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 69
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 

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EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: TRIADIMEFON 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

 

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ii 

TABLE OF CONTENTS 

Abbreviations..................................................................................................iii 

Executive Summary..........................................................................................1 

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

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

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

B. Other Scientifically 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 .....................25 

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

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

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

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

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

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

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

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

LIST OF TABLES: 

 Table 1. Tier 1 Assay for Triadimefon................................................................8 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Triadimefon.................................11 

 Table 3. Androgenic/Anti-Androgenic Pathway for Triadimefon................................17 

 Table 4. Thyroid Pathway for Triadimefon.........................................................23 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................35 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................41 

APPENDIX 3. References Not Utilized in the Triadimefon WoE Analysis..........................61 

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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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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 triadimefon 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 endocrine-related responses 

occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity. 

On February 26, 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 triadimefon with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE 

evaluation in this report are presented by pathway (E, A and then T) beginning with the results of 

the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of 

the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature). 

For the estrogen pathway, the in vitro assays for triadimefon were negative for ER binding, but 

was positive in the ERTA and were positive for alteration of steroidogenesis (inhibition of 

aromatase activity and ↑estradiol production). In the OSRI studies, which satisfied the in vivo

mammalian assays, no estrogen-mediate effects were seen in the absence of systemic toxicity. In 

the mammalian OSRI, when estrogen-related effects were seen they occurred only in the 

presence of systemic/overt toxicity (e.g., body weight loss, decreases in overall body weight gain 

and hepatotoxicity), which significantly confounds the interpretation of these estrogenic effects 

within the context of endocrine disruption. For example, in the modified one-generation 

reproduction toxicity study in females, the delay in VO and disruption in estrus cyclicity was 

accompanied by decreased terminal body weight and increased liver weight. In the twogeneration reproduction toxicity study, decreased fertility was noted along with decreased body 

weights in the P and F1 parents, increased liver weights in the P generation and decreased birth 

and lactation body weights and viability in the F1 and F2 pups. In a bobwhite quail reproduction 

study, at the mid and high treatments (100 and 500 mg/kg-diet), there was an increase in number 

of cracked eggs (but no effects on eggshell thickness) and effects on egg viability and offspring 

survival; no effects were observed in the mallard duck study. 

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For the FSTRA, there was significant decreases in fecundity at the mid (39%; 0.24 mg a.i./L) and 

high (46%; 0.97 mg a.i./L) concentrations, and a significant increase in GSI (49%) at the high 

concentration. However, there were no effects on gonadal histology or plasma VTG in females. 

While there were no clinical signs of toxicity or significant effects on survival noted in the 

FSTRA study for females (male body weight was considered to be significantly decreased at the 

mid and high concentration), test concentrations in this test were similar to concentrations in 

rainbow trout and fathead minnow early life cycle tests where significant effects on survival 

were reported (fathead minnow: effects at 0.57 and 1.1 mg a.i./L; rainbow trout: effects at 0.89 

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

≥0.1 mg/L. Therefore, while distinct overt toxicity was not observed in the FSTRA for females, 

given that overt toxicity has been observed in other fish assays at similar concentrations and 

male body weight in the FSTRA was considered to be significantly decreased, overall, the fish in 

the FSTRA may have been experiencing some stress which may have resulted in decreased 

resources (e.g., energy) for input into reproductive status. In summary, given that the effects in 

the mammalian studies coincided with systemic/overt toxicity and there is a general lack of 

complementarity for the FSTRA along with concerns about potential stress impacting 

reproduction, there is a lack of convincing evidence to suggest the triadimefon is potentially 

interacting with the estrogen pathway. 

For the androgen pathway, the results of the Tier 1 in vitro AR binding were negative, although 

alterations in expression of genes for steroid homeostasis were observed. In the in vivo

mammalian assays, no androgenic or anti-androgenic effects were seen in the Hershberger assay. 

In the OSRI studies which satisfied the male pubertal assay, androgen-related effects were seen 

only in the presence of overt or systemic toxicity (e.g., body weight loss, decreases in overall 

body weight gain and hepatotoxicity). For example, in the modified one-generation reproduction 

toxicity study in males, the observed anti-androgenic effects occurred in conjunction with 

decreased terminal body weights and increased liver weights. In the FSTRA for males, there was 

a significant increase in GSI concentrations at the mid and high concentrations along with a 

reported decrease in fat pad development in the high treatment. There were no effects on male 

gonadal histology. There was a decrease in male body weight at both of these test concentrations 

that was considered to be biologically significant; along with the discussion above regarding test 

concentrations, this confounded the ability to discern a potential androgen-related interaction 

from an overtly/systemically toxic dose. Therefore, overall, there was a lack of convincing 

evidence for a potential interaction with the androgen pathway. 

For the thyroid pathway, in the mammalian OSRI studies which satisfied the male and female 

pubertal and the AMA Tier 1 testing requirements, no thyroid-related effects were seen in the 

absence of systemic toxicity. No changes in thyroid weights were seen in any of the studies. 

Decrease in the circulating levels of thyroid hormones was seen only in the presence of systemic 

toxicity or overt toxicity. For example, in the one-generation mammalian reproduction study in 

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males, there was a decrease in T4 levels in the presence of overt toxicity. In a subchronic 

mammalian study, decreases in T4 and TSH were seen in the presence of systemic toxicity (Wolf 

et al. 2006). Treatment-related histopathological lesions of the thyroid glands were seen in 

multiple species only at the high dose in the presence of systemic toxicity included follicular cell 

proliferations, follicular cell hyperplasia, colloid depletion and follicular cell adenomas. For the 

wildlife, effects on T3 and T4 levels (whole body) were reported along with alterations in gene 

expression for thyroid in newly-hatched larval zebrafish, however, these effects were only 

observed in the presence of systemic toxicity (bent spines and uninflated swim bladders). In an 

organ morphogenesis study (Lenkowski et al., 2010) using Xenopus laevis tadpoles, mortality 

was increased at ≥36.5 mg/L and intestinal and muscle malformations were observed. However, 

this study is limited in its ability to inform potential thyroid interaction based on endpoints 

examined and age of tadpoles (thyroid pathway is still relatively inactive at this stage). 

Therefore it is possible that the interaction of triadimefon with the thyroid hormone system may 

occur only at high doses as a sequel to either primary effects on the liver (i.e., increased hepatic 

clearance of thyroid hormones due to induction of hepatic enzymes PROD and UDP-GT, Wolf et 

al. 2006) or in the presence of systemic/overt toxicity (i.e., body weight/body weight gain 

decreases, spine deformities). 

In summary, the WoE indicates that in mammals, triadimefon may affect circulating levels of 

steroid and/or thyroid hormones only in the presence of systemic toxicity. The available data 

shows that the observed effects are due to increased metabolic activity of the liver, and not direct 

actions on the HPG or HPT axes. In wildlife, effects were also generally observed in the 

presence of systemic/overt toxicity (i.e., body weight decreases, skeletal malformations). 

Therefore, overall, there is a lack of convincing evidence to suggest a potential interaction with 

the E, A or T pathways. 

Based on weight of evidence considerations, mammalian and wildlife EDSP Tier 2 testing are 

not recommended since there was a lack of convincing evidence of potential interaction with the 

estrogen, androgen or thyroid pathways. 

. 

 

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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. 

On February 26, 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 triadimefon with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE 

evaluation in this report are presented by pathway (E, A and then T) beginning with the results of 

the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of 

the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature). 

Triadimefon has a solubility of 64 mg/L in water (20°C), and a vapor pressure of 0.2 – 0.6 mPa 

at 20°C and 25°C, respectively (7.6 × 10-7 mm Hg). Triadimefon is stable to hydrolysis and 

appears to undergo slow degradation in aerobic soils under field conditions. It is also moderately 

soluble and was detected below the soil surface in soil column leaching studies. 

The available information considered in determining the potential interaction of triadimefon with 

the E, A, or T pathways include submitted EDSP Tier 1 assays and/or other scientifically 

relevant information (OSRI) such as general 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. 

II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the 

agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix 

1. 

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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 O-PP (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. Additionally, literature/studies considered but not utilized for the WoE analysis are 

listed in Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

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

substance to interact with endocrine-related processes (i.e., E, A or T hormone pathways) were 

as described in the WoE guidance document (USEPA, 2011) and 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 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 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 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 include a battery of eleven 

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

designed specifically to evaluate a number of key biological events including potential effects on 

receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other 

effects on the HPG and HPT axes. OSRI may 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 consistency 

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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. The extent of expression of responses at higher levels of biological 

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

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

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

first component is whether the data provide 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-related effect from a non-endocrine toxic response. 

This uncertainty in distinguishing whether the responses were endocrine-related was discussed at 

the FIFRA Scientific Advisory Panel (SAP) meeting in July 2013 that evaluated scientific issues 

associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the 

SAP stated that , “In summary, the Panel agreed that little, if any, 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 

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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., changes in organ weights or blood 

chemistry). The circumstances for which a dose/concentration was considered overtly toxic for a 

particular study are described in Section IV.A. 

In summary, EPA considers multiple lines of evidence in including the observed responses in 

the Tier 1 assays and OSRI in the context of a chemical’s physical/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 main 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 considered: 1) uncertainties and their 

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

conflicting data; 4) overall strength of 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 caseby-case analysis which took all available toxicity data into account. 

The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 

assays and OSRI which are depicted in Tables 2 - 4. These tables contain data that are 

considered scientifically and biologically relevant with regard to a treatment-related effect 

which supports a conclusion of whether or not 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 Tier 1 Screening Assays 

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

below in Table 1.

Table 1. Tier 1 Assays for Triadimefon

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay 

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

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID No. 48619605) 

AR Binding Assay 

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

Steroidogenesis Assay 

(Human cell line H295R) 

OCSPP 890.1550; 

OECD 456 

Requirement Satisfied by OSRI 

(MRID No. 48264602) 

Aromatase Assay 

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

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement Satisfied by OSRI 

(MRID Nos. 48264605; 00032541; 

00155075) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement Satisfied by OSRI 

(MRID No. 48619604) 

Pubertal Female Assay (Rat) OCSPP 890.1450 

Requirement Satisfied by OSRI 

(MRID Nos. 48619605; 42153901; 

00032541; 00155075) 

Pubertal Male Assay (Rat) OCSPP 890.1500 

Requirement Satisfied by OSRI 

(MRID Nos. 48619603; 48264602; 

48264607; 00032541; 00155075) 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID No. 486673901) 

Amphibian Metamorphosis Assay 

(Frog) 

OCSPP 890.1100; 

OECD 231 

Requirement Satisfied by OSRI 

(MRID Nos. 48264601; 48264602; 

48264603; 48264604; 48264605; 

48264607; 00155075; 00032541; 

42153901) 

Page 16 of 69
TRIADIMEFON FINAL 

Page 9 of 61 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis 

1. Effects on Estrogen Pathway 

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

The various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as 

to facilitate determination of potential for estrogenic, anti-estrogenic or HPG axis effects. This 

table also includes HPG-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. 

Triadimefon was negative (or not interactive) in the Tier 1 in vitro ER binding assay. In contrast, 

in the Tier 1 ERTA, the mean RPCmax was ≥ PC10 in both runs at approximately 10-4 M, 

triadimefon was considered positive for estrogen receptor transcriptional activation. 

The requirement for the steroidogenesis assay was satisfied by an in vitro and in vivo rat 

steroidogenesis study submitted as OSRI (Goetz et al., 2009). In the in vitro portion of this study, 

H295R cells exposed to triadimefon for 48 hours had increased (approximately 180% of control) 

estradiol levels at 1 and 3 μM, but decreased estradiol levels at higher concentrations. In the in 

vivo portion of this study, male Wistar Han rats fed triadimefon at 126 mg/kg/day had increased 

(↑20%) serum estradiol levels after 30 days of treatment but was not statistically significant. The 

Tier 1 aromatase assay was satisfied by OSRI (aromatase assay validation study, USEPA, 2007). 

Results indicate triadimefon inhibited aromatase activity with a log IC50 of -5.767 M. 

In another study submitted as OSRI (Kojima et al., 2004), triadimefon was negative for estrogen 

receptor transcriptional activation in CHO cells. In a cell proliferation/ER binding assay (Okubo 

et al., 2004), triadimefon induced cell proliferation (maximum at 100 μM) in MCF-7 cells that 

could be blocked by the anti-estrogen ICI 182,780, but did not bind to the ER in a non-guideline 

binding assay. In an ER binding/ERTA/VTG induction study (Petit et al., 1997), triadimefon was 

negative in a trout ER binding assay and a yeast trout ER transcriptional activation assay, but 

positive in a trout hepatocyte VTG induction assay (178% induction at 10-4 M). Finally, in a cell 

proliferation/transcriptional activation study (Vinggaard et al., 1999), triadimefon caused a 2.4-

fold increase in cell proliferation in an MCF-7 cell assay measuring DNA content, but was 

negative in a yeast estrogen screening assay. 

The requirement for the uterotrophic and female pubertal assays were satisfied collectively by 

four studies submitted as OSRI: 1) a modified one-generation rat reproduction toxicity study 

(Rockett et al., 2006); 2) a three-generation reproduction toxicity study (MRID 00032541); 3) a 

two-generation reproduction toxicity study (MRID 00155075); and 4) a combined chronic 

toxicity/carcinogenicity study (MRID 42153901). 

Page 17 of 69
TRIADIMEFON FINAL 

Page 10 of 61 

In the modified one-generation rat reproduction toxicity study (Rockett et al., 2006), VO was 

delayed, estrus cyclicity was initially disrupted and ovarian weight was increased at termination 

on PND 99 in the high-dose (90 mg/kg/day) triadimefon group. However, numerical data were 

not reported in this literature study. No estrogen-specific effects were observed at the lower 

doses (5 and 25 mg/kg/day). In a three-generation reproduction toxicity study (MRID 

00032541), fertility was affected at the high dose (90 mg/kg/day) in all generations. 

Correspondingly, in the two-generation reproduction toxicity study (MRID 00155075), fertility 

was decreased in the high-dose (90/mg/kg/day) F1 parents. These decreases appear to be related 

to reduce mating interest and/or ability of male rats exposed to triadimefon as described in 

Section 2 below. While no estrogen-specific effects were observed in the three-generation study 

at lower doses (2.5 and 15 mg/kg/day), a significant decrease in ovarian weight was observed at 

the 2.5 mg/kg/day in the two-generation study. In the combined chronic toxicity/carcinogenicity 

study (MRID 42153901), no effects were noted on ovary weight or histopathology of the ovary 

or uterus at any dose (2.5, 15, and 90 mg/kg/day). 

In the FSTRA, fecundity was reduced by 39% and 46% at the mid (0.24 mg/L) and high 

(0.97 mg/L) concentrations, respectively, relative to the negative control. Female GSI was 

increased by 49% relative to the negative control at the high concentration. There were no effects 

on female VTG or gonadal histopathology. In a bobwhite quail reproduction study, at the mid 

and high treatments (100 and 500 mg/kg-diet), there was an increase in number of cracked eggs, 

decreases in the number of fertile eggs, viable embryos, hatchlings and 14-day survival; no 

effects on eggshell thickness was observed. The reproduction study with the mallard duck did 

not report any significant effects. In three early life stage studies (one with fathead minnow, two 

with rainbow trout), there were significant effects (p<0.05) on growth at concentrations as low as 

0.116 mg a.i/L (rainbow trout study). There were no significant effects determined on percent 

hatch for all three studies.

