The mean RPC Max for tebuconazole was -0.2% for the first run and -1.6% for the second run; the associated PC Max was 10

− 10 M for the first run and

10

− 5 M for the second run. Because the RPC Max < PC 10 in two assay runs, tebuconazole was considered negative for estrogen receptor tran scriptional activation in this test system.TEBUCONAZOLE FINAL

Page 47 of 81 Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350)

The 21-day fish short-term reproduction assay (MRID 48688301) of tebuconazole (97.5% purity) with fathead minnows ( Pimephales promelas ) was conducted under flow-through conditions. Sixteen spawning groups of adult fish (2 males and 4 females per group; ca. 7 months of age) were exposed to tebuconazole at nomin al concentrations of 0 (water control), 0.0065, 0.013, and 0.026 mg a.i./L, which resulted in me an measured concentrations of <0.00065 (<LOQ), 0.00497, 0.0113, and 0.0195 mg a.i./L, respectiv ely. The mean measured recoveries ranged from 75 to 87% of nominal concentrations . The test system was maintained at 24.7 to 25.6oC and a pH of 7.7 to 7.9.

There were no significant differenc es in survival in the tebucon azole treatment groups compared to the control (overall survival was 100% in the control and 88 -92% in the treatment groups). Additionally, there were no treatment-related clini cal signs of toxicity observed in the treatment groups. No treatment-related effects we re observed on body weight or length.

Spawning occurred at least every 4 days in two of the four control replicates and mean fecundity in controls was 23.7 eggs/female/day/replicat e (replicate range: 15.7-31.9 eggs/female/day);
fertilization success in contro ls was 97.6%. There were no signi ficant effects on fecundity or fertility (fertilization success) in the tebuconazole treatm ent groups. There were no significant
effects on male tubercle score and there were no tubercles observed in females. At the high treatment concentration there was a significant (p<0.05; Jonckheere-Terpstra) decrease of 18.5% in female gonado-somatic index (GSI) and a marginally significan t (p=0.056; JonckheereTerpstra) decrease of 19% in male GSI. Ther e was a significant decrease of 35 and 55% in female plasma vitellogenin (VTG) at the mid and high treatment concen trations, respectively (p<0.05; Jonckheere-Terpstra); th ere were no significant differences in male VTG between the treatment groups and the control.

According to the pathologist’s report, no histop athologic findings observed in the testes of the male fathead were clearly related to tebuconazole treatment. The mean testicular stage scores were decreased in the mid and high treatment gr oups compared to the controls, but the study authors report this decrease is unlikely to be biologically meaningf ul. In the ovary of the female fathead, the histopathologic findi ng of increased granulomatous inflammation (minimal to mild) in the high treatment group compared to the co ntrols was most likely related to tebuconazole treatment. Additional histopathological findings for females included increased prevalence of post-ovulatory follicles and decrease d prevalence of oocyte atresi a in the tebuconazole-treated groups, neither of which were reported as potentia lly treatment-rela ted by the pathologist; in the controls, 47% of the examined females were repo rted to have some level of increased oocyte atresia (2 fish with minimal and 5 fish with mode rate increased atresia) whereas the low, mid and high treatment groups had 40, 20, and 20%, respectively. The mean ovarian stage scores wereTEBUCONAZOLE FINAL

Page 48 of 81 decreased in the high treatment females compar ed to the controls (2.9 vs. 3.2), but the study authors report this decrease is unlikely to be biologically mean ingful. Histological examination
of the liver was also conducted with no substan tially more prevalent findings for treated males compared to the control. However, for male s, the occasional findings of microgranulomas (minimal; mid and high treatment groups) and in dividual hepatocyte necrosis (minimal; high treatment group) in the livers were likely treat ment related; there was also a concentrationrelated decrease in hepatocyte vacuolation co mpared to control. In females, generally concentration-dependent findings in the liver incl uded individual hepatocy te necrosis (occurred in all three treatment groups), multinucleated hepatocytes (mid and high treatment groups), microgranulomas (mid and high treatment groups), hepatocyte megalocytosis (mid and high treatment groups), and decreased hepatocellular vacuolation (all groups). Plasma sex steroids were not measured in this study.

Hershberger Assay (OCSPP 890.1400)

In a Hershberger Assay (MRID 48673702) screen ing for androgenic activity, tebuconazole (97.5% a.i.; Batch # K689052) in 0.5% aqueous methylcellulose 400 + 0.4% Tween 80 was administered daily via oral gavage to groups of six 58-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 75, or 150 mg/kg/day. An androgenic positive control group
consisted of six castrated rats exposed to 0.4 mg/kg/day of te stosterone propionate (TP) by subcutaneous (s.c.) injection.

To screen for potential anti-a ndrogenic activity, tebuconazole in 0.5% aqueous methylcellulose 400 + 0.4% Tween 80 was also administered daily vi a oral gavage to groups of six 58-day old, castrated male Sprague Dawley rats at dose le vels of 0 (vehicle), 35, 75, or 150 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The anti-androgenic positive c ontrol group consisted of six castrated rats exposed to 0.4 mg/kg/day TP and 3 mg/kg/day flutamide (FT) . TP alone was used as the an ti-androgenic negative control.

For both components of the assay, body weights were determined 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- dependent tissues were collected and weighed.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in animals for any dose groups.

In the androgen assay, body we ights throughout the duration of the assay in the tebuconazoledosed groups and positive (vehicle + TP) control group were comparab le to the vehicle controls. Overall (Days 1-10) body weight gains in th e tebuconazole 150 mg/kg/day groups were decreased (p<0.05) by 20% compared to the vehicl e controls. Overall body weight gains in theTEBUCONAZOLE FINAL

Page 49 of 81 positive control group were increased (p<0.05) by 29% compared to the vehicle controls. No treatment-related effects were observed in any of the accessory sex organs of tebuconazole-dosed animals. Rats in the positive control (veh icle + TP) group had accessory sex organ weight increases (p<0.05) as follows: 992% in seminal vesicles; 602% in ventral prostate; 160% in levator ani-bulbocavernosus (LABC); 449% in Cowper’s glands; and 65% in glans penis.

In the anti-androgen assay, body we ights throughout the study and body weight gain from Days 1 to 10 were similar across all groups. At 75 mg/kg/day, mean cumulative body weight gains were decreased (p<0.05) from Days 1 to 3 by 24%. At 150 mg/kg/day, mean cumulative body weight gains were decreased (p<0.05) from Days 1 to 3 by 71%, Days 1 to 4 by 33%, and Days 1 to 6 by 16% compared to the vehicle control; m ean body weights were not affected by the end of the study. The accessory sex organ weights in all tebuconazole treatment groups were comparable to the negative controls. Rats in the positive control (FT + TP) group had accessory sex organ weight decreases (p<0.05) as follows : 71% in seminal vesicles; 69% in ventral prostate; 38% in LABC; 52% in Cowpe r’s glands; and 22% in glans penis.

The dosing in this study was considered adequa te based on the decreased overall body weight gains observed at the hi gh-dose (150 mg/kg/day).

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

Female Pubertal Assay (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48688302), 15 female Crl(CD)SD rats/dose group were treated daily via oral gavage with tebu conazole (97.5% a.i., Lot # K689052) in 0.5% methylcellulose 400 + 0.4% Tween 80 at doses of0 (vehicle), 75 or 150 mg/kg/day from postnatal day (PND) 22 to 42. Animals were examin ed for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainme nt were recorded. Following sacrifice on PND 42, blood was collected for clinical chem istry analyses, and total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels were determ ined using enzyme-linked immunoassay (ELISA) kits. Adrenal, liver, pituitary, thyroid, and urogenital organs were weighed, and microscopic examinations were performed on the ovaries, uterus, t hyroid, and kidneys.

No adverse, treatment-related effects were observed on mortality; estrous cyclicity; gross pathology in the liver, kidneys, or adrenal gl ands; or histopathology in the kidneys. No
treatment-related effect was noted on serum T 4 or TSH levels, or on thyroid weight or morphology.TEBUCONAZOLE FINAL

Page 50 of 81 All rats survived the 21-day test period. Mean daily body weight and body weight gain/day in the 150 mg/kg/day animals was decreased on seve ral days through PND 33, but there were no treatment-related changes in final body weight or cumulative body weight gain (PND 22-42). Compared to the control, the mean age at init ial VO was increased by 1.2 days (not significant, NS) and 2.7 days (p<0.01) at 75 and 150 mg/kg/da y, respectively, and the mean age at complete VO was increased by 1.1 days (NS) and 2.0 days (p<0.05) at 75 and 150 mg/kg/day, respectively. However, there were no signifi cant differences in body we ights between groups at initial and complete VO.

Administration of tebuconazole did not change the day of first es trus compared to the vehicle control group. Animals exhibited similar cycl e lengths and regularity, and the majority of females in the 150 mg/kg/day group were in diestrus at necropsy.

Liver weights and relative (to body) liver weights were increased (p<0.01) by 8% at75 mg/kg/day and by 15-18% at 150 mg/kg/day, and th ere was an increase (p<0.01) of 6% in relative (to body) kidney weights at 150 mg/kg/day. No other tr eatment-related changes were observed in organ weights.

Effects on clinical chemistry parameters included decreased mean creatinine ( ↓ 8%; p ≤ 0.01) and increased total cholesterol ( ↑ 17%; p<0.01) concentrations in the 150 mg/kg/day animals, and decreased blood urea nitrogen ( ↓ 15-17%), total protein ( ↓ 6%), and total bilirubin ( ↓ 100%) concentrations (p<0.05 or 0.01) in female s administered 75 or 150 mg/kg/day.

There were no treatment-re lated changes in serum T 4 and TSH levels or on thyroid weight or morphology associated with tebuconazole treatment.

No overt toxicity was observ ed during treatment, but an earlier range-finding study noted increased salivation at 300 and 150 mg/kg/day, as well as a decrease in body weight gain. The doses tested were determined to be adequate since increased salivati on, and decreased daily body weight and body weight gain we re see at the high dose.

Although increases in the age of initial and complete VO were observed in the tebuconazole treated rats, body weights at VO were similar be tween the control and treated groups, suggesting that the delay in VO for the tebu conazole treated groups resulted from the delayed growth in these groups during the earl y part of the study.

Male Pubertal Assay (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48688302), 15 male Sprague Dawley [Crl:CD (SD)] rats/dose group were treated daily via oral gavage with tebuconazole ( 97.5% a.i., Batch #: K689052) inTEBUCONAZOLE FINAL

Page 51 of 81 aqueous 0.5% methylcellulose 400+0.4% Tween 80 at doses of 0, 75, or 150 mg/kg/day from post-natal day (PND) 23 to 53. Animals were ex amined for preputial separation (PPS) daily beginning on PND 30, and age and weight on the day of attainment was recorded. Following sacrifice on PND 53, blood was colle cted for clinical chemistry and hormone analysis. Total
serum testosterone levels were determined by using an enzyme immunoassay (EIA), and thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels were determined with enzymelinked immunosorbent assays (ELISA). Liver, pituitary, adrena ls, thyroid, and urogenital organs were collected and weighed, and microscopic examinations were conducted on the testes, epididymides, thyroid, and kidneys.

No adverse, treatment-related effects were observed on mortality; clinical chemistry parameters; gross pathology in the liver, kidneys, or adrenal glands; or histopathology in the kidneys. No
treatment-related effect was noted on serum T 4 or TSH levels, or on thyroid weight or morphology.

Clinical signs of toxicity were limited to incr eased salivation in 9/15 males in the 150 mg/kg/day dose group compared to 0/15 in both the contro l and 75 mg/kg/day groups . At 150 mg/kg/day, mean body weight was decreased (p ≤ 0.05 or 0.01) by 6-10% from PND 24 to 53, with a terminal body weight decrease (p ≤ 0.05) of 8%. On PND 24, there was a mean body weight loss (p ≤ 0.01) of 1.5 g compared to a mean body weight gain of 4.9 g in control animals. Differences in cumulative body gain at the high dose lessened throughout the study with an 8% decrease in the final cumulative body weight gain (PND 24-53) . At 75 mg/kg/day, mean body weight was decreased (p ≤ 0.05) on several occasions by 6 to 7%. M ean cumulative body weight gains were decreased by 53% (p ≤ 0.05) on PND 24 and 18% on PND 25, but diminished to an 11% decrease by PND 33. Significant decreases in cumulative body weight gain in the low dose group did not continue past PND 47.

