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 Assessment Type: 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 

::~:E£'~:::e f]g:ams d f 'v--

Environmental Fat~~fec~~~n ~ Office of Pesticide Program 

THROUGH: Jess Rowland ~ S ~~~ Deputy Division Director 

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

EPA has completed its Weight of Evidence (WoE) assessment evaluating results of the 

Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. 

The WoE documents for the 52 chemicals are listed in Attachment A along with the chemical 

and report identifiers. 

Page I of2 

Page 1 of 65
Page 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 

Page 2 of 65
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: MYCLOBUTANIL 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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MYCLOBUTANIL 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 Relevant 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 Assay for Myclobutanil...............................................................8 

 Table 2. Estrogenic/Anti-Estrogenic 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 Myclobutanil WoE Analysis........................58 

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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 Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

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 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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iv 

Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

HPLC/MS/MS High Pressure Liquid Chromatography/Mass Spectroscopy

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

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 Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

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v 

Abbreviation Terminology 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

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vi 

Abbreviation Terminology 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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Page 1 of 57 

Executive Summary 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide 

the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen 

(E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism and 

antagonism at the estrogen and androgen receptor, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary 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 with E, A or T hormone pathways, the number 

and type of effects induced, the magnitude and 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 endocrine related responses 

occurred happened concurrently with general systemic toxicity. 

On February 12, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee 

(T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination 

and Policy (OSCP) conducted a weight-of-evidence (WoE) 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 wildlife studies (40 CFR Part 158 and literature) 

For the estrogen pathway, a positive finding in the estrogen receptor transactivation assay 

(ERTA) was reported a literature study; however, myclobutanil was negative in the Tier 1 

Estrogen Receptor (ER) binding, ERTA, uterotrophic and female pubertal assays. Overall the 

data do not support a potential for myclobutanil to interact with the 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 studies 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 potentially attributed to altered 

steroidogenesis (effects on hormone levels, VTG, gonadal weight and histopathology, and 

fecundity). 

For the androgen pathway, myclobutanil showed positive 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 and 

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Page 2 of 57 

a slight delay in preputial separation (PPS). The androgen-related effects in the male pubertal 

assay occurred at doses that also resulted in increased liver weights and hypertrophy. 

Furthermore, data from OSRI show that alteration of testosterone homeostasis in mammals is 

mediated via activation of constitutive androstane (CAR), pregnane X (PXR) and subsequent 

enzyme induction in the liver. In mammals, effects on androgen endpoints occurred only at doses 

that resulted in increased liver weights, liver hypertrophy, and/or CYP enzyme induction. In the 

FSTRA an increase in male GSI was observed along 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 potential alteration with 

steroidogenesis which may be affecting 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 departure (POD) of 2.5 mg/kg/day is based on decreased testicular 

weights and increased testicular atrophy seen at 9.84 mg/kg/day (LOAEL) in the chronic 

toxicity/carcinogenicity study in rats. This POD is used for deriving the chronic Reference Dose 

(RfD) for chronic dietary risk assessment. 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 study 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 than the lowest dose (200 mg/kg/day) where a 

potentially endocrine mediated effect was observed in the male pubertal rat assay. Consequently, 

the current RfD and MOEs are protective of potentially endocrine-mediated effects observed in 

the pubertal assay. 

Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is NOT 

recommended for myclobutanil since additional testing is unlikely 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 steroidogenesis 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, additional 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. 

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For birds, Part 158 avian reproduction studies with 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 Part 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 myclobutanil, no additional data are recommended for 

amphibians. 

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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 potential of chemicals to interact with the estrogen 

(E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism and 

antagonism at the estrogen and androgen receptor, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary 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 with E, A or T hormone pathways, the number 

and type of effects induced, the magnitude and 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 endocrine related responses 

occurred happened concurrently with general systemic toxicity. 

On February 12, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee 

(T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination 

and Policy (OSCP) conducted a weight-of-evidence (WoE) 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 octanol-water partition coefficient (log Kow) of approximately 3, a 

relatively low propensity to sorb to organic matter and its organic carbon partition coefficient 

(Koc~520 mL/goc), myclobutanil residues are not expected to bioaccumulate. Myclobutanil is 

stable to hydrolysis and to photolysis. Myclobutanil is moderately persistent to persistent 

(DT50>70 days) in aerobic soils and persistent in anaerobic soils. 

The available information considered in determining the potential interaction 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 toxicity studies and other published articles. These 

data are summarized in Sections III.A through III.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 recommendations are presented in Section V. 

. 

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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 shown below in Table 1. Executive Summaries are 

presented in Appendix 1. 

B. Other Scientifically Relevant Information (OSRI) 

In response to the Agency’s Test Orders, data believed 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 literature and/or data submitted to support pesticide 

registration (e.g., Part 158 guideline studies). The Agency’s review of the initial OSRI is 

provided in the Report of the Endocrine Disruptor Review Team on myclobutanil (USEPA, 

2010). Since then, the Agency has also conducted a more recent search of available scientific 

literature for any additional relevant information. Summaries of the available OSRI are presented 

in Appendix 2. 

III. Weight of Evidence (WoE) Evaluation 

The principles, criteria, and approach used in the WoE determination on the potential of a 

substance to interact with endocrine-mediated processes (i.e., E, A, or T hormonal pathways) 

were as described in the WoE guidance document (USEPA, 2011) and presented at the 2013 

FIFRA Scientific Advisory Panel (SAP) (USEPA, 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 conceptual framework of an adverse outcome 

pathway). Broadly, there are four main steps 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 potential endocrine interaction(s). The 

second step is to integrate the data along different levels of biological organization while 

examining the extent of concordance (robustness) of complementarity (i.e., the concordance of 

endpoints within an assay that measures multiple endpoints) and redundancy (i.e., the 

concordance of endpoints/responses across assays) in the observed responses across these 

different levels of biological organization. The third step is to then characterize the main lines of 

evidence as well any conclusions. Finally, the last step is to evaluate whether 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 EDSP Tier 1 data represent a battery of 11 assays 

consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were 

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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 include published literature studies as well as 

studies conducted under USEPA (often referred to as Part 158 data) or OECD guidelines 

submitted in support of pesticide registrations. Each 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 specific endpoint for discerning whether or not the 

chemical has interacted with the endocrine system 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 indication of potential interaction as measured by the study. 

The second step in this WoE process is to formulate hypotheses and integrate 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 support a hypothesis that a chemical 

has the potential to interact E, A, or T pathways are the concepts of complementarity and 

redundancy. These two concepts provide a basis for considering the plausibility, coherence, 

strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are 

meant to evaluate whether or not a chemical can interact with E, A and T consisting of different 

levels of biological organization from a molecular initiating event such as receptor binding 

through potential adverse effects in apical endpoints such as sexual development and fecundity at 

the whole organism level. Transitions to higher levels of biological organization can indirectly 

provide information on potential compensatory capabilities of an individual organism. 

After the data have been assembled and integrated, the third step is to characterize the main lines 

of evidence along with the conclusions; this characterization involves three components. The 

first component is whether the data provide relevant, robust and consistent evidence in terms of 

complementarity and redundancy as well as biological plausibility. Second, is at what level of 

biological organization were the responses observed and whether organisms exhibit 

compensatory responses at higher-levels of biological organization. Finally, 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 effects specifically related to an 

endocrine-mediated effect from a non-endocrine toxic response. This uncertainty in 

distinguishing whether the responses were endocrine-mediated was discussed at the FIFRA SAP 

meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 

screening process. 

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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 toxicity. All effects in 

endocrine sensitive tissues should be evaluated in terms of primary interactions with the 

endocrine system vs. secondary effects related to toxicity in non-endocrine organs or overall 

disruptions in homeostasis” (USEPA, 2013). For these WoE analyses, overt toxicity was 

generally defined in accordance with EPA’s current approach as used by OPP in reviewing 40 

CFR Part 158 studies for both human and ecological risk assessments. Specifically, in these 

analyses, the effects that EPA considered to be potential evidence of overt toxicity included, but 

were not limited to: mortality; clinical signs such as tremors, ataxia and abnormal swimming 

(fish and aquatic-phase amphibians); and body weight decreases of ≥10% in mammals. 

Additionally, other effects including morphological (e.g., organ weights/histopathology), 

biochemical (e.g., blood chemistry), and other clinical signs (e.g., lethargy) were also considered 

when evaluating overt toxicity, especially 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 responses when assessing potential overt toxicity. 