Page 18 of 69
TRIADIMEFON FINAL 

Page 11 of 61 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Triadimefon

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Triadimefon1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight / 

GSI

Ovarian/Gonad 

Staging and 

Histopathology

Pituitary Weight 

Estrous Cyclicity

2

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility/Fecundity 

Vitellogenin 

Systemic Toxicity 

Observed2 

Overt Toxicity 

Observed3 

EDSP Tier 1 

ER Binding (MRID 48619602) N ERTA (MRID 48619605) PAromatase Requirement satisfied by OSRI (MRID 48125203; see below) Steroidogenesis Requirement satisfied by OSRI (MRID 48264602; see below) Uterotrophic Requirement satisfied by OSRI (multiple studies; see below) Female Pubertal Rat Requirement satisfied by OSRI (multiple studies; see below) FSTRA (MRID 486673901) NE 

GSI 

↑49% 

(H) 

N N 

Fcd: 

↓39, 

46% 

(M, H) 

N N
TRIADIMEFON FINAL 

Page 12 of 61 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Triadimefon

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Triadimefon1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight / 

GSI

Ovarian/Gonad 

Staging and 

Histopathology

Pituitary Weight 

Estrous Cyclicity

2

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility/Fecundity 

Vitellogenin 

Systemic Toxicity 

Observed2 

Overt Toxicity 

Observed3 

OSRI 

Aromatase (USEPA, 2007; MRID 48125203) 

P

Steroidogenesis (Goetz et al., 2007; (MRID 48264602)

P P 

AR, ERα, and ERβtranscriptional activation assays (Kojima et al., 2004; MRID 48033008) 

N 

Cell proliferation, ER, and AR binding assays (Okobo et al., 2004; 

MRID 48074205) 

N P 

ER binding, ERTA, and vitellogenin induction assays (Petit et al., 1997) 

N N P 

Cell proliferation and transcriptional activation assays (Vinggaard et al., 1999; MRID 48041733) 

P P 
TRIADIMEFON FINAL 

Page 13 of 61 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Triadimefon

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Triadimefon1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight / 

GSI

Ovarian/Gonad 

Staging and 

Histopathology

Pituitary Weight 

Estrous Cyclicity

2

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility/Fecundity 

Vitellogenin 

Systemic Toxicity 

Observed2 

Overt Toxicity 

Observed3 

Modified one-generation reproduction toxicity (Rockett et al., 2006; 

MRID 48264605) 

 N ↑P(H) N P (H) ↑4d (H) N X (H) N 

Three-generation reproductive toxicity (Rat; MRID 00032541) 

 NE NE N N N ↓P (H) N N 

Two-generation reproductive toxicity (Rat; MRID 00155075) 

NE ↑12% (L) NE NE NE ↓P (H) X (M. H) N 

Combined chronic toxicity/carcinogenicity (Rat; MRID 42153901) 

NE N N NE NE X (H) N 

Developmental neurotoxicity (MRID 47377101) 

 N N X (H) N 

Developmental toxicity (Rat: MRID 00089023) N N 

X 

(M, 

MH, 

H) 

N 

Developmental toxicity (Rat; MRID 00149336) N N N N Developmental toxicity (Rabbit; MRID 41446201) 

 N N N N 

Subchronic toxicity (Rat; MRID 00048624) NE N N N N N 
TRIADIMEFON FINAL 

Page 14 of 61 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Triadimefon

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Triadimefon1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight / 

GSI

Ovarian/Gonad 

Staging and 

Histopathology

Pituitary Weight 

Estrous Cyclicity

2

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility/Fecundity 

Vitellogenin 

Systemic Toxicity 

Observed2 

Overt Toxicity 

Observed3 

Subchronic toxicity (Dog; MRID 00048625) N N N N N N N Carcinogenicity (Mouse; MRID 40752101) 

 NE NE N NE X (H) N 

Chronic toxicity (Dog; MRID 00032539) NE N N N X (H) N Avian reproductive toxicity (Quail; MRID 00110431) 

P 

(M, H)4 N N 

Avian reproductive toxicity (Duck; MRID 42342302) 

 N N N 

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

direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Cha

weight are absolute unless otherwise indicated. 

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

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 

column. For details see Section IV. A 

4. At the mid and high concentrations (100 and 500 mg/kg-diet), there was an increase in number of cracked eggs, and decreases in 

fertile eggs, viable embryos, hatchlings, and 14-day survival. 

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

N Negative findings (in vitro) / No effect (in vivo) 

NE Not examined 
TRIADIMEFON FINAL 

Page 15 of 61 

2. Effects on Androgen Pathway 

The potential for triadimefon 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. 

The Tier 1 in vitro AR binding assay was negative. The Tier 1 steroidogenesis assay was 

satisfied by an in vitro and in vivo rat steroidogenesis study submitted as OSRI (Goetz et al., 

2009). In vitro, triadimefon reduced media levels of testosterone by 40–68% in the adult and 

neonatal testis culture, and altered steroid production in a manner that indicated CYP17A1 

inhibition at the highest concentration. Following 48 hours of exposure, there was an overall 

decrease in testosterone at 10 and 30 M triadimefon in H295R cells. In vivo, triadimefon (126 

mg/kg/d) increased (↑288%; p <0.01) serum testosterone at 30 days and intra-testicular 

testosterone (↑210%; p<0.01). Significant (p<0.05) decreases were seen in absolute epididymis 

(↓5%) and pituitary (↓10%) weights; however, no changes were seen in the relative weights of 

these organs. No treatment-related changes were seen in the absolute or relative weights of the 

testes, seminal vesicle or ventral prostate. Liver weights, both absolute and relative, were 

increased (p<0.01) after 30 days of exposure (↑27% and ↑37%, respectively) and body weights 

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

In the Tier 1 Hershberger assay in the androgen agonist portion of the assay, ventral prostate 

weight was increased (29%, p<0.05) at the high dose (100 mg/kg/day) and in the androgen 

antagonism portion of the assay, LABC weight was decreased (12%) at the high dose. Since only 

one of the five androgen-sensitive tissue weights were altered in the agonism and antagonism 

portion of the assay, triadimefon is considered negative in the Hershberger assay. 

The requirement for the Tier 1 in vivo male pubertal assay was satisfied with five studies 

submitted as OSRI: 1) a modified one-generation rat reproduction toxicity study (Goetz et al., 

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

2009); 3) a gene expression study (Tully et al., 2006); 4) a three-generation reproduction toxicity 

study (MRID 00032541); and 5) a two-generation reproduction toxicity study (MRID 

00155075). A modified one-generation male rat reproduction toxicity study (Goetz et al., 2007), 

triadimefon was administered at 0, 5, 25, and 90 mg/kg/day. No androgen-related effects were 

observed at 5 or 25 mg/kg/day triadimefon. Administration of a high dose of triadimefon (90 

mg/kg/day) resulted in androgen-related effects in the F1 males (increased AGD, delayed PPS, 

decreases in accessory organs weights, increased testis weights) which co-occurred with overt 

toxicity (body weight ↓17-29%). 

Page 23 of 69
TRIADIMEFON FINAL 

Page 16 of 61 

In a study by Tully et al., (2006), adult rats were administered triadimefon at doses of 5, 50 or 

115 mg/kg/day via gavage for 14 days. No adverse, treatment-related effects were seen on sperm 

morphology or motility, testis or epididymis weights, or testis histopathology. Mean serum 

testosterone levels were elevated at the mid and high doses, but the increases did not reach 

statistical significance. Relative liver weights were increased by 27% at the high dose (115 

mg/kg/day) with no effect on body weights. At the mid dose (50 mg/kg/day), 3/5 rats were found 

with mild centrilobular hepatocyte hypertrophy, while high dose animals displayed mild 

centrilobular to panlobular hepatocyte hypertrophy, with 1/5 having moderate mid-zonal 

hepatocyte vacuolation. Gene expression profiles of the liver and testis were also evaluated. 

Triadimefon affected the expression of numerous genes including CYP genes and genes involved 

in steroid hormone metabolism. 

In the three-generation reproduction toxicity study (MRID 00032541), fertility was affected at 

the high dose (90 mg/kg/day) in all generations. In the two-generation reproduction toxicity 

study, fertility was decreased in the high-dose F1 parents. These decreases appear to be related to 

reduced mating interest or ability of male rats exposed to high doses of triadimefon rather than 

an effect on fertility/reproduction. This was further examined in the two-generation reproduction 

toxicity study by cross-mating control F1 parents with high-dose F1 parents (MRID 00155075). 

Control females mated with high-dose males displayed greatly decreased fertility compared to 

high-dose females mated with control males (47.4% vs. 80%). In the absence of sperm 

abnormalities, the decreased fertility was attributed to a decrease in interest or ability to mate in 

the high-dose males. 

In the FSTRA, male gonado-somatic index (GSI) exhibited a significant increase of 33 and 75% 

relative to the negative control at the mid (0.24 mg a.i./L) and high (0.97 mg a.i./L) 

concentrations, respectively. At study termination, fat pads of males were reported as poorly 

developed within three of the four replicate chambers at the high concentration (not statistically 

analyzed). No effects on male gonadal histology were observed. Additionally, there was a 

reduction of 21% and 19% in mean male body weight in the 0.27 and 0.97 mg a.i./L, 

respectively, relative to the negative control, which was not statistically significant but was 

considered to be biologically significant. However, effects on mortality or other clinical signs of 

toxicity were not observed. 

In a bobwhite quail reproduction study, at the mid and high treatments (100 and 500 mg/kg-diet), 

there was an increase in number of cracked eggs, decreases in the number of fertile eggs, viable 

embryos, hatchlings and 14-day survival. There were no effects observed in the reproduction 

study with the mallard duck. 

Page 24 of 69
TRIADIMEFON FINAL 

Page 17 of 61 

Table 3: Androgenic/Anti-Androgenic Pathway for Triadimefon

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Triadimefon1Study 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) 

Age and Weight at 

PPS 

EDSP Tier 1 

AR Binding (MRID 48619603) N Steroidogenesis Requirement satisfied by OSRI (multiple studies; see below) Male Pubertal Rat Requirement satisfied by OSRI (multiple studies; see below) Hershberger (MRID 48619604) N FSTRA (MRID 48673901) NE 

GSI 

↑33% 

(M), 

↑75% 

(H) 

N P (H)4 N 

OSRI 

Steroidogenesis (Goetz et al., 2007; MRID 48264602)

 P P N ↓5% ↓10% N 
TRIADIMEFON FINAL 

Page 18 of 61 

Table 3: Androgenic/Anti-Androgenic Pathway for Triadimefon

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Triadimefon1Study 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) 

Age and Weight at 

PPS 

AR, ERα, and ERβtranscriptional activation assays (Kojima et al., 2004; MRID 48033008) 

N 

Cell proliferation, ER, and AR binding assays (Okobo et al., 2004; 

MRID 48074205) 

P 

Gene expression study (Tully et al., 2006; 

MRID 48264607) 

 E6 N N N NE NE N N 

Gene expression (Goetz and Dix, 2009a and 2009b) 

 P 

Modified one-generation reproductive toxicity (Goetz et al., 2007; MRID 48264603) 

N N N N N N N N N 

Three-generation reproduction toxicity (Rat; MRID 00032541) 

 NE N N N N NE NE P (H) 
TRIADIMEFON FINAL 

Page 19 of 61 

Table 3: Androgenic/Anti-Androgenic Pathway for Triadimefon

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Triadimefon1Study 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) 

Age and Weight at 

PPS 

Two-generation reproduction toxicity (Rat; MRID 00155075) 

 NE N NE N NE NE NE P (H) 

Developmental neurotoxicity (MRID 47377101) 

N 

Subchronic toxicity (Rat; MRID 48264601) NE NE NE NE NE NE Subchronic toxicity (Rat; MRID 00048624) N N N N N N Subchronic toxicity (Dog; MRID 00048625) 

N N N N N N 

Combined chronic toxicity / carcinogenicity (Rat; MRID 42153901) 

NE 

Carcinogenicity (Mouse; MRID 40752101) N N NE N NE NE Chronic toxicity (Dog; MRID 00032539) 

N N NE N N N 

Avian reproductive toxicity (Quail; MRID 00110431) 

P 

(M, H)7 
TRIADIMEFON FINAL 

Page 20 of 61 

Table 3: Androgenic/Anti-Androgenic Pathway for Triadimefon

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Triadimefon1Study 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) 

Age and Weight at 

PPS 

Avian reproductive toxicity (Duck; MRID 42342302) 

 N 

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

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

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

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

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 

details see Section IV. A 

4. At study termination, fat pads of males were reported as poorly developed within three of the four replicate chambers at the high c

statistically analyzed). 

5. There was a reduction of 21% and 19% in mean male body weight in the 0.27 and 0.97 mg a.i./L, respectively, relative to the neg

not statistically significant but was considered to be biologically significant. Effects on mortality or clinical signs of toxicity were 

6. Serum testosterone were elevated at the mid- (50 mg/kg/day) and high-dose (115 mg/kg/day), but not significantly. Data was pre7. At the mid and high concentrations (100 and 500 mg/kg-diet), there was an increase in number of cracked eggs, and decreases in t

viable embryos, hatchlings, and 14-day survival. 

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

N Negative (in vitro) / No effect (in vivo) 

NE Not examined E Equivocal 
TRIADIMEFON FINAL 

Page 21 of 61 

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 triadimefon to interact with thyroid regulation is 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 the potential for thyroid or HPT axis effects. This table also includes 

HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of 

overt toxicity are discussed in the text but are not reported in the table and not considered 

further in the WOE assessment. 

The EDSP Tier 1 in vivo male and female pubertal assays and the AMA were not conducted but 

were satisfied with eight studies submitted as OSRI: 1) a modified one-generation rat 

reproduction toxicity study focusing on males (MRID 48264603); 2) a modified one-generation 

rat reproduction toxicity study focusing on females (MRID 48264605); 3) an in vitro and in vivo

rat steroidogenesis study (MRID 48264602); 4) a gene expression study (MRID 48264607); 

5) a three-generation rat reproduction toxicity study (MRID 00032541); 6) a two-generation rat 

reproduction toxicity study (MRID 00155075); 7) a combined chronic toxicity/carcinogenicity 

study in rats (MRID 42153901); and 8) a subchronic toxicity study in male rats (Wolf et al., 

2006). 

In the modified one-generation rat reproduction toxicity study with focus on males 

(MRID 48264603), total serum T4 levels were decreased by PND 92 by 25% at 90 mg/kg/day; 

however, T3, TSH levels, thyroid and pituitary weights were unaffected by treatment. In the 

modified one-generation rat reproduction toxicity study with focus on females (MRID 

48264605), total serum T4 and TSH levels were not measured, and thyroid pituitary weights were 

unaffected by treatment; however, 2/4 (50%) females had mild diffuse thyroid follicular cell 

hypertrophy at 90 mg/kg/day. In the in vivo portion of the rat steroidogenesis study (MRID 

48264602), pituitary weights were decreased after 30 days by 10% at 126 mg/kg/day of 

triadimefon in the diet, but thyroid weights, serum thyroid hormone levels and thyroid 

histopathology were not examined. In the three-generation rat reproduction toxicity study (MRID 

00032541), the thyroids of the F3b pups were examined microscopically and showed no effects of 

treatment at the low and mid doses (2.5 and 15 mg/kg/day); high-dose (90 mg/kg/day) pups were 

not examined due to reduced fertility of the previous generations. In the two-generation rat 

reproduction toxicity study (MRID 00155075), the thyroid and pituitary glands of the F1 parents 

and F2 pups were unaffected by triadimefon treatment. In the rat combined chronic 

toxicity/carcinogenicity study (MRID 42153901), treatment-related non-neoplastic and 

neoplastic lesions of the thyroid glands were seen at the high dose (90 mg/kg/day). Follicular cell 

Page 29 of 69
TRIADIMEFON FINAL 

Page 22 of 61 

cystic hyperplasia was noted in 4/50 (8%) high-dose females vs. 2/50 (2%) control females; 

however, the increase did not reach statistical significance. Also at the high dose, follicular cell 

adenomas were observed in 3/49 (6%) treated males and 2/50 (4%) females compared to 0/50 

(0%) control individuals of the respective sexes. The incidences of adenomas were increased in 

males (4/49; 8%) which showed a significant trend and exceeded the historical control mean 

(5.2%). In the rat subchronic toxicity study (Wolf et al, 2006; MRID 48264601), thyroid 

follicular cell hypertrophy, increased follicular cell proliferation and colloid depletion was noted 

at 30 days in rats treated with a high dose (90 mg/kg/day) of triadimefon. There was also a dosedependent decrease in T4 levels after 4 and 30 days for treatment, and T3 levels were decreased at 

the high dose of triadimefon after 4 and 30 days. Notably, thyroid hormone levels did not differ 

from control values after 90 days of exposure and TSH levels were not increased in any exposure 

group. 

