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 89
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: TEBUCONAZOLE 

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......................................5 

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

B. Other Scientifically Relevant Information (OSRI)......................................5 

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

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

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis.............................9 

1. Effects on Estrogen Pathway....................................................9 

2. Effects on Androgen Pathway..................................................17 

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

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

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

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

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

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

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

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

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

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

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

LIST OF TABLES: 

 Table 1. Tier 1 Assays for Tebuconazole.............................................................8 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Tebuconazole ................................12 

 Table 3. Androgenic/Anti-Androgenic Pathway for Tebuconazole ...............................19 

 Table 4. Thyroid Pathway for Tebuconazole ......................................................25 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................43 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................55 

APPENDIX 3. References Not Utilized in the Tebuconazole WoE Analysis........................80 

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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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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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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 receptors, 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 tebuconazole 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, the dose(s) at which responses happen and whether overt toxicity was 

present. 

On June 18, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) 

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

(OSCP) conducted a weight-of-evidence (WoE) analysis of the potential interaction of 

tebuconazole 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, although tebuconazole was negative in an estrogen receptor (ER) 

binding assay available in OSRI and in the Tier 1 ER transactivation assay (ERTA), it appears to 

alter estrogen production. In vitro studies showed tebuconazole can potentially alter 

steroidogenesis and inhibits aromatase activity. In the in vivo mammalian studies, a delay in 

vaginal opening (VO) was observed in the female pubertal assay; this finding was corroborated 

by other findings in mammalian OSRI such as decreased number of offspring and 

gestational/delivery alterations in the absence of overt toxicity. Additionally, complementary 

findings were observed in the fish short-term reproduction assay (FSTRA) which included 

effects on plasma vitellogenin (VTG), gonadal weight and histopathology. Furthermore, effects 

on gonadal histopathology were reported in a fish sexual development test. Therefore, the 

majority of the findings suggests that tebuconazole potentially interacts with the estrogen 

pathway both in vitro and in vivo. It is noted that generally, these in vivo findings co-occurred 

with liver effects that included increased weight and/or changes in histopathology. 

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For tebuconazole, effects on testosterone biosynthesis and androgen receptor (AR) interaction 

were consistently noted in the Tier 1 and OSRI in vitro studies. In the mammalian in vivo

studies, anti-androgen effects were observed in young but not adult animals. Testosterone levels 

were decreased in the Tier 1 male pubertal assay, as well as in a reproductive toxicity in the 

published literature; this concurred with decreased weights of androgen-dependent tissues. 

While there were limited findings in male fish in the FSTRA which included a decrease in 

gonadal somatic index (GSI), gonadal histopathological findings were reported in a fish sexual 

development test. The majority of the findings suggests that tebuconazole has anti-androgenic 

activity in vitro and in vivo only in the young, and can interact with the androgen receptor and 

interfere with steroidogenesis. Again, it is noted that these in vivo findings co-occurred with liver 

effects that included increased weight and/or changes in histopathology. 

Based on the available data, there is limited evidence for a direct effect of tebuconazole on the 

thyroid pathway or the HPT axis. Negative responses for the T pathway were seen for 

tebuconazole in the in vivo Tier 1 male and female pubertal assays and six of the seven pertinent 

general toxicity studies which showed no effect from tebuconazole treatment. In the published 

studies, effects were seen in thyroid hormone levels in maternal animals in developmental 

toxicity studies, but these occurred in the presence of systemic toxicity. Developmental delays 

(including NF stage and normalized hind-limb) along with thyroid hypoplasia were observed in 

the amphibian metamorphosis assay (AMA), however, this coincided with a significant reduction 

in growth (length and body weight) and liver effects. Although findings of thyroid gland 

hypoplasia are identified in the AMA guideline as diagnostic criteria, no effects were seen on the 

incidence of follicular cell hypertrophy or hyperplasia, which are expected to be more commonly 

affected in the presence of a thyroid-related developmental delay. Overall, the thyroid and 

developmental findings in the AMA with tebuconazole are inconsistent with a direct, thyroidrelated delay and may be more reflective of treatment-related growth inhibition. 

Based on in vivo and in vitro results, tebuconazole showed potential to alter the steroidogenesis 

pathway which may result in endocrine-related effects in the estrogen and androgen pathways. In 

the androgen pathway, effects seem to have a greater impact on young than in adult mammals. 

It is noted that the effects observed in mammals and fish coincided with liver effects. 

Based on the available data, there was a lack of complementary and redundancy of evidence in 

the available Tier 1 and OSRI data to suggest a potential interaction with the thyroid pathway 

either in mammals or wildlife. 