Additionally, effects which were considered to be signs of systemic 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/chemical properties and its 

known modes of action in its overall characterization of a chemical’s potential to interact with 

the E, A or T pathway. Adequately addressing the three components described above is 

fundamental to the WoE process and in determining whether additional data are needed. In 

addition to characterizing the WoE, reviewers also consider: 1) uncertainties and their potential 

impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting 

data; 4) overall strength of evidence supporting a conclusion; and, 5) what, if any, additional data 

are needed and why. Assessing the need for additional data is based 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 biologically 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 the presence of overt toxicity are discussed in the text for each 

respective pathway (E, A or T) but are not reported in the table for E, A or T. 

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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) 

ERα 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) 

 

Page 16 of 65
MYCLOBUTANIL FINAL 

Page 9 of 57 

B. Effects on Hypothalamic-Pituitary-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-relevant 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 table and not 

considered further in the WOE assessment. 

Myclobutanil was classified as negative (or not interactive) in the Tier 1 in vitro ER binding (rat 

uterine cytosol) assay. Although the ERTA requirement was considered 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 screen for the binding and activation 

of human estrogen receptor (hER-α). In this yeast-based assay, myclobutanil produced an 

estrogenic response with an estrogenic potency relative to 17β-estradiol (E2) of 7x10-6, and an 

estrogenic LOEC of 781 μg/L in this test system. However, when hERα-HeLa-9903 cells were 

used as required in the guideline ER transcriptional activation assay, myclobutanil was negative 

for ERα transcriptional activation up 10-4 M (highest soluble concentration). 

In the Tier 1 aromatase assay (CYP 19), myclobutanil reduced aromatase 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 considered a positive response in this assay. The 

response in this assay is consistent with the results from the two published literature articles 

(Trösken et al., 2004, 2006) that were submitted as OSRI in response to the test order. In the Tier 

1 steroidogenesis assay, 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 

(0.4-0.7-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 weight 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 assay, liver effects including increased liver 

weights and histopathology were seen at the mid and high doses. 

In the FSTRA, female gonadal-somatic index (GSI) 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. Vitellogenin (VTG) was significantly reduced (p<0.05) by 

88% in females at the high dose. In females, there was a significant increase (p<0.05) in serum 

testosterone at the mid-dose, but not at the high dose, and there was a significant reduction 

(p<0.05) of 85% in 17β-estradiol at the high dose. Also, fecundity was significantly reduced by 

Page 17 of 65
MYCLOBUTANIL 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 mortality or behavior) this concentration considered to not be 

overtly toxic. 

In the Part 158 Guideline studies, the high dose (200 mg/kg/day) in the rabbit developmental 

study showed increased resorptions and resorptions/litter and decreased litter size and viability 

index; these effects were observed in the presence of decreased maternal body weights and body 

weight gains. In the 2-generation rat reproduction study at the high dose 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, reproductive effects were 

observed at the highest dose tested in the respective 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 mean total length compared to the control. 

Growth as determined by wet weight was also significantly affected at 4.0 mg/L; 100% mortality 

occurred at 8.5 mg/L. 

Page 18 of 65
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Table 2: Estrogenic/Anti-Estrogenic Pathway for Myclobutanil

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Myclobutanil1

Study Type / Literature Citation 

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 

EDSP Tier 1 Assay 

ER Binding (MRID 48618104) N ERTA (MRID 48618105) 

N

Steroidogenesis (MRID 48618110) 

P

Aromatase (MRID 48618103) 

P

Uterotrophic (MRID 48618111) N Female Pubertal Rat (MRID 48618108) N N N N N N FSTRA (MRID 48618106) 

P5 

↑GSI: 

41, 46%

(M, H)

P

(H) N 

Fcd: 

↓98% 

(H) 

P

(H
MYCLOBUTANIL FINAL 

Page 12 of 57 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Myclobutanil1

Study Type / Literature Citation 

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 

OSRI 

ERTA (Hurst and Sheahan, 2003) 

 P 

Aromatase Trosken et al., (2004 and 2006) 

 P 

Subchronic toxicity (Rat; MRID 00145048) NE N N NE Subchronic toxicity (Mouse; MRID 00165245) 

NE N N NE 

Developmental toxicity (Rat; 00141672) NR N Developmental toxicity (Rabbit; MRID 00164971) 

NR N 

Two-generation reproduction (Rat; MRIDs 00143766, 00149581) 

NE NE N NE NE 

P

(H) 

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

NE N N NE 
MYCLOBUTANIL FINAL 

Page 13 of 57 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Myclobutanil1

Study Type / Literature Citation 

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 

Carcinogenicity (Mouse; MRIDs 00164990, 40244301, 42809102) 

NE N N N 

Chronic toxicity (Dog; MRID 00165248) N N N N Avian Reproduction (Quail; MRIDs 00265744,) 

N 

Avian Reproduction (Duck; MRIDs 00265744,) 

N 

Avian Reproduction (Quail; MRIDs 4340201, 43087901) 

N 

Avian Reproduction (Duck; MRID 43087902) 

N 

1. Key to responses: L=Low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows (↓ or ↑) indi

response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight aotherwise indicated. LW = Liver weight, LH = liver hypertrophy, LE = serum levels of liver enzymes, KW = kidney weight 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OS

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 

details see Section IV. A 

4. 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 significant reduction 

estradiol at the high dose 
MYCLOBUTANIL FINAL 

Page 14 of 57 

6. A marginal, but significant (9%; p=0.022), reduction in female body weight was observed at the high concentration, but, given the 

signs of clinical toxicity (no treatment-related effects on mortality or behavior) this concentration considered to not be overtly toxic 

P Positive findings (in vitro) / No effect (in vivo) 

N Negative findings (in vitro) / No effect (in vivo) NE Not examined NR Not reported 
MYCLOBUTANIL FINAL 

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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 Tier 1 assays are delineated so 

as to facilitate determination of potential for androgenic, anti-androgenic, or HPG axis effects. 

This table also includes HPG-relevant 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 

table and not considered further 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 weak affinity for the androgen-receptor. In the 

steroidogenesis assay, testosterone 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/day) in the Hershberger assay for both androgen agonism and 

antagonism. 

No effects on androgen-related endpoints were observed 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 seminal vesicle + coagulating gland 

(↓15/19%) and ventral prostate (↓16%). At 400 mg/kg/day, tissue weight decreases were noted in 

the seminal vesicle + coagulating gland (↓33/41%), ventral 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) was reported. In addition to the changes in AST weights, relative 

adrenal weights were also increased (15%) at 400 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 hepatocellular hypertrophy was also observed at the doses that showed changes in 

androgen-related endpoints. 

In the FSTRA, testosterone was reported to not be affected in males. Significant increase in male 

GSI (↑80%) along with alterations in the male gonads reported as increased proportions of 

mature sperm relative to spermatogenic 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 studies, testicular effects were 

observed in the mid and mid-high, and high males (10, 39 and 106 mg/kg/day, respectively) and 

included significantly decreased mean absolute testes weights, and decreased absolute and 

relative (to brain) testicular weights, and increased incidences of testicular atrophy at the midhigh and high doses. Histopathological testicular changes included increased incidences of 

bilateral aspermatogenesis, hypospermia and cellular debris in the epididymides at the high dose. 

Page 23 of 65
MYCLOBUTANIL FINAL 

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Liver mixed function oxidase activity was significantly increased in females at 13 mg/kg/day at 3 

months (61%), males at 39 mg/kg/day at 3 and 6 months (34–47%) and females 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%) and 6 months (20%), respectively. 

In an OSRI study, Tully et al. (2006) evaluated gene expression profiles in the liver and testis to 

characterize the toxicity of myclobutanil. In this study rats were 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 disruption of testosterone homeostasis 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 weights were increased 9–13%, at PND 1, 50 and 92. Hepatocellular 

hypertrophy was present at PND 92 at the high dose. Relative pituitary weights were reduced at 

PND 92 in mid and high dose groups. Absolute testis weights were increased at PND 1 and 22 

and relative ventral prostate weights and testosterone levels were increased at PND 92. 

Decreased insemination (134 mg/kg/day) and fertility (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 testes 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 profiles 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 hepatic steroid metabolism and sterol biosynthesis rather than 

modulating steroidogenic gene expression in the testes. 

There were no androgen-related effects determined for the available Part 158 wildlife studies.