Decreases (p ≤ 0.05 or 0.01) were observed in kidney, pituitary and adrenal gland weights. These decreases in weight at 75 and 150 mg/kg/day were dose-dependent for kidneys (7 and 9%, respectively) and pituitary glands (9 and 12%), but not for adrenal glands (18 and 13%). Additionally, relative (to body) liver weight was increased (p ≤ 0.01) by 10% at 150 mg/kg/day.

Compared to the control, mean serum testostero ne was decreased by 17% (not significant, NS) at 75 mg/kg/day and by 53% (p ≤ 0.05) at 150 mg/kg/day. The ages at initial PPS were delayed by ca. 2.5 days (p<0.01) in the high- dose group, but there was no difference in age at complete PPS between groups. The delay in initial PPS in the high-dose group was possibly related to delayed growth, as body weight gains we re reduced in the high-dose gr oup and body weights at initial and complete PPS were similar across groups. Although the age at complete PPS was similar between groups, there was a dose-related trend in the number of males in which the preputia were not completely separated by the end ofthe study (control 3/15, low-dose 4/15, and highTEBUCONAZOLE FINAL

Page 52 of 81 dose 7/15). Therefore, the lack of significant di fferences in age at complete PPS may be masked by the use of PND 54 (termination date, plus one) in calculating averages for complete PPS in the high-dose group.

With the exception of the ventral prostate, there were dose-dependent decreases in the weights of androgen-dependent tissues. At 150 mg/kg/day, decreases (p ≤ 0.05 or 0.01) were observed in the weights of the seminal vesicle and coa gulating gland with and without fluid ( ↓ 23-33%), the dorsolateral prostate ( ↓ 21%), the LABC ( ↓ 24%), and the left and right epididymis ( ↓ 13-14%). In addition, the weights of the te stes were decreased (NS) by 10-11% at the high dose. At 75 mg/kg/day, dorsolateral prosta te weights were decreased (p ≤ 0.05) by 21%, and although not significant, substantial weight de creases were also noted in the seminal vesicle and coagulating gland with and without fluid ( ↓ 17-21%) and the LABC ( ↓ 11%).

Atrophy was observed in the following androgen-depe ndent tissues (# affected/# examined): (i) testes (atrophic/small and/or soft) at 75 (2/15) and 150 (3/15) mg/kg/day compared to control (1/15); (ii) epididymis at 150 mg /kg/day (1/15) compared to cont rol (0/15); (iii) seminal vesicle at 75 (5/15) and 150 (7/15) mg/kg/day compared to control (2/15); (iv) pros tate at 75 (2/15) and 150 (5/15) mg/kg/day compared to control (1); and (v) LABC at 75 (1/15) and 150 (3/15) mg/kg/day compared to control (0 /15). Although the incidences of atrophy were small, it was a common finding in several androgen-dependent tissues, and there was additional evidence of effect on androgen-related systems. Additionally, a red focus was observed in the epididymis at 150 mg/kg/day (2/15) compared to control (0/15). Tubular de generation in the testes was increased at 350 mg/kg/day: unilateral (1/15 treated vs 0/15 contro l) and bilatera l (3/15 treated vs 1/15 control). Bilateral oligospermia in the epididymis was observed at 75 (2/15) and 150 (4/15) mg/kg/day compared to control (1/15).

The high-dose used in the curren t study is considered adequate based on the effects observed on body weight and body weight gains, and the inform ation from the availa ble range-finding study.

Steroidogenesis Assay (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48619404), H295R cells cultured in vitro in 24-well plates
were incubated with tebuconazole (97.5% purity, Batch # AE F069623-01-18) at increasing logarithmic concentrations from 10

− 10 to 10

− 4 M for 48 hours in triplicate for three independent experiments. Dimethyl sulfoxide (DMSO) was used as the vehicl e, at a final concentration of 0.1% (v/v).

Testosterone and estradiol levels were measured using specific EIA kits. A Quality Control (QC) plate was run concurrently with each i ndependent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentrationTEBUCONAZOLE FINAL

Page 53 of 81 levels. The positive controls included a known inhibitor (prochloraz) and a known inducer (forskolin) of estradiol a nd testosterone production.

Guideline acceptability recommendations and requirements were generally met, including lack of cytotoxicity, adequate producti on of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with positive controls, w ith the following exceptions. In the QC plates, th e variability (CV) in the solven t control (SC) between the runs was >30% for estradiol (31%), and for Run 3, the forskolin control re sulted in a 1.80–fold
induction of testoste rone, outside the ≥ 2-fold induction recommendation in the Guideline (average induction across runs was >2-fold).

Precipitation of the test compound was not reported at any concen tration. Cytotoxicity was not observed at any tested concentration. Decreases (p ≤ 0.05) in testosterone concentrations ( ↓ 32- 98%) were observed at ≥ 10

− 6 M, and estradiol production was completely inhibited at ≥ 10

− 5 M.
An effect was not observed at other c oncentrations in these two assays.

Based on the hormone responses in each of the three independent runs, tebuconazole treatment resulted in statistically signi ficant and reproducible decreases in testosterone and estradiol production.

Uterotrophic Assay (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48673703) conducted to screen for potential estrogenic activity, tebuconazole (97.5% a.i., Lot # K689052) in 0.5 % aqueous methylcellulose 400 + 0.4% Tween 80 was administered daily via oral gavage to gr oups of 6 immature female, Sprague-Dawley rats at dose levels of 0 (vehicle), 35, 75, or 150 mg/kg/day on post-natal days (PND) 19-21. A positive control group was treated with 17 α -ethynyl estradiol (EE) in corn oil by gavage at a dose level of 1 μg/kg/day. All animals were terminated and necropsied on PND 22 approximately 24 hours after the final dose to determine wet and blot ted uterine weights.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in any rats in the vehicle control, EE, or ≤ 75 mg/kg/day tebuconazole groups. Five of six animals in the 150 mg/kg/day group were observed to have reduced motor activity on Day 3. Body weights for the 35 and 75 mg/kg/day tebuconazole groups were comparable to the vehicle controls throughout the duration of the assay. Overall (Days 1-3) body weight gains were decreased (p<0.05) in the 75 mg/kg/day ( ↓ 38%) group compared to the vehicle controls. Final body weights were decreased (p<0.01) by 15-24 % on Days 2-3 in the 150 mg/kg/day group compared to controls, resulting in an overall (Days 1-3) body weight loss (p<0.01) of 3.0 g (compared to a gain of 8.2 g in the controls). Uterine weights in the te buconazole treated groups were comparable to the vehicle controls. Ab solute wet and blotted uterus weights for theTEBUCONAZOLE FINAL

Page 54 of 81 positive control (EE) group were increased (p<0.05) as expected by 210% and 202%, respectively. Based on the observed decreases (15-24%) in bo dy weights at 150 mg/kg/day, the dose levels used in this study were adequate.

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

Page 55 of 81 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

ER Binding Assay

In a published study by Laws et al. (2006) , 50 structurally diverse chemicals were tested in the ER binding assay as a part of the then ongoing valid ation of this assay for the EDSP. EPA tested tebuconazole in the rat uterine cystol ER compe titive binding assay as one of the chemicals used to evaluate a set of QSAR models in an unpublished report by Battelle (USEPA, 2002). Tebuconazole was negative for ER binding. The procedures were described in Laws et al., (2006); however, this paper was a follow-up for the positive chemicals. Information concerning this study is available at Laws et al. (2009) http://www.epa.gov/ncct/d sstox/sdf_kierbl.html . The concentration-response data are available from EPA. [Note: This study was not included in Table 2]

Hershberger Assay

In a published study by Taxvig et al. ( MRID 48264606 ), groups of male Wi star rats (one group of intact rats and six groups of castrated rats; six rats/group) we re used to investigate the antiandrogenic potential of tebuconazole. The group of intact rats and one group of castrated rats served as negative controls and were given peanut oil only. A second group of castrated rats was administered TP at 0.5 mg/kg/day s.c. only and se rved as the control gr oup to which all other groups were compared. A third group of castr ated rats received TP with flutamide (10 mg/kg/day p.o.) and served as the anti-androg enic positive control group. The remaining three groups of castrated rats were given subcutaneous injection of TP as above and received tebuconazole (98.0% purity) in pe anut oil via oral gavage at doses of 50, 100 or 150 mg/kg/day for seven consecutive days. After euthanasia, th e testis (from intact groups), ventral prostate, seminal vesicles (with coagulating glands including fluids), LABC, paired bulbourethral glands, pituitary, liver and kidney were collected and weighed. Additionally, serum was collected and luteinizing hormone, rat follicle-stimulating hormone and T 4 hormone levels were analyzed.

No treatment-related effects were seen on the wei ghts of the any of the secondary sex organs or in the hormone levels (LH, FSH, or T 4 ). Body weights were unaffected by treatment. Liver weights were increased (p<0.05) by 23% in the 150 mg/kg/day group.

In Vitro ER and AR Transactivation Assays

In a multi-component study by Kjærstad et al. (2010) , tebuconazole (98.0% purity) was tested for endocrine disrupting effects in three in vitro assays. In an MCF-7 cell proliferation assay, MCF-7 BUS cells were exposed at twenty concentrations of tebuconazole between 0.001 μM and 150 μM to test for estrogenic effects. Anti -estrogenic and estrogen po tentiating effects were tested by co-treating the MCF-7 ce lls with l0 pM estr adiol (which induces half of the maximum response). The combined effect on CYP19 activity (aromatase) and anti-estrogenic effect wasTEBUCONAZOLE FINAL

Page 56 of 81 tested by co-treating the MCF-7 cells with 1 μM testosterone (which is presumed to be converted to estradiol by aromatase and thus induce about 65% of the maximum estradiol response). Effects on the production of estrad iol, progesterone, and testoster one were tested in the human
adrenocortical carcinoma cell lin e H295R. In an AR reporter ge ne assay, AR-transfected CHO cells were exposed at twelve concentrations between 0.025 μM and 50 μM in the presence and absence of R1881 (AR agonist) to te st for AR agonism or antagonism.

Tebuconazole inhibited the estradiol-induced pr oliferation of the MCF-7 cells with an IC 50 value of 49 μM and inhibited the testostero ne-induced proliferation with an IC 50 value of 17 μM. While the effect on testosterone-treated cells may be related by inhibition of aromatase, this was not demonstrated in the study. Tebuconazole inhibited E 2 production in a steroidogenesis assay
with an IC 50 of 1.2 μM. Tebuconazole was also an AR antagonist in the presence of R1881 with

an IC 50 value of 8.1 μM.

In a published study by Orton et al. (2011) , tebuconazole (>97% a.i.) was tested for endocrine disrupting effects in two in vitro ARTA assays. In MDA-kb2 ce ll assay (human breast cancer cells stably transfected with an androgen-response element-containing promoter with a luciferase reporter gene), cells were initially exposed eight serial dilutions of tebuconazole in the presence
and absence of 0.25 nM DHT to test for AR a gonism or antagonism. Cells were initially exposed at concentrations between 0.64 nM and 50 μM. In a yeast anti-androgen screen (YAS), AR-transfected yeast cells were exposed to te buconazole at concentrations between 0.016 μM and 750 μM in the presence of DHT.

In the MDA-kb2 cell assay, tebucona zole had an anti-androgen IC 20 2.89 μM and a cytotoxic

IC 20 of 38.9 μM, and was negative as an androge n agonist. No results were provided for tebuconazole in the YAS, and no explanation was provided.

Steroidogenesis Assays

In a multi-component study, Kjærstad et al. (2010 ) examined the effect of tebuconazole on steroidogenesis in an in vitro assay using H295R cells e xposed to six tebuconazole concentrations from 0.1 and 30 μM. The effects on E 2 , progesterone, and testosterone production were evaluated. Tebuconazole inhibited the pr oduction of both E 2 (IC 50 1.2 μM) and testosterone (IC 50 0.5 μM), and increased progesterone pr oduction (data presen ted graphically).