Tebuconazole showed potential to alter the steroidogenesis pathway which may result in 

endocrine-related effects in the estrogen and androgen pathways. For tebuconazole, the point of 

departure (POD) for human health risk assessment is based on a developmental neurotoxicity 

endpoint observed in offspring in a developmental neurotoxicity study in rats. The basis for the 

POD is alterations in motor activity, decreases in body weight and brain weight and changes in 

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brain morphometrics observed in the offspring at 8.8 mg/kg/day, the lowest observed effect level 

(LOAEL). Since the study did not establish a no-observed-adverse-effect–level (NOAEL), the 

default 10X Uncertainty Factor (UFL) was applied (i.e., for the use of a LOAEL) resulting in an 

extrapolated NOAEL of 0.88 mg/kg/day. This value is used as the current POD for calculating 

the chronic Reference Dose (RfD) for dietary risk assessment and calculating the Margins of 

Exposure (MOEs) for non-dietary exposure risk assessments. This POD is approximately 75-fold 

lower than the lowest doses (75 mg/kg/day) that caused androgen and estrogen-related effects in 

the Tier 1 pubertal assays. Consequently, the chronic RfD and the MOEs are protective of the 

endocrine-related effects seen in the pubertal assays. 

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

recommended for tebuconazole since additional testing will not impact the current EPA 

established regulatory endpoints for human health risk assessments. 

For the wildlife the EDSP Medaka Extended One-Generation Reproduction Test (MEOGRT) is 

recommended. In the FSTRA, effects were observed at 19.5 μg/L. Gonadal histological effects in 

the fish sexual development test (FSDT) were observed at 0.045 and 0.022 μg/L for females and 

males, respectively, in the absence of effects on liver. The current chronic NOAEC for fish is 12 

μg/L, based on survival effects in the rainbow trout early-life cycle. It is noted that no effects on 

fecundity were observed in the sheepshead minnow life-cycle study at concentrations up to 196 

μg/L; effects on survival (F0) and growth (F1) were noted. Since the reproductive effects 

observed in the FSTRA and FSDT occur at concentrations similar or below the current NOAEC, 

additional information on potential reproductive effects is recommended. 

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard 

duck are available for tebuconazole. In both species, effects on reproductive parameters were 

observed. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) 

are considered sufficient for evaluating potential reproductive effects to birds from tebuconazole 

exposure. Relative to the EDSP, additional testing is not recommended. 

 

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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 receptors, 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 tebuconazole 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 June 18, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) 

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

(OSCP) conducted a weight-of-evidence analysis (WoE) of the potential interaction of 

tebuconazole with the E, A or T hormone 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).

Tebuconazole is a systemic triazole fungicide whose mode of action is through inhibition of the 

C-14 demethylation step within fungal sterol biosynthesis. Tebuconazole’s solubility in water is 

36 mg/L at 20°C. The vapor pressure of tebuconazole is 1.3 x 10−8

 mm Hg at 20°C (HSDB, 

2010) and volatilization of tebuconazole is not expected (estimated Henry's Law of 1.4  10−10

atm-m3

/mole). Tebuconazole is stable to hydrolysis and is not expected to undergo photolysis. 

Tebuconazole is expected to have moderate to limited mobility in soil and adsorb to suspended 

solids and sediment. Its log Kow is 3.7. 

The available information considered in determining the potential interaction of tebuconazole 

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

relevant information (OSRI) such as general toxicity studies and other published articles. These 

data are summarized in Sections III.A through III.C. An analysis of the data submitted to the 

agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in 

Section IV. The EDSP Tier 2 study recommendations are presented in Section V. 

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II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

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

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

1. 

B. Other Scientifically Relevant Information (OSRI) 

In response to the Agency’s Test Orders, data 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 tebuconazole (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 (SAP0 (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 11 assays 

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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 

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, under what 

conditions did 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. 

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

the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of 

the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “In summary, the 

Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in the 

presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in terms 

of primary interactions with the endocrine system vs. secondary effects related to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s current 

approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological 

risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential 

evidence of overt toxicity included, but were not limited to: mortality; clinical signs such as 

tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight 

decreases of ≥10% in mammals. Additionally, other effects including morphological (e.g., organ 

weights/histopathology), biochemical (e.g., blood chemistry), and other clinical signs (e.g., 

lethargy) were also considered when evaluating overt toxicity, especially if the effects were 

extreme. In some instances, one parameter (i.e., death or >10% decrease in mammalian body 

weight) was sufficient to consider a dose/concentration to be overtly toxic. However, in other 

instances, more than one parameter was needed to determine overt toxicity. For example, in the 

FSTRA, generally, body weight decreases were considered along with other responses when 

assessing potential overt toxicity. Additionally, effects which were considered to be signs of 

systemic toxicity were also captured and these effects were generally considered as less severe 

forms of toxicity (e.g., 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 

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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. 

 A. EDSP T1 Screening Assays 

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

below in Table 1. 