Page 24 of 65
MYCLOBUTANIL FINAL 

Page 17 of 57 

Table 3: Androgenic/Anti-Androgenic Pathway for Myclobutanil Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Myclobutanil1 Study 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 

Observed2

EDSP Tier 1 Assay 

AR Binding (MRID 48618102) P Steroidogenesis (MRID 48618110) 

P

Hershberger (MRID 48618107) NE N ↑LW (M, H) Male Pubertal Rat (MRID 48618109) 

↓49%↓85%

(M, H)

N N N N N 

↓SV 

(M, H)

↓VP 

(M, H) 

↓LABC 

(H) 

↓DP 

 (H)

PPS age: 

↑1.7 d

(H) 

↑LW, 

↑LH 

(M, H)

LE 

(H) 

FSTRA (MRID 48618106) N 

↑GSI 

79% 

(H) 

P 

(H) N N 

N

OSRI 

Subchronic toxicity (Rat; MRID 00145048) 

 N N NE NE NE NE X (MH, H) 

Subchronic toxicity (Mouse; MRID 00165245) 

 N N NE NE NE NE X (MH, H) 
MYCLOBUTANIL FINAL 

Page 18 of 57 

Table 3: Androgenic/Anti-Androgenic Pathway for Myclobutanil Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Myclobutanil1 Study 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 

Observed2

Developmental toxicity (Rat; 00141672) 

N NE 

Developmental toxicity (Rabbit; MRID 00164971) 

N NE 

Two-generation reproduction (Rat; MRIDs 00143766, 00149581) 

 NE 

P5

(H) NE 

P5

(H) NE NE 

N X (M,H) 

2-yr Chronic toxicity/Carcinogenciity7

(Rat; MRIDs 00165247, 42809101) 

P6

(M,MH 

H) 

P6

(M,

MH, 

H) 

NE 

P7

(H) NE NE X (H) 

Carcinogenicity (Mouse; MRIDs 00164990, 40244301, 42809102) 

N N N N NE NE X (H) 

Chronic toxicity (Dog; MRID 00165248) 

N N NE NE N NE X (M,H) 
MYCLOBUTANIL FINAL 

Page 19 of 57 

Table 3: Androgenic/Anti-Androgenic Pathway for Myclobutanil Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Myclobutanil1 Study 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 

Observed2

Liver and Testis toxicity profiles (Tully et al., 

2006)18 

>2-

fold

(H) 

NE NE 

sperm 

mobility

X 

(H) 

Reproductive Toxicity Mode of Action (Goetz, et al., 2007, 2009a,b)16 



(M,H)



(L, H) 

PND 1 



(M,H) 

VP 

(M) 



(M, H)



AGD 

X 

(H) 

Avian Reproduction (Quail; MRIDs 00265744,) 

NE NE N N 

Avian Reproduction (Duck; MRIDs 00265744,) 

NE NE N N 

Avian Reproduction (Quail; MRIDs 4340201, 43087901) 

 N N 

Avian Reproduction (Duck; MRID 43087902) 

 N N 

1. Key to responses: L=Low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows (↓ or ↑) indi

response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight a
MYCLOBUTANIL FINAL 

Page 20 of 57 

otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (Dbulbocavernosus (LABC), Epididymides (EP). 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 OS

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 

details see Section IV. A 

4. A marginal, but significant (9%; p=0.022), reduction in female body weight was observed at the high concentration, but, given the 

signs 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 necrotic spermatocytes/spermatids or decreased spermatozoa and atrophy of the 

the 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 an

study: 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 stu

P Positive findings (in vitro) / No effect (in vivo) 

N Negative findings (in vitro) / No effect (in vivo) NE Not examined 
MYCLOBUTANIL FINAL 

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

The current EDSP Tier 1 battery 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 detect 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 thyroid pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for thyroid or HPT axis effects. This table also includes 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 table and not considered further in the 

WOE assessment. 

The female pubertal assay was negative for all observed thyroid parameters at all doses evaluated 

(50, 200 and 400 mg/kg/day). No systemic toxicity was noted in the study at doses up to 

400 mg/kg/day; however, there were significant dose-dependent increases 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 weight 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 significant, and no changes were noted in T4 levels. Thyroid weight 

was also unaffected, although non-significant changes in thyroid histopathology 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 and elevated gamma glutamyl transferase levels at the high dose. 

In the amphibian metamorphosis assay there were no treatment-related effects on thyroid 

histopathology. Developmental stage was significantly delayed (p<0.05) on Day 7 (NF stage 52 vs. 

NF 54 in control), and normalized HLL was significantly reduced (p<0.05) by 13% at the high 

dose on Day 7 and at all treatment levels on Day 21 (↓10–24%). Clinical observations were 

transiently noted in all treatment groups with a slightly higher proportion of individuals affected in 

the two highest treatment levels; effects included lying on the surface or the bottom of the tank, 

inverted or irregular swimming and 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 treatment groups, and as such these treatment 

groups were not considered to be overtly toxic. 

In the OSRI, none of the studies evaluated showed 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. 

Page 29 of 65
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Evidence of systemic toxicity was also see at this dose, including increased 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 Myclobutanil1

Study Type / 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage or asynchronous & HLL Growth (BW, SVL) Systemic Toxicity Observed2

Overt Toxicity Observed3

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) 

 N4 

OSRI

Subchronic toxicity 

(Rat; 

MRID 00145048)

N P5

(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 N 

Page 30 of 65
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Table 4. Thyroid Pathway for Myclobutanil.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Myclobutanil1

Study Type / 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage or asynchronous & HLL Growth (BW, SVL) Systemic Toxicity Observed2

Overt Toxicity Observed3

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 the 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 examined 

Page 31 of 65
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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 endocrine mediated processes (i.e., E, A, or T hormonal 

pathways) as well as recommendations 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 peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (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 biological 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 available 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 through an endocrine pathway, therefore non-endocrine 

toxic responses (including but not limited to mortality or body weight changes) are also 

considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated 

with weight of evidence evaluation of the results of the Tier 1 assays 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 toxicity. All effects 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 observed which may be considered overt/systemic 

toxicity are also describe below. Generally, one parameter (i.e., death or >10% decrease in 

mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. 

However, in other instances, more than one parameter was needed to determine overt toxicity. 

Effects which were considered to be signs of systemic toxicity were generally less severe forms of 

toxicity (e.g., organ weights, blood chemistry). 

1. Tier 1 in vivo Assays 

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In the Tier 1 mammalian studies, only the uterotrophic assay showed evidence of overt toxicity. 

In this assay, body weight was decreased (p<0.05) by 15% on Day 3, resulting in a 59% decrease 

(p<0.05) in body weight gain at the high dose (475 mg/kg/day). No treatment-related effects on 

bodyweight were seen at the two lower doses (50 and 150 mg/kg/day). 

In the Hershberger assay, no evidence of systemic 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 portion 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 histopathological lesions in the liver including hepatocellular 

hypertrophy and vacuolization. 

In pubertal female rats, no evidence of systemic toxicity was seen at the low dose (50 mg/kg/day). 

Dose-dependent increases (p<0.05) in absolute, relative, and adjusted liver weights were observed 

at 200 (↑18–19%) and 400 (36–41%) mg/kg/day. Treatment-related 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 incidence and severity of liver histological including hypertrophy 

and number of mitotic figures. Male pubertal rats also showed significant (p<0.05) dosedependent increases in liver weights. Liver weights increased by 12–13% at 200 mg/kg/day and 

27–28% at 400 mg/kg/day, and relative liver weights 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 consisted of hepatocellular hypertrophy with altered 

tinctorial properties in centrilobular and midzonal hepatocytes and 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 significant (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 behavior) 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 control 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 isolated 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. 

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2. OSRI 

Conazoles such as myclobutanil were designed to specifically inhibit CYP51, a sterol 

14-α-demethylase which catalyzes the 14-α-demethylation of lanosterol in the ergosterol 

biosynthesis pathway in fungi. The biosynthesis of ergosterol is critical to the formation of fungal 

cell walls. The lack of normal sterol production slows or stops the growth of the fungus, 

effectively preventing further infection and invasion of host tissues (Ghannoum and Rice, 1999). 

Cyp51 is a conserved gene across many taxi including fungi, protists, mammals, and plants and is 

required for sterol biosynthesis in all eukaryotic systems. Mammalian cholesterol synthesis is 

blocked by conazoles at the step of the sterol 14-α-demethylase, but a much higher concentration 

is required to cause the same degree of inhibition as in fungi (de Brabander et al., 1980; Van de 

Bossche, 1980; Zarn et al., 2003). Myclobutanil has been shown to result in adverse health 

outcomes related to the liver and the endocrine system. The key events leading 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 homeostatic pathways are regulated by the constitutive androstane 

(CAR), pregnane X (PXR), peroxisome proliferator-activated receptor alpha (PPARα), and other 

nuclear receptors in liver. In mechanistic studies conducted by EPA’s Office of Research and 

Development (ORD), relatively high doses of myclobutanil 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 molecular 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 xenobiotics, it may be that CAR and PXR are also 

components of a regulatory network that regulates 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 activity of myclobutanil through the 

pregnane X receptor (PXR) and constitutive androstane 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 genomic data from Goetz et al. (2009a) suggest the induction 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 steroidogenic genes in the testis. 