Puberty Onset and Kiss1 Expression in Female Rats

In a published study by Overgaard et al. (2013) , groups of time-mated female Wistar rats were administered tebuconazole in corn oil by daily oral gavage at dose levels of 0 (9 dams), 12.5 (6 dams), or 50 (4 dams) mg/kg/day from GD 7-21, and then the day after delivery to PND 16. Puberty onset was examined in female offspring, as was mRNA expression of the Kiss1 geneTEBUCONAZOLE FINAL

Page 57 of 81 that encodes for kisspeptin, which is essential fo r activation of the HPG axis and the onset of puberty in rats.

There were no effects of tebuconazole treatment on juvenile (PND 28) or adult body weights, or on onset of puberty. Kiss 1 mRNA expression was not affected by tebuconazole exposure.

Subchronic Toxicity (Rat, OCSPP 870.3100)

In a subchronic toxicity study ( MRID 40700930 ), groups of 10 Wistar (BOR:WISW[SPF Cpb]) rats/sex were fed diets containing tebuconazo le (Batch # 16007/83; 93.4% purity) at 0, 100, 400, or 1600 ppm (equivalent to 0/0, 8.6/ 10.8, 34.8/46.5, and 171.7/235.2 mg/kg/day in males/females) for 13 weeks. In addition to the parameters normally examined, liver

N

-demethylase and cytochrome P-450 levels were determined.

There were no treatment-related effects on clin ical signs, ophthalmic examinations, hematology, urinalysis, or gross pathology. One 1600 ppm male died during Week 1 and one 1600 ppm female died during Week 4. It was stated that both deaths we re probably due to hemorrhagic
diathesis and were considered to be treatment-re lated. Additionally, two control males died; one during Week 4 and the second during Week 12.

At ≥ 400 ppm, male body weights were decreased (p< 0.05) by 5-12% at Week 13; overall (Week 0-13) body weight gains were decreased (p< 0.01) by 12% at 1600 ppm. In the 1600 ppm females, body weights were decreased (p<0.05) by 8% at Week 13; overall body weight gains were decreased (NS) by 12%. Food consumpti on was generally increased at 1600 ppm in both sexes throughout the study. Cr eatinine was decreased (p<0.01) by 22-33% in the 100 ppm and above males at Week 13; however, it was stated that the values of the treated groups were similar to historical control values, and were not considered adverse. N -demethylase activity was increased (p<0.01) in the 1600 ppm males; females were similar to controls. Cytochrome P-450 activity was increased (p<0.05) by 26-107% at ≥ 400 ppm in males, and by 10-19% in females at

≥ 100 ppm. Absolute liver weights were decr eased (p<0.05) by 13-16% in the 400 ppm and above males, and by 11-13% in the 100 and 400 ppm females; absolute liver weights were increased (NS) by 2% in the 1600 ppm females. As there were no corroborating microscopic findings, the decreased liver weight s were considered to be a reflection of the decreased terminal body weights. Absolute kidney weights were d ecreased (p<0.05) by 15% in the 1600 ppm males and by 8-9% in the 400 ppm and above females. As no corroborating microscopic findings were found in the kidney, these decreases were also co nsidered to be a refl ection of the decreased terminal body weights. Vacuole formation in th e zona fasciculata of the adrenal glands was noted in the 1600 ppm males and at ≥ 400 ppm in females, and hemosiderosis was noted in a nondose-dependent manner in males at ≥ 100 ppm and females at 1600 ppm. No treatment-related changes were seen in absolute or relative weight s of the testes and adrena l glands. No treatmentrelated histopathological lesions we re seen in the testes, epididym ides, seminal vesicles, prostate, ovaries, uterus, thyroid, adre nal, or pituitary glands.TEBUCONAZOLE FINAL

Page 58 of 81 The NOAEL is 34.8 mg/kg/day for the males and 10.8 mg/kg/day for the females. The LOAEL in males is 171.7 mg/kg/day based on d ecreased body weight, body weight gain, and histopathological findings, and the LOA EL in females is 46.5 mg/kg/day based on histopathological changes in the adrenal glands.

Subchronic Toxicity (Dog, OCSPP 870.3150)

In a subchronic toxicity study ( MRID 40700934 ), groups of four beagle dogs/sex were fed diets containing tebuconazole (93.4% purity) at 0, 200, 1000, or 5000 ppm (equivalent to 0/0, 73.7/73.4, 368.3/351.8, and 1749.1/1724.8 mg/kg/day in male s/females) for 13 weeks. In addition to the parameters normally ex amined, serum triglycerides and liver N -demethylase and cytochrome P-450 levels were determined.

Serum triglyceride leve ls, urinalysis parameters, and gross pathology were unaffected by treatment. One 5000 ppm female was found dead on Day 2, the cause of death was considered to be acute circulatory collapse a nd moderate pulmonary edema, and was considered possibly due to the test compound. Diarrhea, vomiting, and pa sty stool were observed sporadically in both control and treated dogs throughout the study. A few 1000 ppm males and 5000 ppm males and females appeared thin compared to controls sp oradically from Week 7 to study termination, and two 1000 ppm and five 5000 ppm dogs appeared thin or very thin at termination. This finding was considered compound related. Lens opacity of the eyes was observed in the 5000 ppm group with increasing frequency and severity, such that all 5000 ppm dogs were observed with this finding at Week 10.

Body weights were decreased (not analyzed st atistically) in the 1000 ppm and above males ( ↓ 5- 11%) and females ( ↓ 6-17%) at Week 13, resulting in overall decreases in body weight gain of 25-46% and 19-48% in males and females, re spectively. Week 13 body weights were also decreased by 7% in the 200 ppm females; howev er, the overall body weight gain for this group was similar to controls, and Week

− 1 body weights were 0.7 kg less than controls. Food consumption was slightly decreased throughout the study in the 5000 ppm males and 1000 ppm
and above females.

Platelet counts were increased in the 5000 ppm group, and signs of severe anisocytosis were noted in 6/8 dogs at 5000 ppm. Serum alkaline phosphatase activity was increased at 5000 ppm, and serum albumin was decreased in the 5000 ppm males. Liver N -demethylase activities were
increased at ≥ 200 ppm in the males ( ↑ 32-255%) and females ( ↑ 46-208%); cytochrome P-450
activities were increased to a lesser extent at ≥ 1000 ppm in males ( ↑ 25-73%) and at 5000 ppm in females ( ↑ 97%). Absolute spleen weights were increased by 44% and 19% in the 5000 ppm males and females, respectively. Absolute liver weights were slightly increased at ≥ 1000 ppm in males ( ↑ 8% both doses). No treatment-related changes were seen in absolute or relative weights of the testes, prostate, ovaries, thyroid and adrenal glands.TEBUCONAZOLE FINAL

Page 59 of 81 Microscopic findings in the 5000 ppm group included slight degeneration of the posterior wall of the lens of the eye, cataract-lik e eosinophilic plaques, bi lateral cataracts, and slight siderosis in the liver and spleen. Increased vacuole forma tion was noted in the adrenals of one 200 ppm female, one 1000 ppm female, and two 5000 ppm females. No treatment-related histopathological lesions were seen in the testes, epidi dymides, prostate, ovaries, uterus, mammary, thyroid, and adrena l, or pituitary glands.

The NOAEL is 74 mg/kg/day, and the LOA EL is 368.3/351.8 mg/kg/day (m/f) based on decreases in body weight gain, food consumpti on, and increased liver enzyme activities.

Chronic Toxicity (Dog, OCSPP 870.4100)

In a chronic toxicity study ( MRID 40700940 ), groups of four beagle dogs/sex were fed diets containing tebuconazole (96.9% purity) at 0, 40, 200, or 1000 ppm (equivalent to 15.5, 77.3 and 483 mg/kg/day) for 52 weeks. The high dose was increased to 2000 ppm at Week 40, and remained at this level until study termination. Th e reason for this increase was not provided. In addition to the parameters normally ex amined, serum triglycerides and liver N -demethylase and cytochrome P-450 levels were determined.

No treatment-related effects were observed on mortality, clinical signs, body weights, body weight gains, food consumption, and urinalysis parameters. Se veral findings in the eye were observed in the mid- and high-dose groups. Lenticul ar lesions (opacities, lens stars) were noted in two animals of the mid dose as well as one dog at the high dose. In addition, corn eal opacities (cloudy, extensive) were also apparently elevated in one dog in each of the mid dose and high dose groups.

The liver was a target organ for both sexes, based upon several moderate findings: alkaline phosphatase levels were elevated over the cour se of treatment in the high-dose males and females; liver N-demethylase, but not cytoch rome P-450 activity (both sexes) activity was significantly elevated at the high dose, as well as liver triglycerides; elev ations in absolute and relative liver weights in the mid- and high-dose males; a consistent change in the appearance of
the liver in 3/8 mid-dose and 5/8 high-dose dogs, respectively, as we ll as a slight increase in the presence of siderin in mid- (1/4) and high- dose (3/4) males and ORO-positive lipids in one dog in each of the mid and high-dose groups.

Other potential target organs/t issues are the hematopoietic sy stem (increased presence of moderate anisocytosis in mid and high dose animals), the adrenals (increased incidence of intracytoplasmic vacuoules of zona fasiculata) the kidneys (relative weights elevated in midand/or high-dose males and females) and the sple en (elevation in both absolute and relative weights of the high dose males and females). No treatment-related changes were seen in absolute or relative weights of the testes, pr ostate, ovaries, thyroid a nd adrenal glands. NoTEBUCONAZOLE FINAL

Page 60 of 81 treatment-related histopathological lesions were seen in the te stes, prostate, ovaries, uterus, mammary, thyroid, adrenal, or pituitary glands.

The LOAEL was 77.3 mg/kg/day based on the find ings of ocular lesions and changes in erythrocyte morphology; th e NOAEL was 15.5 mg/kg/day.

In a subsequent chronic toxicity study ( MRID 42030601 ), groups of four beagle dogs/sex were fed diets containing tebuconazo le (96% purity) at 0, 100, or 150 ppm (equivalent to 0/0, 2.96/2.94, and 4.39/4.45 mg/kg/day in ma les/females) for 52 weeks.

There were no effects of treatment on mortality, clinical signs, body wei ghts, body weight gains, food consumption, ophthalmological examinations, hematology, clinical chemistry, urinalysis, organ weights, or gross pathology. One control female was euthanized on Day 70 in extremis

.
No treatment-related changes were se en in absolute or relative wei ghts of the testes, ovaries, and adrenal glands. No treatment-related histopat hological lesions were seen in the testes, epididymides, seminal vesicles, prostate, ovar ies, uterus, and vagina , mammary, thyroid and pituitary glands.

Microscopic pathology demonstrated the adrenal gland to be a ta rget organ at 150 ppm in both sexes. In males there was hypertrophy of adrena l zona fasciculata cells (4/4 treated vs, 0/4 controls). Other adrenal findings in males include d fatty changes in the zona glomerulosa (3/4) and lipid hyperplasia in the cort ex (2/4) at 150 ppm vs. 1/4 in c ontrols. In females there was hypertrophy of zona fasciculata cell s of the adrenal (4/4 treated vs . l/4 controls). Fatty changes in the zona glomerulosa of the adrenal was noted in 2/4 150 ppm females compared to l/4
controls.

The NOAEL is 2.9 mg/kg/day, and the LOAEL is 4.4 mg/kg/day based on adrenal effects in both

sexes.

Chronic Toxicity/Carcinoge nicity (Rat, OCSPP 870.4300)

In a chronic toxicity /carcinogenicity study ( MRID 40700939 ), groups of 50 Wistar (Bor:WISW[SPF Cpb]) rats/sex we re fed diets containing tebucon azole (ca. 95% purity) at 0, 100, 300, or 1000 ppm (equivalent to 0/ 0, 5.3/7.4, 15.9/22.8, and 55.0/86.3 mg/kg/day in males/females) for 24 months. Additionally, gro ups of 10 rats/sex were treated as above and
terminated after 12 months.