Table 1. Tier 1 Screening Assay for Tebuconazole

Tier 1 Assays Test Guideline Test Order Status 

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

(USEPA, 2002) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OSCPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID No. 48619403) 

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied 

(MRID No. 48619401) 

Steroidogenesis Assay (Human cell line 

H295R) 

OSCPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID No. 48619404) 

Aromatase Assay (human recombinant 

microsomes) OSCPP 890.1200 Requirement Satisfied 

(MRID No. 48619402) 

Uterotrophic Assay (Rat) OSCPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID No. 48673703) 

Hershberger Assay (Rat) OSCPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID No. 48673702) 

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied 

(MRID No. 48688302) 

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied 

(MRID No. 48688302) 

Fish Short-term Reproduction Assay OSCPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID No. 48688301) 

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID No. 48673701) 

Page 16 of 89
TEBUCONAZOLE FINAL 

Page 9 of 81 

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

1. Effects on Estrogen Pathway 

The potential interactions of tebuconazole with the estrogen pathway are summarized in Table 2. 

If an effect was observed but considered by WoE not to reflect a direct interaction, then the 

effect is discussed in the text but not presented in the table. The various targets of the estrogen 

pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of 

potential for estrogenic, anti-estrogenic or HPG axis effects. This table also includes HPGrelevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt 

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

the WOE assessment. 

Tebuconazole was classified as not interactive (negative) in the Tier 1 in vitro estrogen receptor 

(ER) binding assay and as negative in the Tier 1 in vitro ERα transcriptional activation assay 

(ERTA). In contrast, tebuconazole was positive in both the aromatase and steroidogenesis Tier 1 

assays, and in a series of in vitro cell proliferation and steroidogenesis assays by Kjærstad et al.

(2010) considered as OSRI. In the Tier 1 aromatase study, tebuconazole inhibited aromatase 

activity with an IC50 of approximately −5.7 log M. Tebuconazole completely inhibited estradiol 

production at 10−5 M and above in the Tier 1 steroidogenesis assay, and inhibited estradiol 

production with an IC50 of 1.2 μM in an steroidogenesis assay by Kjærstad et al. (2010). 

Kjærstad et al. (2010) also found that tebuconazole inhibited proliferation of MCF-7 cells 

induced by estradiol (IC50 = 49 μM) or testosterone (IC50 = 17 μM). While the authors attribute 

the inhibition of testosterone-induced proliferation in part to aromatase inhibition (testosterone is 

converted to estradiol by aromatase), this was not demonstrated in the study. 

Tebuconazole was negative in the Tier 1 uterotrophic assay. In the Tier 1 female pubertal assay, 

a 2-day delay in VO (p ≤ 0.05) was accompanied by a slight increase in body weight at 

attainment of VO (not statistically significant) at 150 mg/kg/day. Also in the pubertal study, 

ovarian weight decreases at low (6%) and high (7%) doses and uterus weight decreases of 15% 

(wet or blotted, not grouped by stage) at the high dose were not statistically significant. These 

effects in pubertal females occurred in the absence of overt toxicity. Liver weights were 

increased in low (8%) and high (15-18%) dose groups, and relative kidney weights were 

increased (6%) at the high dose. 

In the FSTRA, there was a significant decrease of 18.5% in female GSI at the high concentration 

(0.0195 mg a.i./L), and significant decreases of 35 and 55% in plasma vitellogenin at the mid 

(0.0113 mg a.i./L) and high treatment. Gonadal histopathology findings included increased 

incidence of granulomatous inflammation in the high treatment compared to the control; 

observations of oocyte atresia was reported in both controls and treatment groups with no 

indication of treatment-related response. Additionally, microscopic findings in the liver were 

reported including treatment-related hepatocyte necrosis in all treatments. 

Page 17 of 89
TEBUCONAZOLE FINAL 

Page 10 of 81 

In the Part 158 developmental studies, increased resorptions per dam were observed in rats and 

rabbits. In the developmental neurotoxicity study, delivery was delayed on average 0.5 days and 

2/25 dams died as a result of prolonged gestation. In the same study, a 1.6-day increase in age at 

VO was observed at the high dose (125 mg/kg/day); however, this finding co-occurred with 

reduced body weights (↓5-24%; PND 5-72). In the 2-generation reproductive study, at the high 

dose (50 mg/kg/day), F1a, F2a and F2b pup weights were decreased (p≤0.05) compared to 

controls from birth to week 4. There were no estrogen-related effects noted in the other Part 158 

mammalian studies. 

In published literature considered as OSRI, post-implantation loss was increased at 50 mg/kg/day 

in a developmental study by Taxvig et al. (2008) while in another developmental study by the 

same researcher (2007) significant post-implantation loss was observed at 100 but not at 50 

mg/kg/day. Similarly, increased anogenital distance (AGD, a potential sign of virilization) was 

observed in females at 50 mg/kg/day by Hass et al. (2012), and at 100 (but not 50) mg/kg/day by 

Taxvig et al. (2007). Both Hass et al. and Taxvig et al. observed increased incidence of nipple 

retention in males (a sign of feminization) at 50 mg/kg/day. Taxvig et al. also found that delivery 

was delayed an average of 0.9 day and dams had a 7-fold increase in levels of progesterone at 

100 mg/kg/day tebuconazole, compared to controls (2007 study), and a 58% decrease in estradiol 

levels at 50 mg/kg/day (2008 study). Other sporadic findings in literature studies include a delay 

age at VO at 20 but not 60 mg/kg/day (Moser et al. 2001), and one male genital malformation at 

50 mg/kg/day (Hass et al. 2012). Another literature developmental study by Jacobsen et al.