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In the Part 158 mammalian studies, the liver was the primary target organ of toxicity. The 

absolute and relative liver weights were significantly increased in both sexes at >150 mg/kg/day 

in the mouse subchronic study. The liver weight increases were associated with histopathological 

effects including centrilobular and midzonal hepatocellular 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 sexes 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 centrilobular hypertrophy, Kupffer cell pigmentation, 

hepatocellular vacuolation and necrosis were also observed in the liver at the high dose. 

In rats, significant decreases in body weights were 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, absolute and relative liver weights were increased in females at 6 

and 3 months, respectively. In the rat 2-generation reproduction study, increased 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. Increased incidences of hepatocellular 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 were 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 affected at 4.0 mg/L. One hundred percent mortality occurred at 8.5 

mg/L. 

No effects on mortality, body weight, feed consumption 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 transcriptional activation assay (Hurst and Sheahan, 

2003) submitted as OSRI was not corroborated by the negative findings in the EDSP Tier 1 ER 

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binding, ERTA, uterotrophic and female pubertal assays. Overall, the data do not support a 

potential for myclobutanil to interact with the ER. The discrepancy in responses between the 

OSRI and the guideline ERTA assays may be attributed to the use of yeast cells in the Hurst and 

Sheahan study. The response in the 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 effects 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 17β-estradiol and plasma VTG, an increase in GSI as well 

as gonadal histological effects (oocyte atresia), and a 98% reduction in fecundity. Additionally, 

GSI and serum testosterone were significantly increased at the mid (0.327 mg a.i./L) 

concentration. 

While significant changes or alterations in estrogen 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 antagonist 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, significant 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 systemic 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 relative to spermatogenic precursor cells. 

Additionally, 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 in these animals. In the chronic rat study, testicular toxicity was 

observed in the mid and high dose males, and included 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. 

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In other OSRI (Goetz et al., 2007) alterations in androgen-related endpoints were observed 

including changes in AGD, AST, and testosterone levels which occurred in the presence of 

increased liver weights and histopathology. Mechanistic studies (Goetz and Dix (2009a and 

2009b) suggested that myclobutanil alters testosterone homeostasis via disruption of hepatic 

steroid metabolism and sterol biosynthesis rather than modulating steroidogenic gene expression 

in the testes. 

Based on the EDSP Tier 1 data and OSRI, myclobutanil appears to alter steroidogenesis which 

then interferes with the androgen 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-related effects were observed in the absence of 

increased liver weight, hepatocellular hypertrophy or CAR/PXR-mediated CYP induction. 

In contrast to the mammals, data are not available 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 myclobutanil could potentially alter steroidogenesis thereby affecting the 

androgen, as well as the estrogen, pathway. 

D. Thyroid Pathway 

In the male and female pubertal assay there were no significant changes 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 increased incidence of small thyroid 

follicles in males at 300 mg/kg/day; a dose that resulted in significant liver and kidney toxicity. 

In the AMA, since the developmental delays were not corroborated by thyroid histological 

diagnoses that would be suggestive of a potential HPT-mediated effect, the observed 

developmental delays were not considered to be thyroid-mediated. Therefore, the 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 this pathway. 

E. Conclusions 

Based on the weight of the evidence, myclobutanil appears to potentially alter steroidogenesis in 

fish which may affect the androgen, as well as the estrogen, pathways. In mammals, however, the 

alteration in androgen-mediated endpoints appears to be mediated 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 thyroid pathway in mammals and non-mammals. 

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V. EDSP Tier 2 Testing Recommendations 

For myclobutanil, the point of departure (POD) of 2.5 mg/kg/day is based on decreased testicular 

weights and increased testicular atrophy seen at 9.84 mg/kg/day (LOAEL) in the chronic 

toxicity/carcinogenicity study in rats. This POD is used for deriving the chronic Reference Dose 

(RfD) for chronic dietary risk assessment. 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 study 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 than the lowest dose (200 mg/kg/day) where a 

potentially endocrine mediated effect was observed in the male pubertal rat assay. Consequently, 

the current RfD and MOEs are protective of potentially endocrine-mediated effects observed in 

the pubertal assay. 

Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is NOT 

recommended for myclobutanil since additional testing is unlikely 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 steroidogenesis 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, additional 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 with 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 Part 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 myclobutanil, no additional data are recommended for 

amphibians. 

 

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VI. References 

Anderson, D.; O’Hara, G.; Brown, W. (1993). RH-3866: Dietary Oncogenicity Study in Female 

Mice: Lab Project Number: 89P-261:89R-261. Unpublished study prepared by Rohm and Haas 

Co. 642 p. (MRID 42809102). 

Ankley, G.T. and Gray, L.E. (2013). Cross-Species Conservation of Endocrine Pathways: a 

Critical Analysis of Tier 1 Fish and Rat Screening Assays with 12 Model chemicals. Environ 

Toxicol Chem. 32(5):1084–7. 

Bailey, J. P., Gehen, S. C., Kramer, V. J.,. Marty, M. S., Coady, K. K (2012). Myclobutanil: 

Weight of Evidence Evaluation of Potential Interaction with the Estrogen, Androgen or Thyroid 

Pathways Dow AgroSciences LLC, Indianapolis, Indiana 46268, Study Number.120690, (MRID 

No. 48686401).

Brown, U. (1985). Two-Generation Reproduction Study with RH-53,866 Technical in Rats: 

Protocol No. 83P-364, Report No. 84R-117. Unpublished study prepared by Research Pathology 

Services, Inc. 78 p. (MRID 00143766). 

Coady, K.K.; Hutchinson, K.L.; Marino, T.A.; Malowinski, N.A.; and Sura, R. (2011). 

Myclobutanil: The Amphibian Metamorphosis Assay Using the African Clawed Frog (Xenopus 

laevis). Unpublished study performed by The Dow Chemical Company, Midland, Michigan. 

Laboratory project study ID 101124, October 17, 2011. Study sponsored by Dow AgroSciences 

LLC, Indianapolis, Indiana. (MRID 48618101). 

Coady, K.K.; and Sosinski, L.K. (2011). Myclobutanil: Evaluation of Myclobutanil in the Human 

Recombinant Aromatase Assay. Unpublished study prepared by Toxicology & Environmental 

Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project Study 

ID No.: 111015, November 28, 2011. (MRID 48618103). 

Coady, K.K.; Louch, D.W.; Malowinski, N.A.; Marino, T.A.; McFadden, L.G.; Perala, A.W.; 

Sosinski, L.K.; and Sura, R. (2011). Myclobutanil — The Fish Short-Term Reproduction Assay 

with the Fathead Minnow, Pimephales promelas. Performed by Toxicology & Environmental 

Research and Consulting, Midland, Michigan. Submitted by Dow Chemical Company. Project 

ID.: 111020, December 14, 2011. (MRID 48618106). 

Costlow, R.; Kane, W. (1984). Teratology Study with RH-53, 866 in Rabbits: Report No. 83R217. Unpublished study prepared by Rohm and Haas Co. 139 p. (MRID 00164971). 

Costlow, R.; Kane, W. (1984). Teratology Study with RH-53,866 in Rats: Final Report: Report 

No. 83R-024. Unpublished study prepared by Rohm and Haas Co. 123 p. (MRID 00141672). 

Costlow, R.; and Harris, J. (1985). RH-53,866: Two Generation Reproduction Study in Rats: 

Report No. 84R-117. Unpublished study prepared by Rohm and Haas Co. 225 p. 

(MRID 00149581). 

Page 39 of 65
MYCLOBUTANIL FINAL 

Page 32 of 57 

Goetz, A.K., Ren H., Schmid, J.E., Blystone, C.R., Thillainadarajah, I., Best, D.S., Nichols, H.P., 

Strader, L.F., Wolf, D.C., Narotsky, M.G., Rockett, J.C., and Dix, D.J. (2007). Disruption of 

Testosterone Homeostasis as a mode of action for Reproductive Toxicity of Triazole Fungicides 

in the Male Rat. Toxicol. Sci. 95(1): 227–239. 

Goetz, A. K. and Dix, D. J. (2009a). Mode of Action for Reproductive and Hepatic Toxicity 

Inferred from a Genomic Study of Triazole Antifungals. Toxicol. Sci. 110(2):449–462. 

Goetz, A. K. and Dix, D. J. (2009b). Toxicogenomic Effects Common to Triazole Antifungals 

and Conserved Between Rats and Humans. Toxicol Appl Pharmacol. 238(1):80–89. 

Goldman, P.; Harris, J. (1986). RH-3866: Dietary Chronic and Oncogenicity Study in Mice: 

Final Report: Report Number 84R-023. Unpublished study prepared by Rohm and Haas Co. 

3326 p. (MRID 00164990). 