There were no effects of treatment on mortality, clinical signs, ophthalmoscopic examinations, clinical chemistry,

Male body weights were unaffected by treatmen t. Female body weights were decreased (p<0.05) by 3-5% at 300 ppm beginning at Week 15, and by 7- 10% at 1000 ppm beginning atTEBUCONAZOLE FINAL

Page 61 of 81 Week 1. These decreases were observed consiste ntly during the remainder of the study. The 1000 ppm females were also noted to have sli ghtly increased food consumption during dosing. In females, but not males, there was a small but consistent decreases in hemoglobin, hematocrit and altered mean corpuscular concentrations a nd volumes at 79 and 104 weeks of analyses which correlated with an increased deposition of spleni c hemosiderin in the 1000 ppm females. Doserelated decreases in female absolute and relative adrenal weights (statistically significant at all dose levels) were associated with a dose-related decrease in the incidence of adrenal cortical hemorrhagic degeneration (statis tically significant at 300 and 1000 ppm). There was also a doserelated increase in liver microsomal enzyme induction at all dose le vels tested, based on histological examination. In th e 1000 ppm males, gross pathology suggested an increase in the presence of kidney cyst/cystic kidneys and an increase in reddened lymph nodes. Histological examination of the lymph nodes indicated a possi ble elevation of blood- filled sinuses of the mesenteric lymph nodes in the 1000 ppm males. In males, but not females, C-cell thyroid
adenoma and carcinoma were noted in treatedbut not control groups. These were non-doserelated findings and were not significant. Th e incidence of thyroid C-cell hyperplasia was somewhat elevated at 300 and 1000 ppm (1 /50, control vs. 7/50, 300 ppm, 6/50, 1000 ppm) being statistically significant (p< 0.05) at the mid but not high dose level. The pathology report, noted that thyroid C-cell neoplas ia and hyperplasia can be combin ed since the differentiation is arbitrary depending mainly on size of the lesion s. Combination of hyperplasia and neoplasia increased the statistical signifi cance of the hyperplastic findings which resulted in the following respective incidences: 2%, 10%, 20%, and 18%. Historical control data from the Bayer laboratories for spontaneous tumors in Wistar TNO/W 70 rats from eleven studies initiated between 1973-1976 were submitted. The range of t hyroid parafollicular tumors (which included interstitial or C-cell adenomas) was 0-19.3% with an average of 7.4%. Thus, the individual and combined C-cell tumors from the present st udy were essentially within the historical parafollicular control range.

The NOAEL is 15.9 mg/kg/day for the males and 7.4 mg/kg/day for the females. The LOAEL in males is 55 mg/kg/day based on decreased testes weights, and the LOAEL in females is 22.8 mg/kg/day based on decreases in body weight and weight gains, decreased erythrocyte parameters and increased incidence of splenic he mosiderosis (increased er ythrocyte clearance). There was no evidence of carcinogenicity at the doses tested.

Eight-Week Range-Finding Study (Mouse, OCSPP none)

In an eight-week range-finding study ( MRID 40700933, 1986 ), groups of 5 NMRI mice/sex were fed diets containing tebuconazole (purity information not provide d) at 0, 500, or 2000 ppm
for 8 weeks. Parameters examined included mo rtality, clinical signs, body weights, food and water consumption, clinical chemistry, and gr oss and microscopic pathology. A second study was performed using groups of 5 NMRI mice/sex that were fed diets c ontaining tebuconazole atTEBUCONAZOLE FINAL

Page 62 of 81 0, 125, 500, or 2000 ppm for 5 days for determinati on of liver cytochrome P-450, triglycerides,
and N - and O-demethylase levels.

There were no effects of treatment observed on mortality, clinical sign s, or food or water consumption. One control female and one 500 pp m male died during terminal blood collection, due to either overdose or hypovolemic shock. B ody weights were consistently decreased by 1-2 g in the 2000 ppm females during treatm ent; male body weights were unaffected by tebuconazole. In the males, total bilirubin was decreased (p<0.05) by 34% at 2000 ppm, indirect bilirubin was decreased (p<0.05) by 58-62% at 500 and 2000 ppm, and serum iron was increased (p<0.01) by 26% at 2000 ppm.

Absolute liver weights were increased (p<0.05) by 34% and 37% in the 500 ppm males and females, respectively, and by 43% and 34% in the 2000 ppm males and females, respectively. Absolute adrenal weights were decreased (p< 0.05) by 21% in the 2000 ppm females. There were no effects of treatment on testes or ovary weights, or on testes or ovary histopathology.
Gross examinations revealed swollen or incr eased lobulation of the liver in the 500 and 2000 ppm males, and pale livers in the 500 and 2000 pp m males and females. Microscopically, there
was an increase in the incide nce and severity (gra de) of liver cell degeneration, liver cell necrosis, focal necrosis and presen ce of vacuoles and lipid content at either or both doses. At 2000 ppm, there was an increase in the incidence of pigmentati on (iron-related pigment) and round cell infiltrates of the kidneys. The adrenals were also significantly affected as evidenced by an increase in cortical ce ll lipids (500 and 2000 ppm) and si nus dilation (2000 ppm) of the
males.

In the 5-day enzyme induction study, N -demethylase was increased (p<0.01) by 68-156% in the 125 ppm and above females; males were unaffected by treatment. O -demethylase was increased (p<0.01) by 80% in the 2000 ppm males and by 59 -89% in the 500 ppm and above females. Cytochrome P-450 was increased by 52-259% (p< 0.01) in the 125 ppm and above males and by 12-242% (p,0.05) in the 125 ppm and above female s. Finally, triglycerides were increased (p,0.01) by 196-454% in the 125 ppm and above males and by 105-500% in the 500 ppm and
above females.

Carcinogenicity (Mouse, OCSPP 870.4200)

In a carcinogenicity study ( MRID 40700941 ), groups of 50 NMRI (Bor:NMRI[SPF Han])
mice/sex were fed diets contai ning tebuconazole (ca. 95% purity) at 0, 20, 60, or 180 ppm (equivalent to 0/0, 5.9/9.0, 18.2/26.1, and 53.1/80.5 mg/kg/day in males/females) for 91/92 weeks. Additionally, groups of 10 mice/sex were treated as above a nd terminated after 52
weeks.

No treatment-related effects were observed on mortality, clinical signs , food consumption, or hematology. A minimal depression (p<0.05) in male, but not female, mean body weights wasTEBUCONAZOLE FINAL

Page 63 of 81 noted for several weeks during the first third of the dosing period ( ↓ 3-5%) at the high dose. Bilirubin values were increased (p<0.05) in the high dose females at Week 53 ( ↑ 37%) and in all treated females at Week 92 ( ↑ 18-64%). Slight increases (NS) in absolute and relative liver weights were noted in high dose group males ( ↑ 2-7%) and females ( ↑ 9-23%) at Weeks 53 and 92/93, reflective of ability of tebuconazole to induce microsom al enzymes (cytochrome P-450,

N

- and O -demethylase). Gross pathology suggested a ge neral increase in male liver enlargement and the presence of liver nodules. Interim and terminal sacrifice non-ne oplastic histopathology indicated the liver as a primary target organ, usually at the mid and high dose levels in both sexes. In males the most frequent liver lesions were vacuolation (centr ilobular, periportal) and lipid deposition (centrilobular, focal , periportal); in females, hepa tic vacuolation (centrilobular, minimal diffuse), minimal extramedullary hemopoies is, sinusoidal cellularity and increased lipid deposition were noted. In males, the adrenals were affected at the mid and high dose with an increased enlargement and hyperpla sia of cortical cells and ther e was an increase in minimal stomach gastritis in the high dose group. Females al so had apparent elevati ons in the pancreatic interstitial edema and stomach ga stritis at the mid or high dose levels. Although these findings indicate that the high dose resu lted in some toxicity in liver and other organs, the nature and severity of such toxicity s uggested that the high dose was not high enough and thus the MTD was not achieved in this study. No treatment-relate d changes were seen in absolute or relative weights of the testes and adrenal glands. Ov aries were not weighed. No treatment-related histopathological lesions were seen in the testes, epidi dymides, seminal vesicles, prostate, ovaries, vagina, mammary glands, thyroid, adrenal, or pituitary glands. In males, there was an apparent slight elevation in be nign but not malignant liver tumors at 60 and 180 ppm (4%/control vs 8% at the mid dose, 12% at the high dos e), but comparison to combined benign plus malignant liver tumors to previous data indicate d that the combined incidences were within the historical control range (6-18%).

In a subsequent oncogenicity study ( MRID 42175001, 1991 ), groups of 50 NMRI (Bor:NMRI[SPF Han]) mice/sex were fed diets containing tebuconazole (96.2% purity) at 0, 500, or 1500 ppm (equivalent to 0/0, 84.9/103.1, and 279/356.5 mg/kg/da y in males/females) for up to 91 weeks. Additionally, grou ps of 10 mice/sex were treated as above and terminated after
52 weeks.

There were no effects of treatment on mortalit y. Distended abdomen was observed in the 1500 ppm males and females. In males, there were decreases (p<0.05) in body weight at 500 ppm during the first half of the testing period ( ↓ 3-5% from data presente d) and at 1500 ppm up to Week 84 ( ↓ 5-8% from data presented). In females, there were sporadic decreases (p<0.05) in body weight at 500 ppm during th e initial third of the study, and at 1500 ppm, there were decreases (p<0.05) in body weight in 39 of the first 59 weeks a nd in 7 of the last 8 weeks ( ↓ 5-6%
from data presented). Mean food consumption was increased (p<0.05) frequently in males at 500 ppm and generally throughout th e study in both sexes at 1500 ppm.TEBUCONAZOLE FINAL

Page 64 of 81 Significant changes in blood parameters were observed at 1500 ppm in both sexes, though mainly in males. At sacrifice, in the high dos e males, these changes included leucocytosis, and changes consistent with anemia (decreased erythrocytes, hemoglobin, MCHC, and hematocrit values). In addition, high-dose males had increa sed platelet counts and decreased thromboplastin times. In the case of high-dose females, a reduced hematocrit and leucocytosis were observed at 51 weeks, and only an increased pl atelet count at terminal sacrif ice. There were no consistent
effects on differential blood counts. There was a dose-dependent increase in the plasma activity of liver enzymes (GPT, GOT, AP) at the interim and final sacrifices. Except for the interim sacrifice in high-dose females, the increase in plasma activity of liver enzymes reached significance for both the interim and final sacrifices in the 1500 ppm group. In addition, significance was observed at 500 pp m at both sacrifices for GPT in males and for GPT and GOT in females. Cholesterol was statistically signifi cantly decreased vs controls in both sexes at 500 and 1500 ppm for the interim sacrifice, and at 500 ppm for the final sacrifice. Albumin levels were transiently decreased at interi m sacrifice at 500 ppm and 1500 ppm.

Absolute liver weights were increased (p<0.05) at both the interim and terminal sacrifices in the 500 ( ↑ 17-39%) and 1500 ( ↑ 112-254%) ppm males, and in the 1500 ppm females ( ↑ 103-273%). Absolute kidney weights were decreased (p<0.05) in the 500 and 1500 ppm males ( ↓ 12-15%). No treatment-related changes were se en in absolute or relative we ights of the testes and adrenal glands. Ovaries were not weighed.

At the interim sacrifice, the following increases (p<0.05) were noted grossl y: i) liver appearing large at 1500 ppm in both sexes; ii) pale livers in both sexes at 500 and 1500 ppm; and iii) areas of change in the liver in the 1500 ppm females. At the terminal sacrifice, the following increases (p<0.05) were observed grossly: i) liver appearing large at 1500 ppm in both sexes; ii) irregular surfaces in livers at 1500 ppm in both sexes; a nd iii) liver masses in the 1500 ppm females. Microscopically, in the terminal sacrifice males, there were dose -dependent increases (p<0.05) in extramedullary hematopoiesis, focal hyperplasia of hepatocytes, and oval cell proliferation at 1500 ppm, and in hepatic panacinar fine fatty vacuolation at 500 and 1500 ppm. In the 1500 ppm females at terminal sacrifice, there were increases (p<0.05) in focal hyperplasia of hepatocytes, hepatic pa nacinar fine fatty vacuolation, periacinar hepatocyte hypertrophy, oval cell proliferation, eosinoph ilic foci of hepatocyte alteration, and pigment-laden Kupfer cells.

There was also a dose-dependent increase (p<0 .05) in ovarian atr ophy at 500 ppm and 1500 ppm. Also in the uterus, bila teral luteal cell hyperplasia was (p <0.05) in creased at 1500 ppm. No treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicles, prostate, vagina, ma mmary glands, thyroid, adrena l or pituitary glands.