(2012) found that gavage dosing of dams on GD 7 to LD 16 up to 50 mg/kg/day had no effect on 

gestation length or implantation loss, nor on offspring VO, organ weights or histopathology, 

AGD, nipple retention or genital malformations. Finally, in a study to observe puberty onset and 

gene expression by Overgaard et al. (2013), tebuconazole had no effect on attainment of VO, 

however in that study pups were not directly dosed (dams were dosed via gavage). 

In the available Part 158 avian one-generation reproduction toxicity studies (two quail and two 

duck), dietary administration of tebuconazole resulted in reproductive effects in two of the 

studies. In the first bobwhite quail study, no treatment-related reproductive effects or clinical 

signs were noted at dietary levels up to 73.5 ppm. However, in the second bobwhite quail study, 

significant reproductive effects were observed at the high dose (290 ppm), including a reduction 

of 33% in the number of eggs laid, 11% reduction in normal hatchlings, as well as a 37% 

decrease in offspring survival and 7-11% decreases in offspring body weight. Abnormal livers 

(5/18) in females were also reported at this dose. In this study, significant reductions in offspring 

body weight (4-6%) were also reported in the low treatment group (156 ppm). In the first 

mallard duck study, no treatment-related reproductive effects or clinical signs were noted at 

dietary levels up to 76 ppm; however, in the second mallard duck study, while clinical signs were 

not observed, significant decreases in the number of hatchlings per hen were reported at the high 

dose of 611 ppm. 

Page 18 of 89
TEBUCONAZOLE FINAL 

Page 11 of 81 

In a fathead minnow fish sexual development study, there was no effect on hatch but survival 

was significantly reduced at ≥0.045 mg/L with effects on female growth at 0.022 mg/L. There 

was no difference in sex ratio after 54 days of exposure but effects on gonadal histopathology 

were reported at ≥0.011 mg/L in females. However, systemic toxicity was also reported at these 

concentrations including histopathological changes in the liver, pancreas and intestines (≥0.011 

mg/L), kidneys (0.045 mg/L) and spinal deformities (≥0.022 mg/L). No reproductive effects 

were noted in a Part 158 sheepshead minnow life cycle study, but parental survival was 

significantly reduced (day 58 & 177) at the highest treatment tested (0.197 mg/L), and parental 

body length was significantly reduced at the mid-high treatment (0.097 mg/L; highest treatment 

not analyzed due to mortality). Additionally, offspring length was significantly reduced at the 

mid and mid-high concentrations (≥0.044 mg/L). In a Part 158 rainbow trout fish early life-cycle 

study, there were no effects on hatch but survival and growth was significantly reduced at ≥0.025 

mg/L. In another Part 158 fish early life cycle toxicity study with sheepshead minnows, there 

were no effects on hatch or survival, but wet weight was significantly reduced at ≥0.048 mg/L 

and length was reduced at 0.099 mg/L. 

Page 19 of 89
TEBUCONAZOLE FINAL 

Page 12 of 81 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Tebuconazole

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Tebuconazole1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Gene Expression 

Sex Steroid Hormones 

Uterine Weight 

Ovarian Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at VO 

2° Sex Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

EDSP Tier 1 Assay 

ER Binding Assay satisfied by USEPA, 2002; see below. ERTA (MRID 48619403) N Aromatase (MRID 48619402) P Steroidogenesis (MRID 48619404) P Uterotrophic (MRID 48673703) N Female Pubertal Rat (MRID 48688302) N N N N N ↑2.0 days (H) FSTRA (MRID 48688301) NE ↓18.5% (H) P4 (H) N N ↓

OSRI 

ER Binding (USEPA, 2002) N Cell Proliferation and Aromatase Assays (Kjærstad et al., 2010) 

P P 
TEBUCONAZOLE FINAL 

Page 13 of 81 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Tebuconazole

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Tebuconazole1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Gene Expression 

Sex Steroid Hormones 

Uterine Weight 

Ovarian Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at VO 

2° Sex Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Pubert y Onset and Kiss1

Expression (Overgaard et al., 2013) 

N N 

Subchronic Toxicity (Rat; MRID 40700930) NE NE N NE NE Subchronic Toxicity (Dog; MRID 40700934) 

NE N N NE NE 

Chronic Toxicity (Dog; MRID 40700940) NE N N NE NE Chronic Toxicity (Dog; MRID 42030601) NE N N NE NE Chronic toxicity/ carcinogenicity (Rat; MRID 40700939) 

NE N N NE NE 

Eight-Week Range Finding Study (Mouse; MRID 40700933) 

NE N N NE NE 

Carcinogenicity (Mouse; MRID 40700941) 

NE NE N NE NE 

Carcinogenicity (Mouse; MRID 42175001) 

