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 

# &

Health Effects Divison

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# f] g:ams d f 'v--

Environmental

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

THR O UG H: Je ss Rowland

## ~ S

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

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

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

.

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

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

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

CHEMICAL: MYCLOBUTANIL

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYMYCLOBUTANIL 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..................................................15

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

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

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................24

1. Tier 1 in vivo Assays.............................................................24

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

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

C. Androgen Pathway.........................................................................28

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

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

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

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

LIST OF TABLES: Table 1. Tier 1 Assa y for Myclobutanil...............................................................8
Table 2. Estrogenic/Anti-Estroge nic Pathway for Myclobutanil ................................11
Table 3. Androgenic/Anti-Androgenic Pathway for Myclobutanil ..............................17
Table 4. Thyroid Pathway for Myclobutanil ........................................................22

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................35
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................47

APPENDIX 3. References Not Utilized in the Myclobutan il WoE Analysis........................58MYCLOBUTANIL 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 enerationMYCLOBUTANIL 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 ConcentrationMYCLOBUTANIL FINAL

v Abbreviation Terminology

MTD Maximum Tolerated Dose
N Ne g ative
NE Not examined/evaluate d
NF sta g e Nieuwkoo p and Faber’s Sta g in g Atlas NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level NS Not Statisticall y Si g nificant
N R Not Re p orte d OCSPP Office of Chemical Safet y Pollution and Prevention OECD Organization for Economic Co-Operation and Development

OPP Office of Pesticide Programs

ORD Office of Research and Develo p ment OSCP Office of Science Coordination and Polic y OSRI Other Scientificall y Relevant Informatio n P Positive
P Parental g eneratio n PC Positive Control PC 10 Positive Control at 10% of response

PC 50 Positive Control at 50% of response

PND Post-Natal Da y POD Point of De p arture PPS Pre p utial Se p aration PROD Pentaox y resorufin-O-dealk y lase ( or de p ent y lase )
PX R Pre g nane X rece p to r QC Quality Control

RBA Relative Binding Affinity

RBC Red Blood Cells
RfD Reference Dose RPC max Relative to Positive Control at maximum

SAP Scientific Advisor y Panel SC Solvent Control

s.c Subcutaneous SDH Sorbitol deh y dro g enase SDWA Safe Drinking Water Act

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th SV Seminal Vesicles
T Th y roid ( hormonal p athwa y) T1WoERC EDSP Tier 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)MYCLOBUTANIL 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-EvidenceMYCLOBUTANIL FINAL

Page 1 of 57 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen re ceptor, altered steroido genesis, 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 myclobutanil interacts w ith E, A or T hormone pathways, the number and type of effects induced, the magnitude a nd pattern of responses observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whether or not the dose(s) at which endocri ne related responses occurred happened concurrently wi th general systemic toxicity.

On February 12, 2014, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-of-evidence (WoE) analysis of the potential interaction of myclobutanil with the E, A, or T hormone pathways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the
results of the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the
results of the cited OSRI for mammalian and wi ldlife studies (40 CFR Pa rt 158 and literature)

For the estrogen pathway, a positive finding in the estrogen receptor transactivation assay (ERTA) was reported a literatur e study; however, myclobutanil was negative in the Tier 1 Estrogen Receptor (ER) binding, ER TA, uterotrophic and female pubertal assays. Overall the data do not support a potential for myclobutanil to interact with th e ER. The results from the Tier 1 aromatase and steroidogenesis assays indicate that there is a potential for myclobutanil to reduce estradiol production in vitro. There was no convincing evidence of estrogen-related
effects in the mammalian st udies suggesting that the in vitro effects on estradiol production are
not relevant to mammals. In the fish short- term reproduction assay (FSTRA), however, there were effects on estrogen-related endpoints that could be poten tially attributed to altered steroidogenesis (effects on hormone levels, VTG, gonadal weight and histopathology, and fecundity).

For the androgen pathway, myclobutanil showed pos itive binding to the androgen receptor (AR); however, the affinity appeared to be weak and did not manifest in an AR agonist or antagonist

response in vivo in the Hershberger assay. In the male pubertal assay, significant decrease in
testosterone levels correlated with a decrease in multiple androgen-dependent tissue weights andMYCLOBUTANIL FINAL

Page 2 of 57 a slight delay in preputial separation (PPS). Th e androgen-related effects in the male pubertal assay occurred at doses that also resulted in increased liver weights and hypertrophy. Furthermore, data from OSRI s how that alteration of testosterone homeostasis in mammals is
mediated via activation of constitutive androsta ne (CAR), pregnane X (PXR) and subsequent enzyme induction in the liver. In mammals, effect s on androgen endpoints o ccurred only at doses that resulted in increased liver weights, liv er hypertrophy, and/or CYP enzyme induction. In the
FSTRA an increase in male GSI was observed alo ng with alterations in the male gonads reported as increased proportions of mature sperm relative to spermatogenic precursor cells. Therefore, as in the estrogen pathway, the non-mammalian data suggest potenti al alteration with steroidogenesis which may be aff ecting the androgen pathway.

There were limited complementary and redundant responses among the EDSP Tier 1 and OSRI for HPT-mediated effects suggest that myclobutanil is not likely to interact with the thyroid pathway.

For myclobutanil, the point of de parture (POD) of 2.5 mg/kg/day is based on decreased testicular weights and increased tes ticular atrophy seen at 9

. 84 mg/kg/day (LOAEL) in the chronic toxicity/carcinogenicity study in ra ts. This POD is used for deri ving the chronic Reference Dose (RfD) for chronic dietary risk as sessment. A POD of 10 mg/kg/day based on atrophy of the testes and prostate, increased number of still born pups, and a decrease in pup weight gain during lactation at 50 mg/kg/day (LOAEL) in the reproductive toxicity st udy is used for calculating the Margins of Exposure (MOEs) for non-dietary risk assessments. Thus, the PODs used for the
overall risk assessments are 20–80-fold lower th an the lowest dose (200 mg/kg/day) where a potentially endocrine mediated e ffect was observed in the male pubertal rat assay. Consequently, the current RfD and MOEs are protective of poten tially endocrine-mediated effects observed in the pubertal assay.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is NOT recommended for myclobutanil sin ce additional testing is unlikel y to impact the current EPA established regulatory endpoints for human risk assessments.

Medaka Extended One-Generation Reproduction Test (MEOGRT) is recommended since the Tier 1 battery and other OSRI suggested potential alterations to steroi dogenesis and effects on reproduction were reported in the FSTRA. Data on survival and growth on early-life stages (ELS) for fish are available with a NOAEC value of 0.98 mg/L (based on growth), but effects on reproduction were not examined. Effects in the FSTRA were observed at 0.327 and 3.3 mg/L
which are lower than the NOAEC value in the ELS study. Therefore, additio nal data obtained by the MEOGRT will be used to establish dose-response relationships for assessing potential adverse effects on reproduction for fish from myclobutanil exposure.MYCLOBUTANIL FINAL

Page 3 of 57 For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for myclobutanil. In both species, no 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 myclobutanil exposure. Relative to EDSP, additional testing is not recommended.

As there were no thyroid-mediated effects observed in the AMA or convincing evidence of a potential thyroid-mediated interaction for my clobutanil, no additional data are recommended for amphibians.MYCLOBUTANIL FINAL

Page 4 of 57 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen re ceptor, altered steroido genesis, 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 myclobutanil interacts w ith E, A or T hormone pathways, the number and type of effects induced, the magnitude a nd pattern of responses observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whether or not the dose(s) at which endocri ne related responses occurred happened concurrently wi th general systemic toxicity.

On February 12, 2014, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-of-evidence (WoE) analysis of the potential interaction of myclobutanil with the E, A or T hormone pathways.

Myclobutanil is not anticipated to volatilize appreciably, based on vapor pressure and Henry’s Law Constant. Based on a log octano l-water partition coefficient (log K ow ) of approximately 3, a relatively low propensity to sorb to organic matter and its organic carbon partition coefficient (K oc ~520 mL/g oc ), myclobutanil residues are not expect ed to bioaccumulate. Myclobutanil is stable to hydrolysis and to photolysis. Myclobutan il is moderately persistent to persistent (DT 50 >70 days) in aerobic soils and persistent in anaerobic soils.

The available information considered in determ ining the potential intera ction of myclobutanil 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.

.MYCLOBUTANIL FINAL

Page 5 of 57 II. Sources of Scientific Data and Technical Information

A. EDSP T1 Screening Assays

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the agency’s test order for myclobutanil are show n 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 Test Order recipients and/or the public. This included studies published in the open literatu re and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). The Agen cy’s review of the initial OSRI is provided in the Report of the Endocrine Disr uptor Review Team on myclobutanil (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any additional rele vant information. Summaries of th e available OSRI are presented in Appendix 2.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria, and approach used in the WoE determination on the potential of a
substance to interact with endocrine-mediated processes ( i.e., E, A, or T hormonal pathways) were as described in the WoE guidance docum ent (USEPA, 2011) and presented at the 2013 FIFRA Scientific Advisory Panel (SAP) (U SEPA, 2013). The weight of evidence process
identifies how the individual lines of evidence are assembled and integrated along two concepts ( i.e ., complementarity and redundancy) within the c onceptual 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 assemble and evaluate the individual studies for their scientific quality and relevance in evaluating potentia l endocrine interaction(s). The second step is to integrate the data along diffe rent levels of biological organization while examining the extent of concordan ce (robustness) of complementarity ( i.e ., the concordance of endpoints within an assay that measur es multiple endpoints) and redundancy ( i.e ., the concordance of endpoints/responses across assa ys) in the observed responses across these different levels of biological or ganization. The third step is to then character ize the main lines of evidence as well any conclusions. Finally, the last st ep is to evaluate whet her additional testing 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 ED SP Tier 1 data represent a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays wereMYCLOBUTANIL FINAL

Page 6 of 57 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 concordance or consistency (complementarity) 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 collective 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 mol ecular initiating event such as receptor binding through potential adverse effects in apical endpoint s such as sexual develo pment and fecundity at the whole organism level. Transitions to higher le vels of biological orga nization can indirectly provide information on potential compensatory capabilities of an i ndividual 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 rele vant, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher-levels of biol ogical organization. Finally, an evaluation of conditions where the responses occur including consideration of whether the responses were observed in the presence of overt or systemic toxicity. The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-mediated effect from a non-endocrine toxic respon se. This uncertainty in distinguishing whether the responses were endoc rine-mediated was discus sed at the FIFRA SAP meeting that evaluated scientific issues associ ated with the WoE evaluation of the EDSP Tier 1 screening process.MYCLOBUTANIL FINAL

Page 7 of 57 In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in the presence of overt to xicity. All effects in
endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary ef fects related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013). For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s curre nt approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecologi cal risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity included, but were not limited to: mortality; clinical signs such as tremor s, ataxia and abnormal swimming (fish and aquatic-phase amphibian s); and body weight decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g ., organ weights/histopathology), biochemical ( e.g ., blood chemistry), and ot her clinical signs ( e.g., lethargy) were also considered when evaluating overt toxicity, espe cially 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/concentration 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 decreases were considered along with other respons es when assessing pote ntial overt toxicity. Additionally, effects which were co nsidered to be signs of system ic toxicity were also captured and these effects were generally considered as less severe forms of toxicity ( e.g ., organ weights, blood chemistry). The circumstances for which a dose/concentration was considered overtly toxic for a particular study are described in Section IV.A.

Therefore, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addres sing the three component s described above is fundamental to the WoE process and in determ ining whether additional data are needed. In addition to characterizing the WoE, reviewers also consider: 1) uncertain ties and their potential impact to conclusions; 2) discus sion of key studies; 3) descripti on of inconsistent or conflicting data; 4) overall strength of evid ence supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing the need for additional data is ba sed on a case-by-case analysis which include all available toxicity data. In summary, the evaluation of the EDSP Tier 1 screening process and ultimate decision for any additional testing is based on a totality of the
scientific evidence.

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 to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, A, or T pathway. Effects that occurred in th e presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not re ported in the table for E, A or T.MYCLOBUTANIL FINAL

Page 8 of 57 A. EDSP T1 Screening Assays

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

.

Table 1. Tier 1 Assays for Myclobutanil

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48618104)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied by OSRI (Hurst and Sheahan, 2003); however a

Tier 1 ERTA assay was submitted (MRID No. 48618105)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48618102)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48618110)

Aromatase Assay (Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied by OSRI (Trösken et al. , 2004, 2006; however a Tier 1 aromatase assay was submitted (MRID No. 48618103)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48618111)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID No.48618107)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID No. 48618108)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID No. 48618109)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48618106)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 48618101)MYCLOBUTANIL FINAL

Page 9 of 57 B. Effects on Hypothalamic-Pit uitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

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

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

Myclobutanil was classified as negative (or not interactiv e) in the Tier 1 in vitro ER binding (rat uterine cytosol) assay. Although the ERTA requirement was cons idered to be satisfied by a published study by Hurst and Sheahan (2003), a guideline ERTA assay was subsequently submitted to the agency for review. In the OSRI study, Hurst and Sheahan used an in vitro recombinant yeast-based β -galactosidase reporter assay to sc reen for the binding and activation of human estrogen receptor (hER- α ). In this yeast-based as say, myclobutanil produced an estrogenic response with an estr ogenic potency relative to 17 β -estradiol (E2) of 7x10 -6 , and an estrogenic LOEC of 781 μ g/L in this test system. However, when hER α -HeLa-9903 cells were used as required in the guidel ine ER transcriptiona l activation assay, myclobutanil was negative
for ER α transcriptional activation up 10 -4 M (highest soluble concentration).