In other OSRI evaluated to inform potential interaction with the thyroid pathway, there were no 

treatment-related findings affecting the thyroid or pituitary. 

In a study by Liu et al. (2011), zebrafish embryos were exposed from 6 h post-fertilization to 

120 hpf (hatched larvae). The zebrafish exposed to 2 and 4 μg/mL triadimefon exhibited bent 

spine and uninflated swim bladder (results not presented). The T3 content in the larvae (whole 

body) exposed to 2 and 4 μg/mL triadimefon was significantly less than the controls. In contrast, 

the larvae exposed to 2 and 4 μg/mL had significantly higher T4 content than in the controls. The 

beta-subunit of TSH (TSH-β) gene transcription was significantly upregulated (↑4.0- and 

↑5.2-fold) upon exposure to 2 and 4 μg/mL triadimefon, respectively; triadimefon exposure 

significantly downregulated dio1 gene transcription (1.5-fold) in the 2 μg/mL exposure group. 

The thrb gene transcription was significantly down-regulated (↓2.6-fold) after exposure to 

2 μg/mL triadimefon. Given the reported malformations, the reported results appear to have 

coincided with systemic toxicity. 

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Table 4. Thyroid Pathway for Triadimefon.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Triadimefon1

Study Type / Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

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

Overt Toxicity Observed3

EDSP Tier 1 Assays 

Male Pubertal Rat Requirement satisfied by OSRI (see below)

Female Pubertal Rat Requirement satisfied by OSRI (see below)

AMA (Frog) Requirement satisfied by OSRI (see below)

OSRI

Steroidogenesis 

(Goetz et al., 2007; 

MRID 48264602) 

NE NE ↓10%

(H) X N 

Modified one-generation 

reproduction toxicity focusing on 

males 

(Goetz et al., 2007; 

MRID 48264603) 

N N N N N X 

(H) 

X 

(H) 

Modified one-generation 

reproduction toxicity focusing on 

females 

(Rockett et al., 2006; 

MRID 48264605) 

N P N X 

(H) N 

Two-generation reproduction 

(Rat; MRID 00155075) NE N N X 

(H) N 

Three-generation reproduction 

(Rat; MRID 00032541) NE N NE X 

(M, H) N 

Developmental neurotoxicity 

(MRID 47377101) NE NE NE X 

(H) N 

Developmental toxicity 

(Rat: MRID 00089023) NE NE NE 

X 

(M, MH, 

H) 

N 

Developmental toxicity 

(Rat; MRID 00149336) NE NE NE N N 

Developmental toxicity 

(Rabbit; MRID 41446201) NE NE NE N N 

Subchronic toxicity 

(Rat; Wolf et al., 2006; 

MRID 48264601) 

NE P P P NE X 

(H) N 

Subchronic toxicity 

(Rat; MRID 00048624) N N N N N 

Subchronic toxicity 

(Dog; MRID 00048625) N N N N N 

Chronic toxicity 

(Dog; MRID 00032539) N N N X 

(H) N 

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Table 4. Thyroid Pathway for Triadimefon.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Triadimefon1

Study Type / Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

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

Overt Toxicity Observed3

Combined chronic 

toxicity/carcinogenicity 

(Rat; MRID 42153901) 

NE P NE X 

(M, H) N 

Carcinogenicity 

(Mouse; MRID 40752101) NE N NE X 

(H) N 

Development and gene expression 

(Zebrafish; Liu et al., 2011) NE NE ↑(MH, H)4 NE NE NE P X (MH, H) N 

1. Key to responses: L=Low treatment, M=Medium treatment, MH=Medium-High treatment, H=High treatment 

Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not 

routinely evaluated or is not applicable in this assay. 

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

toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI 

is indicated by an X in this column. For details see Section IV. A 

4. Zebrafish exposed to triadimefon at 2 and 4 μg/mL for as embryos through hatch exhibited elevated T4 levels, 

decreased T3 levels, and altered expression of genes involved in the thyroid pathway. Larvae at these levels 

also reported to exhibit bent spines and uninflated swim bladders. 

N Negative finding 

P Positive finding 

NE Not examined 

IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight of evidence (WoE) determination on the 

potential of triadimefon to interact with endocrine related processes (i.e., E, A or T hormonal 

pathways) as well as 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. 

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TRIADIMEFON FINAL 

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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 elicit 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 related through an endocrine pathway, therefore non-endocrine 

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

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

with weight of evidence 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 non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 and OSRI 

studies are described below. In addition to the endocrine-related effects described above for the 

in vivo Tier 1 assays and OSRI, 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., changes in organ weights or blood chemistry).

1. Tier 1 Assays 

In the androgen agonist phase of the Hershberger assay, body weight gains were decreased 

(p<0.05) by 44% in the high-dose group (100 mg/kg/day) compared to the vehicle control. Final 

body weights and body weight gains in the low-dose group were comparable to controls 

throughout the duration of the assay. 

In the anti-androgen phase of the Hershberger assay, final body weight was decreased (↓9%; 

p<0.05) in the high dose group (100 mg/kg/day) and overall body weight gains were decreased 

(p<0.05) in the 50 mg/kg/day (↓29%) and 100 mg/kg/day (↓44%) groups. Final body weights 

and body weight gains in the 25 mg/kg/day group were comparable to controls. 

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TRIADIMEFON FINAL 

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In the FSTRA, there were no significant effects on mortality and no apparent effects on clinical 

signs (i.e., behavioral and other sublethal effects) except for one dark fish observed at 

0.057 mg a.i./L. There was a reduction of 21% and 19% in mean male body weight in the 0.27 

and 0.97 mg a.i./L dose group, respectively, relative to the negative control, but the effect was 

not significant (Mann Whitney U; p>0.05); however, these decreases were considered to be 

biologically significant. 

2. OSRI 

In the modified one-generation rat reproduction toxicity study with focus on males (Goetz et al.,

2007), overt toxicity at the high dose (90 mg/kg/day) manifested as decreases in the body 

weights of F1 male offspring during PND 1–22 (↓17–27%) and weeks 4–13 (↓22–29%). Daily 

food consumption (g/rat/day) was decreased by 9–26% during Weeks 4–12. Relative liver 

weights were increased at PND 1, 50 and 92, but this was a result of the decreased body weights 

at these time points. Liver histology revealed strong panlobular hypertrophy in high-dose 

triadimefon males. 

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

et al., 2006), there was a decrease in body weight and increase in liver weight at the high dose 

(90 mg/kg/day); however, the authors did not report numerical data. 

In the in vivo portion of the rat steroidogenesis study, liver weights were increased 27% after 

30 days of exposure at 126 mg/kg/day. 

In the rat gene expression study, relative liver weights were increased by 27% at 115 mg/kg/day. 

In the same study, 3/5 (60%) rats were found with mild centrilobular hepatocyte hypertrophy at 

50 mg/kg/day, while animals dosed at 115 mg/kg/day displayed mild centrilobular to panlobular 

hepatocyte hypertrophy, with 1/5 (20%) having moderate mid-zonal hepatocyte vacuolation. 

In the two-generation rat reproduction toxicity study, at the high dose (90 mg/kg/day), body 

weights were decreased (p<0.05) in the P males during weeks 4, 5 and 6; in the P females during 

weeks 1–16 and 21–26; and in the F1 parental males and females during weeks 9–40. Terminal 

body weights were decreased (p<0.01) by 12% in the P females. Absolute and relative (to body 

weight) liver weights were increased (p<0.01) in the P males (↑23–29%) and females (↑23-39%). 

In the three-generation rat reproduction study, at 90 mg/kg/d, body weights of the P generation 

parents were decreased (↓15%; p<0.01) during F1b pre-mating, mating, gestatio, and lactation. 

Body weight gains of the F1 generation parental males and females were decreased (p<0.01) 

throughout pre-mating, gestation and lactation for both matings. 

In the rat combined chronic toxicity/carcinogenicity study, at 90 mg/kg/day females, survival 

was slightly decreased (NS) at weeks 78, 90, and 105 (↓9–14%). Decreases (p<0.05) in body 

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TRIADIMEFON FINAL 

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weights were observed from week 1 until study termination (↓5–12%). Cumulative body weight 

gain was decreased at weeks 0–53 (↓15%) and 0–104 (↓23%). In the 90 mg/kg/day group, food 

consumption was increased (p<0.05) throughout the study (↑12–34% males; ↑34–67% females), 

resulting in increased overall mean food consumption (↑19% males; ↑53% females). The liver 

was a target organ. In the 90 mg/kg/day females, increased (p<0.05) serum cholesterol was 

observed at Weeks 26, 53 and 78 (↑24–32%), and absolute and relative (to body) liver weights 

were increased (p<0.05; ↑15% and ↑27%, respectively) at Week 52. At week 104, absolute and 

relative liver weights were dose-dependently increased (p<0.05) in the females at 300 ppm and 

above (↑11–17% and ↑8–32%, respectively). A dose-dependent increase in fat in the hepatocytes 

was observed in the males and females at 90 mg/kg/d (p<0.05; 44–60% treated vs. 6–16% 

controls). 

In the carcinogenicity study in mice, mean body weights and overall body weight gains were 

decreased in high-dose (765 mg/kg/day) males (body weight ↓6–15%; body weight gain ↓22%). 

There were non-neoplastic and preneoplastic changes in the liver as well as increases in liver 

weights and correlating effects on serum enzymes. Hepatocellular hypertrophy was increased 

(p<0.01) in males receiving 300 or 1800 ppm and in females receiving 50, 300 or 1800 ppm. 

Hyperplastic nodules, altered cell foci, and microvesicular fatty changes were increased in both 

sexes (p<0.01) at 1800 ppm. Kupffer cell proliferation, lipofuscin pigment in liver macrophages 

and single cell necrosis were increased significantly (p<0.01) in males receiving 1800 ppm and 

females receiving 300 and 1800 ppm. At study termination, liver weights were increased 

(p<0.01) in mid- and high-dose males (↑19–64%) and in high-dose females (↑69%) when 

compared to controls. Kidney weights were decreased (p<0.01) by 16% in high-dose males. 

Serum alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase were 

increased in both sexes receiving 1800 ppm. There was an increase (p<0.05) in hepatocellular 

adenomas in high-dose males and females; however, there was no increase in liver carcinomas. 

In a rainbow trout fish (Oncorhynchus mykiss) early life stage toxicity study, the 17-day LC50

was 1.270 mg/L (growth not evaluated). In another rainbow trout fish early life stage toxicity 

study, sixty-day cumulative mortality was significantly greater than control mortality at 0.89 

mg/L. Growth in terms of final length and/or weight was significantly reduced in at 0.116, 0.30 

and 0.89 ppb. In the fathead minnow fish (Pimephales promelas) early life stage toxicity study, 

survival at the two highest test concentrations (0.57 and 1.1 mg a.i./L) was significantly reduced 

when compared to the pooled control. At test termination, mean standard length and mean wet 

weight were significantly reduced at the 0.27 mg a.i./L (top two treatments excluded from 

growth analyses). Abnormalities in the two highest test levels were noted primarily during the 

first week following the completion of hatch and were considered dose related. There were no 

reported signs of systemic or overt toxicity reported in the avian reproduction studies. 

Overall, the liver is a target organ in mammals which is consistent with other triazole fungicides. 

In the available data, effects on the liver ranged from increases in liver weight only, up to 

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evidence of hepatotoxicity (histopathology, increased serum alanine aminotransferase). In 

addition, mechanistic studies evaluating the mode of action for triadimefon showed changes in 

CYP expression (gene expression study, Goetz and Dix 2009, Goetz et al. 2007) and induction of 

hepatic enzymes PROD and UDP-GT (subchronic rat study, Wolf et al. 2006). Therefore the 

systemic effects, particularly in the liver are likely influencing endocrine-related endpoints in 

mammals. In addition to effects on the liver, triadimefon is known to cause neurotoxicity and its 

MoA involves blocking the re-uptake of dopamine which leads to increased motor activity and 

hyperactivity in rodents. Triadimefon acts as an indirect dopamine agonist by binding to the 

dopamine transporter and increasing levels of synaptic dopamine. Signs of neurotoxicity seen 

following triadimefon exposure include hyperactivity, increased motor activity, rearing, body 

temperature, habituation and spatial distribution. Additionally, it is noted that in some cases, 

endocrine-related effects coincided with signs of overt toxicity (decreased body weight) in both 

mammals and wildlife. 

B. Estrogen Pathway 

The in vitro assays for triadimefon were negative for ER binding, but was positive in the ERTA 

and were positive for alteration of steroidogenesis (inhibition of aromatase activity and ↑estradiol 

production). In the OSRI studies, which satisfied the in vivo mammalian assays, no estrogenmediate effects were seen in the absence of systemic toxicity. In the mammalian OSRI, when 

estrogen-related effects were seen they occurred only in the presence of systemic/overt toxicity 

(e.g., body weight loss, decreases in overall body weight gain and hepatotoxicity), which 

significantly confounds the interpretation of these estrogenic effects within the context of 

endocrine disruption. For example, in the modified one-generation reproduction toxicity study in 

females, the delay in VO and disruption in estrus cyclicity was accompanied by decreased 

terminal body weight and increased liver weight. In the two-generation reproduction toxicity 

study, decreased fertility was noted along with decreased body weights in the P and F1 parents, 

increased liver weights in the P generation and decreased birth and lactation body weights and 

viability in the F1 and F2 pups. In a bobwhite quail reproduction study, at the mid and high 

treatments (100 and 500 mg/kg-diet), there was an increase in number of cracked eggs (but no 

effects on eggshell thickness) and effects on egg viability and offspring survival; no effects were 

observed in the mallard duck study. 

For the FSTRA, there was significant decreases in fecundity at the mid (39%; 0.24 mg a.i./L) and 

high (46%; 0.97 mg a.i./L) concentrations, and a significant increase in GSI (49%) at the high 

concentration. However, there were no effects on gonadal histology or plasma VTG in females. 

While there were no clinical signs of toxicity or significant effects on survival noted in the 

FSTRA study for females (male body weight was considered to be significantly decreased at the 

mid and high concentration), test concentrations in this test were similar to concentrations in 

rainbow trout and fathead minnow early life cycle tests where significant effects on survival 

were reported (fathead minnow: effects at 0.57 and 1.1 mg a.i./L; rainbow trout: effects at 0.89 

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

≥0.1 mg/L. Therefore, while distinct overt toxicity was not observed in the FSTRA for females, 

given that overt toxicity has been observed in other fish assays at similar concentrations and 

male body weight in the FSTRA was considered to be significantly decreased, overall, the fish in 

the FSTRA may have been experiencing some stress which may have resulted in decreased 

resources (e.g., energy) for input into reproductive status. In summary, given that the effects in 

the mammalian studies coincided with systemic/overt toxicity and there is a general lack of 

complementarity for the FSTRA along with concerns about potential stress impacting 

reproduction, there is a lack of convincing evidence to suggest the triadimefon is potentially 

interacting with the estrogen pathway. 

C. Androgen Pathway 

For the androgen pathway, the results of the Tier 1 in vitro AR binding were negative, although 

alterations in expression of genes for steroid homeostasis were observed. In the in vivo

mammalian assays, no androgenic or anti-androgenic effects were seen in the Hershberger assay. 