NE NE P NE NE 
TEBUCONAZOLE FINAL 

Page 14 of 81 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Tebuconazole

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Tebuconazole1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Gene Expression 

Sex Steroid Hormones 

Uterine Weight 

Ovarian Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at VO 

2° Sex Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Developmental Toxicity (Rat; MRID 40700943) 

NR P6 

Developmental Toxicity (Rabbit; MRID 40700945) 

NR P7 

Developmental Toxicity (Rabbit; MRID 43776201) 

NR N 

Developmental Toxicity (Rat; Taxvig et al., 2008) 

P N P8 

Developmental Toxicity (Rat; Hass et al., 2012) 

P P9 (H) N 

Developmental Toxicity (Rat; Jacobsen et al., 2012) 

N N N N 

Two-Generation Reproduction Toxicity (Rat; MRID 40700946) 

NE N N NE N 

Reproductive Toxicity (Rat; Taxvig et al., 2007) 

P 

(H) N N NE NE P9 (H) P10 (H) 

Developmental Neurotoxicity (Rat; MRID 45074301) 

↑1.6 

days 

(H) 

P11

(H) 
TEBUCONAZOLE FINAL 

Page 15 of 81 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Tebuconazole

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Tebuconazole1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Gene Expression 

Sex Steroid Hormones 

Uterine Weight 

Ovarian Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at VO 

2° Sex Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Reproductive Function (Rat; Moser et al., 2001) 

N N N N 

Avian reproduction (Bobwhite quail; MRID 40700910) 

N 

Avian reproduction (Bobwhite quail; MRID 41624201) 

P12 

(L, H) 

Avian reproduction (Duck; MRID 40700909) 

N 

Avian reproduction (Duck; MRID 41818301) 

P13

(M, 

H) 

Fish Sexual Development Toxicity (Fathead minnow, MRID 48109802) 

P14 

(MMH) N 

Fish Full Life Cycle Toxicity (Sheepshead minnow, MRID 43009601) 

N 

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

liver weight, KW = kidney weight. Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is paraevaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated. 
TEBUCONAZOLE FINAL 

Page 16 of 81 

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

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 b

details see Section IV. A 

4. In the ovary, the microscopic findings included increased granulomatous inflammation (minimal to mild). 

5. Microscopic findings in the liver were reported including treatment-related hepatocyte necrosis in all treatments. 

6. Mean number of live fetuses/dam was decreased at the high dose due to an increase in both early (embryonic) and late (fetal) 

resorptions: 2.8/dam treated vs. 0.6/dam controls). Mean live fetuses/dam were decreased in the high dose group, due to an in

late resorptions (mean total resorptions: 2.8/dam treated vs. 0.6/dam controls). 

7. There was a reduction in live fetuses/dam observed in the high dose group as compared to the controls (6.4 treated vs. 7.4 con

in total (both early and late) resorptions/dam (2.4 treated vi. 0.7 controls). 

8. At 50 mg/kg/day, tebuconazole increased (p<0.05) the frequency of post-implantation loss (11.4%) compared to controls (0%9. Increased incidence of nipple retention in males (a sign of feminization) at 50 mg/kg/day. Increased anogenital distance (AGD

was observed in females at 50 mg/kg/day by Hass et al. (2012), and at 100 (but not 50) mg/kg/day by Taxvig et al. (2007). 

10. Significant post-implantation loss was observed at 100 but not at 50 mg/kg/day. At 100 mg/kg/day, delivery was delayed an a11. At the high dose (1000 ppm), two P females died as a result of prolonged gestation and the number of live fetuses/dam was d

the number of dead fetuses/dam was increased (p<0.01), the stillborn index was increased (p<0.01) and the number of pup de

PND 1-5. Additionally at high dose mean gestation length was increased (23.0 days) compared to the controls (22.5 days), anresult of prolonged gestation. 

12. At high dose (290 ppm) reduction of 33% in the number of eggs laid, 11% reduction in normal hatchlings, 37% decrease in o

11% decreases in offspring body weight. Reductions in offspring body weight (4-6%) in the low treatment group (156 ppm) 

13. Significant decreases in the number of hatchlings per hen were reported at the high dose of 611 ppm 14. No difference in sex ratio; effects on gonadal histopathology were reported at ≥0.011 mg/L 

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

NE Not examined NR Not reported 

 . 
TEBUCONAZOLE FINAL 

Page 17 of 81 

2. Effects on Androgen Pathway 

The potential interactions of tebuconazole with the androgen pathway are summarized in Table 

3. If an effect was observed but considered by WoE not to reflect a direct interaction, then the 

effect is discussed in the text but not presented in the table. The various targets of the 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 HPGrelevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt 

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

the WOE assessment. 