At 1500 ppm, the incidence of hepatocellular ad enomas and hepatocellular carcinomas was increased (p<0.05) in males, and the inciden ce of hepatocellular carcinomas was increased (p<0.05) in females.TEBUCONAZOLE FINAL

Page 65 of 81 Developmental Toxicity (Rat, OCSPP 870.3700)

In a prenatal developm ental toxicity study ( MRID 40700943 ) groups of 25 pregnant Wistar rats
were administered tebuconazole (98.3% purity) in distilled water + 0.5% Cremophor EL by oral gavage (10 mL/kg) at dose levels of 0, 30, 60, or 120 mg/kg/day during GD 6-15. The dams were euthanized on GD 21, and their fetuse s were collected by cesarean section.

There were no treatment-related effects on morta lity, clinical signs, or gross pathology. Mean body weights were slightly decreased (p<0.05) in the high dose dams ( ↓ 5%) on Day 21, and mean body weight gains were decreased ( ↓ 28%) during dosing (GD 6-15). These decreases were accompanied by a reduction in food consumption ( ↓ 15%) during dosing. Mean maternal absolute and relative liver weights we re increased (p<0.05) at the mid ( ↑ 8-9%) and high dose ( ↑ 9-15%). The maternal NOAEL is 30 mg/kg/da y, and the LOAEL is 60 mg/kg/day based on
increased absolute and re lative liver weights.

Pregnancy rates ranged from 88-96%. No abortions or premature deliveries were noted. Mean corpora lutea counts or mean implantations did not differ among the treated groups. Mean live
fetuses/dam were decreased in th e high dose group, due to an increase in both early (embryonic) and late (fetal) resorptions (total resorptions: 2.8/dam treated vs. 0.6/dam controls). Mean fetal weight was (NS) reduced by approximately 11% at the high dose, while sex ratios were unaffected by treatment. The fetal weight depres sion may be treatment-related as a decrease in mean fetal weight was still obs erved despite a reduction in litt er size at the high dose.

Developmental toxicity was ev idenced at both the mid and high dose groups by delays in ossification of thoracic, cervical and sacral vertebrae, the ster num and fore- and hind limbs along with an increase in supernumerary ribs. Frank malformations were observed in two fetuses of two high dose dams as missing tail, agnatha, mi crostomia and anophthalmia. The developmental NOAEL is also 30 mg/kg/day, and the LOAEL is 60 mg/kg/day based on delayed ossification
and increased number of fetu ses with supernumerary ribs.

Developmental Toxicity (Rabbit, OCSPP 870.3700)

In a prenatal developm ental toxicity study ( MRID 40700945 ), groups of 16 pregnant Chinchilla (Kfm:CHIN, hybrids, SPF Quality) rabbits were administered tebuconazole (98.3% purity) in distilled water + 0.5% Cremophor EL by oral gava ge (4 mL/kg) at dose levels of 0, 10, 30, or 100 mg/kg/day during GD 6-18. The dams were euthanized on GD 28, and their fetuses were collected by cesarean section.

There were no effects of treatmen t on mortality, clinical signs, liv er weights, or gross pathology. One high dose doe died, but this wa s attributed to a gavage error. Maternal mean body weight gains were decreased by 38% dur ing dosing at the high dose, a nd there was no evidence of a rebound during the post-dosing peri od. When GD 6-28 body weight gains were corrected forTEBUCONAZOLE FINAL

Page 66 of 81 gravid weights, a slight loss was noted at the high dose. Food consumption was also decreased during dosing ( ↓ 12%) at the high dose, but rebounded during the post-dosing period. The maternal NOAEL is 30 mg/kg/day, and the L OAEL is 100 mg/kg/day based on decreased body weight gain and food consumption.

Pregnancy rates ranged from 88-94%. M ean numbers of corpora lutea/dam and implantations/dam were not different among th e dose groups. There was a reduction in live fetuses/dam observed in the high dose group as co mpared to the controls (6.4 treated vs. 7.4 controls), due to an increase in total (both early and late) resorp tions/dam (2.4 treated vi. 0.7 controls). Mean fetal weights and sex ratios were unaffected by treatment. Small decreases in
the rate of ossification were noted in high dose fetuses in the right and left digits or toes of the fore- and hind-limb. Frank malformations in 8 fetuses of 5 litters (peromelia, palatoschisis, malrotation of right hind-limb, agenesis of cl aws) were noted at th e high dose, but not in
concurrent controls or at the sa me frequency in historical data. These effects were considered treatment-related. The developmental NOA EL is 30 mg/kg/day, and the LOAEL is 100 mg/kg/day.

Developmental Toxicity (Rabbit, OCSPP 870.3700)

In a developmental toxicity study ( MRID 43776201 ), 16 pregnant Chinchilla (CHbb:CH hybrids) rabbits/group were admini stered tebuconazole (96.3-96.8% purity) in distilled water + 0.5% Cremophor EL by oral gavage at nominal doses of 0, 10, 30, or 100 mg/kg/day (analytical doses of 0, 8.8, 26.7, or 96.7 mg/kg/day) on GD 6- 18. On GD 28, all surviving does were
sacrificed and all fetuses were weighed and examined for external, visceral, and skeletal malformation/variations. In a supplementary study, blood was collected from 5 does/group on GD 6, 12, and 19 for hematology and clinical chemistry. On GD 19, these animals were necropsied following blood collection. The adrenals , kidneys, liver, and spleen were weighed and examined histologically. Liver samples we re analyzed for cytochrome P-450 content, N - and

O -demethylase activity, and tr iglyceride content.

No effects of treatment were observed on morta lity, clinical signs of toxicity, maternal body weights, body weight gains, food consumption, or at necropsy. In the supplementary study, single cell necrosis of the liver was seen in 5/5 does examined in all treatment groups, compared
with 1/4 control animals. The maternal L OAEL was 8.8 mg/kg/day, ba sed on histopathology of the liver (single cell necrosis) seen at all doses; the maternal NOAEL was not identified (<8.8 mg/kg/day).

No treatment-related differences were obser ved in the number of corpora lutea/doe, implantations/doe, post-implantation loss, resorptions /doe, fetal body weights, or fetal sex ratios.
No treatment-related skeletal malformations/var iations were observed in any fetuses. The combined incidence rate of litters containing fetu ses with external and visceral malformations was 0/16, 2/15, 5/14 (p less than 0.05), and 5/14 (p less than 0.05), respectively. TheTEBUCONAZOLE FINAL

Page 67 of 81 developmental LOAEL was 26.7 mg/kg/day based on external and visceral malformations/ variations; the developmenta l NOAEL was 8.8 mg/kg/day.

Developmental Toxicity

In a published study by Taxvig et al. ( MRID 48277401, 2007 ), groups of pregnant Wistar (HanTac: WH) rats were administered tebuconazole (98% purity) in corn o il oral via oral gavage at dose levels of 0, 50, or 100 mg/kg/day from GD 7 to PND 16, and their offspring were assessed on GD 21 and PND 16 using histology and hormone measurements. Some dams were
sacrificed on GD 21 for evaluation of the sexual di fferences in the fetuses. Anogenital distance, nipple retention, and hormone leve ls (testosterone and progesterone ) were examined at birth, at PND 13 or PND 16, and semen quality was asse ssed in adults. Organ weights (testis, epididymides, seminal vesicle, ventral prosta te, LABC, bulbourethral gland, ovaries, uterus, thyroid, adrenals, kidneys, and liver) were obtained on PND 16. Se minal vesicles, epididymides, ventral prostate thyroids and adrenal underwent histopathological examinations.

Significant effects (p<0.05) obser ved at the high dose in dams included decreased body weight gain during gestation ( ↓ 29%), mean gestation length increased by 0.94 day, increased post implantation loss (27.3% treated vs. 6.6% contro ls), and body weight lo ss during the lactation period (

− 4.57 g treated vs. +7.53 g cont rols); concomitant changes at the low dose were not statistically significant but sugge st a dose-response. The mean number of implantations and
fetuses and the sex ratio were not affecte d. AGD was increased in both newborn female offspring (1.91 mm treated vs. 1.72 mm controls ) and in female fetuses at GD 21 (2.02 mm treated vs. 1.65 mm controls). When expre ssed in mm, AGD in females was increased on GD 21 at both doses and at birth at the high dose (p< 0.05) but the AGD index, expressed per cubic root body weight, was statistically diffe rent only in the high dose group (p <0.01). An effect (p<0.01) on nipple retention at PND 13 was seen in male pups at both doses.

In plasma from high dose dams at GD 21, there wa s a marked (7-fold) in crease (p<0.05) in the progesterone level as well as decreases in both T 3 ( ↓ 11%; p<0.05) and T 4 ( ↓ 29%; NS) levels. Testis of GD 21 fetuses showed higher levels of 17 α -hydroxyprogesterone ( ↑ 238-330%) and progesterone ( ↑ 127-178%) at both doses and a lower level of testosterone ( ↓ 50%) at the high dose. Progesterone levels were si gnificantly increase (178%) in te stis of fetuses treated at the low dose, but a similar increase (127%) at the hi gh dose was not statistically significant. On PND 16, no statistical differences were noted on testosterone levels in plasma for male pups or ovarian levels of estradiol, or in organ weights at eith er dose level. No effects were seen on semen quality of PND 22 pups at 50 mg/kg/day; semen wa s not evaluated at the high dose due to fetal toxicity. No treatment-related changes in organ weights or histopathologi cal lesions were seen.

In a second published study by Taxvig et al. ( MRID 48264606 ), groups of ten female Wistar
rats were administered tebuconazo le (98.0% purity) in com oil by or al gavage at dose levels of 0 or 50 mg/kg/day during GD 7-21. Dams were sa crificed on GD 21 and bl ood was collected fromTEBUCONAZOLE FINAL

Page 68 of 81 dams and fetuses to measure 17 α -hydroxyprogesterone, progesterone , testosterone and estradiol levels on GD 21. In addition, progesterone and test osterone were measured in fetal testis and
estradiol in fetal ovaries. Ex-v ivo production of testosterone a nd progesterone by fetal testis was
measured. AGD were measured in all fetuses.

Tebuconazole increased (p<0.05) th e percent post-implantation loss to 11.4% compared to 0% in controls. AGD was not affected by treatment. In treated dams, estradiol levels were decreased (p<0.05) by 58%. In the fetuses born to treated dams, testicular progesterone levels were increased (p<0.05) by 238%. Tebuconazole ha d no effect on testicular testosterone concentration or steroid production ex-vivo. It was also reported (dat a not provided) that the fetal plasma testosterone or progesterone levels, or estradiol level in fetal ovaries did not change.

Developmental Toxicity

In a published study by Hass et al. ( 2012 ), groups of female Wistar rats were administered tebuconazole (98.5% purity) in com oil at 0 (22 dams), 12.5 (12 dams), or 50 (10 dams) mg/kg/day during GD 7 through PND 16. Maternal body weights were recorded daily, and dams were allowed to litter normally. Gestati on length, AGD, nipple retention, and genital malformations in male pups were recorded. P up weights were measured on PND 6, 13, and 22. Tebuconazole was also tested in an AR reporter gene assay at 0.03-30 μM, in a T-screen for thyroid receptor activity at 0.01-30 μM, and in a steroidogene sis assay using H295R cells.

There were no effects of treatment on maternal body weight gains from GD 7 to GD 21 or from GD 7 to PD 1. No clinical signs of toxicity were observed in the dams. Gestation length, the number of implantation scars in the uterus, post-implantation loss, and perinatal loss were unaffected by treatment. In the 50 mg/kg/da y group, female AGD was increased (14.5 mm treated vs. 13.6 mm controls; p<0.05), and male nipple retention was increased (1.6 treated vs. 0 controls; p<0.05). One 50 mg/kg/ day male pup was observed with slight genital malformations (NS). Tebuconazole exhibited so me degree of AR antagonism (dat a presented graphically). In the steroidogenesis assay, tebucona zole reduced testosterone and estradiol production and caused a slight increase in progesterone production (data presented graphi cally). Data for the T-screen assay were not presented.