In the Tier 1 aromatase assay (CYP 19), mycl obutanil reduced aromatas e activity by 34, 75 and
96% at concentrations of 10 -7 , 10 -6 and 10 -5 M, respectively, with concentrations ≥ 10 -4 M resulting in >99% inhibition which would be cons idered a positive response in this assay. The response in this assay is consistent with the results from the two publis hed literature articles (Trösken et al. , 2004, 2006) that were submitted as OSRI in re sponse to the test order. In the Tier 1 steroidogenesis assay, d ecreased estradiol levels were observed at 10 -7 M myclobutanil (0.8- fold the solvent control; p ≤ 0.05 in Runs #2 and #3), and in all runs at concentrations ≥ 10 -6 M (0.4-0.7-fold; p ≤ 0.05). The uterotrophic assay was negative at the three doses evaluated (50, 150, and 475 mg/kg/day). Overt toxicity (15% body wei ght decrease) was observed at the high dose. In the Tier 1 female pubertal assay, no effects on estrogen-related endpoints were observed at any dose (50, 200, and 400 mg/kg/day). In that as say, liver effects incl uding increased liver weights and histopathology were seen at the mid and high doses.

In the FSTRA, female gonadal-somatic index (GS I) was significantly (p <0.05) increased 41 and 46% at the mid (0.327 mg a.i./L) and high doses ( 3.3 mg a.i./L), and severe oocyte atresia was noted in females at the high dose. Vitellogeni n (VTG) was significantly reduced (p<0.05) by 88% in females at the high dose. In females, there was a significant incr ease (p<0.05) in serum testosterone at the mid-dose, but not at the high dose, and th ere was a signif icant reduction (p<0.05) of 85% in 17 β -estradiol at the high dos e. Also, fecundity was significantly reduced byMYCLOBUTANIL FINAL

Page 10 of 57 98% at the high dose (p<0.05). At the high dose, female body weight was marginally (9%) but significantly reduced. However, given the lack of other reported signs of clinical toxicity (no
treatment-related effects on mortal ity or behavior) this concen tration considered to not be overtly toxic.

In the Part 158 Guideline studies, the high dos e (200 mg/kg/day) in the rabbit developmental study showed increased resorptions and resorptions/li tter and decreased litter size and viability index; these effects were obser ved in the presence of decreas ed maternal body weights and body weight gains. In the 2-generation rat reproduc tion study at the high dos e of 50 mg/kg/day, an increase in the number of stillborns was observed; also seen at this dose were hepatocyte hypertrophy and increased liver weights. Thus, in both species, repr oductive effects were observed at the highest dose tested in the re spective study accompanied by systemic toxicity.

No effects on avian reproduction were reported in four studies, two studies each with the bobwhite quail and mallard duck. In a Part 158 fish early life cycle test (fathead minnow), at the 2.2 mg/L exposure, there was a 9.7% reduction in m ean total length compar ed to the control. Growth as determined by wet weight was also si gnificantly affected at 4.0 mg/L; 100% mortality occurred at 8.5 mg/L.MYCLOBUTANIL FINALPage 11 of 57Table 2: Estrogenic/Anti-Estrogenic Pathway for MyclobutanilLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Myclobutanil1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt) / Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48618104)NERTA(MRID 48618105)NSteroidogenesis(MRID 48618110)PAromatase(MRID 48618103)PUterotrophic(MRID 48618111)NX(H)4Female Pubertal Rat(MRID 48618108)NNNNNN↑LW↑LH(M, H)LE (H)NFSTRA(MRID 48618106)P5↑GSI:41, 46%(M, H)P(H)NFcd:↓98%(H)P(H)N6MYCLOBUTANIL FINALPage 12 of 57Lines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Myclobutanil1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt) / Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRIERTA(Hurst and Sheahan,2003)PAromataseTroskenet al.,(2004 and 2006)PSubchronic toxicity(Rat; MRID 00145048)NENNNEX(MH,H)X(H)Subchronic toxicity(Mouse; MRID00165245)NE N N NEX(MH,H)X(H)Developmental toxicity(Rat; 00141672)NRNNENDevelopmental toxicity(Rabbit;MRID 00164971)NRNNENTwo-generationreproduction(Rat; MRIDs 00143766,00149581)NENENNE NEP(H)X(M, H)N2-yr Chronic toxicity(Rat; MRIDs 00165247,42809101)NENNNEX(H)NMYCLOBUTANIL FINALPage 13 of 57Lines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Myclobutanil1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt) / Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Carcinogenicity(Mouse; MRIDs00164990, 40244301,42809102)NENNNX(H)NChronic toxicity(Dog; MRID 00165248)NNNNX(M,H)NAvian Reproduction(Quail; MRIDs00265744,)NN NAvian Reproduction(Duck; MRIDs00265744,)NN NAvian Reproduction(Quail; MRIDs 4340201,43087901)NN NAvian Reproduction(Duck; MRID43087902)NN N1. Key to responses: L=Low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows (↓or↑) indicate the direction of theresponse. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unlessotherwise indicated. LW = Liver weight, LH = liver hypertrophy, LE = serum levels of liver enzymes, KW = kidney weight2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Body weight was decreased 15%.5. A significant increase (p<0.05) in serum testosterone at the mid-dose, but not at the high dose, and there was a significantreduction (p<0.05) of 85% in 17βestradiol at the high doseMYCLOBUTANIL FINALPage 14 of 576. A marginal, but significant (9%; p=0.022), reduction in female body weight was observed at the high concentration, but, given the lack of other reportedsigns of clinical toxicity (no treatment-related effects on mortality or behavior) this concentration considered to not be overtly toxicP Positive findings (in vitro) / No effect (in vivo)N Negative findings (in vitro) / No effect (in vivo)NE Not examinedNR Not reportedMYCLOBUTANIL FINAL

Page 15 of 57 2. Effects on Androgen Pathway

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

Myclobutanil was classified as a binder in the AR binding assay; however, based on the analysis of the response curves, myclobutanil showed a w eak affinity for the androgen-receptor. In the steroidogenesis assay, testoster one production was significantly decreased in all three runs by 0.8-fold at myclobutanil concentrations of 10 -4 M. Myclobutanil was negative at all doses evaluated (25, 125 and 500 mg/kg/da y) in the Hershberger assay for both androgen agonism and antagonism

.

No effects on androgen-related endpoints were ob served at the low dose (50 mg/kg/day) in the male pubertal assay. Serum testosterone levels were significantly decreased by 49% at the mid (200 mg/kg/day) and 85% at high dose (400 mg/kg/day). At 200 mg/kg/day, androgen-sensitive tissue (absolute/relative) weight decreases were noted in the semi nal vesicle + coagulating gland ( ↓ 15/19%) and ventral prostate ( ↓ 16%). At 400 mg/kg/day, tissue weig ht decreases were noted in the seminal vesicle + coagulating gland ( ↓ 33/41%), ventra l prostate ( ↓ 27/29%), dorsolateral prostate ( ↓ 25/27%) and LABC complex ( ↓ 16/18%). At 400 mg/kg/day, a slight delay (1.7days) in preputial separation (PPS) wa s reported. In addition to the ch anges in AST weights, relative adrenal weights were also increased (15%) at 40 0 mg/kg/day and absolute /relative liver weights were increased at 200 mg/kg/day (  12/21%) and 400 mg/kg/day (  27/37%). An increased incidence of hepatocellu lar hypertrophy was also observed at th e doses that showed changes in androgen-related endpoints.

In the FSTRA, testosterone was repor ted to not be affected in males. Significant increase in male GSI ( ↑ 80%) along with alterations in the male gona ds reported as increased proportions of mature sperm relative to spermatoge nic precursor cells were observed.

In a Part 158 two-generation reproduction rat study, multifocal or diffuse testicular atrophy, increased necrotic spermatocytes/spermatids or decreased spermatozoa and atrophy of the prostate were observed at 50 mg/kg/ day. In the chronic rat toxicity st udies, testicular effects were observed in the mid and mid-high, and high male s (10, 39 and 106 mg/kg/day, respectively) and included significantly decreased mean absolute testes weights, and decreased absolute and relative (to brain) testicular we ights, and increased incidences of testicular atrophy at the midhigh and high doses. Histopathol ogical testicular changes incl uded increased incidences of bilateral aspermatogenesis, hypospermia and cellular de bris in the epididymides at the high dose.MYCLOBUTANIL FINAL

Page 16 of 57 Liver mixed function oxidase activit y was significantly increased in females at 13 mg/kg/day at 3 months (61%), males at 39 mg/kg/day at 3 a nd 6 months (34–47%) and fe males at 52 mg/kg/day at 3 months (78%). Relative and absolute liver weights were significantly increased in females at 52 mg/kg/day at 3 months (13%) a nd 6 months (20%), respectively.

In an OSRI study, Tully et al. (2006) evaluated gene expression prof iles in the liver and testis to characterize the toxicity of my clobutanil. In this study rats we re administered myclobutanil by gavage for 14 days (PND 60–74) at 10, 75 or 150 mg /kg/day. Serum testosterone was elevated >2X and decreased sperm mobility was observed at the high dose. A 13% increase in relative liver weight was also seen at the high dose. There was no effect on epididymal weights at any
dose. Goetz et al. , (2007) evaluated the disr uption of testosterone hom eostasis as a potential mode of action for the reproductive toxicity of three triazole fungicides (including myclobutanil). In this study male Wistar Han rats were fed myclobutanil at 0, 100, 500 or 2000 ppm (0, 6, 33, 134 mg/kg/day) from gestation day (GD) 6 to postnatal day (PND) 120. Following exposure, anogenital distance (AGD) was significantly (p<0.001) increased at the high dose (134 mg/kg/day). Relative liver wei ghts were increased 9–13%, at PND 1, 50 and 92. Hepatocellular hypertrophy was present at PND 92 at the high dose . Relative pituitary we ights were reduced at PND 92 in mid and high dose groups. Absolute te stis weights were increased at PND 1 and 22 and relative ventral prostate weights and testosterone leve ls were increased at PND 92. Decreased insemination (134 mg/kg/day) and fertil ity (33 and 134 mg/kg/day) were also reported in the males treated with myclobutanil.

Additional mechanistic studies conducted by Goetz and Dix (2009a and 2009b) evaluated the potential mode of action for reproductive and hepatic toxicity by triazole fungicides. The authors conducted gene expression profiling on liver and te stes from male rats exposed to triazole fungicides including myclobutanil at 500 and 2000 ppm (33 and 134 mg/kg/day) from GD 6 to PND day 92. The results of the expression prof iles showed differentially expressed genes involved in pathways for fatty acid and steroid metabolism in the liver. No common disrupted pathways were identified in the testes. These data suggest that myclobutanil alters testosterone homeostasis via disruption of he patic steroid metabolism and ster ol biosynthesis rather than modulating steroidogenic gene expression in the testes.

There were no androgen-related effects determin ed for the available Pa rt 158 wildlife studies.MYCLOBUTANIL FINALPage 17 of 57Table 3: Androgenic/Anti-Androgenic Pathway for MyclobutanilLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Myclobutanil1Study Type /Literature Citation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48618102)PSteroidogenesis(MRID 48618110)PHershberger(MRID 48618107)NEN↑LW(M, H)NMale Pubertal Rat(MRID 48618109)↓49%↓85%(M, H)NNNNN↓SV(M, H)↓VP(M, H)↓LABC(H)↓DP(H)PPSage:↑1.7 d(H)↑LW,↑LH(M, H)LE(H)NFSTRA(MRID 48618106)N↑GSI79%(H)P(H)NNNN4OSRISubchronic toxicity(Rat;MRID 00145048)N N NE NE NE NEX(MH, H)X(H)Subchronic toxicity(Mouse;MRID 00165245)N N NE NE NE NEX(MH, H)X(H)MYCLOBUTANIL FINALPage 18 of 57Table 3: Androgenic/Anti-Androgenic Pathway for MyclobutanilLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Myclobutanil1Study Type /Literature Citation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Developmentaltoxicity(Rat; 00141672)NNE NDevelopmentaltoxicity(Rabbit;MRID 00164971)NNE NTwo-generationreproduction(Rat; MRIDs00143766,00149581)NEP5(H)NEP5(H)NE NENX(M,H)N2-yr Chronictoxicity/Carcinogenciity7(Rat; MRIDs00165247,42809101)P6(M,MHH)P6(M,MH,H)NEP7(H)NENEX(H)NCarcinogenicity(Mouse; MRIDs00164990,40244301,42809102)NNNNNENEX(H)NChronic toxicity(Dog;MRID 00165248)NN NE NE NNEX(M,H)NMYCLOBUTANIL FINALPage 19 of 57Table 3: Androgenic/Anti-Androgenic Pathway for MyclobutanilLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Myclobutanil1Study Type /Literature Citation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Liver and Testistoxicity profiles(Tullyet al.,2006)18>2-fold(H)NENEspermmobilityX(H)NReproductiveToxicity Mode ofAction(Goetz,et al.,2007, 2009a,b)16(M,H)(L, H)PND 1(M,H)VP(M)(M, H)AGDX(H)NAvianReproduction(Quail; MRIDs00265744,)NENENN NAvianReproduction(Duck; MRIDs00265744,)NENENN NAvianReproduction(Quail; MRIDs4340201,43087901)NN NAvianReproduction(Duck; MRID43087902)NN N1.Key to responses: L=Low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows (↓or↑) indicate the direction of theresponse. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unlessMYCLOBUTANIL FINALPage 20 of 57otherwise indicated. Abbreviations for androgen sensitive tissues:Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Levator anibulbocavernosus (LABC), Epididymides (EP). LW= Liver weight, LH = liver hypertrophy, LE = serum levels of liver enzymes2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. A marginal, but significant (9%; p=0.022), reduction in female body weight was observed at the high concentration, but, given the lack of other reportedsigns of clinical toxicity (no treatment-related effects on mortality or behavior) his concentration considered to not be overtly toxic.5. Multifocal or diffuse testicular atrophy, increased necroticspermatocytes/spermatids or decreased spermatozoa and atrophy of the prostate were observed atthe high dose (50 mg/kg/day).6. Decreased absolute and relative (to brain)testicular weights, and increased incidences of testicular atrophy were seen at the mid and mid-high doses (firststudy: 10 and 40 mg/kg/day, respectively) and high dose (second study: 106 mg/kg/day)7. Increase incidences of bilateral aspermatogenesis, hypospermia and cellular debris in the epididymides at the high dose (second study: 106.08 mg/kg/day).P Positive findings (in vitro) / No effect (in vivo)N Negative findings (in vitro) / No effect (in vivo)NE Not examinedMYCLOBUTANIL FINAL

Page 21 of 57 C. Effects on Hypothalamic-Pitui tary-Thyroidal (HPT) Axis

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

The potential for myclobutanil to interact with thyroid regulation are summarized in Table 4. The various targets of the th yroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid orHPT axis effects. This table also includes HPTrelevant 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 tabl e and not considered further in the

WOE assessment.