Tebuconazole was classified as a binder in the Tier 1 in vitro AR binding assay, as average 

radioligand binding was reduced to <50% at concentrations of 10-3 M and 10-4 M. In the Tier 1 

steroidogenesis assay, tebuconazole reduced testosterone production by 32-98% at 10−6

 M and 

above. Four published studies (OSRI) also examined effects of tebuconazole on AR 

transactivation and/or steroidogenesis. In an AR reporter gene assay using AR-transfected CHO 

cells, tebuconazole was an AR antagonist in the presence of R1881 (a strong androgen agonist), 

and inhibited testosterone production in a steroidogenesis assay (Kjærstad et al., 2010). In an 

AR-transcription activation assay using MDA-kb2 cells with and without dihydrotestosterone 

(DHT), tebuconazole was also an AR antagonist (Orton et al., 2011). In a study by Hass et al.

(2012), tebuconazole also exhibited some antagonistic action in an AR reporter gene assay and 

reduced testosterone production in a steroidogenesis assay. 

Tebuconazole was negative in the Tier 1 Hershberger assay, as well as another Hershberger 

assay from the published literature (Taxvig et al., 2008). Conversely, in the male pubertal assay, 

tebuconazole reduced serum testosterone levels in a dose dependent manner (17% NS at low 

dose and 53% p≤0.05 at high dose). Weights of several androgen-target organs also decreased in 

a dose-dependent manner (seminal vesicle 17-33%, dorsolateral prostate 21%, LABC 11-24%, 

epididymis 2-14%) although the change at the low dose was statistically significant only for the 

dorsolateral prostate. A 10% testis weight decrease was not statistically significant. Pituitary 

weights decreased (p≤0.05) at the low (9%) and high (12%) doses. Age at initial preputial 

separation (PPS) was delayed 2.5 days (p≤0.05), however a similar 2.4 day delay in age at 

complete PPS was not statistically significant. A small increase in incidence of gross atrophy 

was observed in testis (1/2/3 of 15 in control/low/high dose), seminal vesicles (2/5/7) and LABC 

(0/1/3). Microscopically, increased incidences of tubular degeneration in the testes (1/0/4) and 

oligospermia in the epididymis (1/2/4) were also observed. These androgen-related effects 

occurred in the absence of overt toxicity but co-occurred with decreases in serum bilirubin (75% 

at low and not detected at high dose) and urea nitrogen (10% at high dose), decreased organ 

weights of kidneys (7-9%) and adrenals (18-13% not dose-dependent) and a 10% increase in 

liver weight (high dose, relative only). 

Page 25 of 89
TEBUCONAZOLE FINAL 

Page 18 of 81 

In the FSTRA, a 19% decrease in GSI was marginally significant (p=0.056) at the high 

concentration (0.0195 mg/L). For the liver, the occasional findings of microgranulomas 

(minimal) and individual hepatocyte necrosis (minimal) in the livers were likely treatmentrelated (based on findings in females); there was also a dose-related decrease in hepatocyte 

vacuolation compared to control. 

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

In addition to the Part 158 toxicity studies, five published studies considered as OSRI examined 

effects of tebuconazole on development and reproductive parameters in rodents. In a couple of 

rat developmental toxicity studies by Taxvig et al. (2007 and 2008), tebuconazole did not affect 

fetal serum testosterone levels following exposure from GD 7 through 21 at 50 mg/kg/day, but 

testicular levels were decreased in the 2007 study at 100 mg/kg/day. Increased male pup nipple 

retention was observed postnatally, without changes in male AGD, in the 2007 Taxvig et al.

study and another similar study by Hass et al. (2012) at ≤ 50 mg/kg/day. However, neither the 

Taxvig et al. nor the Hass et al. studies found treatment-related changes in postnatal organ 

weights or histopathology, and there were no effects on semen quality. In an additional rat 

developmental toxicity study by Jacobsen et al. (2012), male pups dosed from GD 7 through 

PND 16 showed no effects of tebuconazole treatment at ≤ 50 mg/kg/day on reproductive organ 

weights, semen quality or age at PPS. Finally, in a rat reproductive toxicity study (Moser et al., 

2001) examining effects from pre- and post-natal exposure (GD 14 to PND 7) to tebuconazole at 

6, 20 or 60 mg/kg/day, high-dose male pups at PND 45 had reduced epididymides weights 

(↓17%) that may have been related to decreased body weight; no effects were noted on AGD, 

age to PPS, testes weight, or sperm counts or mobility. 

In a bobwhite quail study submitted as OSRI, while not specific to the androgen-pathway, a 

significant effect on percent normal hatchlings and subsequent offspring survival and weight was 

reported at the high dose (290 ppm); there were no androgen-related endpoints affected in the 

other avian reproduction studies. In a fathead minnow fish sexual development study, there was 

no difference in sex ratio but effects on male gonadal histopathology were reported at ≥0.022 

mg/L for males. However, histopathological changes in other tissues were also reported at these 

concentrations including the liver, pancreas and intestines (≥0.011 mg/L), kidneys (0.045 mg/L) 

and spinal deformities (≥0.022 mg/L). No effects on androgen-related endpoints were reported in 

the fish life-cycle study. 