Goldman, P.; Harris, J. (1986). Correction Pages for RH-3866: Dietary Chronic and 

Oncogenicity Study in Mice: Laboratory Project ID: 84R-023. Unpublished study prepared by 

Rohm and Haas Co. 7 p. (MRID 40244301). 

Goldman, P.; Harris, J.; Frantz, J. (1986). RH-3866: One Year Dietary Study in Beagle Dogs: 

Protocol No. 84P-203: Report No. 84R-078. Unpublished study prepared by Rohm & Haas Co. 

775 p. (MRID 00165248). 

Goldman, P.; Harris, J.; Lampe, K. (1986). RH-3866: A Three-month Dietary Toxicity Study in 

Mice: Report No. 83R-136. Unpublished study prepared by Rohm & Haas Co. 522 p. 

(MRID 00165245). 

Hestor, S.D., Wolf, D.C., Nesnow, S., and Thai S.F. (2006). Transcriptional profiles in liver from 

rats treated with tumorigenic and non-tumorigenic triazole conazole fungicides: Propiconazole, 

triadimefon and myclobutanil. Toxicol. Pathol., 34(7):879–894. 

Hurst M.R. and Sheahan, D.A. (2003). The potential for oestrogenic effects of pesticides in 

headwater streams in the UK. Sci Total Environ 301: 87-96. (MRID 48242003). 

LeBaron, M.J.; Schisler, M.R.; and Visconti, N.R. (2011). Evaluation of Myclobutanil in an 

in vitro Androgen Receptor Binding Assay. Unpublished study prepared by Toxicology & 

Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. 

Laboratory Study No.: 111113, December 5, 2011. (MRID 48618102). 

LeBaron, M.J.; Schisler, M.R.; and Visconti, N.R. (2011). Evaluation of Myclobutanil in an 

in vitro Estrogen Receptor Binding Assay. Unpublished study prepared by Toxicology & 

Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. 

Laboratory Study No.: 111118, December 14, 2011. (MRID 48618104). 

LeBaron, M.J.; Kan, H.L. (2011). Evaluation of Myclobutanil in an in vitro Estrogen Receptor 

Transcriptional Activation Assay in Human Cell Line hERα-HeLa-9903. Unpublished study 

prepared by Toxicology & Environmental Research and Consulting, The Dow Chemical 

Page 40 of 65
MYCLOBUTANIL FINAL 

Page 33 of 57 

Company, Midland, MI. Laboratory Report No.: 101193, December 12, 2011. 

(MRID 48618105). 

LeBaron, M.J.; Kan, H.L.; Perala, A.W. (2011). Evaluation of Myclobutanil in the in vitro 

Steroidogenesis Assay. Unpublished study prepared by Toxicology & Environmental Research 

and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Study No.: 101192, 

December 5, 2011. (MRID 48618110). 

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, The Dow Chemical Company, Midland, MI. 

Laboratory Study No.: 101131, October 21, 2011. (MRID 48618107). 

Marty, M.S.; Zablotny, C.L.; and Stebbins, K.E (2011). Myclobutanil: Pubertal Development and 

Thyroid Function in Intact Juvenile/Peripubertal Female CrL:CD(SD) Rats. Unpublished study 

by Dow Chemical Company, Midland, MI. Laboratory Project Study ID: 111089, December 13, 

2011. (MRID 48618108). 

Marty, M.S.; Andrus, A.K.; and Stebbins, K.E. (2011). Myclobutanil: Pubertal Development and 

Thyroid Function in Intact Juvenile/Peripubertal Male Crl:CD(SD) Rats. Unpublished study 

prepared by Toxicology & Environmental Research and Consulting, The Dow Chemical Co., 

Midland, MI. Laboratory Study ID: 111090, December 12, 2011. (MRID 48618109). 

Marty, M.S.; Brooks, K.J. (2011). Myclobutanil: Uterotrophic Assay in the Immature Female 

Crl:CD(SD) Rat. Unpublished study prepared by Dow Chemical Company, Toxicology & 

Environmental Research and Consulting, Midland, MI. Laboratory Project Study ID.: 101172, 

June 20, 2011. (MRID 48618111). 

McAllister, William A.; McLain, Terri; Seidel, Alan; Franklin, Brenda B. (1986). Early Life 

Stage Toxicity of RH-3866 to Fathead Minnow (Pimephales promelas) in a Flow-Through 

System. Report No. 34538, October 15, 1986. Test performed by Analytical Bio-Chemistry 

Laboratories, Inc. and submitted by Rohm & Haas Company. (MRIDs 00164986, 40409201, and 

40480401). 

O’Hara, G.; DiDonato, L. (1984). RH-3866: Three Month Dietary Toxicity Study in Rats: Final 

Report: Report No. 83R-068. Unpublished study prepared by Rohm and Haas Co. 702 p. 

(MRID 00145048). 

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, 

Page 41 of 65
MYCLOBUTANIL FINAL 

Page 34 of 57 

Neillsville, WI. Sponsor by Rohm and Haas Co.·Research Laboratories, 727 Norristown Rd., 

Spring House, PA. Laboratory Report ID: BLAL No. 111-010-08. (MRID 43087902). 

Shellenberger, T.; Billups, L. (1986). Chronic Toxicity and Oncogenicity Study with RH 3866 in 

Rats: Final Report: Project No. 8342. Unpublished study prepared by Rohm & Haas Co. Project 

No. 85RC-61 prepared by Tegeris Laboratories, Inc. 3536 p. (MRID 00165247). 

Tegeris Laboratories, Inc. (1985). RH 3866, 24-Month Chronic Toxicity-Oncogenicity Study in 

the Rats: Interim Reports: Project No. 8342. Unpublished incomplete study. 110 p. 

(MRID 00149582). 

Trosken, E., Scholz, K., Lutz, R., Volkel, W., Zam, J., and Lutz, W. (2004). Comparative 

Assessment of the Inhibition of Recombinant Human CYP19 (Aromatase) by Azoles Used in 

Agriculture and as Drugs for Humans. Endocrine Res. 3:387–394. (MRID 48242001). 

Trosken, E.R., Fischer K., Volkel W., Lutz W.K. (2006). Inhibition of human CYP19 by azoles 

used as antifungal agents and aromatase inhibitors, using a new LC-MS/MS method for the 

analysis of estradiol product formation. Toxicol 219:33–40. (MRID 48242002). 

USEPA. (2010). Myclobutanil: Report of the Endocrine Disruptor Review Team — Test Order 

#s: EDSP-128857-158 and EDSP-128857-159. December 8, 2010. Office of Chemical Safety and 

Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC. 

USEPA (2011). Endocrine Disruptor Screening Program — Weight of Evidence: Evaluating 

Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical 

Safety and Pollution Prevention Science Coordination and Policy. U.S. Environmental Protection 

Agency, Washington, DC. 

USEPA (2013) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting 

held July 30 - August 1, 2013 on Weight-of-Evidence: Evaluating Results of EDSP Tier 1 

Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection 

Agency, Washington, DC. 

Wolfe, G. (1993). RH-3866 Technical (Myclobutanil) 104-Week Dietary Oncogenicity Study in 

Rats: Final Report: Lab Project Number: 417-471: 89RC-260. Unpublished study prepared by 

Hazleton Washington, Inc. 1649 p. (MRID 42809101). 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay; (OCSPP 890.1100) 

The 21-day assay (MRID 48618101) of Myclobutanil (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) concentrations 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 did 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 surfacing activity, and being nonresponsive 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 isolated 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 low and high treatment groups on Day 21 (20 and 

22%%, respectively, p<0.01). Similarly, snout-vent length (SVL) was significantly reduced in the 

mid and high treatment groups on Day 7 (16 and 19%, respectively, p <0.05) and significant 

reductions were observed in the low and high treatment groups on Day 21 (6%, p <0.01). There 

were also significant reductions in non-normalized hind limb length (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 significantly 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 reported on Day 7 or 21. A small proportion of test 

animals (<20%) were ‘late-stage’ (classified as >NF 60 on Day 21), and these tadpoles were not 

included in the analysis of growth (body weight, SVL) and HLL measurements. There were no 

treatment-related effects on thyroid gland histopathology at any treatment level. 

Androgen Receptor (AR) Binding Assay; (OCSPP 890.1150)

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In an androgen receptor (AR) binding assay (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 appropriate affinity for the radiolabeled ligand. 

Saturation binding data were not presented in the study report; however, saturation binding data 

were presented in the study profile submitted separately by the test order recipient (study profile, 

MRID 48618102). The competitive binding experiment was conducted to measure the binding 

of a single concentration of [3H]-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. Three independent competitive binding assays were 

performed. 