The female pubertal assay was negative for all obse rved thyroid parameters at all doses evaluated (50, 200 and 400 mg/kg/day). No systemic toxic ity was noted in the study at doses up to 400 mg/kg/day; however, there were significant dose-dependent increas es in absolute/relative liver weights at the mid ( ↑ 18/19%) and high ( ↑ 36/41%) doses as well as changes in hepatocellular histopathology.

In the male pubertal assay, decreased pituitary we ight was observed at all dose levels, but the effect was not dose dependent. Dose dependent increases were noted in serum TSH levels, but the changes were not statistically signifi cant, and no changes were noted in T 4 levels. Thyroid weight was also unaffected, although non-si gnificant changes in thyroid hi stopathology were seen at the high dose ( ↑ follicular cell height, ↓ colloidal area). Consistent with the female pubertal rats, liver weights were increased at the mid ( ↑ 12–21%) and high ( ↑ 27–37%) doses, accompanied by hepatocyte hypertrophy in males a nd elevated gamma glutamyl transf erase levels at the high dose.

In the amphibian metamorphosis assay there were no treatment-related effects on thyroid histopathology. Developmental st age was significantly delayed (p<0.05) on Day 7 (NF stage 52 vs

. NF 54 in control), and normalized HLL was si gnificantly reduced (p<0.05) by 13% at the high dose on Day 7 and at all tr eatment levels on Day 21 ( ↓ 10–24%). Clinical observations were transiently noted in all treatment groups with a sl ightly higher proportion of individuals affected in the two highest treatment levels ; effects included lying on the su rface or the bottom of the tank, inverted or irregular swimming a nd being non-responsive to stimulus . However, these observations did not persistent in the study and were isolated to one tadpole occasionally displaying lethargy or surfacing from Day 8-21 in both the control and tr eatment groups, and as such these treatment groups were not considered to be overtly toxic.

In the OSRI, none of the studies evaluated show ed changes in thyroid weights. A subchronic toxicity study in rats showed an increase in small follicles in males at 300 mg/kg/day and higher.MYCLOBUTANIL FINAL

Page 22 of 57 Evidence of systemic toxicity was also see at this dose, including in creased liver and kidney weights; hypertrophy and necrosis in the liver; and pigmentation in the convoluted kidney tubules.

Table 4. Thyroid Pathway for Myclobutanil.

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

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

EDSP Tier 1 Assays

Male Pubertal Rat (MRID 48618109) N N N N ↓ 8–10% (L, M, H) N ↑ LW, ↑ LH (M, H)

 LE (H) N

Female Pubertal Rat (MRID 48618108) N N N N N N ↑ LW, ↑ LH (M, H)

 LE (H) N

AMA (Frog; MRID 48618101) N Day 7: ↓ NF stage & ↓ 13%
NHLL (H); Day
21: ↓ 10- 24% HLL (L,M,H) Day 7: ↓ 16,19 % SVL (M,H), ↓ 38,45 % BW (M,H); Day 21:

↓ 6% SVL (L,H), ↓ 20,22 % BW (L,H) N 4

OSRI Subchronic toxicity (Rat; MRID 00145048) N P 5 (males) NE X (MH, H) X (H)

Subchronic toxicity (Mouse; MRID 00165245) N N NE X (M, H) X (H)

Developmental toxicity (Rat; 00141672) NE NE NE NE N

Developmental toxicity (Rabbit; MRID 00164971) NE NE NE NE NMYCLOBUTANIL FINAL

Page 23 of 57 Table 4. Thyroid Pathway for Myclobutanil.

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

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

Two generation reproduction (Rat; MRIDs 00143766, 00149581) NE NE NE X (M,H) N

2-yr Chronic toxicity (Rat; MRIDs 00165247, 42809101) N N NE N N

Carcinogenicity (Mouse; MRIDs 00164990, 40244301, 42809102) NE N NE N X (H)

Chronic toxicity (Dog; MRID 00165248) N N N X (M,H) N

1 Key to responses: L=Low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.. Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. LW = Liver weight, LH = liver hypertrophy, LE = serum levels of liver enzymes 2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 3. The overt toxicity in the Tier 1 assays are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 4. While clinical signs of toxicity were observed in th e top two treatment groups from Day 1 to 7 in a greater proportion than the control and low treatment, these behavior did not persist in the study and was isolated to a few observations from Day 8-21 in the control and treatment groups. 5 Increase incidence of small thyroid follicles was seen in males at ≥ 3000 ppm (300 mg/kg/day). P Positive findings N Negative findings NE Not examinedMYCLOBUTANIL FINAL

Page 24 of 57 IV. Committee’s Assessment of Weight of Evidence

This section of the document will describe the weight of evidence (WoE) determination on the potential of myclobutanil to interact with endocri ne mediated processes ( i.e., E, A, or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated analysis of those lines of evidence. The WoE evaluation includes considerations of biologi cal plausibility of the findings from the different lines of evidence by examining the consistency, coherence, and interrelationships among the measured endpoints within and across studies. The availabl e findings from standard toxicology studies on the substance may contribute to the WoE evaluation in helping elucidate if effects seen in the Tier 1 assay are related to perturbations of the endocrine system per se or alternatively a sequelae of systemic effects. Endocrine modes of action may evoke a number of phenotypic consequences other than those evaluated in the Tier 1 assays.

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

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

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

vivo Tier 1 assays and OSRI, other effects obser ved which may be considered overt/systemic toxicity are also describe belo w. Generally, one parameter (i.e ., death or >10% decrease in mammalian body weight) was sufficient for a dose/concen tration to be considered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of syst emic toxicity were generally less severe forms of toxicity (e.g., organ weights, blood chemistry).

1. Tier 1 in vivo AssaysMYCLOBUTANIL FINAL

Page 25 of 57 In the Tier 1 mammalian studies, only the uterot rophic assay showed evid ence of overt toxicity. In this assay, body weight was decreased (p<0.0 5) by 15% on Day 3, resulting in a 59% decrease (p<0.05) in body weight gain at the high dose (4 75 mg/kg/day). No treatm ent-related effects on bodyweight were seen at the two lo wer doses (50 and 150 mg/kg/day).

In the Hershberger assay, no evidence of sy stemic toxicity was observed at the low dose (25 mg/kg/day). Systemic toxicity was observed at the higher doses in both the androgenic and anti-androgenic parts of the assay. For the androgenic por tion of the study, absolute liver weights were increased by 20% and 53% at 125 and 500 mg/kg/day myclobutanil, respectively. In the anti-androgenic portion of the assay, a 59% increase in absolute liver weight was seen in animals given 500 mg/kg/day myclobutanil with testosterone proprionate (TP). The liver weight increases at the high dose correlated with histopathologi cal lesions in the liver including hepatocellular hypertrophy and vacuolization.

In pubertal female rats, no evidence of systemic toxicity was seen at th e low dose (50 mg/kg/day). Dose-dependent increases (p<0.05) in absolute, re lative, and adjusted liver weights were observed at 200 ( ↑ 18–19%) and 400 (36–41%) mg/kg/day. Treatment-r elated increases (p<0.05) in alanine transaminase (ALT) ( ↑ 24%) and gamma-glutamyl transpeptidase (GGT) (130%) were also observed at 400 mg/kg/day. In addition, females in the 200 and 400 mg/kg/day groups displayed dose-dependent increase in the inci dence and severity of liver hi stological including hypertrophy and number of mitotic figures. Male pubertal rats also showed significant (p<0.05) dosedependent increases in liver weights. Liver weights increas ed by 12–13% at 200 mg/kg/day and 27–28% at 400 mg/kg/day, and relative liver we ights increased by 21% and 37% at 200 and 400 mg/kg/day, respectively. Concomitant with the increases in liver weight, there were histological changes at 200 and 400 mg/kg/day, which consiste d of hepatocellular hy pertrophy with altered tinctorial properties in centrilobul ar and midzonal hepatocytes a nd a slight increase the numbers of mitotic figures in hepatocytes at 400 mg/kg/ day. A significant increase (53%) in GGT was also noted at 400 mg/kg/day.

In the FSTRA, at the 3.3 mg a.i. /L level, a marginal, but sign ificant (9%; p=0.022) reduction in female body weight was observed but given the lack of other reported signs of clinical toxicity (no treatment-related effects on mortality or beha vior) this concentration not considered to be overtly toxicity.

In the AMA, some abnormal behaviors including floating on the surface, lying on the bottom of the tank, inverted or irregular swimming, lack of surfacing activity and being nonresponsive to stimulus were transiently noted among contro l and exposed tadpoles. A greater proportion of
these abnormal behaviors were observed in the two highest treatment groups. However, these observations did not persistent in the study and were isolat ed to one tadpole occasionally displaying lethargy or surfacing from Day 8-21. There was no mortality in the control group and it did not exceed 7% in any treatment level.MYCLOBUTANIL FINAL

Page 26 of 57 2. OSRI

Conazoles such as myclobutanil were designe d 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. The biosynthesis of ergosterol is crit ical to the formation of fungal cell walls. The lack of normal sterol producti on slows or stops the gr owth 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 includi ng fungi, protists, mammals, and plants and is required for sterol biosynthesis in all eukaryotic systems. Ma mmalian 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). Myclobutanil has been show n 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 myclobutanil 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 my clobutanil appeared to perturb fatty acid and steroid metabolism in the male rat liver predominantly through the CAR and PXR signaling pathways (Goetz et al. , 2007; Goetz and Dix 2009 a, b).

There are uncertainties with respect to the mo lecular initiating event(s) for the liver and reproductive effects for myclobutanil. Exposure to myclobutanil induced CAR/PXR stimulated pathways which are involved in xenobiotic metabolism and clearance in the liver (Goetz et al.

, 2007; Plant and Gibson, 2003; Sun et al. , 2005, 2006; Tully et al. , 2006). Since both CAR and PXR regulate the expression of CYP genes and the associated enzyme activity of oxidative
metabolism of steroid hormones as well as xenobi otics, it may be that CAR and PXR are also components of a regulatory network that regu lates circulating hormone levels. Whereas myclobutanil is predominantly metabolized by the CYP2C and 3A sub-families, myclobutanil appears to induce CYP2B and CYP3A. (Sun et al. 2004, 2005, 2006; Goetz et al. 2007; Tully et
al. 2006; Barton et al. , 2006). This presumably reflects ac tivity of myclobutanil through the pregnane X receptor (PXR) and constitutive andros tane receptor (CAR) receptors coordinately altering expression of a number of xenobiotic-metabolizing enzymes, including CYP2B1 (Barton

et al ., 2006; Sun et al. 2004; Tully et al. 2006).

The genom ic data from Goetz et al. (2009a) suggest the inducti on of CAR and PXR nuclear receptor-mediated genes following triazole exposure results in increased testosterone metabolism in the liver and does not appear to be due to modulation of steroidogeni c genes in the testis.MYCLOBUTANIL FINAL

Page 27 of 57 In the Part 158 mammalian studies, the liver wa s the primary target organ of toxicity. The absolute and relative liver weight s were significantly increased in both sexes at >150 mg/kg/day in the mouse subchronic study. The liver weight incr eases were associated with histopathological effects including centrilobular and midzonal he patocellular hypertrophy and vacuolation were
seen in males at ≥ 150 mg/kg/day and in females at ≥ 450 mg/kg/day. Increases in ALT were seen in males at 150 mg/kg/day and in both sexe s at 450 mg/kg/day and above. In the mouse carcinogenicity study, absolute and relative liver weights were increased in males (10%) and females (12–13%) at 3 months at the high dose (70.2 and 85.2 mg/kg/day, respectively). Increased incidence and severity of centr ilobular hypertrophy, Kupf fer cell pigmentation, hepatocellular vacuolation and necrosis were also observed in the liver at the high dose.

In rats, significant decreases in body weights we re seen in the subchronic study in males (25%) and females (14%) at 500 mg/kg/day. Liver and kidney weights were significantly increased in both sexes at 150 and 500 mg/kg/day. Effects at 150 and 500 mg/kg/day also included elevated ALT, GGT, cholesterol, and blood urea nitrogen (BUN) levels. In the chronic toxicity/carcinogenicity studies, ab solute and relative liver weights were increased in females at 6 and 3 months, respectively. In the rat 2-genera tion reproduction study, increas ed liver weights and hepatocellular hypertrophy were seen in the F0 males at 10 mg/kg/day and in both sexes and in the F0 and F1generations at 50 mg/kg/day.

In the dog subchronic study, absolute liver weights were significantly increased in males at 29.1 and 56.8 mg/kg/day and in males and in females at 58 mg/kg/day. Relative liver weights were increased in both sexes at the high dose. Increas ed incidences of hepa tocellular hypertrophy were
seen in males at ≥ 7.3 mg/kg/day in males and at 32.4 and 58 mg/kg/day in females. Increased liver weights and hepatocellular hypertrophy we re observed in the chronic dog study in both sexes at doses of 10 mg/kg/day and 40 mg/kg/day. Increased levels of alkaline phosphatase (AP), ALT (males only) and GGT (females only) were seen at 40 mg/kg/day.