In the OSRI studies which satisfied the male pubertal assay, androgen-related effects were seen 

only in the presence of overt or systemic toxicity (e.g., body weight loss, decreases in overall 

body weight gain, and hepatotoxicity). For example, in the modified one-generation reproduction 

toxicity study in males, the observed anti-androgenic effects occurred in conjunction with 

decreased terminal body weights and increased liver weights. In the FSTRA for males, there was 

a significant increase in GSI concentrations at the mid and high concentrations along with a 

reported decrease in fat pad development in the high treatment. There were no effects on male 

gonadal histology. There was a decrease in male body weight at both of these test concentrations 

that was considered to be biologically significant; along with the discussion above regarding test 

concentrations, this confounded the ability to discern a potential androgen-related interaction 

from an overtly/systemically toxic dose. Therefore, overall, there was a lack of convincing 

evidence for a potential interaction with the androgen pathway. 

D. Thyroid Pathway 

For the thyroid pathway, in the mammalian OSRI studies which satisfied the male and female 

pubertal and the AMA Tier 1 testing requirements, no thyroid-related effects were seen in the 

absence of systemic toxicity. No changes in thyroid weights were seen in any of the studies. 

Decrease in the circulating levels of thyroid hormones was seen only in the presence of systemic 

toxicity or overt toxicity. For example, in the one-generation mammalian reproduction study in 

males, there was a decrease in T4 levels in the presence of overt toxicity. In a subchronic 

mammalian study, decreases in T4 and TSH were seen in the presence of systemic toxicity (Wolf 

et al. 2006). Treatment-related histopathological lesions of the thyroid glands were seen in 

multiple species only at the high dose in the presence of systemic toxicity included follicular cell 

proliferations, follicular cell hyperplasia, colloid depletion and follicular cell adenomas. For the 

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wildlife, effects on T3 and T4 levels (whole body) were reported along with alterations in gene 

expression for thyroid in newly-hatched larval zebrafish, however, these effects were only 

observed in the presence of systemic toxicity (bent spines and uninflated swim bladders). In an 

organ morphogenesis study (Lenkowski et al., 2010) using Xenopus laevis tadpoles, mortality 

was increased at ≥36.5 mg/L and intestinal and muscle malformations were observed. However, 

this study is limited in its ability to inform potential thyroid interaction based on endpoints 

examined and age of tadpoles (thyroid pathway is still relatively inactive at this stage). 

Therefore it is possible that the interaction of triadimefon with the thyroid hormone system may 

occur only at high doses as a sequel to either primary effects on the liver (i.e., increased hepatic 

clearance of thyroid hormones due to induction of hepatic enzymes PROD and UDP-GT, Wolf et 

al. 2006) or in the presence of systemic/overt toxicity (i.e., body weight/body weight gain 

decreases, spine deformities). 

E. Conclusions 

In summary, the WoE indicates that in mammals, triadimefon may affect circulating levels of 

steroid and/or thyroid hormones only in the presence of systemic toxicity. The available data 

shows that the observed effects are due to increased metabolic activity of the liver, and not direct 

actions on the HPG or HPT axes. In wildlife, effects were also generally observed in the 

presence of systemic/overt toxicity (i.e., body weight decreases, skeletal malformations). 

Therefore, overall, there is a lack of convincing evidence to suggest a potential interaction with 

the E, A or T pathways. 

V. EDSP Tier 2 Testing Recommendations 

Based on weight of evidence considerations, mammalian and wildlife EDSP Tier 2 testing are 

NOT recommended since there was a lack of convincing evidence of potential interaction with 

the estrogen, androgen or thyroid pathways. 

 

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EDSP 109901-15. December 20, 2010. 

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. 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. 

Vinggaard, A., Breinholt, V., and Larsen, J. (1999). Screening of selected pesticides for estrogen 

receptor activation in vitro. Published: Food Addit. Contam. 16(12): 533–542. 

(MRID 48041733). 

Willoughby, J.A. (2009). Triadimefon: estrogen receptor transcriptional activation (human cell 

line (HeLa-9903). Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory 

Study No.: 9077V-100376ERTA, December 14, 2011. (MRID 48619605). 

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Willoughby, J.A. (2011a). Triadimefon: Androgen Receptor Binding (Rat Prostate Cytosol). 

Unpublished study conducted by CeeTox Inc., Kalamazoo, MI. Laboratory study number 90770-

100376ARB, December 14, 2011. (MRID 48619603). 

Willoughby, J.A., Sr. (2011b). Triadimefon: Estrogen receptor binding (rat uterine cytosol). 

Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9077V100376ERB, December 14, 2011. (MRID 48619602). 

Wolf, D., Allen, J., George, M., et al. (2006). Toxicity profiles in rats treated with tumorigenic 

and nontumorigenic triazole conazole fungicides: propiconazole, triadimefon, and myclobutanil. 

Toxicol. Path. 34: 895–902. (MRID 48264601). 

York, D. (2012). Triadimefon — short-term reproduction assay with the fathead minnow 

(Pimephales promelas) following OPPTS 890.1350 and OECD 229 Guidelines. Laboratory 

Study No. 13798.6262. Unpublished study by Smithers Viscient, Wareham, MA. (MRID 

48673901). 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100) 

Requirement satisfied by OSRI (MRID Nos. 48264601; 48264602; 48264603; 48264604; 

48264605; 48264607; 00155075; 00032541; 42153901); See Appendix 2. 

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

In an androgen receptor (AR) binding assay (MRID 48619603), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment, 

which was conducted to measure the binding of a single concentration of [3

H]-R1881 (1 nM) in 

the presence of increasing concentrations of triadimefon (logarithmic increase from 10−11 to 10−4

M). Dimethyl sulfoxide (DMSO) was used as the vehicle at a final assay concentration of 

approximately 3.2%. The assay included dexamethasone as a weak positive control, and R1881 

as the ligand reference standard. 

The saturation binding experiment was conducted to demonstrate that the AR in the rat prostate 

cytosol was present in reasonable numbers and was functioning with appropriate affinity for the 

radiolabeled reference ligand prior to routinely conducting AR competitive binding experiments. 

Saturation binding data were not originally provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. Separate cytosolic preparations were used for each of the 

three competitive binding runs, so data from three saturation binding experiments were 

evaluated. For the cytosolic preparation conducted on March 30, 2011, the mean dissociation 

constant (Kd) for [3H]-R1881 was 0.614±0.065 nM, and the estimated Bmax was 1.080±0.109 

fmol/100 μg protein for the single batch of prostate cytosol that was prepared. For the cytosolic 

preparation conducted on July 23, 2011, the Kd for [3

H]-R1881 was 1.080 ± 0.690 nM, and the 

estimated Bmax was 1.071 fmol/100 μg protein (±0.167) for the single batch of prostate cytosol 

that was prepared. For the cytosolic preparation conducted on September 24, 2011, the 

dissociation constant (Kd) for [3

H]-R1881 was 0.613 ± 0.041 nM, and the estimated Bmax was 

0.817 fmol/100 μg protein (±0.049) for the single batch of prostate cytosol that was prepared. 

The mean and individual Kd values were generally below the range reported in the EPA 

validation program (0.685 to 1.57 nM), although two individual values were within the range and 

one value was above the range. Confidence in these numbers is generally high according to the 

goodness of fit (R2

 = 0.880-0.992) and the small variation in R2 and Kd estimates among runs 

(although greater variability was observed in the July 23, 2011 saturation binding data). 

In the competitive binding experiment, precipitation of triadimefon was visually observed at the 

highest concentration (10−3

 M) in the first run; therefore, the 10−3 M data were not included in 

the evaluation and the highest concentration in the second and third runs was 10−4 M triadimefon. 

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In all three runs, he specific [3H]-R1881 AR binding was >75% at all soluble concentrations of 

triadimefon tested. An IC50 and relative binding affinity (RBA) could not be calculated for 

triadimefon. 

Mean log IC50s for R1881 and the weak positive control (dexamethasone) were −9.3 and −4.4 M, 

respectively, and the mean RBA for the weak positive control was 0.0012%. Confidence in 

these numbers is high due to the small variation. All performance criteria were generally met; 

however, the top percent binding for triadimefon was low (77.7 – 82.9%) at 10−4

 M in the first 

run, but specific binding in the presence of triadimefon was consistently >80% at concentrations 

≤10−5 in the first run and at all concentrations in the other two runs. As this deviation was minor 

and was not considered to have affected the results of the study, all three runs were considered 

valid. 

Based on the results from the three runs, triadimefon is classified as a Non-Binder in the 

Androgen Receptor Binding Assay. 

Aromatase Assay; (OCSPP 890.1200) 

Requirement satisfied by OSRI (USEPA 2007; MRID No. 48125203); See Appendix 2. 

Estrogen Receptor (ER) Binding Assay; (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48619602), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiments. A saturation binding 

experiment was performed to demonstrate that the ER in the rat uterine cytosol was present in 

reasonable numbers and was functioning with appropriate affinity for the radiolabeled reference 

estrogen prior to conducting ER competitive binding experiments. The competitive binding 

experiment was conducted by measuring the binding of a single concentration of [3

H]-17βestradiol (1 nM) in the presence of increasing concentrations of triadimefon (logarithmic increase 

from 10−10 to 10−3

 M). DMSO was used as a vehicle at a final concentration of 2%. The assay 

included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, 

and 17β-estradiol as the natural ligand reference material. 

Raw saturation binding data were not provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. The mean dissociation constant (Kd) for [3

H]-17β-estradiol 

was 0.303 ± 0.090 nM and the mean estimated Bmax was 0.143 ± 0.003 nM (33.59 ± 0.70 

fmol/100 μg of cytosol protein) for the uterine cytosol preparation used in this study. The Kd and 

Bmax were within the range recommended in the Test Guideline. 

In the competitive binding experiment, triadimefon had no effect on [3

H]-17β-estradiol binding 

to the ER at concentrations of ≤10−4

 M in all runs. Precipitation was observed at 10−3 M for all 

runs; therefore, the data from the 10−3 M triadimefon concentration were not evaluated. A mean 

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log IC50 was not calculated for triadimefon as competitive binding was not observed in any run. 

The mean IC50 was 1.2 × 10−9 M for the natural ligand, 17β-estradiol, and 2.95 × 10–6 M for the 

weak positive control, which also had a relative binding affinity (RBA) of 0.041%. 

Based on the results from the three runs, triadimefon 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 48649605), hERα-HeLa-9903 

cells cultured in vitro were exposed to triadimefon (99.20% a.i., Batch #2009020210/2010-

006597 at concentrations of 10−4 to 10−10.3 M in DMSO (0.1%). The experiments were 

performed using 96-well plates and each triadimefon concentration was tested in six wells per 

plate. Cells were exposed to the test agent for 24 ± 2 hours to induce a luciferase reporter gene. 

Luciferase expression in response to activation of the estrogen receptor was measured using a 

proprietary method. 

Performance criteria values for reference chemicals were generally below the expected ranges, 

indicating increased sensitivity of the assay. Triadimefon was tested up to the limit of solubility 

and cytotoxicity, 10−4 M. The RPCMax was 26.6% and 29.7% in runs 1, and 2, respectively; the 

associated PCMax were 10-4.3 and 10-4 M, respectively. The response was dose-dependent. 

Because the RPCMax > PC10 in two assay runs, triadimefon was considered positive for estrogen 

receptor transcriptional activation in this test system. 

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

The 21-day short-term reproduction assay of triadimefon with fathead minnows (Pimephales 

promelas) was conducted under flow-through conditions. Adult fish, 16 spawning groups (2 

males and 4 females in each group; 5-6 months old) were exposed to triadimefon (99.2% purity) 

at nominal concentrations of 0 (control), 0.063, 0.25, and 1.0 mg a.i./L; mean-measured 

concentrations were <LOQ (<0.0046), 0.057, 0.24, and 0.97 mg a.i./L. The test system was 

maintained at 24 to 26o

C and a pH of 6.9 to 8.0. 

There were no significant effects on mortality (p>0.05) and no apparent effects on clinical signs 

(i.e., behavioral and other sublethal effects) except for one dark fish observed in the low 

treatment group. There was a reduction of 21 and 19% in mean male body weight in the mid and 

high treatment groups, respectively, relative to the negative control, which was not statistically 

significant (Mann Whitney U; p>0.05) but was considered to be biologically significant. There 

were no significant differences in body weight for female fish or in body length for male or 

female fish. 

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Spawning frequency and fecundity in controls, respectively, were at least every 4 days and 22 

eggs/female/day; fertilization success in controls was 97%. There was a significant (JonckheereTerpstra; p<0.05) reduction in fecundity of 39% and 46% at the mid and high treatment levels, 

relative to the negative control. While male vitellogenin (VTG) exhibited a significant 

(Jonckheere-Terpstra; p<0.05) increase of 359% in the median value at the high concentration, 

relative to the negative control, it is noted that variability in VTG levels appeared to be greater in 

the treatment groups, and the significant increase in the highest treatment appeared to be due to 

two male fish with higher VTG values and not an increased treatment-related response from the 

fish in that treatment. Male and female gonado-somatic index (GSI) exhibited a significant 

increase (Jonckheere-Terpstra; p<0.05) relative to the controls with males exhibiting a 33% and 

75% increase in GSI at the mid and high treatment levels and females exhibiting a 49% increase 

at the high treatment level. There were no significant effects on fertility, female VTG, or male 

tubercle scores (no tubercles detected in females) when treated groups were compared to the 

negative control (p>0.05). Plasma testosterone and 17β-estradiol (sex steroids) were not 

measured in this study. 

Gonadal histopathology was not statistically analyzed, and there were no notable treatment 

related findings. In males, there were findings of minimal severity of increased proportion of 

spermatogonia, and interstitial cell hyperplasia/ hypertrophy; and findings of minimal to mild 

severity of asynchronous gonad development with no consistent pattern among concentrations. 

In females, there were findings of minimal to moderate severity of increases oocyte atresia and 

egg debris in the oviduct, and findings of minimal severity of granulomatous inflammation with 

no indication of treatment-related effects. At study termination, fat pads of males were poorly 

developed within three of the four replicate chambers at the high treatment level (not statistically 

analyzed). There were no other treatment-related effects on secondary sex characteristics or 

gonadal staging. 

Hershberger Assay; (OCSPP 890.1400)

In a Hershberger assay (MRID 48619604) screening for androgenic activity triadimefon (99.2% 

a.i., lot/batch# 20009020210/2010-006597) in polyethylene glycol (PEG; molecular weight 400) 

was administered daily via oral gavage to groups of eight 59- to 60-day old, castrated male 

Sprague Dawley rats at dose levels of 0 (vehicle), 50, or 100 mg/kg/day. An androgenic positive 

control group consisting of eight castrated rats exposed to 0.4 mg/kg/day of testosterone 

propionate (TP) by subcutaneous (s.c.) injection was also included. 

To screen for potential anti-androgenic activity, triadimefon (99.2% a.i., lot/batch# 

20009020210/2010-006597) in PEG was administered daily via oral gavage to groups of eight 

59- to 60-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 25, 50, or 

100 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by 

s.c. injection. The anti-androgenic positive control group consisting of eight castrated rats 

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exposed to 0.4 mg/kg/day TP and 3 mg/kg/day flutamide (FT) was also included. TP alone was 

used as the anti-androgenic negative control. 

For both components of the assay, body weights were determined daily. The animals were dosed 

for 10 consecutive days and terminated approximately 24 hours after the final dose 

administration. At necropsy, the five androgen-dependent tissues and liver were collected and 

weighed. 

For the androgen agonist assay, one animal in the high-dose group was observed to have 

hunched posture beginning on Day 7 and was moribund on Day 9; this animal was euthanized on 

Day 9. For the anti-androgen assay, one animal in the low-dose group died on Day 7 as a result 

of a dosing error. All other rats survived until scheduled sacrifice. The death of these animals 

does not affect the validity of this screening assay because no other animals from any dosing 

group showed signs of moribundity. Other than the euthanized rat in the androgen agonist assay, 

no animals exhibited any clinical signs of toxicity. There were no treatment-related gross 

pathological findings. 