In the saturation binding experiment, the maximum binding capacity (Bmax) was 3.245 fmol/100 

μg protein and the dissociation constant (Kd) was 0.4641 nM. Although the Bmax fell slightly 

below the expected range of 7 to 16 fmol/100 μg protein and the Kd was below the expected range 

of 0.685 to 1.57 nM, confidence in these results is high because of the goodness of fit (r2 = 0.9871 

– 0.9937) and the reproducibility among the runs. With the exception of the high concentration of 

10 nM in one assay with a 24.6% nonspecific binding, non-specific binding as a percent of total 

binding was <20% across the entire 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 saturation binding experiment were conducted. 

In the competitive binding experiment, myclobutanil reduced the mean specific binding of the 

radiolabeled ligand at the two highest concentrations in a concentration-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. However, these results may be an artifact of the data when using an 

undefined bottom plateau for the curve fitting. When the data were re-analyzed by constraining 

the bottom of the response curves to zero, the results of all three independent runs were similar 

with acceptable r2 values and standard errors. 

The mean estimated log IC50 was -3.5 M for myclobutanil for the analysis using a constrained 

bottom plateau. The range of RBA values for myclobutanil across the three runs was 0.00034% 

to 0.00037% indicating a weak interaction with the AR. The mean log IC50s for the strong 

positive control R1881 and the weak positive control (dexamethasone) were -9.0 and -4.4 M, 

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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 [3H(N)]-androst-4-ene-3,17-dione; [3H]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). Three runs were conducted and each run included a full 

activity control, a background activity control, a positive control series (10–10 to 10–5) with a 

known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), 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 IC50, and coefficient of variation for replicates of each concentration, with some minor 

deviations for Run #1. All performance criteria were met in Run #2 and #3, with the mean 

performance criteria values within 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 IC50 values across the three 

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independent runs did not differ significantly. The mean goodness of fit (R2) value was 0.99, and 

the mean IC50 of the test material was –6.6 M. The average dose-response curve indicated that 

the aromatase activity of the test material at concentrations ranging from 10−10 M to 10−8 M was 

essentially equivalent to the activity observed in the full activity controls. At myclobutanil 

concentrations ≥10–7 M, aromatase activity was substantially inhibited. Aromatase activity was 

inhibited by 34, 75 and 96% at myclobutanil 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) Binding Assay; (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48618104), uterine cytosol from Sprague 

Dawley rats was used as the source of ER to evaluate the potential for myclobutanil to displace 

the bound reference estrogen, radiolabeled [3

H]-17β-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. Saturation binding data were not presented in the study 

report; however, saturation binding data were presented in the study profile submitted separately 

(MRID 48672404). The competitive binding experiment was conducted to measure the binding 

of a single concentration of [3

H]-17β-estradiol (1 nM) in the presence 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 17β-estradiol as the natural ligand reference 

material. 

In the saturation binding experiment, the maximum binding capacity (Bmax) was 59.28 fmol/100 

μg protein and the dissociation constant (Kd) was 0.1032 nM. The Bmax fell within the expected 

range of 10 to 150 fmol/100 μg protein and the Kd for the run was within the expected range of 

0.03 to 1.5 nM. Nonspecific binding as a percent of total binding was 1.7%-8.6% across the 

entire concentration range in the saturation binding 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, myclobutanil 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 IC50 and RBA were not calculated for myclobutanil because it did not show competitive 

binding. 

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The performance criteria were met for the competitive binding experiment and the reference 

compounds generally performed as expected. The negative control, octyltriethoxysilane, had no 

effect on binding, and there were concentration-dependent decreases in labeled estradiol binding 

for the natural ligand and the weak positive control. The mean IC50 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%, compared to the natural ligand. The mean Hill 

Slope for the natural ligand was −0.9792, indicating that increasing concentrations of unlabeled 

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

Based on the results of the three runs, myclobutanil is classified as Not Interactive in the Estrogen 

Receptor Binding Assay. 

ERα Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300)

In an estrogen receptor transcriptional activation assay (MRID 48618105), hERα-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 triplicate (3 wells/plate) in each run. Cells were 

exposed to the test agent for approximately 24 hours to induce reporter (luciferase) gene products. 

Luciferase expression in response to activation 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 performance criteria for all of 

the four reference chemicals, but these deviations did not negatively affect the interpretation of 

the study. The log PC50 and log PC10 for 17α-methyltestosterone were lower than the acceptable 

values, but this indicates increased sensitivity of the cells to weak agonists. 

The RPCMax was 6.4% for the first run, 2.8% for the second run and 7.3% for the third run, with 

an associated PCMax of 10−5

 M for all three runs. Because the RPCMax < PC10 in at least two assay 

runs, myclobutanil was considered negative for estrogen receptor transcriptional activation in this 

test system. 

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

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The 21-day short-term reproduction assay (MRID 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 concentrations of 0 (control), 0.032 0.32, and 3.2 mg 

a.i./L; time-weighted average (TWA)-measured 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 effects on mortality (p>0.05) and while effects on secondary sex 

characteristics (fat pad development) and clinical signs (including missing caudal fin) were 

observed, they did not appear to be treatment-related. At the high treatment level, a significant 

reduction (9%; p=0.022) in female body weight was observed. 

Spawning in the negative control occurred at least every 4 days and fecundity ranged from 21 to 

33 eggs/female/day/replicate. Fecundity was 25.6, 20.1, 22.6 and 0.550 eggs/female/day/replicate 

in the control, low, mid, and high treatment levels, 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 fertilization success. Fertilization rate in the 

control was 99%, compared to 99.5, 99.1 and 100% in the 

low, mid, and high treatment groups. Female gonado-somatic index (GSI) exhibited a significant 

(Jonckheere-Terpstra; p<0.05) increase of 41% and 46% at the mid and high treatment levels, 

while male GSI was significantly (Jonckheere-Terpstra; p<0.001) increased 79% at the high 

treatment level. There were no significant differences 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 significant 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 17β-estradiol concentrations at the high treatment level was observed. 

Histopathology on female gonads reported severe oocyte atresia at the high treatment level 

relative to the control. Male fish gonads were reported as having a higher incidence of increased 

proportions of mature sperm (gonadal stage 3) in the high treatment group, relative to the control. 

Hershberger Assay; (890.1400)

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In a Hershberger assay (MRID 48618107) testing for androgenic activity, 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. The study included an androgenic positive control 

group of seven castrated male rats dosed daily with 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 positive control group consisted 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 consumption were measured; serum hormone 

concentrations were not determined. Animals were euthanized approximately 24 hours after the 

final dose administration. The five androgen-dependent tissues, adrenals, 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 antagonist assays on mortality, 

clinical signs of toxicity, body weights, body weight gains, food consumption, adrenals and 

kidneys weights, or gross pathology. The increased liver weights and microscopic liver findings at 

500 mg/kg/day indicated that the animals were dosed to a level that showed some toxicological 

effects. 

For the androgenic portion of the study, there were no statistically significant increases in the 

weights of accessory sex tissue at any dose of myclobutanil. For the TP positive control group, 

there were significant (p<0.05) increases in the weights of the Cowper’s glands (688%), glans 

penis (77%), levator ani-bulbocavernosus (LABC; 194%), ventral prostate (917%), and seminal 

vesicles with coagulating glands (1105%), indicating that the test system was sensitive to an 

androgenic response. 

For the anti-androgenic portion of the study, there was a statistically 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 prostate, Cowper’s gland or glans penis 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%), ventral prostate (75%), and seminal vesicles with 

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coagulating glands (81%) relative to weights in the “TP only” treated group. These data indicate 

that the test system was sensitive to an anti-androgenic response. 

Liver weights were increased by myclobutanil treatment in both the androgenic and antiandrogenic portions of the study. For the androgenic 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 increases correlated with histopathologic lesions in 

the liver, which included hypertrophy, 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 were 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 androgenicity and anti-androgenicity in the Hershberger 

assay. 

Female Pubertal Assay; (890.1450) 

In a Female Pubertal Assay (MRID 48618108), 16 Sprague 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 beginning on PND 22, and age and weight at day of attainment was 

recorded. Following sacrifice on PND 42, total thyroxine (T4) and thyroid stimulating hormone 

(TSH) levels were determined using chemiluminescent immunoassay and radioimmunoassay, 

respectively. At termination, weights were determined for liver, kidneys, thyroid, adrenals, 

pituitary, and urogenital organs. Microscopic examinations were performed on the ovaries, 

uterus, thyroid, and kidneys from the control and high-dose groups and on livers from all dose 

groups. 

There were no effects of treatment on mortality, clinical signs of toxicity, body weights, body 

weight gains, age of attainment of VO, body weight at VO, mean age at first estrus, mean cycle 

length, percent cycling, percent regular cycling, organ weights, serum T4 and TSH levels, or gross 

pathology. 