In a Part 158 fish early life cycle test (fathead minnow), at the 2.2 mg/L exposure, there was a 9.7% reduction in mean total length compared to the control. Growth as determined by wet weight was also significantly a ffected at 4.0 mg/L. One hundred pe rcent mortality occurred at 8.5 mg/L.

No effects on mortality, body weight, feed consumpti on or clinical signs were reported in the Part 158 bobwhite quail and mallard duck avian reproduction assays.

B. Estrogen Pathway

The positive response observed in the ER transc riptional activation assa y (Hurst and Sheahan, 2003) submitted as OSRI was not corroborated by the negative findings in the EDSP Tier 1 ERMYCLOBUTANIL FINAL

Page 28 of 57 binding, ERTA, uterotrophic and female pubertal assays. Overall, the data do not support a potential for myclobutanil to inte ract with the ER. The discrepa ncy in responses between the OSRI and the guideline ERTA assays may be attri buted to the use of yeas t cells in the Hurst and Sheahan study. The response in th e yeast cells may have resulted from an indirect mechanism
rather than from a direct interaction with the ER.

Results from the Tier 1 (steroidogenesis and aromatase assays) and OSRI (Trosken et al. , 2004, 2006) studies suggest a potential for myclobutanil to alter steroidogenesis in vitro . However, there is no convincing evidence of estrogen-related ef fects in the female pubertal assay or Part 158 mammalian toxicity studies. However, there were effects observed in the FSTRA that could be
attributed to alterations in estradiol levels. In the females, at the high concentration (3.3 mg a.i./L), there was a reduction in serum 17 β -estradiol and plasma VTG, an increase in GSI as well as gonadal histological effects (oocyte atresia), and a 98% redu ction in fecundity. Additionally, GSI and serum testosterone were significan tly increased at the mid (0.327 mg a.i./L)
concentration.

While significant changes or alterations in estr ogen related responses were not reported in the

in vivo mammalian studies, the results are consistent with differences in sensitivities between fish and rats regarding chemicals that appear to alter steroidogenesis (Ankley and Gray, 2013).

C. Androgen Pathway

Although myclobutanil showed a weak affinity for the AR in the AR binding assay, the in vitro binding did not manifest in an agonist or an tagonist response in the Hershberger assay. Myclobutanil was positive in the steroidogenesis assay resulting in a decrease in testosterone production in vitro . In the male pubertal assay, signifi cant decreases in testosterone ( ↓ 49–85%) correlated with a decrease in multiple androgen-dependent tissue weights ( ↓ 15–41%) and a slight delay (1.7 day) in PPS. These responses, however, occurred in the presence of systemic toxicity which included increased liver weight and hepatocellular hypertrophy. No androgen-related responses were observed in the absence of system ic toxicity in the male pubertal assay. While testosterone was reported to not be affected in males in the FSTRA, the study did report a significant increase in male GSI ( ↑ 80%) along with alterations in the male gonads reported as increased proportions of mature sperm rela tive to spermatogenic precursor cells.

Additionally, in a Part 158 two-generation reprod uction rat study, multifocal or diffuse testicular atrophy, increased necrotic spermatocytes/sperma tids or decreased spermatozoa and atrophy of the prostate were observed in these animals. In the chronic rat study, te sticular toxicity was observed in the mid and high dose males, and in cluded significantly decreased mean absolute testes weights, and decreased absolute and relative (to brain) testicular weights. Incidences of testicular atrophy were increased at the mid and mid high doses. Additionally, liver mixed function oxidase activity was significantly increased in males at 3 and 6 months.MYCLOBUTANIL FINAL

Page 29 of 57 In other OSRI (Goetz et al. , 2007) alterations in androgen-re lated endpoints were observed including changes in AGD, AST, and testosterone levels which occurred in the presence of increased liver weights and hi stopathology. Mechanistic studie s (Goetz and Dix (2009a and 2009b) suggested that myclobutanil alters testosterone homeostas is via disruption of hepatic steroid metabolism and sterol biosynthesis rather than modulating steroi dogenic gene expression
in the testes.

Based on the EDSP Tier 1 data and OSRI, myclobut anil appears to alter steroidogenesis which then interferes with the androge n signaling pathway and the HPG axis. In mammals, however, the
alteration in testosterone levels appears to be mediated via CAR/ PXR activation. In the Tier 1 and OSRI mammal studies evaluated, no androgen-rela ted effects were observed in the absence of increased liver weight, hepatocellular hypertr ophy or CAR/PXR-mediated CYP induction.

In contrast to the mammals, data are not availa ble by which to understand the mode of action for myclobutanil for fish, therefore, the effects observed in the FSTRA along with the effects in the in

vitro assays suggest that myclobutan il could potentially alter steroidogenesis th ereby affecting the androgen, as well as the estrogen, pathway.

D. Thyroid Pathway

In the male and female pubertal assay there were no significant change s in thyroid-mediated endpoints; pituitary weight was decreased at all doses in the male pubertal assay. The only thyroid-related effect observed in the Part 158 OSRI was an increas ed incidence of small thyroid follicles in males at 300 mg/kg/day; a dose that resu lted in significant liver and kidney toxicity. In the AMA, since the developmental delays we re not corroborated by thyroid histological diagnoses that would be s uggestive of a potential HPT-me diated effect, the observed developmental delays were not considered to be thyroid-mediated. Therefore, the limited complementary and redundant responses among th e EDSP Tier 1 and OSRI for HPT-mediated effects suggest that myclobutanil is not likely to interact w ith this pathway.

E. Conclusions

Based on the weight of the evidence, myclobutanil a ppears to potentially a lter steroidogenesis in fish which may affect the androgen, as well as th e estrogen, pathways. In mammals, however, the alteration in androgen-mediated endpoints appears to be mediat ed via CAR/PXR activation and subsequent enzyme induction in the liver. There is no convincing evidence of an interaction with the estrogen pathway in mammals or the thyr oid pathway in mammals and non-mammals.MYCLOBUTANIL FINAL

Page 30 of 57 V. EDSP Tier 2 Testing Recommendations

For myclobutanil, the point of de parture (POD) of 2.5 mg/kg/day is based on decreased testicular weights and increased tes ticular atrophy seen at 9

. 84 mg/kg/day (LOAEL) in the chronic toxicity/carcinogenicity study in ra ts. This POD is used for deri ving the chronic Reference Dose (RfD) for chronic dietary risk as sessment. A POD of 10 mg/kg/day based on atrophy of the testes and prostate, increased number of still born pups, and a decrease in pup weight gain during lactation at 50 mg/kg/day (LOAEL) in the reproductive toxicity st udy is used for calculating the Margins of Exposure (MOEs) for non-dietary risk assessments. Thus, the PODs used for the
overall risk assessments are 20–80-fold lower th an the lowest dose (200 mg/kg/day) where a potentially endocrine mediated e ffect was observed in the male pubertal rat assay. Consequently, the current RfD and MOEs are protective of poten tially endocrine-mediated effects observed in the pubertal assay.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is NOT recommended for myclobutanil sin ce additional testing is unlikel y to impact the current EPA established regulatory endpoints for human risk assessments.

Medaka Extended One-Generation Reproduction Test (MEOGRT) is recommended since the Tier 1 battery and other OSRI suggested potential alterations to steroi dogenesis and effects on reproduction were reported in the FSTRA. Data on survival and growth on early-life stages (ELS) for fish are available with a NOAEC value of 0.98 mg/L (based on growth), but effects on reproduction were not examined. Effects in the FSTRA were observed at 0.327 and 3.3 mg/L
which are lower than the NOAEC value in the ELS study. Therefore, additio nal data obtained by the MEOGRT will be used to establish dose-response relationships for assessing potential adverse effects on reproduction for fish from myclobutanil exposure.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for myclobutanil. In both species, no 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 myclobutanil exposure. Relative to EDSP, additional testing is not recommended.

As there were no thyroid-mediated effects observed in the AMA or convincing evidence of a potential thyroid-mediated interaction for my clobutanil, no additional data are recommended for amphibians.MYCLOBUTANIL FINAL

Page 31 of 57 VI. References

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Marty, M.S.; Marshall, V.A.; Jeong, YC.; and Sura, R. (2011). Myclobutanil: Hershberger Assay in Castrated Male Crl:CD(SD) Rats. Unpublished study prepared by Toxicology & Environmental Research and Consulting, Th e Dow Chemical Company, Midland, MI. Laboratory Study No.: 101131, Oc tober 21, 2011. (MRID 48618107).

Marty, M.S.; Zablotny, C.L.; and Stebbins, K.E ( 2011). Myclobutanil: Pubertal Development and Thyroid Function in Intact Juve nile/Peripubertal Female CrL: CD(SD) Rats. Unpublished study by Dow Chemical Company, Midland, MI. La boratory Project Study ID: 111089, December 13, 2011. (MRID 48618108).

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Pedersen, C. A.; and Lesar, C.L. (1993). RH -3866 Technical (Myclobutanil): Toxicity and Reproduction Study in Bobwhite Quail. Bio- Life Associates, Ltd., .N6230 County Road G, Neillsville, WI. Sponsored by Rohm and Haas Co. Research Laboratories, 727 Norristown Rd., Spring House, PA. Laboratory Report ID: BLAL No. 111-010-07. (MRIDs 43402001 and 43087901).

Pedersen; C.A.; ! and Lesar, C.L. (1993). RH-3866 Technical (Myclobutanil): Toxicity and Reproduction Study in Mallard Ducks. Bio-Life Associates, Ltd. -! N6230 County Road G,MYCLOBUTANIL FINAL

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USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
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Page 35 of 57 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosi s Assay; (OCSPP 890.1100)

The 21-day assay (MRID 48618101) of Myclob utanil (93.7% purity) on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 per control and treatment group) were exposed to a negative control and nominal concentrations of 0.100, 0.500, and 2.50 mg a.i./L. Time-weighted average (TWA) c oncentrations were <0.00474 (<LOQ; control), 0.0964, 0.438, and 2.04 mg a.i./L. The test system was maintained at 20.1 to 22.9°C and a pH of

7.0 to 7.7.

There was no mortality in the control group and it di d not exceed 7% in any treatment level. From Day 1-7, some abnormal behaviors (floating on the surface, lying on the bottom of the tank, inverted or irregular swimming, lack of surfaci ng activity, and being nonr esponsive to stimulus) were transiently noted among control and exposed tadpoles, with a greater proportion of these abnormal behaviors observed among those in the two highest treatment groups. However, these observations did not persistent in the study and were isolat ed to one tadpole occasionally displaying lethargy or surfacing from Day 8-21 in both the control and treatment groups.

Myclobutanil significantly reduced wet weight in the mid and high treatment groups on Day 7 (38 and 45%, respectively, p <0.05) and in the lo w and high treatment groups on Day 21 (20 and 22%%, respectively, p<0.01). Similarl y, snout-vent length (SVL) was significantly reduced in the mid and high treatment groups on Day 7 (16 an d 19%, respectively, p <0.05) and significant
reductions were observed in th e low and high treatment groups on Day 21 (6%, p <0.01). There were also significant reductions in non-normalized hind limb le ngth (HLL) in the mid and high treatment groups on Day 7 ( ≥ 20%, p <0.05) and in all treatment groups on Day 21 ( ≥ 15%, p

≤ 0.01).

On Day 7, developmental stage was significantly (p <0.05) delayed at the high treatment level (NF stage 52 vs. NF 54 in control). Normalized HLL on Day 7 was significantly decreased at the high treatment level (13%, p= 0.025) and was significan tly decreased at all treatment levels on Day 21 (10%, 24%, and 23% at the low, mid, and high treatment levels, respectively, p <0.05). There was no evidence of asynchronous development reporte d on Day 7 or 21. A small proportion of test animals (<20%) were ‘late-stage’ (classified as >NF 60 on Day 21), and th ese tadpoles were not included in the analysis of growth (body wei ght, SVL) and HLL measurements. There were no treatment-related effects on thyroid gla nd histopathology at any treatment level.

Androgen Receptor (AR) Bi nding Assay; (OCSPP 890.1150)MYCLOBUTANIL FINAL

Page 36 of 57 In an androgen receptor (AR) binding a ssay (MRID 48618102), ventral prostate cytosol homogenate from Sprague Dawley rats was used as the source of AR to evaluate the potential of myclobutanil (93.7%) to displace the bound reference androgen, radiolabeled [ 3 H] -R1881 (methyltrienolone) from the AR. A saturation binding experiment was conducted prior to the competitive binding experiment to demonstrate that the AR in the cytosol preparation was present in adequate numbers and functioning with the a ppropriate affinity for the radiolabeled ligand. Saturation binding data were not presented in the study report; how ever, saturation binding data were presented in the study profile submitted separately by the test order recipient (study profile, MRID 48618102). The competitive binding experi ment was conducted to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations of myclobutanil (logarithmic increase from 10

− 10 to 10

− 3 M). Ethanol was used as a vehicle at a final assay concentration of <3%. The assay included dexamethasone as a weak positive control, and R1881 as the ligand reference standard. Thr ee independent competitive binding assays were performed.

In the saturation binding experime nt, the maximum binding capacity (B max ) was 3.245 fmol/100 μg protein and the di ssociation constant (K d ) was 0.4641 nM. Although the B max fell slightly below the expected range of 7 to 16 fmol/100 μg protein and the K d was below the expected range of 0.685 to 1.57 nM, confidence in these results is high because of the goodness of fit (r 2 = 0.9871 – 0.9937) and the reproducibility among the runs. W ith the exception of the high concentration of 10 nM in one assay with a 24.6% nonspecific bi nding, non-specific binding as a percent of total binding was <20% across the entir e concentration range for the saturation binding assay. All other performance criteria and the competitive binding assays indicated acceptable performance of the assay. Two runs of the satu ration binding experiment were conducted.