In the androgen agonist assay, body weight gains were decreased (p<0.05) by 44% in the highdose group compared to the vehicle control. Final body weights and body weight gains in the 

low-dose group were comparable to controls throughout the duration of the assay. Final body 

weights were increased (p<0.05) by 9% in the positive control group, resulting in increased 

(p<0.05) overall body weight gains of 66%. At 100 mg/kg/day, ventral prostate weight was 

significantly increased 29% (p<0.05). There were no treatment-related effects in seminal 

vesicles, levator ani/bulbocavernosus (LABC) muscle complex, Cowper’s gland, or glans penis, 

and multivariate analysis of all five androgen-dependent organ weights did not show an overall 

statistically significant difference with the administration of triadimefon. Liver weight was 

significantly increased (p<0.05) in the 100 mg/kg/day group compared to the control (↑25%), 

and relative liver weight was significantly increased (p<0.05) in the 50 and 100 mg/kg/day 

groups (↑15 and ↑30%). Animals in the TP group had increased (p≤0.05) accessory sex organ 

weights as follows: 1079% in seminal vesicles; 1360% in ventral prostate; 223% in LABC; 

573% in Cowper’s gland; and 79% in glans penis. The performance criteria indicated that this 

assay was performing as expected. 

In the anti-androgen assay, final body weight was decreased (p<0.05) in the 100 mg/kg/day 

group (↓9%) and overall body weight gains were decreased (p<0.05) in the 50 and 100 

mg/kg/day groups (↓29% and ↓44). Final body weights and body weight gains in the 25 

mg/kg/day group were comparable to controls. Body weight gains were decreased (p<0.05) by 

20% in the positive control group. At 100 mg/kg/day, LABC weight was significantly decreased 

12% (p<0.05). There were no treatment-related effects in seminal vesicles, ventral prostate, 

Cowper’s gland, or glans penis, and multivariate analysis of all five androgen-dependent organ 

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weights did not show an overall statistically significant difference with the administration of 

triadimefon. Absolute and relative liver weights were similar to the controls in the triadimefon 

dose groups and the positive control group. Animals dosed with TP + FT (positive control) had 

decreased (p<0.05) accessory sex organ weights as follows: 78% in seminal vesicles; 77% in 

ventral prostate; 54% in LABC; 65% in Cowper’s gland; and 28% in glans penis. The 

performance criteria indicated that this assay was performing as expected. 

Percent coefficient of variations (CVs) for the vehicle control and the 100 mg/kg/day group were 

compared to the performance criteria in the guideline. All %CV values were less than the 

maximum recommended Guideline values.

The high-dose level selected for this study (100 mg/kg/day) was considered to be adequate based 

on a dose range-finding study showing reduced body weight and body weight gain and increased 

relative liver weights after 10 days of dosing at 120 mg/kg/day. 

Statistically significant changes were not seen in two or more of the five androgen sensitive 

tissue weights. Triadimefon was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

Female Pubertal Assay; (OCSPP 890.1450) 

Requirement satisfied by OSRI (MRID Nos. 48619605; 42153901; 00032541; 00155075); see Appendix 

2. 

Male Pubertal Assay; (OCSPP 890.1500) 

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

Appendix 2. 

Steroidogenesis Assay; (OCSPP 890.1550) 

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

Uterotrophic Assay; (OCSPP 890.1600) 

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

 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Published literature and Part 158 studies considered relevant for determining the potential 

interaction of triadimefon with the endocrine system are summarized below. Given the breath of 

toxicity data available on most pesticides, the Part 158 studies discussed in this appendix are 

those that fulfill current guideline requirements (where available) and/or are directly relevant to 

assessing potential endocrine effects (i.e. multigeneration rodent reproductive toxicity studies, 

developmental toxicity studies, chronic toxicity studies, etc.).

Aromatase Assay 

The purpose of Integrated Summary Report (ISR) for Aromatase (MRID No. 48125203) was to 

summarize the development and validation of the microsomal aromatase assay (OPPTS Test 

Guideline No. 890.1200) which had been proposed as an alternative in vitro assay for the Tier-1 

screening battery in the Endocrine Disruptor Screening Program (EDSP). Triadimefon was 

included in supplementary blind testing of the recombinant microsomal assay. The purpose of 

this study was to test the protocol using a set of 16 diverse chemicals for which evidence of 

aromatase effects had been reported; triadimefon, an azole fungicide, had been reported to be a 

weak to moderate aromatase inhibitor. The testing was conducted according to the same protocol 

that was used in all method development and validation studies included in the report. The 

substrate used for the assays was a mixture of non-radiolabeled ASDN (purity 100%) and 

radiolabeled 3

H-ASDN (radiochemical purity >97%; specific activity 25.3 Ci/mmol). Aromatase 

activity was measured in an assay solution containing sodium phosphate buffer, propylene 

glycol, microsomal protein, 3

H-ASDN, and NADPH. The following controls were included: 

a full enzyme activity control (assay solution + solvent vehicle), a background activity control 

(full enzyme activity control except NADPH), and a positive control, 4-OH ASDN (purity 99%; 

in assay solution), which is a known aromatase inhibitor that binds irreversibly to aromatase. The 

report indicated that the vehicle control was either ethanol or DMSO, depending on the reference 

chemical used; no specific information was provided by chemical. 

The full aromatase control activity for the triadimefon runs was 0.393–0.431 nmol/mg 

protein/min; uncorrected background activity was 0.001–0.002 nmol/mg protein/min, 

corresponding to ~0.3–0.5% of the full activity control. These results were consistent with the 

respective performance criteria of ≥0.100 nmol/mg protein/min for the full activity control and 

≤1% of full activity control for the background control. The positive control results for the study 

indicated that there was not significant variation between technicians conducting the assays; 

based on the concentration response curve for triadimefon, there was minimal variation across 

the three runs for this reference chemical. For the three triadimefon runs, the four model 

parameters for positive controls were: Bottom = 0.6% to 0.9%; Top = 97.7% to 103%; 

LogIC50 = -7.22 to -7.24; and slope = -1.0 to -1.2. These were within the ranges of the 

performance criteria for the positive control of -5% to 6% for Bottom, 100 ± 10% for 

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Top, -7.3 to -7.0 for LogIC50, and -1 ± 0.2 for slope. The authors also provided data indicating 

that even the bounds of the 95% confidence intervals were within the performance criteria ranges 

for triadimefon. 

Aromatase activity results for the reference chemicals were normalized to percent of control by 

dividing by the full enzyme activity for the run; results were not reported in terms of aromatase 

activity per se (nmol/mg protein/min). Mean percent of control values increased with decreasing 

concentration of the test substance from 2.02% at 10-4 M to 100.06% at 10-8 M (CVs 8.22 

decreasing to 3.11). The percent of control data were fit to the four- and two-parameter models 

using nonlinear regression methods to calculate Bottom, Top, LogIC50, and slope values. Results 

for triadimefon using the 4-parameter model were Bottom = -0.002% to -0.292%; Top = 97.55% 

to 101.8%; LogIC50 = -5.74 to -5.79; and slope = -0.9238 to -0.9571. For the 2-parameter model 

(Bottom = 0; Top = 100), results were: LogIC50 = -5.728 to -5.829 and slope = -0.8861 

to -0.9801. The results for all runs using the four-parameter model were within the performance 

criterion of Top = 100 ± 10%. 

Under the chemical classification criteria for aromatase inhibition used in the assay, triadimefon 

was unequivocally classified as an aromatase inhibitor at the highest tested concentration of 

10-4 M (i.e., the most inhibited level lies below 50% of the full activity control). Comparison of 

best fit estimates for the four- and two-parameter models yielded LogIC50 values of -5.77 for 

both models. Percent control values spanned the entire range of 0–100% for all concentrations of 

triadimefon, confirming the validity of the LogIC50 calculated from the data. 

AR and ER Transcriptional Activation Assays 

Kojima et al. (MRID 48033008) screened 200 chemicals (including triadimefon) for estrogen 

and androgen activity by conducting transcriptional activation assays for hERα, hERβ and hAR 

using Chinese hamster ovary (CHO) cells with a luciferase reporter. The authors stated that 

cytotoxicity was not observed in any of the tested compounds at the selected concentration range 

(data not shown). It was reported that triadimefon was negative for both estrogen and androgen 

receptor transcriptional activation from 10-8 to 10-5 M in this test system (data not shown).

Cell Proliferation, ER, and AR Binding Assays 

Okubo et al. (MRID 48074205) tested twenty pesticidal chemicals (including triadimefon) for 

estrogenic and anti-estrogenic activity using a MCF-7 cell proliferation assay. Chemicals were 

also tested for AR and ER binding using the Ligand Binding Screening System, a proprietary 

screening system by Toyobo. Anti-estrogenic activity was assessed by measuring the suppression 

of cell proliferation induced by 30 pM of 17β-estradiol. Triadimefon was reported to not be 

cytotoxic in the range tested. 

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Triadimefon was tested from 1 μM to 1 mM in an MCF-7 cell proliferation assay and a 

proprietary ERα and AR binding assay. Triadimefon resulted in maximal cell proliferation at 

100 μM. The anti-estrogen ICI 182,780 blocked cell proliferation. In addition, triadimefon was 

shown to be an anti-estrogen at 100 to 1000 μM when tested with 30 pM of E2. In contrast, 

Okubo reported that triadimefon did not bind in the ER binding assay (data not shown). In the 

AR binding assay, the authors reported an IC50 of 902 ± 143 μM for triadimefon and a relative 

binding affinity (RBA) of 0.005. 

ER Binding, ER Transcriptional Activation and Vitellogenin Induction Assays

In this study, Petit et al. (1997) used a trout ER binding assay, trout hepatocyte cultures, and 

yeast p-galactosidase reporter gene assays transfected with trout ER to study the estrogenic 

effects of 49 chemical compounds (including triadimefon). The yeast cell walls were removed, 

thus removing the cell wall barrier. Triadimefon was tested at concentrations arranging from 10-8

to 10-4 M in the transcriptional activation and binding assays. The mean induction values at 

maximum response for the transcriptional activation and vitellogenin induction assays were 

reported with E2 being 100%. Triadimefon was negative in the yeast transcriptional activation 

assay and did not promote cell proliferation. However, it was tested at 10−4

 M in the vitellogenin 

assay and found to be positive (178% induction). Data were displayed graphically for the ER 

binding assay and vitellogenin assays. The results of the experiments were explained as follows: 

the negative results in binding and transcriptional activation assays, but positive in the trout 

hepatocyte assay indicated that either a metabolite of triadimefon is an ER binder or that 

vitellogenin induction was occurring through an indirect gene activation pathway. 

Cell Proliferation and Transcriptional Activation Assays 

In a study by Vinggaard, Breinholt, and Larsen (MRID 48041733), at total of 20 pesticides, 

including triadimefon, were tested in an MCF-7 cell proliferation assay and in a yeast estrogen 

screening (YES) assay. MCF-7 cells were incubated at 0.001, 0.1, 1.0 and 10 μM with 

triadimefon, and proliferation was determined by measuring DNA content. Triadimefon was 

toxic to yeast above 30 μM. Triadimefon caused a 2.4-fold increase in cell proliferation with a 

relative proliferation efficiency of 53%, indicating it was a partial estrogen agonist. Triadimefon 

was also run in the MCF-7 assay at 10 μM and measured as positive by the resazurin metabolic 

reduction method. Triadimefon was negative in the YES screen, but no data were provided. 

Steroidogenesis Study (in vitro and in vivo) in Rats

In an in vivo and in vitro steroidogenesis study (Goetz et al. 2009; MRID 48264602), 

triadimefon was tested for effects on steroid production. Hormone production in vitro was 

measured in testis organ cultures from untreated adult (PND 90–100) and neonatal (PND 1) 

Sprague-Dawley rats following exposures to 1, 10 or 100 μM triadimefon. The media was 

collected at 0.5, 1.5, and 2 hours following exposure. A rat to human comparison was examined 

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using the H295R human adrenocortical cell line exposed to 1, 3, 10, 30, or 100 μM triadimefon. 

Cells were examined microscopically to evaluate cell viability and number. Cell viability was 

calculated using a commercially available viability kit. For the in vivo portion of the study, male 

Wistar Han rats (PND 60) received triadimefon at 126 mg/kg/day in the diet for 30 days. On 

PND 74 (14 days after initiation of dosing), tail blood was collected for testosterone 

measurements. On PND 90 (30 days after initiation of dosing), rats were sacrificed and trunk 

blood was collected for evaluation of serum testosterone, estradiol, luteinizing hormone (LH), 

and prolactin. The weights of the testes, epididymis, ventral prostate, seminal vesicles, pituitary 

gland and the liver were determined. 

In vitro, triadimefon reduced media levels of testosterone levels by 40–68% in the adult and 

neonatal testis culture, and altered steroid production in a manner that indicated CYP17Al 

inhibition at the highest concentration. In the H295R cells following 48 hours of exposure, there 

was an overall decrease in estradiol, progesterone, and testosterone. No effects on cell growth 

were observed cells following treatment with triadimefon. A decrease was seen in testosterone 

concentrations at 10 and 30 M of triadimefon. An increase in estradiol production was observed 

at 1 and 3 μM; however, a decrease in production was seen at the higher test concentration. 

In vivo, serum testosterone levels were not increased after 14 days but were increased (↑290%; 

p<0.01) at 30 days as was intra-testicular testosterone (↑210%; p<0.01). Serum levels of 

estradiol, LH and prolactin were somewhat elevated at 30 days, but increases were not 

statistically significant. Liver weights, both absolute and relative, were significantly increased 

after 30 days of exposure (↑27% and ↑37%, respectively). Significant (p<0.05) decreases were 

seen in the absolute epididymis (↓5%) and pituitary (↓10%) weights; however, no changes were 

seen in the relative weights of these organs. No treatment-related changes were seen in the 

absolute or relative weights of the testes, seminal vesicle or ventral prostate. 

Modified One-Generation Reproduction Toxicity Study in Rats 

In a study by Goetz et al., 2007 (MRID 48264603), triazole fungicides associated with a range of 

reported male reproductive effects in experimental animals were selected to assess potential toxic 

modes of action. In a modified one-generation reproduction toxicity study , groups of timed 

pregnant female Wistar Han rats were fed diets containing either 100, 500, or 2000 ppm 

myclobutanil, 100, 500, or 2500 ppm propiconazole, or 100, 500, or 1800 ppm (5, 25 or 90 

mg/kg/d) triadimefon from GD 6 through lactation. Dams were allowed to litter normally, and 

litters were culled on PND 8 to eight pups/dam (males retained preferentially). Pups were fed the 

same diet as their dam beginning at weaning (PND 23) until termination. Pups were maintained 

on their appropriate diet up to PND 120. One male/dose group/litter was necropsied on PND 1, 

22, 50, or 92. Measurements included anogenital distance (AGD) at PND 0, weekly body weight 

and food consumption, age and weight at PPS, organ weights (brain, hypothalamus, 

hippocampus, pituitary, thyroid, liver, testes, epididymides, ventral prostate, and seminal 

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vesicle), organ histopathology (brain, pituitary, thyroid, liver, testes, epididymides, and ventral 

prostate), serum estradiol, testosterone, T3, and T4 levels, sperm morphology and motility, and 

fertility and fecundity via mating assays using PND 78 or older treated males paired with 

untreated females. 

High-dose triadimefon (1800 ppm; 90 mg/kg/day) caused overt toxicity in the F1 males. Weekly 

body weights were decreased during PND 1–22 (↓17–27%) and Weeks 4–13 (↓22–29%), and 

mean daily food consumption (g/rat/day) was decreased by 9–26% during Weeks 4–12. AGD 

was increased by high-dose triadimefon, suggestive of hyper-virilization. This dose level delayed 

PPS (PND 47.4 treated vs. PND 42.5 controls) with a concomitant decrease in body weight at 

PPS (↓13%), consistent with delayed puberty. Relative liver weights were increased at PND 1, 

50, and 92, but this was a result of the decreased body weights at these time points. Liver 

histology revealed strong panlobular hypertrophy in high-dose triadimefon males. Pituitary 

weights were unaffected by treatment. Absolute testis weights were increased by 21% at PND 22 

and by 14% at PND 50 by high-dose triadimefon treatment. At PND 92, absolute epididymides 

weights were decreased by 8% and absolute seminal vesicle weights were decreased by 19%. 