At 400 mg/kg/day, treatment-related increases (p<0.05) were observed in ALT (↑24%) and GGT 

(↑130%). Dose-dependent increases (p<0.05) in absolute, 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 very slight to slight 

hypertrophy with altered tinctorial properties (increased cytoplasmic eosinophilia) of centrilobular 

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and midzonal hepatocytes, very slight multifocal chronic inflammation, and very slight to slight 

increased number of mitotic figures of hepatocytes. These findings showed a dose-dependent 

increase in incidence and severity, and were indicative of hepatotoxicity 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 hepatic 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 Sprague-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) daily beginning on PND 30, and age and weight at 

day of attainment was recorded. Following sacrifice on PND 53, total serum testosterone, 

thyroxine (T4), and thyroid stimulating hormone (TSH) levels were analyzed using 

radioimmunoassays (TSH) or chemiluminescent assays (testosterone and T4). Liver, kidney, 

pituitary, adrenals, thyroid, and urogenital organ weights were recorded, and microscopic 

examinations of the thyroid, testes, epididymides, 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 weight, body weight grain, weight at attainment of PPS, or serum 

T4 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 (unadjusted and adjusted) and 

relative (to body) liver weight, which were dose-dependent. Liver weights 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 changes at 200 and 400 mg/kg/day, which consisted of 

slight to very slight hypertrophy with altered tinctorial properties 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 hepatocytes (2/15 control, 9/15 at 400 mg/kg/day). A 

significant increase (53%) was also noted in gamma glutamyl transferase at the high dose. 

Other significant changes in organ weights included a 15% increase in relative adrenal weight at 

400 mg/kg/day, and 8-10% decreases in pituitary weight at all dose levels that were not dosedependent. No significant weight 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 statistically significant (NS). In addition, dose-dependent increases 

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in serum TSH levels of 23%, 28%, and 63% were noted at 50, 200, and 400 mg/kg/day, 

respectively, although these increases were not significant. No changes were noted in serum T4 

levels. 

Significant, dose-dependent decreases were also observed in the weights 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 were noted in the seminal vesicle + coagulating gland (↓15-19%) 

and the ventral prostate (↓16%). At 400 mg/kg/day, weight decreases were noted in the seminal 

vesicle + coagulating gland (↓33-41%), ventral prostate (↓27-29%), dorsolateral prostate (↓25-

27%), and LABC complex (↓16-18%). Concomitant with the weight decreases in these androgen 

sensitive tissues, there were significant, dose-dependent decreases in serum testosterone levels of 

49% at 200 mg/kg/day and 85% at 400 mg/kg/day. 

Other significant changes in clinical 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, there 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 adequate based on the results from 

a range finding assay that showed increased liver weights and microscopic liver findings at a 

myclobutanil dose of 500 mg/kg/day. 

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (MRID 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 triplicate in three independent experiments. Dimethyl sulfoxide 

(DMSO) was used as a vehicle, at a final concentration of 0.1%. 

Testosterone and estradiol levels were measured using LC-APPI-MS/MS. A Quality Control 

(QC) plate was run concurrently with each independent run of a test chemical plate to 

demonstrate that the assay responded properly to positive control agents at two concentration 

levels. The positive controls included a known inhibitor (prochloraz) and a known inducer 

(forskolin) of estradiol and testosterone production. 

Guideline acceptability recommendations and requirements were generally met, including lack of 

cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low 

%CV), and appropriate induction and inhibition with positive controls, with 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 estradiol level in the SC of Run 1 was 35.5 pg/mL 

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(less than the guideline limit of 40 pg/mL); and (iii) the inhibition of estradiol 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 solvent control values in all 3 

runs at 10−4 M myclobutanil. In Run 2, there were significant 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 the three independent runs, myclobutanil treatment 

resulted in statistically significant and reproducible decreases in testosterone and estradiol 

production. 

Uterotrophic Assay; (OCSPP 890.1600)

In a uterotrophic assay (MRID 48618111) conducted to screen for potential 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 17α-ethynyl estradiol (EE) at 10 μg/kg/day by oral gavage. Body weights were 

determined daily. All animals were examined for precocious vaginal opening, and then 

terminated and necropsied on PND 22 approximately 24 hours after the final dose to determine 

wet and blotted uterine weights. 

All animals survived until scheduled termination. No clinical signs 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 (Days 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-dose 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. 

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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 IC50 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 results of this assay with other methods. Myclobutanil, tested in the 

DBF assay as noted above had an IC50 of 0.47 μM compared with 47 μM in the testosterone / LSMS/MS assay. The two order of magnitude difference in IC50 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 carrying 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 μg/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 results in this assay provided information on the ability of myclobutanil to 

interact with the estrogen receptor and activate 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 diets 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 depressed at 3,000 ppm and 

above in the male groups and at 10,000 ppm and above in the female groups. There was no 

correlation of growth to food consumption in the 3,000-ppm male groups, but there was body 

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weight suppression that correlated with reduced food consumption in the 10,000- and 30,000-ppm 

groups of both sexes. In the 10,000-ppm group, the 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 sexes and in 3,000 and 10,000 ppm groups. The hemosiderosis was more 

severe in the high dose males. Nevertheless, the red 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 kidney. Mixed function oxidase activity 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 prominent architecture at 1,000 ppm 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 occurred in the 1,000-ppm female group. Increased relative kidney weights 

were present in the 3,000 and 10,000 ppm doses in both sexes. Many of the other relative weight 

values were a result of decreased terminal body weights and are not toxicologically significant. 

Histopathological changes in the liver consisted of hepatocellular necrosis 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 was seen in the male thyroids at 3,000 ppm and 

above. 

In 1994, the EPA concluded in an RfD/Peer Review Report of Myclobutanil that the NOAEL for 

this study was 1000 ppm based on increased liver and kidney weights; hypertrophy and necrosis 

in the liver; pigmentation in convoluted kidney tubules; and vacuolation in the adrenal cortex at 

3000 ppm. 

90-Day (Subchronic) Oral Toxicity 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 reduction 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 liver. Absolute and relative liver weights were 

significantly increased in both sexes at ≥1000 ppm, and gross enlargement of the liver was noted 

in both sexes at ≥3000 ppm. Microscopic changes in the liver were seen in males at ≥1000 ppm 

and in females at ≥3000 ppm. These observations included centrilobular and midzonal 

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hepatocytic hypertrophy, swollen-vacuolated centrilobular hepatocytes, single large hepatocytic 

vacuoles, centrilobular hepatocytic necrosis, and centrilobular necrotic hepatitis. 

No treatment related changes in weight or histology were reported for the testes, ovaries, 

parathyroids or thyroid. No weight changes were noted for the adrenals, but cytoplasmic 

eosinophilia and/or hypertrophy of the zona fasciculata 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 effects, 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/day 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 cervical ribs was observed at 312.6 and 468.9 mg/kg/day. No other 

developmental effects were observed at any dose level tested. The developmental toxicity 

LOAEL was 312.6 mg/kg/day and the developmental 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 Non-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 Zealand White rabbits (18/group) received 0 

(water control), 0 (Hi-Sil control) 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 gestation. The test material was prepared with the 

solid adsorbent (carrier) Hi-Sil 233 and with 1.0% methylcellulose (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) with a rebound during the 

post-dosing period. Other effects included irregular feces, bloody urine, bloody urogenital or anal 

area and blood and/or aborted material in the drop 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, clinical signs of toxicity and possibly abortions. The maternal 

toxicity NOAEL was 60 mg/kg/day. 

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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/litter, decreases in litter 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 toxicity 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 ppm in the diet throughout the study (0, 2.5, 

10 or 50 mg/kg/day by standard conversion factors). The animals 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 was made when the pups were 25 days of age, and 

the mated animals in the study were approximately 81days of age at mating. 

At 200 ppm, centrilobular hepatocellular hypertrophy was observed in the F1 males. This was 

supported by slight but statistically significant increases in absolute 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 generations. Slight but statistically significant increases 

in absolute and relative liver weights in both F0 and F1 males (107–114%) and females 

(106-109%). The parental (systemic) toxicity LOAEL is 200 ppm and the parental (systemic) 

toxicity NOAEL is 50 ppm based on hepatocellular hypertrophy and increases in liver weights. 