In the competitive binding experiment, myclobutan il reduced the mean sp ecific binding of the radiolabeled ligand at the two highest concentrations in a co ncentration-dependent manner; however, the myclobutanil binding curve decreased from approximately 84% to approximately 14% over a single log unit of concentration (from 10 -4 to 10 -3 M) and had steep Hill slopes in runs 2 and 3, indicating that the data did not fit the four-parameter , nonlinear regression model for analysis of one-site binding. Howe ver, these results may be an artifact of the data when using an undefined bottom plateau for the curve fitting. Wh en the data were re-analyzed by constraining the bottom of the response curves to zero, the results of all thre e independent runs were similar with acceptable r 2 values and standard errors.

The mean estimated log IC 50 was -3.5 M for myclobutanil for the analysis using a constrained bottom plateau. The range of RBA values fo r myclobutanil across the three runs was 0.00034% to 0.00037% indicating a weak interacti on with the AR. The mean log IC 50 s for the strong positive control R1881 and the weak positive cont rol (dexamethasone) were -9.0 and -4.4 M,MYCLOBUTANIL FINAL

Page 37 of 57 respectively, and the mean RBA for the weak positive control was 0.0028%. All performance criteria for the competitive binding assays indicated acceptable performance of the assay.

Based on the results from the three runs, myclobutanil is classified as a Binder, albeit with weak affinity, in the Androgen Receptor Binding Assay.

Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48618103) , myclobutanil (93.7±1.3% a.i., lot# 627, TSN027818-0019) was incubated with human recombinant aromatase and tritiated androstenedione (1- β [ 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) in ethanol for 15 minutes to assess the potential of myclobutanil to inhibit aromatase activity. Myclobutanil was tested at logarithmic concentrations from 10 –10 M to 10

− 3 M in 3 independent runs.

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC). Thr ee runs were conducted and each run included a full activity control, a background activity c ontrol, a positive control series (10 –10 to 10 –5 ) with a known inhibitor (4-hydroxyandrostenedione; 4-OH AS DN), and the test chemical series (10 –10 to
10 –3 ) with 3 repetitions per concentration.

Aromatase activity in the full activity controls ranged from 0.090 to 0.174 nmol

· mgprotein

− 1

· min

− 1 for the three test runs, with a mean and standard deviation of 0.136±0.016
nmol

· mg-protein

− 1

· min

− 1 . Activity in the background controls ranged from 0.13% to 0.60% of the overall average of the background adjusted full activity controls. The responses of the full activity and background controls were acceptable.

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.145±0.017
nmol

· mg-protein

− 1

· min

− 1 at the lowest tested concentration (10

− 10 M) and 0.0016±0.0001
nmol

· mg-protein

− 1

· min

− 1 at the highest tested concentration (10

− 5 M). These results were generally within the recommended ranges for the top of the curve, bottom of the curve, Hill Slope, log IC 50 , and coefficient of variation for replicat es of each concentration, with some minor deviations for Run #1. All performance criteri a were met in Run #2 and #3, with the mean performance criteria values w ithin the recommended ranges.

For myclobutanil, aromatase activity averaged 0.127±0.013 nmol

· mg-protein

− 1

· min

− 1 at the lowest tested concentration (10

− 10 M) and 0.00017±0.00004 nmol

· mg-protein

− 1

· min

− 1 at the highest tested concentration (10

− 3 M). The overall %CV for the 10 –3 and 10 –4 M concentrations (

− 303% and 83.9%, respectively) exceeded the recommended limit of 15%. The data for myclobutanil were modeled for all runs; the best fit Hill Slope and log IC 50 values across the threeMYCLOBUTANIL FINAL

Page 38 of 57 independent runs did not differ signi ficantly. The mean goodness of fit (R 2 ) value was 0.99, and
the mean IC 50 of the test material was –6.6 M. The average dose-response cu rve indicated that the aromatase activity of th e test material at concen trations ranging from 10

− 10 M to 10

− 8 M was essentially equivalent to the activity observed in the full activ ity controls. At myclobutanil
concentrations ≥ 10 –7 M, aromatase activity was substantia lly inhibited. Aromatase activity was inhibited by 34, 75 and 96% at mycl obutanil concentrations of 10 –7 , 10 –6 and 10 –5 M, respectively, with concentrations ≥ 10 –4 M resulting in >99% inhibition of activity.

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

Estrogen Receptor (ER) Bindi ng Assay; (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48618104), uterine cytosol from Sprague Dawley rats was used as the source of ER to evaluate the potential fo r myclobutanil to displace the bound reference estrogen, radiolabeled [ 3 H]-17 β -estradiol from the ER. A saturation binding experiment was conducted prior to the competitive binding experiment to demonstrate that the ER in the uterine cytosol was present in adequate numbers and functioning with the appropriate affinity for the radiolabeled ligand. Saturati on binding data were not presented in the study report; however, saturation binding data were pres ented in the study profile submitted separately (MRID 48672404). The competitive binding experime nt was conducted to measure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presen ce of increasing concentrations of myclobutanil (logarithmic increase from 10

− 10 to 10

− 3 M). Ethanol was used as the vehicle at a final concentration of <3%. The assay included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17 β -estradiol as the na tural ligand reference
material.

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

In the competitive binding experiment, my clobutanil did not significantly displace the radiolabeled estradiol from the ER at concentrations up to 10

− 3 M. Across all three runs, specific labeled estradiol binding averaged 94.5 – 102.9% at myclobutanil concentrations of 10

− 10 to 10

− 3

M. An IC 50 and RBA were not calculated for myclobutanil because it did not show competitive binding.MYCLOBUTANIL FINAL

Page 39 of 57 The performance criteria were met for the competitive binding experiment and the reference compounds generally performed as expected. Th e negative control, octyltriethoxysilane, had no effect on binding, and there were concentration-dependent decrease s in labeled estradiol binding for the natural ligand and the weak positive control. The mean IC 50 was 1.14 × 10

− 9 M for the natural ligand and 3.518 × 10

− 6 M for the weak positive control. The relative binding affinity (RBA) for the weak positive control was 0.032%, comp ared to the natural ligand. The mean Hill Slope for the natural ligand was

− 0.9792, indicating that increasing co ncentrations of unlabeled

17 β -estradiol displaced [ 3 H]-17 β -estradiol in a manner consiste nt with one-site binding.

Based on the results of the three runs, myclobutanil is classified as Not Inte ractive in the Estrogen Receptor Binding Assay.

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

In an estrogen receptor transcript ional activation assay (MRID 48618105), hER α -HeLa-9903 cells
cultured in vitro were exposed to myclobutanil (Lot# 627, TSN027818-0019, 93.7% a.i.) at logarithmically increasing concentrations from 10

− 10 to 10

− 4 M in DMSO (0.1%) for 24 hours in three independent runs. The experiments were performed using 96-well plates and each myclobutanil concentration was tested in tripli cate (3 wells/plate) in each run. Cells were exposed to the test agent for approximately 24 hours to induce reporter (lucif erase) gene products. Luciferase expression in response to activa tion of the estrogen receptor was measured upon addition of a luciferase substrate and detection with a luminometer with acceptable sensitivity.

Three valid independent runs of the assay were performed. Myclobutanil was tested up to the limit of solubility, 10

− 4 M. The responsiveness of cells to the required laboratory proficiency chemicals was not reported. There were deviations from expected performa nce criteria for all of the four reference chemicals, but these deviations did not negatively affect the interpretation of the study. The log PC 50 and log PC 10 for 17 α -methyltestosterone were lower than the acceptable values, but this indicates increased sens itivity of the cells to weak agonists.

The RPC Max was 6.4% for the first run, 2.8% for the s econd run and 7.3% for the third run, with
an associated PC Max of 10

− 5 M for all three runs. Because the RPC Max < PC 10 in at least two assay runs, myclobutanil was considered negative for estr ogen receptor transcripti onal activation in this
test system.

Fish Short Term Reproduction Assay (FSTRA); (OCSPP 890.1350)MYCLOBUTANIL FINAL

Page 40 of 57 The 21-day short-term reproduction assay (M RID 48618106) of myclobutanil with fathead minnows ( Pimephales promelas ) was conducted under flow-through conditions. Adult fish, 16 spawning groups (2 males and 4 females in each group; ca. 5.75 months old), were exposed to myclobutanil (93.7% purity) at nominal concen trations of 0 (contro l), 0.032 0.32, and 3.2 mg a.i./L; time-weighted average (TWA)-m easured concentrations were <LOQ (<0.00510), 0.0338, 0.327, and 3.30 mg a.i./L. The test system was maintained at 24.6 to 25.4oC and a pH of 7.2 to 7.5.

There were no significant eff ects on mortality (p>0.05) and while effects on secondary sex characteristics (fat pad development) and clin ical signs (including missing caudal fin) were observed, they did not appear to be treatment-re lated. At the high treatme nt level, a significant reduction (9%; p=0.022) in female body weight was observed.

Spawning in the negative control occurred at leas t every 4 days and fecundity ranged from 21 to 33 eggs/female/day/replicate. Fecundity wa s 25.6, 20.1, 22.6 and 0.550 eggs/female/day/replicate in the control, low, mid, and high treatment leve ls, respectively. Fecundity at the high treatment level was 98% lower than the control, and was statistically significant (Mann Whitney-U; p<0.05). No significant effect was detected for fe rtilization success. Ferti lization rate in the control was 99%, compared to 99.5, 99.1 and 100% in the low, mid, and high treatment groups. Female gona do-somatic index (GSI) exhibited a significant (Jonckheere-Terpstra; p<0.05) increase of 41% a nd 46% at the mid and high treatment levels, while male GSI was significantly (Jonckheere-Te rpstra; p<0.001) increas ed 79% at the high treatment level. There were no significant diffe rences in male nuptial tubercle scores, and no nuptial tubercles were observed on female fish.

Female vitellogenin (VTG) was significantly reduced (Jonckheere- Terpstra; p=0.001) 88% relative to control at the high treatment level, while no effect was detected for male VTG. Additionally, there was a significan t increase (p=0.019) in female testosterone levels at the mid treatment level, relative to the control, and a statistically significant (p <0.05) 85% reduction in female blood plasma 17 β -estradiol concentrations at th e high treatment level was observed. Histopathology on female gonads reported severe oocyte atresia at the high treatment level
relative to the contro l. Male fish gonads were reported as having a higher incidence of increased proportions of mature sperm (gonadal stage 3) in the high treatment group, re lative to the control.

Hershberger Assay; (890.1400)MYCLOBUTANIL FINAL

Page 41 of 57 In a Hershberger assay (MRID 48618107) testin g for androgenic activ ity, myclobutanil (93.7% a.i., Lot # 627, TSN027818-0019) was administered daily in corn oil via oral gavage to groups of seven 55-day old castrated male Sprague Dawley rats/group at dose levels of 0 (vehicle), 25, 125, or 500 mg/kg/day for 10 consecutive days. Th e study included an androgenic positive control group of seven castrated male rats dosed daily wi th testosterone propionate (TP) at 0.4 mg/kg/day via subcutaneous (s .c.) injection.

To screen for possible anti-androgenic activity, additional groups of seven 55-day old castrated male Sprague Dawley rats/group were dosed daily via oral gavage with myclobutanil in corn oil at 25, 125, or 500 mg/kg/day for 10 consecutive days in conjunction with a daily s.c. injection of TP at 0.4 mg/kg/day. The control group consisted of seven castrated male rats dosed daily with TP (0.4 mg/kg/day) by s.c. injection, and the pos itive control group consis ted of seven castrated male rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg/day for 10 days in conjugation with a daily s.c. dose of TP (0.4 mg/kg/day).

Body weights, body weight gains, and food c onsumption were measured; serum hormone
concentrations were not determined. Animals we re euthanized approximately 24 hours after the
final dose administration. The fi ve androgen-dependent tissues, ad renals, kidneys, and liver were weighed and macroscopically examined. Liver samples from the control and high dose animals were examined microscopically.

No treatment-related effects were observed in either the agonist or antagoni st assays on mortality, clinical signs of toxicity, body weights, body weight gains, food consumption, adrenals and kidneys weights, or gross pathol ogy. The increased liver weights a nd microscopic liver findings at 500 mg/kg/day indicated that the an imals were dosed to a level that showed some toxicological
effects.

For the androgenic portion of the study, there were no statistically significant incr eases in the weights of accessory sex tissue at any dose of myclobutanil. For the TP positive control group, there were significant (p<0.05) in creases in the weights of the Cowper’s glands (688%), glans penis (77%), levator ani-bulbocav ernosus (LABC; 194%), ventral prostate (917%), and seminal vesicles with coagulating glands (1105%), indica ting that the test system was sensitive to an androgenic response.

For the anti-androgenic porti on of the study, there was a statistica lly significant decrease in one of the five target accessory sex tissues. A 20% decrease in LABC weight was observed at 500 mg/kg/day of myclobutanil + TP. No significant changes in organ weights were seen in the seminal vesicle, ventral prosta te, Cowper’s gland or glans pe nis as any dose. For the TP+FT positive control group, there were treatment-related decreases in the weights of the Cowper’s glands (62%), glans penis (26%), LABC (54%), vent ral prostate (75%), and seminal vesicles withMYCLOBUTANIL FINAL

Page 42 of 57 coagulating glands (81%) relative to weights in the “TP only” treated group. These data indicate that the test system was sensitiv e to an anti-androgenic response.

Liver weights were increased by myclobutanil treatment in both the androgenic and antiandrogenic portions of the study. For the androgen ic portion of the study, absolute liver weights were increased by 20% and 53% at 125 and 500 mg /kg/day myclobutanil, respectively. For the anti-androgenic portion of the study, there was a 59% increase in absolute liver weight in animals given 500 mg/kg/day with TP. These weight incr eases correlated with hi stopathologic lesions in the liver, which included hypertr ophy, vacuolization, and necrosis.

The performance criteria for the assay were met as the %CVs for weights accessory sex tissues in the control and treated groups we re within the recommend values specified in the guideline.