Developmental Toxicity

In a published study by Jacobsen et al. ( 2012 ), groups of female Wistar rats were administered tebuconazole (98.5% purity) in com oil at 0 (22 dams), 12.5 (12 dams), or 50 (10 dams) mg/kg/day during GD 7 through PND 16. Maternal body weights were recorded daily, and dams were allowed to litter normally. Gestati on length, AGD, nipple retention, and genital malformations in male pups were recorded. P up weights were measured on PND 6, 13, and 22. On PND 16, 1-3 pups/sex/litter were weighed, de capitated, and trunk blood was collected for hormone analysis. Uterus, ovaries , thyroid, and liver were collect ed from one female pup/litter;TEBUCONAZOLE FINAL

Page 69 of 81 testes, male accessory sex organs, liver, and thyr oid were collected from one male pup/litter. Testes, thyroids, and any organ showing a significant weight change were examined microscopically. On PND 22, 1-3 pups/sex/litte r were weaned; the dams and any unselected pups were killed and blood collected for hormone analysis. The number of uterine implantations
were also counted in the dams . The day of VO or PPS was determined in all weaned pups. Motor activity and habituation were measured in weaned pups on PND 28 and 59; learning and memory was examined at an age of 2-5 months ; and male mating behavior was observed at an age of 2-9 months. Female offspring were necr opsied at approximately 6 months of age, blood was collected for hormone analysis, and the th yroid was examined microscopically. Male offspring were necropsied at approximately 9 mo nths of age. Semen motility was analyzed in spermatozoa were obtained from the distal ca uda epididymis. The following organs were excised and weighed: right and left testis, ventral prostrate, semi nal vesicles with seminal fluid, epididymis, bulbourethral glands, LABC, liver and thyroid. The right or left testis was fixed, processed, and stained with hematoxylin and eosin. The following organs were fixed and processed: caput epididymis, vent ral prostrate, seminal vesicles and liver. In ventral prostate, the degree of epithelial atrophy, ep ithelial infolding and inflammation was scored. Additionally, prostates were evaluated for intraepithelial v acuolation, acini with c oncretions in the lumen, presence of areas with mu ltilayering of epithelial cells , and focal acinar atrophy.

There were no effects of treatment on maternal body weight gains from GD 7 to GD 21 or from GD 7 to PD 1. No clinical signs of toxicity were observed in the dams. Gestation length, the number of implantation scars in the uterus, post-implantation loss, and perinatal loss were unaffected by treatment. 50 mg/kg/day tebuconazole caused a liver weight increase (p<0.05) of 7% in the males; females liver weights were unchanged. Male and female reproductive organs and thyroid were unaffected by treatment. Seme n quality was unchanged by treatment. Age at VO or PPS was unaffected by treatment. There we re no effects of treatment on motor activity, learning and memory, or male mating behavior.

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a two-generation repr oduction toxicity study ( MRID 40700946 ), tebuconazole (95.2% purity)
was administered to 25 Wistar rats/sex/dose vi a the diet at dose levels of 0, 100, 300, or 1000 ppm (equivalent to 0, 5, 15 and 50 mg/kg/day) during a pre-ma ting period of 120 days; with exposure continuing in dams through gestation, la ctation and weaning. The P generation rats were mated twice to produce F 1a and F 1b litters. Twenty-five pups/sex were selected from the F 1b litters at PND 28, fed the same dietary concentra tions as their parents fo r approximately 70 days, and then mated to produce F 2a and F 2b litters.

There were no effects of treatment on mortality, food consumption, or gross pathology. Clinical signs of toxicity noted at the high dose included loss of hair in the P and F 1b females, inflammation/changes of the eye in the P males, and ill/bad condition in the F 1b males. Loss ofTEBUCONAZOLE FINAL

Page 70 of 81 hair was also increased in the mid dose F 1b females. Body weights we re consistently decreased (p<0.05) at the high dose in both sexes in both generations ( ↓ 4-15% males; ↓ 5-13% females from data provided). F 1b parents showed decreases (p< 0.05) in terminal body weights ( ↓ 9%) and absolute liver ( ↓ 12%), kidney ( ↓ 10%), and adrenal ( ↓ 11%) weights in the F 1b males, and in terminal body weights ( ↓ 5%) and kidney ( ↓ 7%) weights in the F 1b females. The NOAEL for parental/systemic toxicity is 15 mg/kg/day, and the LOAEL is 50 mg/kg/day based on the increased incidence and severity of spleni c hemosiderosis and decreased body weights.

A significant (p <0.05) decrease in viab ility index (live pups at PND 5 ÷ PND 0 x 100) at the low (90.3%) and high (88.1%) doses was observed for F 1a pups when compared to controls (98.5%), but not at the mid dose; similar changes in viability index observed in F 1b litters were not statistically significant. The lactation index (live pups at 4 weeks ÷ PND 5 x 100) of F 1a litters was decreased compared to contro l (86.3% vs. 95.2%) and that of F 1b litters at the low and high doses (76.1-80.3% vs. 90.9%) altho ugh not in a dose-dependent manner, and was increased at the mid dose (98.2%). No such findings were reported for the F 2 litters. Given the lack of dose response and consistency within generations, these effects on viability and lactation indices are
not considered to be trea tment-related. High dose F 1a , F 2a and F 2b pup weights were decreased (p ≤ 0.05) compared to controls from birth to week 4. No treatment-related changes were seen in the absolute or relative weights of the testes or ovaries of the F 1b parents. No treatment-related histopathological lesions were seen in the testes, epididymides , seminal vesicle, prostate, ovaries, uterus, vagina, mammary glands, ad renal, or pituitary glands.

The reproductive NOAEL is 50 mg/kg/day, and a LOAEL was not determined. For offspring toxicity, the NOAEL is 15 mg/kg/day and the LOAEL is 50 mg/kg/day based on decreased pup body weight.

Developmental Neurotoxicity (Rat, OCSPP 870.6300)

In a developmental neurotoxicity study ( MRID 45074301 ), groups of 25 pregnant Sprague Dawley (Crl:CD®BR VAF/Plus®) rats were administered te buconazole (96-96.9% purity) in
corn oil via the diet from GD 6 to LD 11 at dose levels of 0, 100, 300 or 1000 ppm (equivalent to 0/0, 8.8/16.3, 22.0/41.3, and 65.0/125.4 mg/ kg/day [GD/LD]). P dams were allowed to deliver naturally. On PND 5, litters were standardized to a maximum of 10 pups/litter. Pups were assigned to one of 5 Subsets ( 20 pups/sex/dose/subset). Physic al development landmarks were evaluated for all subsets (including surface righting, eye opening, pinna unfolding, acoustic startle response, and pupil constriction); sexual maturation was evaluated in subsets 2-4. Subset 1 pups were sacrificed on PND 12, all brains were weighed, and histopa thological evaluations and morphometric analyses were performed. Subset 2 pups were eval uated for learning and memory (passive avoidance and Water M-maze). Subset 3 pups were evaluated for motor activity and for auditory startle habituation. Subset 4 pups received detailed weekly clinical evaluations. In addition, Subset 4 pups were sacrificed on PND 83, brains were weighed, andTEBUCONAZOLE FINAL

Page 71 of 81 histopathological evaluati ons and morphometric analyses were performed. Subset 5 pups were sacrificed and necropsied on PND 22.

At 1000 ppm, two P females died as a result of pr olonged gestation. Body we ights were slightly decreased (p<0.01) in the P females during gestation ( ↓ 4-8%) and early lactation ( ↓ 6-12%). A body weight loss was noted during GD 6-9 (

− 9.3 g); body weight gains were decreased (p<0.05) during GD 6-21 ( ↓ 22%) and 0-21 ( ↓ 16%). A body weight loss was also noted during LD 1-4 (

− 3.6 g); body weight gains were decreased (p<0.05) during LD 4-7 ( ↓ 55%). Both absolute (g/animal/day) and relative (g/kg/day) matern al food consumption were reduced (p<0.05) beginning on GD 6, and continuing through early l actation (up to LD 12). There was also an increase in alopecia in high dose dams. The num ber of live fetuses/dam was decreased (p<0.01);
while the number of dead fetuses/dam was incr eased (p<0.01). No treatment-related findings were observed in dams at 300 or 100 ppm. Th e maternal LOAEL was 1000 ppm (equivalent to 65.0/125.4 mg/kg/day [GD/LD]) based on decreased body weights, body weight gains, and food consumption, prolonged gestation with mortality, and an increased number of dead fetuses. The maternal NOAEL was 300 ppm (equivalent to 22.0/41.3 mg/kg/day [GD/LD]).

There was a slight increase in m ean gestation length at the high dose (23.0 days) compared to the controls (22.5 days). A slight delay in VO was noted at 1000 ppm (PND 33.2) compared to controls (PND 31.6). No treatment-related changes were seen in the age at PPS. At 1000 ppm,
the stillborn index was increas ed (p<0.01) and the number of pup deaths was increased during PND 1-5. Body weights were decreased (p<0.01) in the males during PND 5-86 ( ↓ 7-23%) and in the females during PND 5-72 ( ↓ 5-24%). Pinna unfolding was s lightly delayed (p<0.01) at 1000 ppm (PND 3.8) compared to controls (PND 3.2). There were decreases in several morphometric measurements of the brain, in cluding decreased (p<0.01) thickness of the
cerebellum in the males and females on PND 12 and 83, and an increas ed thickness of the germinal layer of the cerebellar cortex in th e PND 12 males (p<0.01). Absolute brain weights were decreased in the PND 12 and 83 animals (p< 0.05). Relative (to body) brain weights were increased (p<0.05) in the PND 12 males and females. There were also changes in motor activity on PND 14 and 22, and changes in auditory startle amplitude on PND 23 and 63.

At 300 ppm, there were also decreases in body weight ( ↓ 3-7%) and body weight gain ( ↓ 4-16%, PND 5-23 and 72-86 in males, PND 5-51 in fema les). Pinna unfolding was slightly delayed (p<0.01) at 300 ppm (PND 3.7) compared to c ontrols (PND 3.2). There were changes in auditory startle amplitude in both sexes: a dose-related decrease in females on PND 23, and a
dose-related increase in males on PND 63. In a ddition, there was a decrease in absolute brain weight in both sexes on PND 12, and in ante rior/posterior cerebrum measurements.

At 100 ppm, there were d ecreases in body weight ( ↓ 3-7%) and body weight gain ( ↓ 5-13%) (PND 5-37 in males, PND 5-51 in females). There we re decreases in motor activity on PND 14 and 18 in males, and changes in auditory startle amp litude on PND 14 and 63. There was also a decreaseTEBUCONAZOLE FINAL

Page 72 of 81 in absolute brain weight in both sexes on P ND 12, and in anterior/posterior cerebrum

measurements.

The LOAEL for offspring toxicity was 100 ppm ba sed on decreases in b ody weights, decreases in absolute brain weights and measurements, and decreases in motor activity. The NOAEL was
not determined.

Reproductive Function Study

In a published study by Moser et al. ( MRID 45486901 ), groups of pregnant Sprague-Dawley (Tac:N[SD]fBR) rats (P generati on) were administered tebucon azole (97.4% purity) in 0.7% methylcellulose via oral gavage at dose levels of 0, 6, 20, or 60 mg/kg/day from GD 14 to PND 7. Beginning on PND 7, F 1 pups were dosed daily by oral gavage at the same dose levels as their
dams until PND 42. In a subset of these pups, PPS and VO were evaluated beginning on PND 35 and PND 25, respectively. As adults, these offspring were ma ted with untreated animals to produce F 2 litters that were reared to PND 10 to assess normal maturation. Another second F 1 subset (11-12 pups/sex/dose group) was sacr ificed on PND 45/46 for assessment of organ weights and histopathology (tes tes, epididymides, uterus, liv er, and adrenal). Blood was collected for hematology and clinical chemistry parameters. Testes and epididymides were processed for sperm count and motility. (Note: in this study, a second cohort of rats were used for neurotoxicity and immunotoxicity studies. As endpoints from these studies are not useful in determining endocrine systems disruption, th ey are not discussed in this summary.)