Page 26 of 89
TEBUCONAZOLE FINAL 

Page 19 of 81 

Table 3: Androgenic/Anti-Androgenic Pathway for TebuconazoleLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Tebuconazole1Study Type/ Literature Citation 

AR Binding/ARTA 

Steroidogenesis 

Sex Steroid hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility/Fecundity 

Age and Weight at 

PPS 

Vitellogenin 

EDSP Tier 1 Assay 

AR Binding (MRID 48619401) P Steroidogenesis (MRID 48619404) P Hershberger (MRID 48673702) NE N Male Pubertal Rat (MRID 48688302) 

↓53% 

(H) N P (L, H) ↓13-14% (H) P (H) ↓ 9, 12% (L, H) P4 (H) N 

FSTRA (MRID 48615506) NE ↓19% (H) N N N N 

OSRI 

ARTA/ Steroidogenesis assays (Kjærstad et al., 2010) 

P P 

ARTA assays (Orton et al., 2011) P Hershberger (Rat; Taxvig et al., 2008) NE N Subchronic Toxicity (Rat; MRID 40700930) 

N N NE N NE NE 

Subchronic Toxicity (Dog; MRID 40700934) 

N N NE N NE NE 
TEBUCONAZOLE FINAL 

Page 20 of 81 

Table 3: Androgenic/Anti-Androgenic Pathway for TebuconazoleLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Tebuconazole1Study Type/ Literature Citation 

AR Binding/ARTA 

Steroidogenesis 

Sex Steroid hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility/Fecundity 

Age and Weight at 

PPS 

Vitellogenin 

Chronic Toxicity (Dog; MRID 40700940)

N N NE NE NE NE 

Chronic Toxicity (Dog; MRID 42030601)

N N NE N NE NE 

Chronic toxicity/ carcinogenicity (Rat; MRID 40700939)

N N NE N NE NE 

Eight-Week Range Finding Study (Mouse; MRID 40700933)

N N NE NE NE NE 

Carcinogenicity (Mouse; MRID 40700941)

N N NE N NE NE 

Carcinogenicity (Mouse; MRID 42175001)

N N NE N NE NE 

Developmental Toxicity (Rat; Taxvig et al., 2007) 

N N NE N N P (L, H) 

Developmental Toxicity (Rat; Taxvig et al., 2008) 

P7 N 

Developmental Toxicity (Rat; Hass et al., 2012) 

P P P 
TEBUCONAZOLE FINAL 

Page 21 of 81 

Table 3: Androgenic/Anti-Androgenic Pathway for TebuconazoleLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Tebuconazole1Study Type/ Literature Citation 

AR Binding/ARTA 

Steroidogenesis 

Sex Steroid hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility/Fecundity 

Age and Weight at 

PPS 

Vitellogenin 

Developmental Toxicity (Rat; Jacobsen et al., 2012) 

N N N N N N 

Two-Generation Reproduction Toxicity (Rat; MRID 40700946) 

N N NE N 

Developmental Neurotoxicity (Rat; MRID 45074301) 

N 

Reproductive Function (Rat; Moser et al., 2001) 

N N ↓17% (H) N N N 

Avian reproduction (Bobwhite quail; MRID 40700910) 

 N 

Avian reproduction (Bobwhite quail; MRID 41624201) 

 P8 

Avian reproduction (Duck; MRID 40700909) 

 N 

Avian reproduction (Duck; MRID 41818301) 

 N 

Fish Sexual Development Toxicity (Fathead minnow, MRID 48109802) 

P9 N 
TEBUCONAZOLE FINAL 

Page 22 of 81 

Table 3: Androgenic/Anti-Androgenic Pathway for TebuconazoleLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Tebuconazole1Study Type/ Literature Citation 

AR Binding/ARTA 

Steroidogenesis 

Sex Steroid hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility/Fecundity 

Age and Weight at 

PPS 

Vitellogenin 

Fish Full Life Cycle Toxicity (Sheepshead minnow, MRID 43009601) 

N 

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

or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicChanges in weight are absolute unless otherwise indicated. as Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (Dani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP), and tubercle score (TS). 

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

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 

details see Section IV. A 

4. Weights of several androgen-target organs also decreased in a dose-dependent manner (seminal vesicle 17-33%, dorsolateral p

24%, epididymis 2-14%) although the change at the low dose was statistically significant only for the dorsolateral prostate. 

5. These androgen-related effects occurred in the absence of overt toxicity but co-occurred with decreases in serum bilirubin (75% 

at high dose) and urea nitrogen (10% at high dose), decreased organ weights of kidneys (7-9%) and adrenals (18-13% not doseincrease in liver weight (high dose, relative only). 

6. For the liver, the occasional findings of microgranulomas (minimal) and individual hepatocyte necrosis (minimal) in the livers 

related (based on findings in females); there was also a dose-related decrease in hepatocyte vacuolation compared to control 

7. In GD 21 fetal testis, levels of testosterone decreased 50% at 100 mg/kg/day, while levels of 17α-hydroxyprogesterone increas

and 238% at 100 mg/kg/day. Plasma testosterone levels were not changed from controls in male fetuses or dams. 