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

assay.

Female Pubertal Assay; (890.1450)

In a Female Pubertal Assay (MRID 48618108), 16 Sp rague Dawley [Crl:CD(SD)] rats/dose group were treated daily via oral gavage with myclobutanil (93.7% a.i., Lot# 627) in corn oil at doses of 0, 50, 200, or 400 mg/kg/day from post-natal day (PND) 22 to 42. Animals were examined for vaginal opening (VO) daily begi nning on PND 22, and age and weight at day of attainment was recorded. Following sacrifice on PND 42, total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels were determined using chem iluminescent immunoassay and radioimmunoassay, respectively. At termination, weights were de termined for liver, kidneys, thyroid, adrenals, pituitary, and urogenital organs. Microscopic examinations were performed on the ovaries, uterus, thyroid, and kidneys fr om the control and high-dose groups and on livers from all dose

groups.

There were no effects of treatment on mortalit y, clinical signs of t oxicity, body weights, body weight gains, age of attainment of VO, body weight at VO, mean age at first estrus, mean cycle length, percent cycling, pe rcent regular cycling, or gan weights, serum T 4 and TSH levels, or gross pathology.

At 400 mg/kg/day, treatment-related increa ses (p<0.05) were observed in ALT ( ↑ 24%) and GGT ( ↑ 130%). Dose-dependent increases (p<0.05) in ab solute, relative, and adjusted liver weights were observed at 200 ( ↑ 18-19%) and 400 ( ↑ 36-41%) mg/kg/day. Animals in these dose groups also displayed treatment-related histopathological effects consisting of ve ry slight to slight hypertrophy with altered tinctorial properties (increased cytoplasmic eosinophilia) of centrilobularMYCLOBUTANIL FINAL

Page 43 of 57 and midzonal hepatocytes, very slight multifocal chronic inflammation, and very slight to slight
increased number of mitotic fi gures of hepatocytes. These findings showed a dose-dependent
increase in incidence and severi ty, and were indicative of hepa totoxicity at 400 mg/kg/day.

The high dose in the current study is considered to be an adequate high dose, based on the liver effects seen in this assay and on evidence of hepa tic necrosis at 500 mg/kg/ day in a concurrently submitted Hershberger study (MRID 48618107),

Male Pubertal Assay; (OCSPP 890.1500)

In a male pubertal assay (MRID 48618109), 16 Sp rague-Dawley (Crl:CD[SD]) rats/dose group were treated daily via oral gavage with myclobutanil (93.7%, Lot # 627, TSN027818-0019) in corn oil at doses of 0, 50, 200, or 400 mg/kg/day from post-natal day (PND) 23 to 53. Animals were examined for preputial separation (PPS) da ily beginning on PND 30, and age and weight at day of attainment was recorded. Following sacrifice on PND 53, total serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were analyzed using radioimmunoassays (TSH) or chemilumi nescent assays (testosterone and T 4 ). Liver, kidney, pituitary, adrenals, thyroid, a nd urogenital organ weights were recorded, and microscopic examinations of the thyroid, testes, epidi dymides, kidneys, and liver were performed.

At myclobutanil doses up to 400 mg/kg/day, no treatment-related effects were observed on mortality, clinical signs, body we ight, body weight grain, weight at attainment of PPS, or serum

T 4 levels. However, age at PPS was slightly delayed (p<0.05) in the high dose group (PND 44.0 vs. PND 45.7).

Significant (p<0.05) increases were observed in liver weight (unadjus ted and adjusted) and relative (to body) liver weight, which were dose- dependent. Liver weig hts increased by 12-13% at 200 mg/kg/day and 27-28% at 400 mg/kg/day, and relative liver weights increased by 5%, 21% and 37% at 50, 200 and 400 mg/kg/day, respectively. Concomitant with the increases in liver weight, there were minor histological change s at 200 and 400 mg/kg/da y, which consisted of slight to very slight hypertr ophy with altered tinctorial propert ies in centrilobular and midzonal hepatocytes (0/16 control, 16/16 at 200 mg/kg/day, and 15/15 at 400 mg/kg/day) and a very slight increase the numbers of mitotic figures in hepa tocytes (2/15 control, 9/15 at 400 mg/kg/day). A significant increase (53%) was also noted in ga mma glutamyl transferase at the high dose.

Other significant changes in orga n weights included a 15% increase in relative adrenal weight at 400 mg/kg/day, and 8-10% decreases in pituitary weight at all dos e levels that were not dosedependent. No significant wei ght changes were noted in the thyroid, but increased thyroid follicular cell heights and decreased colloid area were noted at 400 mg/kg/day, although the
observed differences were not stat istically significant (NS). In a ddition, dose-dependent increasesMYCLOBUTANIL FINAL

Page 44 of 57 in serum TSH levels of 23%, 28%, and 63% were noted at 50, 200, and 400 mg/kg/day, respectively, although these increas es were not significant. No changes were noted in serum T 4 levels.

Significant, dose-dependent decreases were also observed in the we ights of the four accessory sex tissues at doses of 200 mg/kg/day (2/4 tissues ) and 400 mg/kg/day (4/4 tissues). At 200 mg/kg/day, weight decreases we re noted in the seminal vesicle + coagulating gland ( ↓ 15-19%) and the ventral prostate ( ↓ 16%). At 400 mg/kg/day, weight decr eases were noted in the seminal vesicle + coagulating gland ( ↓ 33-41%), ventra l prostate ( ↓ 27-29%), dorsolateral prostate ( ↓ 25- 27%), and LABC complex ( ↓ 16-18%). Concomitant with the wei ght decreases in these androgen sensitive tissues, there were significant, dose-depe ndent decreases in serum testosterone levels of 49% at 200 mg/kg/day and 85% at 400 mg/kg/day.

Other significant changes in cl inical chemistry parameters observed at 200 and 400 mg/kg/day included: 21-32% increase in creatinine; 27 % decreased in blood urea nitrogen; and a 17-21% decrease in potassium. At 400 mg/kg/day, ther e were also significant changes in alkaline phosphatase ( ↓ 26%), total protein ( ↑ 4%), and chloride ( ↓ 3%),

It appears the high dose (400 mg/kg/ day) used in this assay is ad equate based on the results from a range finding assay that showed increased liver weights and microscopi c liver findings at a myclobutanil dose of 500 mg/kg/day.

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48618110), H295R cells cultured in vitro in 24-well plates were incubated with myclobutanil (93.7±1.3% purity, Lot # 627) at log concentrations from 10

− 10

to 10

− 4 M for 48 hours in trip licate in three independent expe riments. Dimethyl sulfoxide (DMSO) was used as a vehicle, at a final concentration of 0.1%.

Testosterone and estradiol levels were meas ured using LC-APPI-MS/MS. A Quality Control (QC) plate was run concurrently with each i ndependent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels. The positive controls included a known inhibitor (prochloraz) and a known inducer (forskolin) of estradiol a nd testosterone production.

Guideline acceptability recommendations and requir ements were generally met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptabl e reproducibility (low %CV), and appropriate induction and inhibition with positive controls, w ith the following exceptions: (i) the testosterone level in the solvent control (SC) of Run 2 was 470 pg/mL (less than the guideline limit of 500 pg/mL ); (ii) the estradio l level in the SC of Run 1 was 35.5 pg/mLMYCLOBUTANIL FINAL

Page 45 of 57 (less than the guideline limit of 40 pg/mL); and (iii ) the inhibition of estrad iol in Run 1 with 1 μM prochloraz was 0.6-fold (greater than the guideline limit of ≤ 0.5-fold).

Testosterone levels were decreased (p ≤ 0.05) to 0.3- to 0.4-fold the so lvent control values in all 3

runs at 10

− 4 M myclobutanil. In Run 2, there were signi ficant increases in testosterone production (1.3- to 1.5-fold change) at concentrations from 10

− 8 to 10

− 5 M. Decreased estradiol levels were
observed at 10

− 7 M myclobutanil (0.8-fold the solvent control; p ≤ 0.05 in Runs #2 and #3), and in
all runs at concentrations ≥ 10

− 6 M (0.4- to 0.7-fold; p ≤ 0.05). Decreased (p ≤ 0.05) estradiol (0.8- fold the solvent control) was also noted at the lowest level tested in Run 2.

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

Uterotrophic Assay; (OCSPP 890.1600)

In a uterotrophic assay (MRID 48618111) conducted to screen for potenti al estrogenic activity, myclobutanil (93.7% a.i., Lot # 627) in corn oil was administered daily via oral gavage to groups of six immature female Sprague Dawley rats at dose levels of 0 (vehicle), 50, 150, or 475 mg/kg/day on post-natal days (PND) 19-21. A positive control group was treated with a daily
dose of 17 α -ethynyl estradiol (EE) at 10 μ g/kg/day by oral gavage. Body weights were determined daily. All animals were examin ed for precocious vagi nal opening, and then terminated and necropsied on PND 22 approximate ly 24 hours after the final dose to determine wet and blotted uterine weights.

All animals survived until schedul ed termination. No clinical si gns of toxicity were observed, and no precocious vaginal opening was noted in the myclobutanil-treated females. At 475 mg/kg/day, body weights were decreased (p <0.05) by 15% on Day 3, resulting in a 59% decrease (p<0.05) in overall (Day s 1-4) body weight gains. Uterine weights at this dose were comparable to the vehicle controls. Absolute wet and blotted uterus weights for the positive control (EE) group were increased (p< 0.05) by 891% and 524%, respectively.

The 59% decrease in overall body weight gains in the high-dos e group indicated that these animals were dosed at adequate levels.

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

Page 46 of 57 APPENDIX 2: Other Scientifically Relevant Information (OSRI)

Aromatase Inhibition

In a published study, Trösken et al. (MRID 48242001 ) tested 22 azole compounds (including myclobutanil) for aromatase inhibition using a fluorimetric assay based on human recombinant CYP19 enzyme and using dibenzylfluorescein (DBF) as the substrate. In this assay, myclobutanil was reported to have an IC 50 of 4.7 × 10 -7 M.

In a subsequent study ( Trösken et al. , MRID 48242002 ) tested several azole fungicides in a recombinant aromatase assay using testosterone as the substrate and LC-MS/MS as the method of detection, and compared the result s of this assay with other met hods. Myclobutanil, tested in the DBF assay as noted above had an IC 50 of 0.47 μ M compared with 47 μ M in the testosterone / LSMS/MS assay. The two order of magnitude difference in IC 50 values was said to be due to the higher affinity of testosterone for the enzyme than DBF.

ER Transcriptional Activation (ERTA) Assay

In a published study by Hurst and Sheahan (MRID 48242003 ), myclobutanil was tested in the yeast estrogen screen (YES) assay. This system uses yeast cells expressing the human estrogen receptor (hER) together with a plasmid carryi ng the estrogen-responsive element (ERE) and LacZ reporter. The relative estrogenic potency of myclobutanil was reported to be 7 × l0 -6 (relative to E2) with an LOEC of 781 μ g/L. While EPA does not generally regard yeast assays as the equivalent of Tier 1 ERTA due to concerns about the ability of the test chemical to penetrate the cell wall, the positive resu lts in this assay provid ed information on the ability of myclobutanil to interact with the estrogen receptor and activat e transcription, although at relatively high concentrations (~10 -3 M).

90-Day Subchronic Oral Toxicity in Rodents (Rat); (OCSPP 870.3100)

A subchronic toxicity study in COBS-CD(SD) BR rats ( MRID 00145048 ) was conducted on Myclobutanil (81.1% a.i.). Groups of 10 rats/sex were fed control di ets or test diets containing 5, 15, 50, 150, 500, 1500, 5000 or 15000 ppm myclobutanil. During Weeks 3 and 4, doses were increased to 7, 21, 70, 210, 700, 2100, 7000, or 21000 ppm. During Weeks 5–13, doses were again increased to 10, 30, 100, 300, 1000, 3000, 10000, or 30000 ppm. The high dose resulted in total mortality by week eight.

There was no effect on mortality and no compound -related clinical signs were observed at doses of 10,000 ppm or below. Body weight, however, was significantly depr essed at 3,000 ppm and above in the male groups and at 10,000 ppm a nd above in the female groups. There was no correlation of growth to food consumption in the 3,000-ppm male groups, but there was bodyMYCLOBUTANIL FINAL

Page 47 of 57 weight suppression that correla ted with reduced food consum ption in the 10,000- and 30,000-ppm groups of both sexes. In the 10,000-ppm group, th e decreased HGB and HCT counts together
with decreased HCV values and increased RBC counts indicated that there was red cell destruction with compensatory red cell production. There was mild hemosiderosis in the spleens
of the controls of both sexe s and in 3,000 and 10,000 ppm groups. The hemosiderosis was more severe in the high dose males. Nevertheless, the re d cell destruction was very slight and of little toxicological significance with the exception of the high-dose males where hemosiderosis was evident. The clinical chemical changes were many and varied, and many of these point to an
adverse effect on the liver and ki dney. Mixed function oxidase activ ity was increased in males at doses of 300 ppm or greater and at doses of 1,000 ppm or greater in females. Gross necropsy findings were limited to the liver and were more pronounced in the males. They consisted of dark discolorations, enlargement, and/or prominen t architecture at 1,000 pp m and above. Increased absolute and relative liver weights were seen at 3,000 and 10,000 ppm in both sexes and increased relative liver weight o ccurred in the 1,000-ppm female group. Increased relative kidney weights were present in the 3,000 and 10,000 ppm doses in bot h sexes. Many of the other relative weight values were a result of decreased terminal body weights and are not toxi cologically significant. Histopathological changes in the liver consisted of hepatocellular necros is and hepatocellular hypertrophy at 3,000 ppm and above. At 3,000 or 10,000 ppm, pigmented convoluted tubules of the kidney were observed in males only. The adrenal cortex was vacuolated in both sexes at 3,000 and 10,000 ppm. An increase in small follicles wa s seen in the male t hyroids at 3,000 ppm and
above.