At 60 mg/kg/day, maternal body weights were decreased by 4% on GD 21, with body weight gains during gestation decreased by 16%. No treatment-related effect was observed on AGD for males or females on PND 1, or on age of PPS or VO. The acceleration (p<0.05) of VO for the mid-dose group was considered unrelated to treatm ent, due to the lack of a difference for the high-dose group. For F 1 females mated with untreated males, no overall effects on reproductive systems were observed. Post-mating necropsy of F 1 males showed reduced (p<0.05) absolute epididymides ( ↓ 17%) and kidney ( ↓ 16%) weights at 60 mg/kg/day. These decreases were likely due to a decreased (p<0.05) terminal body weight ( ↓ 10%); however, relative kidney and epididymides weights were not reported. Sperm counts for the testis and epididymis were
unaffected in F 1 males. No effects on estrous cycl e length, the number of female becoming pregnant or delivering a litter, or litter size were observed. The number of dead pups/litter was increased (p<0.05; 2.2 treated vs. 0.4 controls) at 60 mg/kg/day. No effect was observed on relative uterine weight. At the PND 46 necrops y, female terminal body weight was unchanged (157.1 vs. 155.7 g for control), and relative (but not absolute) liver weig ht was increased by 12% in the high-dose females; tissue histology wa s unchanged. The PND 46 male necropsy data
showed few differences. Ma le body weight was unchanged. The high-dose group had a 26% increase in relative spleen wei ght, a 10% increase in relative liver weight, and an 8% decrease in relative kidney weight.TEBUCONAZOLE FINAL

Page 73 of 81 Avian Reproduction Toxicity (Bobwhite Quail, OCSPP 850.2300)

In an avian reproduction toxicity study (MRID 40700910), groups of 18 bobwhite quail/sex ( Colinus virginianus ) were fed diets containing tebuconazo le (97.4% purity) at 0, 5, 20, or 80 ppm (mean measured concentrations 0, 4.96, 18.4, and 73.5 ppm). The birds were fed the diets prior to the onset of egg laying (13 weeks), and then during egg la ying (10 weeks) for a total of
24 weeks. The birds were housed with one ma le and female per pen, with 18 pens per dose

group.

Incidental mortality occurred in all groups due to cage mate aggression (2 females in each of the control and 18.4 ppm group and one female in th e 4.96 and 73.5 ppm). There was one female in each of the 4.96 and 18.4 ppm groups and one male in the 73.5 ppm group that had a nervous disorder affecting the carriage of the head and at times affected the birds’ general coordination. This effect was not considered compound-related since this same observation had been noted in previously untreated birds. These birds app eared to have normal reproduction tendencies and
there were no observable treatment-related lesion s noted in any of the birds at necropsy. The 73.5 ppm dietary level male and fema le birds showed a slight wei ght reduction (NS) at the time
of the adult sacrifice. There were no diffe rences in feed consumption. There were no differences in any of the reproductive paramete rs examined. There was a slightly lower percentage of 21-day embryo survivors and sli ghtly lower hatch percen tage at the 75.8 ppm dietary level. This difference, however, was not statistically differe nt from controls. There were no significant differences between the control gr oup and treated groups in mean hatch weights, 14-day survivor weights or percent survival. There were no significant treatment-related differences shown by any of the treatment levels in either shell strength or shell thickness. Based on the results of this study, the NOAEC for tebuconazole on bobwhite quail reproduction is 73.5 ppm, the highest level tested.

In a second avian reproduction toxicity study (MRID 41624201), groups of 18 bobwhite quail/sex ( Colinus virginianus ) were fed diets containing tebuc onazole (97% purity) at 0, 170, or 340 ppm (mean measured concentrations 0, 156, a nd 290 ppm). The birds were fed the diets prior to the onset of egg laying (duration of treatment not provi ded), and then during egg laying (duration of treatment not provided) for a total of 21 weeks. The birds were housed with one male and female per pen, with 18 pens per dose group.

This study showed no effects on adult body weig ht or feed consumption; however, when necropsy examinations were performed, treatme nt related lesions (a bnormal livers) were observed in the birds of the 290 ppm concentrat ion. Effects on reproducti on were also observed at 290 ppm. These effects included reduction in: i) the number of eggs laid (33%); ii) normal hatchlings of live 3-wk embryos (11%); iii) hatchling (7%) and 14- d survivor (11%) body weights; iv) 14-day survival of the offspring (37%); and v) incr eased eggshell strength. The only effect seen at 156 ppm was a sign ificant reduction in hatchling (4%) and 14-d offspring survivorTEBUCONAZOLE FINAL

Page 74 of 81 (6%) body weight; however, there was no difference in survivorship of the offspring. Based on the results of this study, the NOAEC was <156 ppm based on depressed hatchling and 14-day survivor body weights.

Avian Reproduction Toxicity (Mallard duck, OCSPP 850.2300)

In an avian reproduction toxicity study (MRID 40700909), groups of 15 Mallard ducks/sex ( Anas platyrhyncos ) were fed diets containing tebuconazole (97.4% purity) at 0, 5, 20, or 80 ppm (mean measured concentrations 0, 4.6, 18.8, and 75.8 pp m). The birds were fed the diets prior to the onset of egg laying (10 weeks), and then duri ng egg laying (10 weeks). The total duration of the study was 20 weeks. The birds were housed with one male and one female per pen, with 15 pens per dose group.

There were no treatment-related mortalities over the course of the study. One 18.8 ppm pair and two 75.8 ppm pairs were euthanized because the females began producing eggs prior to being dosed for the required 10 weeks. No overt signs of toxicity were noted during the study. There were no grossly observable treatme nt-related lesions noted in any of the birds. There were no differences in body weights or feed consumption. There were no differences in any of the reproductive parameters examined. There was a s light dose-related trend toward a lower hatch percentage and eggs cracked of eggs laid, wi th the greatest difference in the 75.8 ppm group; however, these differences were not significantly different fr om controls. There were no differences in mean hatch weights, 14-day surviv or weights or percent survival, and there were no differences in mean eggshell strength or thic kness. Based on the resu lts of this study, the NOAEC on mallard duck reproduction is 75.8 ppm, the highest level tested.

In a second avian reproduction toxicity st udy (MRID 41818301), groups of 16 Mallard ducks/sex ( Anas platyrhyncos ) were fed diets containing tebuconazo le (96.9% purity) at 0, 170, 340, or 680 ppm (mean measured concentrations 0, 157, 320, and 611 ppm). The birds were fed the diets prior to the onset of egg laying (10 weeks), a nd then during egg laying (9 weeks). The total duration of the study was 19 weeks. The birds we re housed with one male and one female per pen, with 16 pens per dose group.

One female in the 611 ppm group died during Week 15 after appearing listle ss and anorexic the previous day. Gross pathological examination re vealed petechial hemorrhaging in the skin, a very hard liver, a dark-colored spleen, and an enlarged heart. It was concluded these
observations were incidental to tr eatment. No clinical signs were noted in any of the survivors during the study. No abnormal findings were noted at necropsy of the surviving ducks. No differences in body weights were obs erved at test initiation, prior to egg laying (test week 7), or at termination. No differences in food consumption were recorded.

In the reviewer’s analysis, reported a signifi cant (p<0.05) reduction in the number of eggs hatched per hen in the 611 ppm gr oup. No other statistical differen ces, with regard to other eggTEBUCONAZOLE FINAL

Page 75 of 81 parameters, were noted during the study. Although so me statistically significant differences in mean body weights were recorded for certain groups within hatches, these intergroup differences
were not considered to be c onsistent or dose related. The NOAEC is 320 ppm and the LOAEC is 611 ppm.

Fish Early Life Stage Toxici ty (Rainbow Trout, OCSPP 850.1400)

In a fish early life stage toxicity study (MRID 40700914), groups of rainbow trout ( Salmo gairdneri ) were exposed to tebuconazole at nominal concentrations of 0 (negative and solvent [100 μL/L acetone] controls), 0.015, 0.030, 0.060, 0.120, or 0.240 mg/L (mean measured concentrations 0, 0.012, 0.025, 0.061, 0.120, and 0.230 mg/L). At study initiation, unfertilized eggs and sperm were mixed, left undisturbed for 45 minutes, and then randomly selected and distributed to 28 embryo inc ubation cups (50 eggs/cup; 4 cu ps/concentration). On Day 15, twenty viable embryos/replicate/group were sele cted and further incubated to produce larvae. Following completion of hatching on Day 23, twenty larvae/replicate/group were released into their respective aquaria for the 60-day post-hatch exposure. Expos ure was continuous for a total of 83 days. Behavior and appearance of larvae were observed daily and the number of surviving larvae was counted twice weekly. At 60 days post-hatch exposure (test termination), the percentage larval survival was determined. Th e larvae were measured and weighed individually
to calculate the mean and standard de viation of total length and wet weight.

Exposure to all concentrations tested did not adve rsely affect the embryo viability or survival of organisms at hatch. By Day 42, (19 days post-ha tch) nearly all exposed larvae in treatment
levels of ≤ 0.061 mg/L and the controls completed thei r development to the swim-up stage. Development of larvae in concentrations of 0.120 and 0.230 mg/L appeared to be delayed by several days as compared to th e larvae in the contro ls. Throughout the post-hatch exposure period, larvae at the four highe st test concentrations exhibi ted abnormal appearance (dark coloration) and behavior (lethargic , partial or complete loss of equi librium). At test termination,
larval survival in the four highest test concentrations (i.e., 0.025, 0.061, 0.120, and 0.230 mg/L) was significantly affected compared to pooled cont rol. Because larval survival was significantly reduced at the four highest concentrations, the gr owth data at these levels was not statistically compared to the growth of the control organisms. However, growth at th ese levels appeared to be adversely affected. Larval growth in the remaining treatment (0.012 mg/L) was not significantly different from that of the pooled controls. Based on these results, the NOAEC for
rainbow trout survival and growth is 0.012 mg/L.

Fish Early Life Stage Toxicity (Sheepshead Minnow, OCSPP 850.1400)

In a fish early life stage toxicity study (MRID 42038202), groups of sheepshead minnow ( Cyprinodon variegatus ) were exposed to tebuconazole at nom inal concentrations of 0 (negative and solvent [25 μL/L dimethylformam ide] controls), 0.00625, 0.0125, 0.025, 0.050, or 0.100 mg/L (mean measured concentrations 0, 0.00504, 0.0920, 0.0219, 0.0475, and 0.0993 mg/L).TEBUCONAZOLE FINAL

Page 76 of 81 Unfertilized eggs were obtained by stripping adult females and fertilized with a sperm suspension. Twenty embryos were used per con centration. Exposure was continuous for at least 36 days.

Viability of embryos was verified 3 days after fertilization. Viability of negative and solvent control embryos was 91%; viabil ity of treated embryos was 90- 96%. Hatching in the negative controls was completed in four days, while the solvent controls was completed in nine days. Hatching was 85% in the negative controls, 66% in th e solvent controls (p<0.05 compared to negative controls), and 71-85% in the treated groups. Survival of juvenile fish was 72-93% in the treated groups, and 88% and 87% in the nega tive and solvent controls, respectively. Abnormalities were few with no apparent concen tration relationship and included two-headed fry and spinal deformations. Mean lengths of fish ranged from 12.7-14.1 mm. Mean wet weights of fish ranged from 57.3- 85.0 mg. Growth, as measured by length and wet weight, was significantly reduced in the negati ve controls compared to the so lvent controls. Mean length at 0.0993 mg/L was significantly reduced compared to the solvent controls. Mean weight at 0.0475 and 0.0993 mg/L was significantly reduced compared to the solvent controls.

Fish Full Life Cycle Toxicity (Sheepshead Minnow, OCSPP 850.1500)

In a fish full life cycle toxicity study (MRID 43009601), groups of 200 sheepshead minnow ( Cyprinodon variegatus ) embryos (50 embryos/cup; 4 cups/dose level) were exposed to
tebuconazole at nominal concentrations of 0 (negative and solvent [ 20.55 μL/L triethylene glycol] controls), 0.0125, 0.025, 0.050, 0.100, or 0.200 mg/L under flow-through conditions for 203 days. Mean measured concentrations were 0.0092, 0.019, 0.0436, 0.0967, and 0.1969 mg ai/L for the F0 phase; 0.0092, 0.0219, 0.0490, 0.0986, and 0.196 mg ai/L for the reproductive phase, and 0.0096, 0.0184, 0.0469, 0.0978, and 0.192 mg ai/L for the F1 phase. At study initiation, the embryos were <24 h post-fertilization. On Day 58, 25 fish/dose level were randomly selected and maintained until Day 113 for reproduction. Mature adult fish (generally 2 males and 5 females) were selected and placed in a spawning chamber. Four spawning session, each lasting 14 days, were conducted. At the end of the spawning sessions, the adult fish were sacrificed for length and weight measurements and submitted for residue analysis.