Serum testosterone was increased at PND 50 and PND 92/99 by triadimefon. At 90 mg/kg/day, 

triadimefon affected several reproductive parameters including pup death in 18 of 21 litters, with 

nine total litter losses, resulting in a 47% survival rate, compared to 96% survival in the control 

litters. Untreated females mated with high-dose triadimefon males had an insemination index of 

8% with no females becoming pregnant (fertility index of 0%). Additionally, no copulatory plugs 

were found, and there was a complete lack of sperm in the uterine horns after the mating period. 

However, there were no observed differences between treated males and controls in sperm 

morphology or motility. In addition to the reproductive system effects, total serum T4 levels were 

decreased at PND 92 by high-dose triadimefon (↓25%); T3 and TSH levels were unaffected by 

treatment. These reproductive effects are consistent with the disruption of testosterone 

homeostasis as a key event in the mode of action for triazole-induced reproductive toxicity. 

Modified One-Generation Reproduction Toxicity Study in Rats

Three triazole fungicides were evaluated for effects on female rat reproductive development 

(Rockett et al., 2006; MRID 48264605). In a modified one-generation reproduction toxicity 

study rats were exposed via feed to 100, 500, or 2500 ppm propiconazole, 100, 500, or 2000 ppm 

myclobutanil, or 100, 500, or 1800 ppm (5, 25 or 90 mg/kg/d) triadimefon from gestation day 6 

to postnatal day (PND) 98. Body weight and AGD at PND 0, age and BW at vaginal opening 

(VO), estrous cyclicity, and body and organ weight at necropsy were measured. 

BW at PND 0 was unaffected by treatment. AGD was increased by 2000 ppm myclobutanil. VO 

was delayed by high-dose myclobutanil and triadimefon (PND 36.75 treated vs. PND 32.8 

controls). Estrous cyclicity was initially disrupted by 500 and 2500 ppm propiconazole and 

1800 ppm triadimefon, but later normalized. At PND 99, there was a decrease in body weight at 

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90 mg/kg/day triadimefon (numerical data not reported), an increase in liver weight by high-dose 

propiconazole and triadimefon (numerical data not reported), and an increase in ovarian weight 

by high-dose myclobutanil and triadimefon (numerical data not reported). Thyroid glands of 2/4 

rats exposed to 1800 ppm triadimefon showed mild diffuse follicular cell hypertrophy; no 

thyroid lesions were seen in the other animals. It is concluded that exposure to propiconazole, 

myclobutanil, and triadimefon adversely impacted female rodent reproductive development. 

Three-Generation Reproductive Toxicity Study in Rats

In a multi-generation reproduction toxicity study (MRID 00032541) triadimefon, (purity not 

reported) was administered continuously in the diet to SPF Wistar W. 74 rats (10 males: 20 

females/dose) at nominal dose levels of 0, 50, 300, or 1800 ppm (0, 2.5 15 or 90 mg/kg/day). The 

P animals were given test article diet formulations for approximately 100 days prior to mating to 

produce the F1a litters. After weaning, F1a animals were sacrificed, and a second mating of the P 

animals was conducted to produce the F1b litters. After weaning, 10 males:20 females/dose from 

the F1b litters were selected to become the parents of the F2 generation and were given the same 

concentration test formulation as their dam for approximately 4 weeks prior to mating. After 

weaning, F2a animals were sacrificed, and a second mating of the F1b animals was conducted to 

produce the F2b litters. This process was repeated for a total of three generations. The final 

generation (F3b) was sacrificed at the end of lactation. 

At 90 mg/kg/day, fertility was affected in all generations. In the P generation, no differences 

occurred in the fertility index during the first mating; however, during the second mating the 

fertility index was slightly decreased compared to controls (17/20 treated vs. 19/20 controls, not 

statistically significant). Fertility values were significantly (p<0.05) lower in both matings of the 

F1 generation. Only one female became pregnant in the mating to produce the F2a litters; no 

females became pregnant in the mating to produce the F2b litters. Therefore, mating of the F1

generation resulted in no F2 pups. The number of offspring per dam at birth was decreased 

(p<0.05) in the first litters of both the P generation and the F1 generation (↓16% each). As a 

result of low fertility, there were no animals in the 1800 ppm group of the F2 generation to 

evaluate. 

Additionally at 90 mg/kg/d, body weights of the P generation parents were decreased (↓15%; 

p<0.01) during F1b pre-mating, mating, gestation, and lactation. Body weight gains of the F1

generation parental males and females were decreased (p<0.01) throughout pre-mating, 

gestation, and lactation for both matings. 

Due to low fertility in the 1800 ppm parental generations, only one F2 litter and no F3 litters were 

produced at this dose. 

In the F1 pups, at 90 mg/kg/d, the number of offspring per dam was decreased (p<0.05) in the F1a

and F1b litters (↓20-31%) at PND 5. At PND 28, viability was decreased (p<0.01) in the F1a

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(90.7% treated vs. 97.8% controls) and F1b (56.4% treated vs. 84.2% controls) pups. 

Additionally, body weight gains of the F1a pups were decreased (↓10%; p<0.05) during lactation. 

At15 mg/kg/d, body weight gains of the F2b pups were decreased (↓15%; p<0.05) during PNDs 

5, 7, 14, and 21. Body weight gains were also decreased (↓10%; p<0.05) in the F3b pups at PND 

28. 

Two-Generation Reproductive Toxicity Study in Rats 

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

92.6% a.i.) was administered continuously in the diet to bor:wisw (SPF/Cpb) rats 

(10 males : 20 females/dose) at nominal dose levels of 0, 50, or 1800 ppm (0, 2.5, or 

90 mg/kg/day). The P animals were given test article diet formulations from 100 days prior to 

mating to produce the F1 generation. After weaning, F1 animals were selected 

(10 males: 20 females/dose) to become the parents of the F2 generation and were fed the same 

concentration test formulation as their dam; the P animals were sacrificed. Treatment of the F1 

animals followed the same pattern as the P animals; however, only the 50 ppm F1 parents were 

sacrificed when the F2 litters were weaned. The treatment of the 1800 ppm F1 groups was 

stopped; and the 1800 ppm F1 males were mated with the control F1 females, while the 1800 ppm 

F1 females were mated with control F1 males. This cross-mating procedure produced a second F2

litter and was intended to provide further information regarding the fertility of the F1 parents. For 

all matings, the day that sperm was detected in a vaginal smear was designated as gestation day 

(GD) 0. 

No parental mortalities occurred. Sperm motility, sperm morphology, gestation length, and sex 

ratios were unaffected by treatment. 

At 1800 ppm, fertility was decreased (35% treated vs. 85% controls; p<0.01) in the F1 parents. 

This resulted in decreases in the total number of litters (7 treated vs. 17 controls) and the total 

number of pups (37 treated vs. 196 controls). In the F1 cross-mating, fertility was 47.4% from the 

mating of 1800 ppm males with the control females and 80% from the mating of the 1800 ppm 

females with the control males. In the absence of sperm abnormalities, it was concluded that the 

observed decrease in fertility was due to a decreased interest of the males in copulation. 

Copulation was not observed in 6/10 of the 1800 ppm males during the 3-week cross-mating 

with control females. 

Additionally at 1800 ppm, body weights were decreased (p<0.05) in the P males during Weeks 4, 

5, and 6; in the P females during Weeks 1–16 and 21–26; and in the F1 parental males and 

females during Weeks 9–40. Terminal body weights were decreased (p<0.01) by 12% in the 

P females. Absolute and relative (to body weight) liver weights were increased (p<0.01) in the 

P males (↑23–29%) and females (↑23–39%). 

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At 50 ppm, body weights were decreased (p<0.01) in the F1 dams on GD 1, 6, and 15 by 6–7%. 

Absolute and relative (to body weight) ovary weights were increased (p<0.05) by 12% in the 

P dams (organ weights were not determined for the F1 adults). The biological significance of 

these findings is considered to be equivocal. 

At 1800 ppm, body weights were decreased (p<0.05) at birth and lactation by 12–13% in the F1

and F2 pups. Viability was decreased (p<0.05) in the F1 pups during PND 0–5 (51.4% treated vs. 

87.1% controls) and PND 5–28 (79.2% treated vs. 93.1% controls), and in the F2 pups during 

PND 0–5 (37.8% treated vs. 92.9% controls) and PND 5–28 (71.4% treated vs. 89.7% controls). 

Additionally, litter size was decreased (p<0.01) in the F2 pups (5.3 treated vs. 11.5 controls). 

Gene Expression Study 

In a study by Tully et al. (MRID 48264607) to evaluate gene expression profiles in liver and 

testis, groups of male Sprague-Dawley rats (15/dose group) received triadimefon by oral gavage 

at dose levels of 0, 5, 50, or 115 mg/kg/day from PND 60 to 74. Following exposure, trunk blood 

was collected for measurements of serum testosterone, LH, FSH, and estradiol. Livers, left and 

right testes, thyroids, adrenals, brains, left and right epididymides, seminal vesicles, spleens, and 

ventral prostates were removed; liver, adrenals, brains, left and right epididymis, seminal 

vesicles, spleens, and ventral prostates were weighed; and liver and testes samples were collected 

for histology, enzyme biochemistry, or gene expression profiling. Thyroid histopathology was 

also examined. Sperm morphology and motility were also examined. DNA microarrays and 

quantitative RT-PCR were used to measure gene expression. 

No adverse, treatment-related effects were seen on sperm morphology or motility, testis or 

epididymis weights, or testis or thyroid histopathology. Relative liver weights were increased by 

27% at 115 mg/kg/day with no effect on body weights. At 50 mg/kg/day, 3/5 rats were found 

with mild centrilobular hepatocyte hypertrophy, while animals dosed at 115 mg/kg/day displayed 

mild centrilobular to panlobular hepatocyte hypertrophy, with 1/5 having moderate midzonal 

hepatocyte vacuolation. Mean serum testosterone levels were elevated at 50 and 115 mg/kg, but 

did not reach statistical significance. No changes in LH, FSH or estradiol were observed. 

Triadimefon affected the expression of numerous genes including CYP genes and genes involved 

in steroid hormone metabolism. 

Developmental Neurotoxicity Study (Rat) 

In a developmental neurotoxicity study (MRID 47377101), triadimefon (95.7% a.i.) was 

administered in the diet to groups of 30 mated female Wistar rats/dose at nominal dose levels of 

0, 100, 300, or 800 ppm (equivalent to 0, 8.0, 23.9, and 71.3 mg/kg/day) from GD 6 through 

LD 21. Doses were adjusted during lactation to achieve a more consistent dosage throughout 

exposure. Dams were allowed to deliver naturally and were killed on lactation day (LD) 21, 

following weaning of their respective litters. Any females that were found to be sperm positive 

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and/or with a vaginal plug, but did not deliver, were sacrificed on GD 24. On PND 4, litters were 

standardized to 8 pups/litter (4/sex); the remaining offspring and dams were sacrificed and 

discarded without further examinations. Subsequently, 1 pup/litter/group (at least 

10 pups/sex/dose when available) was allocated to subsets for FOB, motor activity, acoustic 

startle response, learning and memory evaluation, and neuropathological examination. 

No treatment-related effects were observed on mortality, clinical signs of toxicity, food 

consumption, FOB parameters, or reproductive parameters in the dams. At 800 ppm, treatmentrelated effects were limited to decreases in body weights on GD 13 and LD 0 (5% each, p<0.05), 

and body weight gain during GD 0–20 (6%, not significant). No treatment-related effects on 

viability/survival, litter parameters, pre-weaning clinical signs, sexual maturation, age of onset of 

surface righting or pupil constriction, FOB parameters, motor activity, water maze, 

ophthalmoscopic lesions, gross or microscopic lesions, or brain morphometric measurements 

were observed in the F1 pups. 

At 800 ppm, deviated snout was noted in 3 females and 1 male during post-weaning. Preweaning body weights (males and females combined) were decreased (not significant, NS) by 

4-5% on PND 11–21; combined body weight gains were decreased (p<0.05) by 6–7% on 

PND 4–11 and 4–17; and overall (PND 0–21) combined body weight gains were decreased by 

5%. Post-weaning body weights were decreased (p<0.05) by 5–7% in the males during the last 

2 weeks of the study, and by 8% (NS) at termination. During the auditory startle reflex 

habituation test, a treatment-related increase (p<0.05) in total session (blocks 1–5) peak 

amplitude was observed in the females (54%) on PND 60. Increased (47–70%) peak amplitude 

was noted during all test blocks in this group compared to controls; however, statistical 

significance (p<0.05) was only achieved during Block 5 (↑70%). During the passive avoidance 

test, an increase (p<0.05) in the mean number of trials to criterion (2.8 treated vs. 2.1 controls) 

was noted in the females during the retention phase on PND 30. 

Developmental Toxicity Study (Rat)

In a developmental toxicity study (MRID 00089023), groups of 19–21 presumed pregnant 

female Sprague-Dawley rats were administered triadimefon in 0.5% Cremophor EL emulsion 

daily by oral gavage (10 mL/kg) from GD 6–15 at 0, 10, 25, 50, or 100 mg/kg/day. All rats were 

observed daily for clinical signs of toxicity, and body weight and food consumption were 

measured. The rats were sacrificed on GD 20 and the uterus was removed along with the ovaries. 

The numbers of corpora lutea, live fetuses, resorption sites, dead fetuses, sex ratio of fetuses, 

weights of live fetuses and placentae, deformed fetuses, and the number types of external 

morphological abnormalities were recorded. One half of the live fetuses from each litter were 

fixed and prepared for skeletal examination. The remaining half were fixed and examined for 

visceral abnormalities. 

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Maternal toxicity was evident at dosage levels of ≥25 mg/kg/day; toxicity was manifested as 

increased motor activity and/or depression of maternal weight gain. The degree and duration of 

these increased activity episodes were dose-related. The decreases (p<0.05) in body weight gain 

in the 50 and 100 mg/kg/day groups during the entire course of pregnancy did not differ 

significantly between any treated group and the control group. Food consumption was unaffected 

by treatment. No treatment-related effects were observed on the numbers of corpora lutea, 

implants and live fetuses, sex ratio, placental weights and fetal mortality. No external 

abnormalities were observed. Dilatation of the renal pelvis was observed in all groups at low 

frequencies, and did not appear to be treatment-related as it also occurred in the control group. 

Two fetuses from two different litters in the 100 mg/kg/day group were found to have cleft palate 

(a total of 139 fetuses were examined). The 50 and 100 mg/kg/day groups showed significantly 

increased incidence of the presence of fourteenth (lumbar) ribs (49% and 92% treated vs. 11% 

controls). The authors also stated that there was a treatment-related significantly higher degree of 

skeletal ossification (coccygeal vertebrae) in the 100 mg/kg/day group. 

In another developmental toxicity study (1982, MRID 00149336), groups of 26 presumed 

pregnant female Sprague-Dawley rats were administered triadimefon in 0.5% Cremophor EL 

emulsion daily by oral gavage (10 mL/kg) from GD 6–15 at 0, 10, 30, or 90 mg/kg/day. Each 

dam was observed twice daily for adverse reactions or toxicological responses to the test 

substance. Dams were weighed on GD 6, 13, 15, and at termination. On GD 20, the dams were 

killed and their viscera examined grossly. The uterine contents and ovaries were examined and 

the number of corpora lutea were counted. All fetuses were counted, numbered, weighed, and 

checked for viability. Live fetuses were examined for external abnormalities. One half of the 

viable fetuses from each litter were dissected and examined for visceral anomalies. The other 

half of the fetuses from each litter ware processed for skeletal examination. 