In 1994, the EPA concluded in an RfD/Peer Re view Report of Myclobut anil that the NOAEL for this study was 1000 ppm based on increased liver and kidney weights; hy pertrophy and necrosis in the liver; pigmentation in convol uted kidney tubules; and vacuola tion in the adrenal cortex at 3000 ppm.

90-Day (Subchronic) Oral Toxi city in Rodents (Mouse); (OCSPP 870.3100)

In a 90-day oral toxicity study (MRID 00165245) , myclobutanil (81.1% a.i.) was administered in the diet to groups of Crl:CD-1 (ICR) BR mice ( 10//sex/dose group) at levels of 0, 3, 10, 30, 100, 300, 1000, 3000 or 10000 ppm for 13 weeks. Food consumption was generally reduced in the 10000 ppm dose group for both sexes, although the reduc tion was transient in male mice. Over 13 weeks, body weights were significantly reduced by 19 and 8% in males and females at the highest dose (10000 ppm), and by ~7% in males at 3000 ppm.

The principle target organ in mice was the liv er. Absolute and relative liver weights were significantly increase d in both sexes at ≥ 1000 ppm, and gross enlargemen t of the liver was noted
in both sexes at ≥ 3000 ppm. Microscopic changes in th e liver were seen in males at ≥ 1000 ppm
and in females at ≥ 3000 ppm. These observations incl uded centrilobular and midzonalMYCLOBUTANIL FINAL

Page 48 of 57 hepatocytic hypertrophy, swollen-va cuolated centrilobular hepatocytes, single large hepatocytic vacuoles, centrilobular hepa tocytic necrosis, and centril obular necrotic hepatitis.

No treatment related changes in weight or hi stology were reported for the testes, ovaries, parathyroids or thyroid. No we ight changes were noted for the adrenals, but cytoplasmic eosinophilia and/or hypertrophy of the zona fascic ulata cells were noted in the adrenals at ≥ 1000 ppm in males and ≥ 3000 ppm in females. The LOAEL is 1000 ppm based on hepatocytic hypertrophy and other liver eff ects, and the NOAEL is 300 ppm

Prenatal Development in Rodents (Rat); (OCSPP 870.3700)

In a developmental toxicity study ( MRID 00141672 ), 25 Sprague-Dawley [Crl:CD-(SD)BR] rats/dose group received either 0, 31.26, 93.77, 312.58 or 468.87 mg/kg/day technical myclobutanil (84.5% a.i.) by oral gavage from gestation day 6 through 15, inclusive. The maternal toxicity LOAEL was 312.6 mg/kg/da y and the maternal toxicity NOAEL was 93.8 mg/kg/day based on clinical signs of toxicity. A significant increase in the incidences of 14th rudimentary ribs and 7th cer vical ribs was observed at 312.6 and 468.9 mg/kg/day. No other developmental effects were observed at any dos e level tested. The developmental toxicity LOAEL was 312.6 mg/kg/day and the developmen tal toxicity NOAEL was 93.8 mg/kg/day based
on increased incidences of 14th rudimentary and 7th cervical ribs . No effects were reported on reproductive organs.

Prenatal Development in No n-Rodents (Rabbit); (OCSPP 870.3700)

Technical myclobutanil (90.4%) was tested in a developmental toxicity study in rabbits ( MRID 00164971 ). Five groups of female New Zeala nd White rabbits (18/group) received 0 (water control), 0 (Hi-Sil c ontrol) or 20.0, 60.0 or 200.0 mg/kg/ day a.i. myclobutanil by oral gavage (5 mL/kg b.w.) on days 7 through 19 of gest ation. The test material was prepared with the solid adsorbent (carrier) Hi-Sil 233 and with 1.0% met hylcellulose (vehicle).

No maternally toxic effects were observed at either 20 or 60 mg/ kg/day. At 200 mg/kg/day, decreases in maternal body weight and body weight gain were observed during the dosing period (0.03 and -0.02 kg in the controls versus -0.28 kg in the treated group) wi th a rebound during the post-dosing period. Other effects in cluded irregular feces, bloody urine, bloody urogenital or anal
area and blood and/or aborted material in the dr op pan. Three does aborted their litters, however, 2 of those litters were totally resorbed (early). There were no effects noted at necropsy. The maternal toxicity LOAEL was 200 mg/kg/day based on reduced body weight and body weight gain during the dosing period, clin ical signs of toxicity and possibly abortions. The maternal toxicity NOAEL was 60 mg/kg/day.MYCLOBUTANIL FINAL

Page 49 of 57 No developmentally toxic effects were observed at either 20 or 60 mg/kg/day. At 200 mg/kg/day, there were increases in resorptions, resorptions/l itter, decreases in li tter size (p< 0.05) and a decrease in the viability index (p< 0.05). The LOAEL for developmental toxicity was 200 mg/kg/day and the NOAEL for developmental t oxicity is 60 mg/kg/day based on increases in resorptions, decreases in litter size and a decrease in the viability index.

Two-Generation Reproduction in Rodents (Rat); (OCSPP 370.3800)

Technical myclobutanil (purity 84.5 %) was tested in a 2-generation reproduction study with male and female CRL:CD(SD)BR rats ( MRID 00143766 and 00149581 ). Twenty-five animals/sex/dose group received 0, 50, 200 or 1000 pp m in the diet throughout the study (0, 2.5, 10 or 50 mg/kg/day by standard conversion factors). The animal s were mated on a one to one ratio in the F0 parental groups and were given test diets for 8 weeks before they were mated. Selection of the parents for the F1 generation wa s made when the pups were 25 days of age, and the mated animals in the study were a pproximately 81days of age at mating.

At 200 ppm, centrilobular hepatocellu lar hypertrophy was observed in the F1 males. This was supported by slight but statistically significant increases in absolu te and relative liver weights in both F0 and F1 males (104–114%). At 1000 ppm, centrilobular hepatocellular hypertrophy was observed in both sexes in the F0 and F1 generati ons. Slight but statistically significant increases
in absolute and relative li ver weights in both F0 and F1 males (107–114%) and females (106-109%). The parental (systemi c) toxicity LOAEL is 200 ppm and the parental (systemic) toxicity NOAEL is 50 ppm based on hepatocellula r hypertrophy and increases in liver weights.

At 1000 ppm, an increase in the number of s tillborn or % born dead was observed in both generations (4.9–5.3% treated versus 0–1.9% in controls). Multifocal or diffuse testicular atrophy were observed in males in the F1 generation. Increased necrotic spermatocytes / spermatids or decreased spermatozoa and atrophy of the prosta te were also observed in these animals.

The reproductive toxicity LOAEL was 1000 ppm and the reproductive toxicity NOAEL was 200 ppm based on an increased incide nce in the number of stillborns and atrophy of the testes and prostate. At 1000 ppm, there was an apparent decrease in pup wei ght gain during lactation (83.3% to 89.7% of the controls). The developm ental toxicity LOAEL is 1000 ppm and the developmental toxicity NOAEL is 200 ppm based on a decrease in pup body weight gain during
lactation.

Chronic Oral Toxicity/Carcinogenici ty in Rodents (Rat); (OCSPP 870.4300)

In two 2-year chronic feeding/carcinogenicity studies ( MRID 00149582, 00165247, and

42809101 ), Sprague-Dawley rats were administer ed technical myclobutanil. The second studyMYCLOBUTANIL FINAL

Page 50 of 57 was conducted because the high dose in the first st udy was not at or near the Maximum Tolerated

Dose.

In the first study ( MRID 00149582 and 00165247 ), myclobutanil (90.4% and 91.4%) was administered to groups of 110 male and 110 fe male rats at concentrations of 0, 25/35/50, 100/140/200 or 400/560/800 ppm in the di et. The first, second and third dose levels in each series were administered for weeks 1–2, 3–4 and 5–term, respectively. The overall reported mean daily compound consumption was 0, 2.49, 9.84 or 39.21 mg/kg/day for the males and 0, 3.23, 12.86 or 52.34 mg/kg/day for the females. Of the 110 rats/ dose group, 10/sex/dose were sacrificed at 3 and 6 months, 20/sex/dose were sacrificed at 12 mont hs, and 18 males/dose and 10 females/dose were sacrificed at 17 months. A sentinel animal pr ogram was also conducted separately in which 30
untreated rats/sex were evalua ted at 3, 6 and 12 months. Study evaluations included clinical observations and mortality, body weight, f ood and water consumption, ophthalmological examinations, hematology, clinical chemistr y, hepatic mixed function oxidase (MFO) and peroxisomal beta-oxidation activities, urin alysis, gross necropsy and histopathology.

Testicular toxicity was observed in the middle and high dose males. Tes ticular effects included significantly (p<0.05) decreased mean absolute testes weights (77% of controls at termination) at 9.84 mg/kg/day, and decreased absolute and relative (to brain) tes ticular weights (75 and 79% of controls at termination) at 39.21 mg/kg/day. The incidences of te sticular atrophy were increased at 9.84 and 39.21 mg/kg/day; total in cidences of bilateral testic ular atrophy were 5/110, 7/110, 16/110 and 31/110 in the control, 2.49, 9.84 and 39.21 mg/kg/day groups, respectively. Liver mixed function oxidase activity was significantly increased in females at 12.86 mg/kg/day at 3 months (61%), males at 39.21 mg/kg/day at 3 and 6 months (34–47%), and females at 52.34 mg/kg/day at 3 months (78%). Relative and absolute liver weights were significantly increased in females at 52.34 mg/kg/day at 3 mo nths (13%) and 6 months (20%), respectively. With the exception of the testicular lesions, non-ne oplastic histologic findings were incidental to

treatment.

Based on testicular atrophy, the LOAEL for syst emic toxicity in males was 9.84 mg/kg/day. The NOAEL for systemic toxicity in males was 2.49 mg/kg/day. The NOAEL for systemic toxicity for females was 52.34 mg/kg/day.

In the second 2-year feeding/carcin ogenicity study (MRID 42809101, 1993), technical myclobutanil (92.9%) was administered to group s of 50 male and 50 female Sprague-Dawley
Crl:CD BR VAF/Plus rats at concentrations of 0 or 2500 ppm. The overall mean compound consumption for the treated group was 106.08 mg /kg/day for males and 135.62 mg/kg/day for
females. An additional 10 rats/sex were added to each group and sacrificed at 52 weeks. A third group of 15 rats/sex were kept in the study without treatment as sentinel an imals and sacrificed at
52 weeks. Evaluations included clinical observat ions and mortality, body weight, food and waterMYCLOBUTANIL FINAL

Page 51 of 57 consumption, hematology, gross necropsy and hi stopathology. Clinical chemistry and urine analyses were not performed.

Non-neoplastic effects were observed in the trea ted rats in this sec ond study. These included statistically significant increases in liver weight (absolute and/or relative) in treated males ( ↑ 20-28%) and females ( ↑ 14–22%) at the interim sacrifice, but not at the terminal kill (males ↑ 4.5–8.4%; females ↑ 4.2–8.4%). Increased incidences of centr ilobular to midzonal hepatocellular enlargement and vacuolization were observed in both sexes at the 52-week and terminal sacrifices. Absolute and relative testes weights we re significantly lower in treated males at both the interim sacrifice ( ↓ 32–42% in testes) and terminal sacrifice ( ↓ 15–27%). Histopathological testicular changes included in crease incidences of bilateral aspermatogenesis, hypospermia and cellular debris in the epididymid es and, in rats that died or were sacrificed in extremis, arteritis/periarteritis. Ther e were no apparent compound-related effects on survival.

The LOAEL for systemic toxicity is 2500 ppm (106.08 and 135.62 mg/kg/day for males and females, respectively) based on bilateral asperm atogenesis and hepatocellular enlargement. A NOAEL for systemic toxicity was not established.

Carcinogenicity in Rodents (Mouse); (OCSPP 870.4200)

In two feeding/carci nogenicity studies ( MRID 00164990 and 42809102 ), Crl:CD ® -1 (ICR) BR mice were administered technical myclobutan il. The second study was conducted because the high dose in the first study was not at or near the Maximum Tolerated Dose.

In the first study ( MRID 00164990 ), technical mycl obutanil (90.4%) was administered to 110
male and 110 female Crl:CD ® -1(ICR)BR mice in the diet for a peri od of 24 months at dose levels of 0, 20, 100 or 500 ppm. The overall calculate d mean daily compound consumption was 0, 2.7, 13.7 and 70.2 mg/kg/day for males and 0, 3.2, 16.5 and 85.2 mg/kg/day for females, respectively. Of the 110 animals/dose group, 10/sex/dose group were sacrificed at 3 and 6 months and 20/sex/dose group were sacrificed at 12 months. A sentinel anim al program was also conducted separately in which 25 animals/se x were evaluated at 24 months.

At 100 ppm, an increase in hepa tic mixed-function oxidase (MFO ) was observed at 3 months in females (by 29% per mg pr otein, by 34% per g liver; p ≤ 0.05). At 500 ppm, an increase in MFO was observed in both sexes at 3 months (119% in males and 61% in females above control per total liver) and 6 months (212% in males and 210% in females a bove control per total liver) and in females at 12 months (266% above control per total liver). An increas e in SGPT values was
observed in females at 3 months (59% above controls) and an incr ease in absolute and relative liver weights was reported in males (10%) and in females (12% and 13%, respectively) at 3 months. In addition, the following treatment-re lated changes were observed microscopicallyMYCLOBUTANIL FINAL

Page 52 of 57 after 3, 6 and 12 months of treatment in males: in creased incidence and seve rity of centrilobular hepatocytic hypertrophy, Kupffe r cell pigmentation, periporta l punctate vacuolation and individual hepatocellular necros is. Following 24 months of treatme nt, increased incidences of focal hepatocellular alterations and multifocal he patocellular vacuolation were observed in both sexes. These were not associated with any hypert rophy, hyperplasia or neoplastic proliferations in the liver. At the time the DER was written, the LOAEL was considered to be 100 ppm (16.5 mg/kg/day) based on an increase in hepatic mixed-function oxidase at 3 months in females. The NOAEL was 20 ppm (3.2 mg/kg/day). Upon further re view, the increase in MFO at 3 months in females was not considered to be a significant en ough effect to establish an LOAEL. Therefore, the LOAEL was raised to 500 pp m (70.2 mg/kg/day for males a nd 85.2 mg/kg/day for females) based on increases in MFO in both sexes, increases in SGPT values in females, increased absolute and relative liver weights in both sexes at 3 months, increased incide nce and severity of centrilobular hepatocytic hypertr ophy, Kupffer cell pigmentation, periportal punctate vacuolation and individual hepatocellular necr osis in males, and increased incidences of focal hepatocellular alterations and multifocal hepa tocellular vacuolation in both sexes. The NOAEL was 100 ppm (16.5 mg/kg/day).