The F 1 phase of the study was initiated on Day 163. Where possible, 25 embryos, obtained during the period between spawning sessions 3 and 4, were distri buted to each of two embryo incubation chambers per replicate tank (i.e., 50 embryos/replicate,100 embryos/treatment). Survival was recorded until hatching was comp lete. Approximately two weeks after hatching,
all fish were transferred into sc reen retention chambers within th e same test chamber. Survival and sublethal effects were monitored daily. On Day 28, all fish were sacrificed for length and weight measurements and then submitted for residue analysis.

Hatching success (%) of treated F0 embryos was not significantly different from the pooled controls (negative controls and solvent cont rols were 86.5% and 89.0% , respectively). F0TEBUCONAZOLE FINAL

Page 77 of 81 survival on Day 58 was reduced (p<0.05) at 0.19 69 mg/L compared to the pooled controls (negative controls and solvent controls were 93.6% and 89.3%, respectively). F0 survival on Day 177 was reduced (p<0.05) at 0.1969 mg/L co mpared to the pooled controls (negative controls and solvent controls were 76% and 56%, respectively). Due to the significant survival effects, the 0.1969 mg/L exposure group was not incl uded in the growth analyses. F0 length on Day 33 was reduced (p<0.05) at 0.0967 mg/L compared to the pooled controls. F0 growth (i.e., length and weight) on Days 58 and 177 was not significantly reduced at any exposure concentration compared to the poole d controls or to the solvent c ontrols. The residue in tissues of the F0 sheepshead minnows generally increased with concentrations of test material in water. The mean tissue residue ranged from 0.12 mg/kg for fish in the lowest te st level to 12.87 mg/kg for fish in the highest test level. The exposur e solutions showed no significant effect on the reproductive parameters when compared to the control data. For spaw ning sessions 1 and 2, reproductive data at 0.0092 mg/L wa s substantially lower than the pooled control. However, this difference was not statistically significant a nd was not confirmed by the results at higher exposure concentrations.

Hatching success (%) of F 1 embryos exposed to 0.0184 mg/L was significantly reduced when compared to that of the pooled controls and the solvent controls; however, this difference was regarded as spurious. There we re no significant differe nces at any other exposure concentration. Hatching success of the negative controls and solvent controls were 93.7% and 100%, respectively. F 1 survival of the treated groups were highe r than the solvent controls at 28 days post-hatch. Survival of the ne gative controls and solvent c ontrols on Day 28 post-hatch was 95.5% and 86.0%, respectively. F 1 growth (i.e., length and weight) at 28 days post-hatch was reduced (p<0.05) at 0.0978 mg/L when co mpared to the pooled controls. F 1 length at 28 days post-hatch was also reduced (p<0.05) at 0.0436 mg/L when compared to the pooled controls The
residue in F 1 sheepshead minnow tissues generally increa sed with concentra tions of the test material in water. The mean tissue residue rang ed from 0.31 mg/kg for fish in the lowest test level to 2.96 mg/kg for fish in the highest test level.

Fish Sexual Developmental Test (F SDT) (Fathead Minnow, OECD 234)

A fish sexual developmental test (FSDT) (MRID 48109802, 2007) with Tebuconazole using fathead minnows ( Pimephales promelas) was studied under flow-thr ough conditions. The study was conducted on freshly fertilized eggs (<24hrs old) for 122-125 days. Test organisms were exposed to tebuconazole at nominal c oncentrations of 0 (control), 0.00313, 0.00625, 0.0125, 0.0250, 0.050 and 0.100 mg a.i./L; mean-measured concentrations were <LOQ (<0.00035), 0.00265, 0.00583, 0.0112, 0.0221, 0.045 and 0.0896 mg a.i./L. The test system was maintained

at 23.2 to 26.4  C and a pH of 6.9 to 7.2.

In this study, early life stage survival, growth and sexual development were examined. At the juvenile fish (study days 30 to 52) and young adult fi sh (study days 53 to test termination) stage,TEBUCONAZOLE FINAL

Page 78 of 81 very few clinical signs (e.g, underdeveloped fish) were reported for control fish and fish at test levels up to 0.0112 mg a.i./L, with the exception of one fish in the 0.0112 mg a.i./L level which had a deformed spine and other symptoms starti ng on day 55 until test termination. However, multiple sublethal effects, incl uding spinal deformations, were observed in fish at the 0.0221, 0.045 and 0.0896 mg a.i./L levels during both the j uvenile and young adult stages of the study.

The percent of egg survival at study days 0-5 (post hatch day 1) was 91, 86, 87, 90, 91, 90 and 88% in the mean-measured 0 (con trol), 0.00265, 0.00583, 0.0112, 0.0221, 0.045 and 0.0896 mg a.i./L treatment levels, respectiv ely. There were no si gnificant differences between treated and control for egg survival, and time to hatch/hatc hing success. On study da y 54, all individuals at the highest test concentration (0.089 6 mg a.i./L) had died. Mean survival at test termination was 85, 85, 83, 88, 87, 31 and 0% in the mean-measured 0 (control), 0.00265, 0.00583, 0.0112, 0.0221, 0.045 and 0.0896 mg a.i./L treatment levels, resp ectively. The mean overall survival at test termination for the 0.045 and 0.0896 mg a.i./L treatment levels was significantly decreased compared to the negative control (p<0.05). As survival was significantly affected in the 0.045 ,mg a.i./L treatment group which may have affect ed loading rates and environmental conditions, data from this treatment were excluded from analysis of growth parameters.

There was a statistically signifi cant decrease in fe male body weight and length at the 0.0221 mg a.i./L treatment level (p<0.01; Jonckheere). There were no signif icant differences in male body weight and body length or the sex ratio of male/female fish (p>0.05).

According to the study report, changes in the liv er were observed at 0.00583 mg a.i./L and above in males and at 0.0112 mg a.i./L and above in females and consisted of hepatocellular hypertrophy, minimal to slight pleomorphism of li ver cells and nuclei; the study author regarded these findings as adaptive and not adverse. Ho wever, at 0.0112 mg a.i./L and above for both males and females, liver cell degeneration wa s observed and at 0.0221 mg a.i./L and above significant inflammations in th e liver was also observed.

In males, maturation of the test es was more advanced (more adult and less juvenile fishes) at 0.0221 and 0.045 mg a.i./L. Also at 0.045 mg a.i./L, observations of increased spermatozoa in the sperm ducts and significant increases in spermatogonia in the testes were reported.

In females, reduced yolk accumulation in oocytes was recorded in some fishes at 0.0112 mg a.i./L and above. Also at 0.045 mg a.i./L reduced maturity of follicles (less late vitellogenic oocytes) as indicated by staging of the ovary were reported. Add itionally, degeneration of early- /previtellogenic follicles was significantly increased at 0.045 mg a.i./L. While at 0.00583 ug a.s./L and above a significantly reduced incidence of degenerati on of late and mid vitellogenic follicles was observed but it was not dose-responsive.

Additionally, findings were reported in the panc reas (0.0112 mg a.i./L and above), the kidneys (0.045 mg a.i./L) and spinal column/cord deform ations (0.0221 mg a.i./L and above). FungalTEBUCONAZOLE FINAL

Page 79 of 81 growth was reported in the lumen in the intestin es in fish at 0.0112 and 0.0221 mg a.i./L without any associated histopathological findings, whereas a decrease in th e incidence of inflammation in
the caudal intestine was reduced at 0.0221 mg a.i./L and above.

In regards to performance criteria, criteria re lating to dissolved oxygen were met; data was not available to determine if temperature differed by no more than 1.5°C for all chambers at one
time. The variation of the measured test concen trations were greater th an ±20% of the mean
measured value for several test concentrations ; the ratio of the maximum/minimum measured concentrations ranged from 1.30 to 1.54 except for the second lowest treatment which was 1.91. However, this study was conducted using a fish sp ecies that does not have specific performance criteria outlined in the OECD 234 guideline. Ho wever, the survival and growth of the control fish appear to be appropriate.

This study was conducted in accordance with OECD FSDT Draft Proposal (version as of July 26, 2006 and adopted July 28, 2011) and therefore is di fferent than the guidance as outlined in
U.S. EPA OCSPP 890.1350: “Fish Short-Term Re productive Assay” and OECD 229 (2009). This study was designed to assess the toxicity of the test item during the early, juvenile and sexual maturation life stages. The two prim ary endpoints analyzed were phenotypic sex expression via gonad tissue and gonad staging. This study does not evaluate reproductive success measured by fecundity and fertilizati on success. Additionally, this study did not measure plasma vitellogenin levels.

Thyroid Endocrine Di sruption in Fish

In a published study by Yu et al. (2013), zebrafish embryos were exposed to tebuconazole at concentrations of 0, 1, 2, or 4 mg/L from shortly after spawning to 120 h post-fertilization. T 3 and T 4 levels were measured in extracts from 300 larvae/ concentration using commercially available ELISA kits. Additionally mRNA was is olated and gene expression in the HPT axis
was examined.

There were no significant effect s on hatching, malformations, or survival. In the 4 mg/L group,

T 4 levels were decreased (p<0.05) by 10%, and T 3 levels were increased (p<0.05) by 28%. The following HPT axis genes appeared to be upregulated by tebuconazole treatment: corticotrophin-releasing hormone , thyroid stimulating hormone, sodium/iodide symporter, transthyretin, uridine diphosphate glucuronosyltransferase, thyroni ne deiodinase 2, and thyroid hormone receptor β . Thyronine deiodinase 1, thyroid hormone receptor α , and thyroglobulin did not change significantly.TEBUCONAZOLE FINAL

Page 80 of 81 APPENDIX 3. References Not Utilized in the Tebuconazole 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 tebuconazole but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the compound of interest wa s not used in the study; 3) the test material was not adequately describe d; 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.

Andersen, H, Andersson, A-M, Arnold, S, et al. (1999) Comparison of sh ort-term estrogenicity
tests for identification of hormone-dis rupting chemicals. Published: Environ. Health Perspect. 107 (Suppl 1): 89-108.

Desai, Y (2006) Acute toxicity study of tebuconazole tec hnical in rainbow trout, Onchorhynchus mykiss . Jai Research Foundation, Gujrat, India. Laboratory Project Number: 5744, January 31, 2006. Sponsored by Punjab Chemicals & Crop Protection Ltd., Mumbai, India. MRID 46919204. Unpublished.TEBUCONAZOLE FINAL

Page 81 of 81 Horwitz, K, Costlow, M, and McGuire, W (1975) MCF-7: a human breast cancer cell line with estrogen, androgen, progesterone, and gluc ocorticoid receptors. Published: Steroids : 26 (6) : 785-795.

Jacobsen, P, Christiansen, S, Boberg, J, et al. (2010) Combined exposur e to endocrine disrupting pesticides impairs parturition, causes pup mortalit y, and affects sexual differentiation in rats. Int.
J. Androl. 33 : 434-442.

Jensen, B, Petersen, A, Christiansen, S, et al. (2013) Probabilistic assessment of the cumulative dietary exposure of the popul ation of Denmark to endocrine disrupting pesticides. Food. Chem.
Toxicol. 55 : 113-120.

Kjærstad, M, Taxvig, C, Andersen, H, et al. (2010) Mixture effects of endocrine disrupting compounds in vitro

. Int. J. Androl

. 33 : 425-433.

Orton, F, Rosivatz, E, Scholze, M, et al. (2012) Competitive androgen receptor antagonism as a factor determining the predicta bility of cumulative antiandrog enic effects of widely used pesticides. Environ. Health Perspect. 120 (11) : 1578-1584.

Surprenant, D (1987) Acute toxicity of HWG- 1608 (Technical grade) to rainbow trout ( Salmo
sairdneri ) under flow-through conditions. Springborn Life Sciences, Inc., Wareham, MA. Laboratory Report No. 87-5-2394, completion date not provided. Sponsored by Mobay Corporation, Stilwell, KS. MRID 40700911. Unpublished.