All dams survived to termination, and there were no treatment-related clinical signs of toxicity. 

At necropsy, hydronephrosis involving one or both kidneys was the most commonly observed 

finding in all groups. Pregnancy rates were unaffected by treatment. No differences in body 

weight gain were noted in any group prior to dosing. The 90 mg/kg/day group showed a decrease 

(p<0.05) in body weight gain during the dosing period, but rebounded to approximately the same 

level as the controls post-dosing. The mean number of corpora lutea, uterine implantations, 

nonviable implants, late resorptions, viable fetuses, litter sizes, and sex ratio were comparable for 

all groups examined. No dead fetuses were recorded. Mean fetus weights were comparable 

across all groups. Fetuses treated with 90 mg/kg/day were found to have increased (p<0.05) 

incidences of rib anomalies and distended urinary bladder. 

Developmental Toxicity Study (Rabbit) 

In a developmental toxicity study (MRID 41446201), groups of 20 artificially inseminated 

female American Dutch rabbits, were administered triadimefon in 0.5% carboxymethylcellulose 

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+ 0.4% Tween-80 by daily oral gavage (5 mL/kg) during GD 6–18 at dose levels of 0, 20, 50, or 

120 mg/kg/day. All rabbits were checked daily for mortality and clinical signs of toxicity. 

Individual body weights were recorded on GD 6, 10, 14, 18, 21, and 28. Food consumption was 

recorded on GD 1, 6, 7, 12, 15, 19, 23, and 28. All does were sacrificed on GD 28, and subjected 

to Caesarean section and subsequent gross necropsy. The following maternal 

examinations/observations were made: inspection of ovaries, recording of individual intact 

gravid uterine weights, gross examination of thoracic and abdominal cavities, recording of the 

number of corpora lutea, the number/placement of implantations, early/late resorptions, and the 

number of 1ive/dead fetuses. The placentas were blotted dry and weighed. Tissues/organs with 

gross lesions were preserved in 10% modified Millonig’s buffered formalin for possible future 

histologic evaluation. All fetuses were removed from gravid uteri and were subjected to the 

following external observations: recording of individual weight, sex, and gross abnormalities 

(frontal, dorsal, and lateral views of head; size/position of pinnae; bulging of eyes; irregular 

contour and visceral herniations on torso; limbs position; number of digits on fore/hind paws). 

This was followed by internal observations of the abdominal and thoracic viscera and of the sex. 

All fetuses were finally processed for skeletal examination, and the skull, vertebrae, ribs, pelvis, 

appendages, scapulae, clavicles, and sternum were examined. 

No treatment-related mortalities or clinical signs of toxicity were observed. A marginal, transient 

(GD 6–10), but significant loss of body weight and a very transient (GD 7) but significant 

decrease in food consumption were observed in the 120 mg/kg/day group. The biological 

significance of the former effect was considered equivocal since it only occurred during the first 

four days of dosing and was unaccompanied by any concomitant decrease in body weight. The 

latter effect was too short lasting to have any real biological significance. No treatment-related 

gross pathological alterations were observed. The pregnancy rates, gestation indexes, average 

numbers of corpora lutea, implantations, percentages of pre- and post-implantation losses, 

resorptions, fetuses/litter, viable number of fetuses/doe, male and combined fetal body weight, 

and percentage of male fetuses/doe were comparable between groups. 

Developmental toxicity was observed at 50 and 120 mg/kg/day. Dose-related increases in both 

the fetal and litter incidences of several skeletal alterations were observed at 50 mg/kg/day. 

These included incomplete ossification of the pelvic pubes and the anterior and posterior 

phalanges, and irregular spinous process formation. The increases in incidence of each fetal 

alteration was statistically significant (litter incidence increases were not statistically analyzed). 

Significant decreases in placental weight and fetal female body weight, and significant increases 

in the incidences of many external and skeletal alterations were observed at 120 mg/kg/day. 

External alterations consisted of rudimentary or missing tails (2 litters; 10 fetuses). Skeletal 

alterations with only a fetal incidence increase included incomplete ossification of the skull 

bones and enlargement of the fontanelles. Those with increases in both fetal and litter incidences 

included extra ribs, malformations of the arches and centra of the caudal vertebrae, 

incomplete/non ossification of the 1st, 2nd, 5th sternebrae, posterior talus, and posterior 

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phalanges. Those with dose-related increases in both fetal and litter incidences were mentioned 

earlier. The increases in the incidence of each fetal alteration was significant. Except for extra 

ribs, the litter incidences increases were not statistically analyzed. 

Subchronic Toxicity Study (Rat)

In a subchronic toxicity study (MRID 00048624), groups of 15 rats/sex were administered 

triadimefon in the diet at 0, 50, 200, 800, or 2000 ppm (equivalent to 0, 2.5, 10, 40 and 100 

mg/kg/day) for 12 consecutive weeks. Daily physical observations were performed, and body 

weights and food consumption were measured weekly. Hematology, clinical chemistry and 

urinalysis were performed prior to dosing, at Week 6, and at termination. At the end of the study, 

autopsies were performed, and tissues were saved for histopathological examinations. 

No animals died due to the administration of the test material. No clinical signs of toxicity were 

observed. Body weights and food consumption were decreased in the high dose group; this was 

considered to have resulted from palatability issues with the high dose dietary formulation. There 

were no treatment-related effects observed on hematology, clinical chemistry, or urinalysis. No 

treatment-related changes were seen in absolute or relative weights of the testes, ovaries, thyroid, 

adrenal or pituitary glands. No treatment-related histopathological lesions were seen in the testes, 

thyroid, adrenal, or pituitary glands. 

In another subchronic rat toxicity study (Wolf et al., 2006; MRID 48264601), male Wistar/Han 

rats were treated with 100, 500, 1800 ppm triadimefon, 100, 500, 2500 ppm propiconazole, or 

100, 500, 2000 ppm myclobutanil in the feed for four, 30, or 90 days. The rats were evaluated for 

clinical signs, body and liver weight, histopathology of thyroid and liver, hepatic metabolizing 

enzyme activity, and serum T3, T4, TSH, and cholesterol levels. 

There was a dose-dependent increase in liver weight but not body weight for all treatments. The 

indication of cytochrome induction, pentoxyresorufin O-dealkylation (PROD) activity, had doserelated increases at all time points for each conazole. Uridine diphopho-glucuronosyl transferase 

(UDPGT), the T4 metabolizing enzyme measured as glucuronidation of 1-naphthol, was induced 

to the same extent after 30 and 90 days for all three conazoles. Livers from all high dose 

(90 mg/kg/day) treated rats had centrilobular hepatocyte hypertrophy after four days, while only 

triadimefon and propiconazole treated rats had hepatocyte hypertrophy after 30 days, and only 

triadimefon treated rats had hepatocyte hypertrophy after 90 days. Thyroid follicular cell 

hypertrophy, increased follicular cell proliferation, and colloid depletion were present only after 

30 days in rats treated with the high dose of triadimefon. A dose-dependent decrease in T4 was 

present after 4 days with all three compounds but only the high doses of propiconazole and 

triadimefon produced decreased T4 after 30 days. T3 was decreased after high-dose triadimefon 

after four days and in a dose-dependent manner for all compounds after 30 days. Thyroid 

hormone levels did not differ from control values after 90 days and TSH was not increased in 

any exposure group. A unique pattern of toxic responses was not identified for each conazole and 

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the hypothesized mode of action for triadimefon-induced thyroid gland tumors was not supported 

by the data. 

Subchronic Toxicity Study (Dog)

In a subchronic toxicity study (MRID 00048625), groups of four beagle dogs/sex were 

administered triadimefon in the diet at 0, 150, 600, or 2400 ppm (equivalent to 0, 3.75, 15 and 60 

mg/kg/day) for 13 weeks. Daily physical observations were performed, and body weights were 

measured weekly. Ophthalmoscopic examinations, hematology, clinical chemistry and urinalysis 

were performed prior to dosing, at Week 6, and at termination. At the end of the study, autopsies 

were performed, and tissues were saved for histopathological examinations. 

All dogs survived to scheduled termination. No clinical signs of toxicity were observed. Body 

weights and food consumption were decreased in the high dose group; this was considered to 

have resulted from palatability issues with the high dose dietary formulation. At 60 mg/kg/day, 

hematocrit, erythrocyte count and hemoglobin volume were decreased, and alkaline phosphatase 

was increased. It was stated that the necropsies were unremarkable, and that there were no 

histopathological findings indicative of liver damage. No treatment-related changes were seen in 

absolute or relative weights of the testes, prostate, ovaries, thyroid, or adrenal glands. No 

treatment-related histopathological lesions were seen in the testes, epididymides, prostate, 

ovaries, uterus, thyroid, adrenal, or pituitary glands. 

Combined Chronic Toxicity/Carcinogenicity in Rats 

In a combined chronic/carcinogenicity study (MRID 42153901) triadimefon (94.4% a.i.) was 

administered in the diet to 50 Wistar Bor:WISW rats/sex/dose at dose levels of 0, 50, 300, or 

1800 ppm (equivalent to 2.5, 15, and 90 mg/kg/day in males/females) for up to 104 weeks. In 

addition, an interim group of 10 rats/sex/dose was similarly treated for 52 weeks. 

In the 90 mg/kg/day females, survival was slightly decreased (NS) at Weeks 78, 90, and 105 

(↓9–14%). Decreases (p<0.05) in body weights were observed from Week 1 until study 

termination (↓5–12%). Cumulative body weight gain was decreased at Weeks 0–53 (↓15%) and 

0–104 (↓23%). In the 90 mg/kg/day group, food consumption was increased (p<0.05) throughout 

the study (↑12–34% males; ↑34–67% females), resulting in increased overall mean food 

consumption (↑19% males; ↑53% females). 

The liver was a target organ. In the 90 mg/kg/day females, increased (p<0.05) serum cholesterol 

was observed at Weeks 26, 53, and 78 (↑24–32%), and absolute and relative (to body) liver 

weights were increased (p<0.05; ↑15% and ↑27%, respectively) at Week 52. At Week 104, 

absolute and relative liver weights were dose-dependently increased (p<0.05) in the females at 

300 ppm and above (↑11–17% and ↑8–32%, respectively). A dose-dependent increase in fat in 

the hepatocytes was observed in the males and females at 90 mg/kg/day (p<0.05; 44–60% treated 

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vs. 6–16% controls). Additionally, a positive trend was found in the incidence of fat in the 

hepatocytes of females. In the 90 mg/kg/day males, alanine aminotransferase was increased 

(p<0.05) by 22% at Week 26 and by 37% at Week 104. 

Treatment-related non-neoplastic and neoplastic lesions of the thyroid glands seen at the high 

dose were: follicular cell cystic hyperplasia in 4/50 (8%) at the high dose females vs. 2/50 (2%) 

in the control females; however, the increase did not reach statistical significance. Follicular cell 

adenomas were observed in treated males 3/49 (6%) compared to 0/50 (0%). The incidences in 

males showed a significant trend and exceeded the historical control mean (5.2%). 

Carcinogenicity Study (Mouse)

In a carcinogenicity study (MRID 40752101), groups of 60 NMRI mice/sex were administered 

triadimefon in the diet at dose levels of 0, 50, 300, or 1800 ppm (equivalent to 0/0, 13.5/19.6, 

76.0/119.4, and 550.1/765.0 mg/kg/day in males/females) for up to 21 months. Mice were 

observed for mortality and morbidity twice daily; detailed physical examinations were performed 

weekly. Body weights were recorded at study initiation, weekly during Weeks 1–12, and 

bimonthly from Week 15 to study termination. Food and water consumption were recorded at the 

same time at the body weight measurements. Hematology and clinical chemistry parameters 

were measured at Weeks 53 and 90/91. All animals that died or were sacrificed on schedule were 

necropsied. A standard suite of organs/tissues were collected, weighed, and examined 

microscopically. 

There was an increase (p<0.05) in hepatocellular adenomas in high-dose males and females; 

however, there was no increase in liver carcinomas. There was no effect of dosing on survival, 

clinical signs, food or water consumption, or hematology parameters. Mean body weights and 

overall body weight gains were decreased in high-dose (765 mg/kg/day) males (BW ↓6–15%; 

BWG ↓22%). There were nonneoplastic and preneoplastic changes in the liver as well as 

increases in liver weights and correlating effects on serum enzymes. Hepatocellular hypertrophy 

was increased (p<0.01) in males receiving 300 or 1800 ppm and in females receiving 50, 300, or 

1800 ppm. Hyperplastic nodules, altered cell foci, and microvesicular fatty changes were 

increased in both sexes (p<0.01) at 1800 ppm. Kupffer cell proliferation, lipofuscin pigment in 

liver macrophages, and single cell necrosis were increased significantly (p<0.01) in males 

receiving 1800 ppm and females receiving 300 and 1800 ppm. At study termination, liver 

weights were increased (p<0.01) in mid- and high-dose males (↑19–64%) and in high-dose 

females (↑69%) when compared to controls. Kidney weights were decreased (p<0.01) by 16% in 

high-dose males. Serum alkaline phosphatase, aspartate aminotransferase, and alanine 

aminotransferase were increased in both sexes receiving 1800 ppm. The increases were 

significant compared to controls for all enzymes at 91 weeks and for some at 53 weeks. No 

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related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, 

ovaries, uterus, mammary glands, thyroid, adrenal or pituitary glands. 

Chronic Toxicity Study (Dog)

In a chronic toxicity study (MRIDs 00032539 and 00126261), MEB 6447 (triadimefon; 88.9–

92.7% a.i.) was administered to four beagle dogs/sex/dose in the diet for up to 24 months at 

nominal doses of 0, 100, or 330 ppm (equivalent to 0, 3.03/3.49, and 11.42/11.96 in 

males/females). In addition, the dogs were treated with a high dose of 1000 ppm during Weeks 

1–54 (equivalent to 34.70/33.67), that was subsequently increased to 2000 ppm (equivalent to 

68.80/60.42) during Weeks 55–104. 

No treatment-related effects were observed on mortality, clinical signs, ophthalmology, 

hematology, and histologic (including tumors) pathology. At 2000 ppm during Weeks 54–62, 

body weight losses of 0.7 kg and 0.4 kg were noted in males and females, respectively, resulting 

in decreased overall body weight gains for both males (↓20%) and females (↓11%). At the high 

dose, decreased food consumption was observed in females throughout the study (↓5–18%). 

Increased alkaline phosphatase levels were observed at 1000 ppm in males (↑12–92%) and 

females (↑17–55%) and at 2000 ppm in males (↑352–491%) and females (↑121–393%) 

throughout the respective treatment periods. Bile duct concretions were observed in 3/4 high 

dose males. Increased absolute and relative (to body) liver weights (↑16–25%) were observed in 

the males. Cholesterol was increased in the 1000 (↑8–27%) and 2000 (↑31–63%) ppm females. 

Gene Expression Profiling Studies

In a gene expression profiling study (Goetz and Dix, 2009a), key biological pathways affected 

by triazoles in the liver and testis following exposure from gestation through adulthood were 

identified in order to better understand triazole-induced disruptions in testosterone homeostasis. 

Using tissue samples from a previous study (Goetz et al., 2007; MRID 48264603), gene 

expression changes were assessed in the male rat liver and testis following exposure to 

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

(PND) 92 to investigate if triazoles disrupt testosterone homeostasis by increasing expression of 

genes involved in testosterone synthesis in the testis and decrease expression of genes involved 

in testosterone metabolism within the liver. 