At 1000 ppm, an increase in the number of stillborn 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 prostate 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 incidence in the number of stillborns and atrophy of the testes and 

prostate. At 1000 ppm, there was an apparent decrease in pup weight gain during lactation (83.3% 

to 89.7% of the controls). The developmental 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/Carcinogenicity in Rodents (Rat); (OCSPP 870.4300)

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

42809101), Sprague-Dawley rats were administered technical myclobutanil. The second study 

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was conducted because the high dose in the first study 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 female rats at concentrations of 0, 25/35/50, 

100/140/200 or 400/560/800 ppm in the diet. 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 months, and 18 males/dose and 10 females/dose were 

sacrificed at 17 months. A sentinel animal program was also conducted separately in which 30 

untreated rats/sex were evaluated at 3, 6 and 12 months. Study evaluations included clinical 

observations and mortality, body weight, food and water consumption, ophthalmological 

examinations, hematology, clinical chemistry, hepatic mixed function oxidase (MFO) and 

peroxisomal beta-oxidation activities, urinalysis, gross necropsy and histopathology. 

Testicular toxicity was observed in the middle and high dose males. Testicular 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) testicular weights (75 and 79% of 

controls at termination) at 39.21 mg/kg/day. The incidences of testicular atrophy were increased 

at 9.84 and 39.21 mg/kg/day; total incidences of bilateral testicular 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 months (13%) and 6 months (20%), respectively. 

With the exception of the testicular lesions, non-neoplastic histologic findings were incidental to 

treatment. 

Based on testicular atrophy, the LOAEL for systemic 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/carcinogenicity study (MRID 42809101, 1993), technical 

myclobutanil (92.9%) was administered to groups 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 animals and sacrificed at 

52 weeks. Evaluations included clinical observations and mortality, body weight, food and water 

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consumption, hematology, gross necropsy and histopathology. Clinical chemistry and urine 

analyses were not performed. 

Non-neoplastic effects were observed in the treated rats in this second 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 centrilobular to midzonal hepatocellular 

enlargement and vacuolization were observed in both sexes at the 52-week and terminal 

sacrifices. Absolute and relative testes weights were significantly lower in treated males at both 

the interim sacrifice (↓32–42% in testes) and terminal sacrifice (↓15–27%). Histopathological 

testicular changes included increase incidences of bilateral aspermatogenesis, hypospermia and 

cellular debris in the epididymides and, in rats that died or were sacrificed in extremis, 

arteritis/periarteritis. There 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 aspermatogenesis and hepatocellular enlargement. A 

NOAEL for systemic toxicity was not established. 

Carcinogenicity in Rodents (Mouse); (OCSPP 870.4200)

In two feeding/carcinogenicity studies (MRID 00164990 and 42809102), Crl:CD®-1 (ICR) BR 

mice were administered technical myclobutanil. 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 myclobutanil (90.4%) was administered to 110 

male and 110 female Crl:CD®-1(ICR)BR mice in the diet for a period of 24 months at dose levels 

of 0, 20, 100 or 500 ppm. The overall calculated 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 animal program was also conducted 

separately in which 25 animals/sex were evaluated at 24 months. 

At 100 ppm, an increase in hepatic mixed-function oxidase (MFO) was observed at 3 months in 

females (by 29% per mg protein, 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 above control per total liver) and 

in females at 12 months (266% above control per total liver). An increase in SGPT values was 

observed in females at 3 months (59% above controls) and an increase 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-related changes were observed microscopically 

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after 3, 6 and 12 months of treatment in males: increased incidence and severity of centrilobular 

hepatocytic hypertrophy, Kupffer cell pigmentation, periportal punctate vacuolation and 

individual hepatocellular necrosis. Following 24 months of treatment, increased incidences of 

focal hepatocellular alterations and multifocal hepatocellular vacuolation were observed in both 

sexes. These were not associated with any hypertrophy, 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 review, the increase in MFO at 3 months in 

females was not considered to be a significant enough effect to establish an LOAEL. Therefore, 

the LOAEL was raised to 500 ppm (70.2 mg/kg/day for males and 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 incidence and severity of 

centrilobular hepatocytic hypertrophy, Kupffer cell pigmentation, periportal punctate vacuolation 

and individual hepatocellular necrosis in males, and increased incidences of focal hepatocellular 

alterations and multifocal hepatocellular 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 group were sacrificed at 52 weeks. An additional 

20 animals were added to the study as sentinels and also sacrificed at 52 weeks. This study was 

conducted at the request of the HED Carcinogenicity Peer Review Committee because it was 

determined that the previous study (reviewed above) was not conducted at sufficiently high dose 

levels. 

At 2000 ppm, there were statistically significant decreases in body weight (2–12%) and body 

weight gain (12–26%) and increases in liver weights (22.9–33.2%). Related histopathological 

findings include hepatocellular hypertrophy; hepatocellular vacuolation; necrosis of single 

hypertrophied hepatocytes; yellow-brown pigment in the Kupffer cells and cytoplasmic 

eosinophilia and hypertrophy of the cells of the zona fasciculata area 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)

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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 observed in both sexes. Increased liver weights (131% absolute, 

128% relative) were observed in females. At 1600 ppm, hepatocellular hypertrophy in both sexes 

and “ballooned” centrilobular hepatocytes were observed in females. In addition, increased 

absolute and relative liver weights were observed in males (130–144%) and females (152–154%), 

along with increased alkaline phosphatase (160–243% 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 evaluate the disruption of testosterone 

homeostasis as a potential mode of action for the reproductive toxicity of three 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 day (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 significantly (p<0.001) increased at the high 

dose (2000 ppm). Relative liver weights were 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 prostate weights and testosterone levels 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 the 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 changes in the male rat liver and testis following 

exposure to myclobutanil, propiconazole, or triadimefon from gestational day (GD) 6 through 

postnatal day (PND) 92. 

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The study authors concluded that numerous common gene transcripts and biological pathways 

were identified for triazoles in the liver, defining common biological processes modulated by all 

three triazoles and supporting the interpretation of a common mode 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 was concluded that the genomic data demonstrate 

triazole disruption of pathways of key biological functions in the liver, including energy 

homeostasis, biological membrane fluidity, and CYP and other metabolic activities. These 

pathways are critical to liver-mediated steroid homeostasis, and the authors proposed that it is the 

perturbation of these critical pathways that leads to the observed reproductive 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 myclobutanil, 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, consistent, and conserved gene expression 

changes for characterization of the modes 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 involved in early fatty acid catabolism, and to a 

lesser degree on Phase III transporters 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 lipogenesis 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 concluded that the results from these genomic 

analyses provide new leads for understanding the modes of action responsible for triazole 

toxicities. 

Avian Toxicity and Reproduction (OCSPP 850.2200/850.2300)

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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 group. 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 observed 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% relative humidity. Weekly, the eggs were placed in an 

incubator. Prior to incubation, all 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 average body weight of 

representative hatchlings determined. Hatchlings were housed in battery brooders, and weighed at 

14 days old. The following reproductive endpoints were determined: eggs laid, eggs cracked, 

eggs set, viable embryos, live 3-week embryos, normal 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 deaths appeared to be treatment 

related. Gross necropsies did not reveal any consistent 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 significant 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% acetone) 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 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 observed 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% relative 

humidity. Weekly, the eggs were placed in an incubator. Prior to incubation, all eggs were 

candled to detect cracks; on Day 12 of incubation, 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 endpoints 

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were determined: eggs laid, eggs cracked, eggs set, viable embryos, live 3-week embryos, normal 

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 deaths appeared to be treatment 

related. Gross necropsies did not reveal any consistent 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 significant effects on any reproductive endpoints, 

except body weight of 14-day old hatchlings. Hatchling 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 endpoint up to and including the highest 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 replicates, 60 embryos per concentration) to 

myclobutanil (91.9%) at nominal concentrations of 0.5, 1, 2, 4, and 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 measurements 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 mean total length compared 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. 

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MYCLOBUTANIL FINAL 

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APPENDIX 3: References Not Utilized in the Myclobutanil WoE Analysis 

In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing 

Test Orders for the EDSP Tier 1 screening battery was made available in a Federal Register 

Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted 

as “other scientifically relevant 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 registration/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 additional relevant information for their weight of evidence (WoE) 

evaluations. These articles were evaluated in accordance with the agencies Evaluation Guidelines 

for Ecological Toxicity Data in Open Literature, May 2011 

(http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species_r

eregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance for 

considering and Using Open Literature Toxicity Studies to Support Human Health Risk 

Assessment (http://www.epa.gov/pesticides/science/lit-studies.pdf). 

The following published and unpublished references were considered for use in the WoE analysis 

for Myclobutanil but were not utilized due to one or more of the following reasons: 1) the article 

was not available in English; 2) the compound of interest was not used in the study; 3) the test 

material was not adequately described; 4) a formulated end-use product or mixture of chemicals 

was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the 

experimental conditions were not adequately described; 7) only an abstract of the study was 

available; 8) the reference is a review article or book chapter and does not contain primary study 

data; 9) insufficient information was available to adequately assess the validity of the study 

results; 10) the 40 CFR Part 158 guideline study was 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) the study contained only data on invertebrates. 

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