In a second 18-month carcinogenicity study ( MRID 42809102 ), technical myclobutanil (92.9%)
was administered to 60 female Crl:CD ® -1(ICR)BR mice at dose levels of 0 or 2000 ppm (393.5 mg/kg/day) in the diet. Ten animals from each gr oup were sacrificed at 52 weeks. An additional 20 animals were added to the study as sentinels a nd also sacrificed at 52 weeks. This study was conducted at the request of the HED Carcinogeni city Peer Review Committee because it was determined that the previous study (reviewed ab ove) was not conducted at sufficiently high dose
levels.

At 2000 ppm, there were statistically signifi cant decreases in body weight (2–12%) and body weight gain (12–26%) and increases in liver weights (22.9–33.2%). Rela ted histopathological findings include hepatocellular hypertrophy; hepa tocellular vacuolation; necrosis of single hypertrophied hepatocytes; yell ow-brown pigment in the Kupffer cells and cytoplasmic eosinophilia and hypertrophy of the cel ls of the zona fasciculata ar ea of the adrenal. With the exception of the pigment in the Kupffer cells, these lesions were present at the 12-month sacrifice. In this study, the NOAEL could not be established because there were effects at the only dose
level tested.

Myclobutanil was not carcinogenic when tested on Crl:CD ® -1(ICR)BR mice under the conditions of the study.

Chronic Oral Toxicity in Non-Rodents (Dog); (OCSPP 870.4100)MYCLOBUTANIL FINAL

Page 53 of 57 Technical myclobutanil (91.4%) was fed in the diet to 6 male and 6 female Beagle dogs per group at dose levels of 0, 10, 100, 400 or 1600 ppm for one year ( MRID 00165248 ). At 400 ppm, hepatocellular hypertrophy was obser ved in both sexes. Increased liver weights (131% absolute, 128% relative) were observed in females. At 160 0 ppm, hepatocellular hy pertrophy in both sexes and “ballooned” centrilobular hepatocytes were observed in females. In addition, increased absolute and relative liver weights were obs erved in males (130–144%) and females (152–154%), along with increased alkaline phosphatase (160–2 43% in males; 296–381% in females); increased SGPT in males (124–137%) and increased GGT in females (200%). Slight changes in hematological parameters were also observed in males (decreases in RBC, 89.6-90.7%; increases in platelets, 125.3–161.3%; slight increases in MCH, 104–106%; and MCV, 102–103%). There were also slight increases in serum phosphorus in males (114–133%) and a slight decrease in albumin in males (88–91%) and females (91–94%).

The LOAEL was 400 ppm (14.28 mg/kg/day) and the NOAEL was 100 ppm (3.09 mg/kg/day) based on hepatocellular hypertrophy , increases in liver weights, “ballooned” hepatocytes and increased alkaline phosphatase, SGPT and GGT.

Special Mechanistic Studies — Gene Effects in Rat Liver and Testis

Goetz et al. , (2007) conducted a study in male rats to ev aluate the disruption of testosterone homeostasis as a potential mode of action for the reproductive toxicity of th ree triazole fungicides (including myclobutanil). In this study Wistar Han rats were fed myclobutanil at 0, 100, 500 or 2000 ppm from gestation day 6 to postnatal da y (PND) 120. A single male from each litter was sacrificed on PND1, 22, 50 or 92. Anogenital distance (AGD) was measured on PND0. Other measurements included body and organ weights, serum hormone levels, age at PPS, sperm morphology and motility, and fertility. AGD was si gnificantly (p<0.001) in creased at the high dose (2000 ppm). Relative liver weights we re increased 9–13%, at PND1, 50 and 92. Hepatocellular hypertrophy was present at PND92 at the high dose. Relative pituitary weights were reduced at PND92 in mid and high dose groups . Absolute testis weights were increased at PND 1 and 22. Relative ventral prosta te weights and testosterone le vels were increased at PND92. Decreased insemination and fertility were also reported in the males treated with myclobutanil.

Goetz and Dix (2009a) also evaluated testosterone homeostasis in animals from above study (Goetz, et al, 2007) by measuring th e expression of genes involved in testosterone synthesis in the testis and decrease expression of genes involved in testosterone metabolism within the liver, Goetz and Dix examined gene expression change s in the male rat liver and testis following exposure to myclobutanil, propic onazole, or triadimefon from gestational day (GD) 6 through postnatal day (PND) 92.MYCLOBUTANIL FINAL

Page 54 of 57 The study authors concluded that numerous comm on gene transcripts and biological pathways
were identified for triazoles in the liver, defining common biol ogical processes modulated by all three triazoles and supporting th e interpretation of a common m ode of action. In contrast, the small number of differentially expressed genes and affected pathways in the testis indicated that the observed reproductive effects were not due to modulations of gene expression within the testis, and that the testis was not a target organ for triazole reproductive toxicity. All three triazoles had a significant impact on lipid metabolism, including fatty acid and steroid metabolism, as well as lipid transport. It wa s concluded that the genomic data demonstrate triazole disruption of pathways of key biologi cal functions in the li ver, including energy homeostasis, biological membra ne fluidity, and CYP and othe r metabolic activities. These pathways are critical to liver-med iated steroid homeostasis, and th e authors proposed that it is the perturbation of these critical pathways that lead s to the observed reproduct ive and hepatic toxicity
of the triazoles.

In another study by Goetz and Dix (2009b), gene expression profiling was conducted on livers from rats treated with myclobutan il, propiconazole or triadimefon in adulthood by oral gavage or prenatal through adulthood via the feed. Gene expressions from the rat liver studies were then compared to in vitro data from rat and human primary hepatocytes exposed to the myclobutanil, propiconazole, or triadimefon. The study was designed to compare rat in vivo and in vitro models
to human in vitro models and identify common, consis tent, and conserved gene expression changes for characterization of the mode s of action for triazole toxicities.

The study authors concluded that comparisons among in vivo rat liver and rat and human primary hepatocytes showed a targeted effect on genes in volved in early fatty acid catabolism, and to a lesser degree on Phase III trans porters and sterol metabolism. Several consistently perturbed metabolic pathways were common to all three triazoles and conserved between rat and human models. The subsets of genes involved in fatty acid catabolism/lipid homeostasis, Phase III transporters, and metabolism of testosterone are robust candidates for biomarkers defining potential mechanisms of action for disruption of testosterone homeostasis. Furthermore, these gene expression changes indicate shifts in lipoge nesis to fatty acid oxidation for increased energy production, and increased expression of Phase III transporters for the uptake and excretion of triazoles in the hepatocytes; both of these are mechanisms of triazole toxicity which were common across the triazoles. Overall, it was c oncluded that the results from these genomic analyses provide new leads for understanding th e modes of action responsible for triazole
toxicities.

Avian Toxicity and Reproduction (OCSPP 850.2200/850.2300)MYCLOBUTANIL FINAL

Page 55 of 57 In an avian reproduction study ( MRID 43087902 ), 17-week old Mallard ducks ( Anas platyrhynchos ) were dosed with myclobutanil (94.2% a.i.) via the feed at nominal concentrations of 75, 130, 180, and 260 ppm for up to 22 weeks. The study included a solvent control (1% acetone) group, but no negative control. Each dose group consisted of 16 ducks/sex, with a single drake and hen being paired in 16 pens/dose gr oup. The photoperiod was eight hours of light per day during the first 7 weeks (pre egg-laying), and was increased to 17 hours of light per day for the remainder of the study. All adult birds were obs erved daily for mortality, signs of toxicity, and abnormal behavior. Body weights were recorded at study initiation and at Weeks 2, 4, 6, 8, and at study termination, but not during egg-laying. Feed consumption was measured every 2 weeks. Once every 2 weeks during egg-laying, eggs were measured for eggshell thickness. Eggs were collected daily and stored at 19°C and 83% relativ e humidity. Weekly, the eggs were placed in an incubator. Prior to incubation, a ll eggs were candled to detect cracks; on Day 14 of incubation, embryo viability was measured; on Day 24, eggs were placed in a hatcher; on Day 27, all hatchlings, unhatched eggs, and eggshells were removed and the av erage body weight of representative hatchlings determ ined. Hatchlings were housed in battery brooders, and weighed at 14 days old. The following reproduc tive endpoints were determined: eggs laid, eggs cracked, eggs set, viable embryos, live 3-week embryos, nor mal hatchlings, 14-day old survivors, egg shell thickness, total food consumption, and body weights by sex.

Selected mortalities occurred in each dose group, but none of the deat hs appeared to be treatment related. Gross necropsies did not reveal any cons istent treatment-related abnormalities any test group. No statistically significant effects were seen at any test level on adult body weight or average feed consumption, and there were no si gnificant effects on any reproductive endpoints. The NOAEC was 260 ppm, the highest dose tested, and a LOAEC was not observed.

In another concurrent avian reproduction study ( MRID 43402001 and 43087901 ), 18-week old Northern Quail ( Colinus virginianus ) were dosed with myclobutanil (94.2% a.i.) via the feed at nominal concentrations of 75, 130, 180, and 260 ppm for up to 22 weeks. The study included a solvent control (1% ace tone) group, but no negative control. Each dose group consisted of 16 birds/sex, with a single cock and hen being paired in 16 pens/dose group. The photoperiod was eight hours of light per day dur ing the first 7 weeks (pre egg- laying), and was increased to 17 hours of light per day for the remainder of the study. All adult birds were observed daily for mortality, signs of toxicity, and abnormal be havior. Body weights were recorded at study initiation and at Weeks 2, 4, 6, 8, and at study termination, but not during egg-laying. Feed consumption was measured every 2 weeks. Once every 2 weeks during egg-laying, eggs were measured for eggshell thickness. Eggs were collec ted daily and stored at 19°C and 83% relative humidity. Weekly, the eggs were placed in an incubator. Prior to incubation, all eggs were candled to detect cracks; on Day 12 of inc ubation, embryo viability was measured; on Day 21, eggs were placed in a hatcher; on Day 25, all hatchlings, unhatched eggs, and eggshells were removed and the average body weight of representative hatchlings determined. Hatchlings were housed in battery brooders, and weighed at 14 days old. The following reproductive endpointsMYCLOBUTANIL FINAL

Page 56 of 57 were determined: eggs laid, eggs cracked, eggs set, viable embryos, live 3-week embryos, normal hatchlings, 14-day old survivors, egg shell th ickness, total food consumption, and body weights by sex.

Selected mortalities occurred in each dose group, but none of the deat hs appeared to be treatment related. Gross necropsies did not reveal any cons istent treatment-related abnormalities any test group. No statistically significant effects were seen at any test level on adult body weight or average feed consumption, and there were no si gnificant effects on any reproductive endpoints, except body weight of 14-day old hatchlings. Hatc hling body weights were significantly reduced at 75, 180 and 260 ppm, but the effect was not dose-dependent. The NOAEC was 260 ppm, the highest dose tested, and a LOAEC was not observed.

One additional study for each of the mallard duck and bobwhite quail (MRID 00265744) showed no significant effects on any endpoi nt up to and including the highe st concentration tested of 60

ppm.

Early Life Stage Toxicity Test with Fathead Minnow; (OCSPP 850.1400)

In an early life-stage fish study, Fathead minnow embryos ( MRIDs 00164986, 40409201, and

40480401 ) were exposed (15 per replicate, 4 rep licates, 60 embryos per concentration) to myclobutanil (91.9%) at nominal concentrations of 0.5, 1, 2, 4, a nd 8 mg/L. Untreated controls and solvent controls (acetone ) were used. Mean measured concentrations were 0.45, 0.98, 2.2, 4.0, and 8.5 mg/L. Exposure continued until 30 days after hatching was 95% complete.

Based on standard length measurem ents of juvenile fish at end of 35 days of exposure (embryo, larval fish, and juvenile) the NOAEC was 0.98 mg/L and the LOEAC was 2.2 mg/L. At the 2.2 mg/L exposure, there was a 9.7% reduction in m ean total length compar ed to the control. Growth as determined by wet weight was also significantly affected at 4.0 mg/L. One hundred percent mortality occurred at 8.5 mg/L.MYCLOBUTANIL FINAL

Page 57 of 57 APPENDIX 3: References Not Utilized in the Myclobutanil 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 conducted a more recent search (2009 to 2014) of available scientific literature for any a dditional relevant information for their weight of evidence (WoE)
evaluations. These articles were ev aluated 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_r eregistration_workgroup/PDF_rot/e sa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature Toxi city Studies to Support Human Health Risk Assessment ( http://www.epa.gov/pesticides /science/lit-studies.pdf).

The following published and unpublished references we re considered for use in the WoE analysis for Myclobutanil but were not utili zed due to one or more of the following reasons: 1) the article was not available in English; 2) the compound of interest was not used in the study; 3) the test material was not adequately described; 4) a fo rmulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequately described; 7) only an abstract of the study was available; 8) the reference is a review articl e or book chapter and does not contain primary study data; 9) insufficient information was available to adequately assess 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.