Gene expression profiles indicated that all three triazoles significantly perturbed the fatty acid, 

steroid, and xenobiotic metabolism pathways in the male rat liver. In addition, triadimefon 

modulated expression of genes in the liver from the sterol biosynthesis pathway. Although 

individual gene expression was affected, there were no common pathways modulated by all three 

triazoles in the testis. The pathways identified in the liver included numerous genes involved in 

phase I–III metabolism (Aldh1a1, Cyp1a1, Cyp2b2, Cyp3a1, Cyp3a2, Slco1a4, Udpgtr2), fatty 

acid metabolism (Cyp4a10, Pcx, Ppap2b), and steroid metabolism (Ugt1a1, Ugt2a1), for which 

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expression was altered by the triazoles. These differentially expressed genes form part of a 

network involving lipid, sterol, and steroid homeostatic pathways regulated by the constitutive 

androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated alpha, 

and other nuclear receptors in liver. The relatively high dose and long-term exposures to triazole 

antifungals appeared to perturb fatty acid and steroid metabolism in the male rat liver 

predominantly through the CAR and PXR signaling pathways. 

In a second gene expression profiling study (Goetz and Dix, 2009b), biological pathways 

consistently modulated across multiple time points and various study designs were identified. 

Gene expression profiling was conducted on rat livers from three separate studies with triazole 

treatment groups ranging from 6 hours after a single oral gavage exposure, to prenatal to adult 

exposures via feed. To explore conservation of responses across species, gene expression from 

the rat liver studies were compared to in vitro data from rat and human primary hepatocytes 

exposed to the triazoles. Toxicogenomic data on triazoles from 33 different treatment groups and 

135 samples (microarrays) identified thousands of probe sets and dozens of pathways 

differentially expressed across time, dose, and species. Many of these were common to all three 

triazoles, or conserved between rodents and humans. Common and conserved pathways included 

androgen and estrogen metabolism, xenobiotic metabolism signaling through CAR and PXR, 

and CYP related metabolism. Differentially expressed genes included the Phase I xenobiotic, 

fatty acid, sterol and steroid metabolism genes Cyp2b2 and CYP286, Cyp3al and CYP3A4, and 

Cyp4a22 and CYP4A11; Phase II conjugation enzyme genes Ugtlal and UGT1Al; and Phase III 

ABC transporter genes Abcbl and ABCBI. Gene expression changes caused by all three triazoles 

in liver and hepatocytes were concentrated in biological pathways regulating lipid, sterol and 

steroid homeostasis, identifying a potential common mode of action conserved between rodents 

and humans. 

Avian Toxicity and Reproduction (OCSPP 850.2200/850.2300) 

In an avian reproduction study (MRID 00110431), bobwhite quail were fed triadimefon in the 

diet at nominal concentrations of 20, 100 and 500 ppm, along with a control. The birds were 34 

weeks old and weighed 160–200 g at the beginning of the study. Each test level had 24 cages 

with one female and one male per cage. Fresh diet was prepared each week and stored frozen 

until used. Body weights were recorded at initiation, at Weeks 2, 4, 6, 8, and at the end of the 

study. Food consumption was measured weekly. Eggs were collected for incubation twice daily 

during Weeks 10–25. On incubation Day 21, the eggs were placed in a hatcher. On Day 26, 

hatchlings were removed from the hatcher, weighed and then housed according to parental test 

group. Observation of quail continued for 14 days post-hatching. Egg shell thickness was 

measured from eggs taken from each laying female during Weeks 1, 3, 5, 7, and 9 of egg laying. 

Gross postmortem examinations were performed on birds that died during the study and on 20 

randomly selected pairs from each test group. 

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Five birds died during the study, these were not considered to be compound-related. At 100 and 

500 ppm, there was an increase in number of cracked eggs, and decreases in the number of fertile 

eggs, viable embryos, hatchlings, and 14-day survival. 

In another avian reproduction toxicity study (MRID 42342302), groups of pen-reared, unmated 

mallard ducks were housed in 15 pens (1 duck/sex/pen) and exposed to triadimefon in the diet at 

nominal concentrations of 50, 200, or 800 ppm from test initiation until terminal sacrifice. Adult 

birds were observed at least once daily throughout the study for signs of toxicity, injuries, or 

illness. Mortalities occurring prior to terminal adult sacrifice were recorded and necropsied. 

Adult body weights were measured at study initiation, weekly through Week 8, and at terminal 

sacrifice (Week 20). Feed consumption was measured by cage weekly throughout the adult phase 

of the study. Eggs were collected twice daily, labeled according to pen of origin, and washed to 

prevent pathogen contamination. The eggs were removed from the egg cooler weekly and 

candled for shell cracks. Cracked eggs were recorded and discarded. Eggs not cracked or used 

for eggshell thickness measurements were placed in an incubator. Eggs were candled again on 

incubation Day 14 to determine fertility and embryo viability, and on Day 21 to determine 

embryo surviva1. Eggs were collected every other week for eggshell strength and thickness 

measurements. Eggs were then opened, the contents removed, the shell washed, and allowed to 

air dry. The average thickness of the dried shell plus membrane was determined. Hatchlings were 

removed from the hatcher on Days 27 and 28 of incubation, banded, weighed, and housed 

according to week of hatch and parental treatment group. Hatchlings were identified by 

numbered wing bands and placed in batteries of brooding pens from the time of hatch through 

Day 14 post-hatch. Hatchlings were fed untreated diet. Hatchling body weights were measured 

and recorded at hatch and on Day 14. Feed consumption was not monitored. Hatchlings were 

observed twice daily throughout the 14- day period for signs of toxicity, injuries or illness. 

Mortalities occurring prior to the end of the 14-day period were recorded and discarded. 

Hatchlings surviving to Day 14 were sacrificed, weighed, and discarded. 

Mean measured concentrations were 75%, 96%, and 86% of nominal concentrations for the 50-, 

200-, and 800-ppm diets, respectively, after storage for 7 days at room temperature. Only one 

mortality occurred during the study, and it was not treatment related. No treatment-related adult 

mortality occurred in any test group during the course of the study. Occurrences of feather loss 

and lacerations were observed, but were attributed to normal laboratory pen housing. No overt 

signs of intoxication were observed during the study in any test group. Post-mortem observations 

revealed anomalies however, they occurred in a small number of birds and no dose-response was 

indicated. There were no apparent treatment-related effects upon adult body weight at any of the 

concentrations tested. There was a significant decrease in feed consumption during Weeks 5 and 

14 in the 185 ppm group, but these findings were incidental. When compared to the control 

group, there were no statistically significant differences in reproductive parameters at any 

concentration tested. There were no significant differences in the overall mean eggshell thickness 

and strengths of control eggs compared to treatment eggs. The overall mean hatch weight and the 

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overall 14-day survivor weights were not significantly lower for birds in treatment groups when 

compared to controls. Mean hatch weight for hatchlings in the 185 ppm group was significantly 

higher than controls, but this difference was not considered treatment-related due to the lack of a 

distinct dose-response relationship. There were no apparent effects on adult ducks fed 

triadimefon-treated diets. The LOAEC for mallard ducks is greater than 780 ppm of triadimefon. 

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

In a fish early life stage toxicity study (MRID 00110433), rainbow trout embryos (230 to 243) 

were exposed to triadimefon at nominal concentrations of 0, 50, 150, 450, 1350, and 4050 ppb 

under flow-through conditions. The embryos at each level were divided into two groups and 

tested in separate 5 gallon containers. The containers were aerated. Temperature was measured 

twice daily. The flow rate was 75 L/vessel per day. The concentrations were verified by chemical 

analysis periodically. The photoperiod was 16 hours light/8 hours dark. The embryos were 

exposed for 3 to 9 days before they hatched. 

Although the test did not follow the accepted protocol for fish embryo-larvae study, it indicated 

that triadimefon is highly toxic to trout in the early life stages, with a 17-day LC50 of 1270 ppb. 

In another fish early life stage toxicity study (MRID 00131105), rainbow trout embryos were 

exposed to triadimefon at nominal concentrations of 0, 10, 30, 90, 270 and 810 ppb. The 

concentrations were verified by chemical analysis at intervals throughout the test. The mean 

analytical concentrations were 23, 41, 116, 300, and 890 ppb. For each concentration 100 eggs 

were used, divided between four test chambers. The exposure period was 60 days. 

Although there was no concentration-related effect on hatching success, incubation time was 

decreased by approximately one-half day in the highest concentration. There was no effect on 

time to swim up. Sixty-day cumulative mortality was significantly greater than control mortality 

only for the 890 ppb group. Median lethal concentration was 717 ppb. On Days 19 and 20, 

mortality was unusually high (62% for the 2 days) in the 810 ppb group. Dissolved oxygen 

reached its lowest concentration on those two days (65%) but remained at levels not ordinarily 

lethal to juvenile trout. Clinical signs observed included increased or decreased pigmentation, 

abnormal swimming, spinal curvature, tremors, edema, loss of equilibrium fin erosion, partial 

agnathism and. general weakness. Of the 60-day survivors, those in the 890, 300 and 116 ppb 

groups were significantly lighter than controls, and the magnitude of the difference was 

concentration- related. Only the two highest concentrations produced significantly shorter 

survivors. Triadimefon had no discernable effect on hatching success. Growth in terms of final 

length and/or weight was significantly reduced in at 116, 300 and 890 ppb of triadimefon. The 

lowest lethal concentration was 890 ppb. The observed LOAEC was 0.116 mg/L based on 

reduced growth, and the NOAEC was 0.041 mg/L. 

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Fish Early Life Stage Toxicity Study (Fathead Minnow) (OPPTS 850.1400)

In a fish early life stage toxicity study ( MRID 41922103), fathead minnow embryos were 

exposed to triadimefon at nominal concentrations of 0.075, 0.15, 0.3, 0.6, or 1.2 mg a.i./L, and 

the mean measured concentrations were 0.077, 0.17, 0.27, 0.57, and 1.1 mg a.i./L, respectively 

(90–113% of the nominal exposure concentrations). A dilution water control and a solvent 

control (0.012 mL of DMF/L, equal to the highest concentration of solvent at the highest test 

level) were also included. Twenty embryos (approximately 24 hours post-spawn) were 

impartially distributed in groups of two to each incubation cup in each replicate chamber (i.e., 80 

embryos/concentration). Egg mortality in each cup was recorded and dead eggs removed daily. 

Once hatching was complete (Day 7), the fry were released into the test chambers. Test 

chambers were inspected daily and observations of abnormal physical changes, abnormal 

behavior, and mortality were recorded. Dead fry were discarded. At termination (Day 35), 

surviving fish were sacrificed, and blotted wet weight and standard length for individual fish was 

recorded. Hatchability and total survival data were collected along with growth measurements. 

Water quality parameters were measured on Days 0, 1, 7, 14, 21, and 35. Analytical 

concentrations were determined for on Days 0, 1, 7, 14, 21, 28, and 35. 

The egg hatch began on Day 3 and was complete by Day 7, and was not affected by treatment. 

No significant reduction in hatch at any test level was indicated when compared to the pooled 

control. At test termination, survival ranged from 65 to 95% in the control and 80 to 95% in the 

solvent control. No significant difference was detected between the two controls. Survival at the 

two highest test concentrations (0.57 and 1.1 mg a.i./L) was significantly reduced when 

compared to the pooled control. At test termination, mean standard length and mean wet weight 

were significantly reduced only at the 0.27 mg a.i./L test level when compared to those of the 

pooled control. Growth data for the two highest test concentrations (0.57 and 1.1 mg a.i./L) were 

excluded from the statistical growth analysis, since survival was significantly reduced at these 

concentrations and no data were collected from the highest test concentration since mortality was 

100%. In general, morphological and behavioral abnormalities were sporadically observed in the 

control, vehicle blank and the three lowest test levels and involved only a few fish. 

Abnormalities in the two highest test levels were noted primarily during the first week following 

the completion of hatch and were considered dose related. This week was also a period of heavy 

fry mortality for these two levels. Based on these results for survival and growth, the NOEAC 

was 0.17 mg/L and the LOEAC was the 0.27 mg/L. 

Thyroid Hormone Levels and Gene Expression (Zebrafish) 

In this study, Liu et al. (2011) exposed embryo-larval wild-type (AB stain) zebrafish, raised 

under standard laboratory conditions, to varying concentrations of triadimefon. Embryos at a 

stage of 6 hours post-fertilization) were transferred into glass beakers containing 500 mL of 

triadimefon solution (0, 0.5, 1, 2, or 4 μg/mL) until 120 hours post-fertilization. There were 

3 replications for each exposure concentration. 

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The zebrafish exposed to 2 and 4 μg/mL triadimefon exhibited bent spine and uninflated swim 

bladder from (results not presented). The T3 content in the larvae (whole body) exposed to 

2 μg/mL triadimefon was 1.22 ± 0.06 ng/g, which was significantly less than the controls 

(1.55 ± 0.02 ng/g). In Figure 2, T3 whole body content in larvae at 4 μg/mL triadimefon was also 

significantly less than controls. In contrast, the embryos exposed to increasing triadimefon 

concentrations had significantly higher T4 content at 2 and 4 μg/mL than in the controls at 

120 hpf (whole body values; graphically shown in Figure 2). The expression of genes in the HPT 

axis of the zebrafish larvae were determined at 120 hours post-fertilization. The beta-subunit of 

TSH (TSH-β) gene transcription was significantly upregulated (↑4.0- and ↑5.2-fold) upon 

exposure to 2 and 4 μg/mL triadimefon, respectively. Two isoforms of deiodinases (dio1 and 

dio2) were identified. Triadimefon exposure significantly downregulated dio1 gene transcription 

(1.5-fold) in the 2 μg/mL exposure group. However, the levels of dio2 gene transcription were 

not altered in any other triadimefon-treated groups. The mRNA expressions of the genes for the 

two isoforms of the thyroid hormone nuclear receptors (thraa and thrb) were evaluated. There 

were no significant changes in the mRNA expression of thraa. However, the thrb gene 

transcription was significantly down-regulated (↓2.6-fold) after exposure to 2 μg/mL 

triadimefon. 

Organ Morphogenesis Study (Frog)

In an organ morphogenesis study (Lenkowski et al., 2010), Xenopus laevis tadpoles (NF stage 

41) were cultured in modified Marc’s Modified Ringer’s solution (MMR) at a density of 13–18 

tadpoles in 10 mL of MMR maintained at 18°C. Groups of tadpoles were exposed in triplicate to 

triadimefon at working concentrations of 7.29, 21.88, 36.47, 51.05 mg/L for 48 hours, with static 

renewal at 24 hours. Tadpoles were also exposed to atrazine, 2-4, D, and glyphosate, but these 

chemicals will not be discussed in this summary. Tadpoles were humanely euthanized, fixed, and 

examined for intestinal malformations, edemas, and skeletal muscle degeneration. 

Mortality was 0% in the 7.29 and 21.88 mg/L triadimefon exposures, but significantly increased 

at 36.47 and 51.05 mg/L (8.5%, p<0.005; 25.4%, p<0.0001; respectively). Mortality occurred 

between 24 and 48 hours. Triadimefon exposure caused a significant increase in intestinal 

malformations and edemas at 21.88, 36.47, and 51.05 mg/L. Parallel malformations are further 

illustrated by the disruption of differentiated skeletal muscle in exposed animals. Cranial muscles 

in triadimefon exposed tadpoles were reduced as compared to non-treated controls. Additionally, 

the net-like structure of the hypaxial muscle normally encases the intestine and was disrupted in 

exposed tadpoles. These anomalies varied in severity depending on the degree of malformation 

in an individual tadpole. Therefore, higher concentrations of chemical exposure exhibited more 

dramatic disruption of these muscles. 

 

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APPENDIX 3: References Not Utilized in the Triadimefon 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

_reregistration_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 triadimefon 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. 

Hassold, E. and T. Backhaus (2009). Chronic Toxicity of Five Structurally Diverse 

Demethylase-Inhibiting Fungicides to the Crustacean Daphnia magna: A Comparative 

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

Iyer, V.V., Ovacik, M.A., Androulakis, I.P., Roth, C.M., and Ierapetritou, M.G. (2010) 

Transcriptional and metabolic flux profiling of triadimefon effects on cultured hepatocytes. 

Toxicol Appl Pharmacol. 248(3):165-177 

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