8. At high dose, 290 ppm, 11% reduction in normal hatchlings, 37% decrease in offspring survival and 7-11% decreases in offsp

Reductions in offspring body weight (4-6%) in the low treatment group (156 ppm) 

9. No difference in sex ratio but effects on male gonadal histopathology were reported at ≥0.022 mg/L for males 

P Positive findings N Negative findings (in vitro)/No effect (in vivo) NE Not Examined
TEBUCONAZOLE FINAL 

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

The current EDSP Tier 1 battery does not have a specific in vitro assay to detect chemicals with 

the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it 

does include three in vivo assays that provide data to detect changes in the HPT axis, i.e., the 

pubertal male and female (rat) assays, and the AMA (frog). 

The potential interactions of tebuconazole with thyroid regulation are summarized in Table 4. 

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

to facilitate determination of potential for thyroid or HPT axis effects. This table also includes 

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. 

There were no effects of tebuconazole noted on serum T4 or TSH levels, thyroid weights, or 

thyroid histopathology in either the male pubertal or female pubertal assays. In the male pubertal 

study, pituitary weights were decreased by 9-12% (75 and 150 mg/kg/day); these decreases 

occurred in the absence of overt toxicity but co-occurred with 7-9% decrease (dose-dependent) 

in mean kidney weights and 13-18% decrease (not dose-dependent) in mean adrenal weight at 

the low and high doses, and 10% increase relative liver weight at the high dose. There were no 

effects of treatment on pituitary weights in the female pubertal study. 

In the AMA at the highest test concentration (0.341 mg/L), mild thyroid hypoplasia was 

observed in all tadpoles, and a significant 56% reduction in normalized hind-limb length (HLL) 

on Day 21. Additionally, a significant delay of median Nieuwkoop-Faber (NF) developmental 

stage by one stage was observed at Day 7 at all treatment groups (0.017, 0.067 & 0.341 mg/L), 

and significant delays at Day 21 at the mid (by one stage) and high (by 5 stages) concentrations. 

Asynchronous development was not observed. Late-stage tadpoles (NF Stage >60) were 

observed in the study in the negative control (55%) and low dose group (38%), whereas latestage tadpoles accounted for 7% and 0% of survivors in the middle and high dose groups. 

Significant reductions in snout-vent length (SVL) and body weight (BW) were observed at the 

high concentration on both Day 7 (SVL: ↓16%; BW: ↓24%) and 21 (SVL: ↓24%; BW: ↓59%) 

and in the low treatment on Day 21 (SVL: ↓6%; BW: ↓12%). In the high treatment, two tadpoles 

were reported as pale while two more tadpoles showed erratic behavior/loss of equilibrium. 

Additionally, histological findings in the liver were reported in the mid and high treatment levels. 

A total of thirteen mammalian Part 158 guideline studies on tebuconazole were submitted as 

OSRI. There were no HPT effects observed in six of these studies, which included a rat 

subchronic toxicity study, a dog subchronic toxicity study, two dog chronic toxicity studies and 

two mouse oncogenicity studies. In a rat chronic toxicity/carcinogenicity study, the incidence of 

thyroid C-cell (i.e. parafollicular cells) hyperplasia was somewhat elevated at the mid (16 

mg/kg/day; p<0.05) and high (55 mg/kg/day) dose males; however these cell functions relate to 

Page 31 of 89
TEBUCONAZOLE FINAL 

Page 24 of 81 

calcium homeostasis and are not involved in thyroid hormone production or homeostasis. 

Systemic toxicity was also observed in female rats, along with increased activity of several 

hepatic metabolic enzymes at all doses tested. 

Three journal articles relevant to the thyroid pathway were submitted as OSRI. In a Hershberger 

assay (Taxvig et al., 2008), serum T4 levels were measured but were unaffected by treatment. In 

a developmental toxicity study (Taxvig et al., 2007), dam serum thyroid hormones were 

decreased at the high dose (100 mg/kg/day): T3 levels by 11% (p ≤ 0.05); and T4 levels by 29% 

(NS). However, systemic toxicity was also observed at this dose, including decreased body 

weight gain during gestation (↓29%) and body weight loss during the lactation period (−4.57 g 

treated vs. +7.53 g controls), increased gestation length and increased post implantation loss. In 

an embryonic zebrafish study, serum T3 levels were increased by 28% while whole body T4

levels were decreased by 10% at the highest dose tested (4 mg/L) (Yu et al., 2013). 

Additionally, the following HPT axis genes appeared to be upregulated by tebuconazole 

treatment: corticotrophin-releasing hormone, thyroid stimulating hormone, sodium/iodide 

symporter, transthyretin, uridine diphosphate glucuronosyltransferase, thyronine deiodinase 2 

and thyroid hormone receptor β. 

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Table 4. Thyroid Pathway for Tebuconazole.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Tebuconazole1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

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

Overt Toxicity Observed3

EDSP Tier 1 Assay 

Male Pubertal Rat 

(MRID 48688302) N N N N ↓9, 12% (L, H) 

P4

(L, H) N 

Female Pubertal Rat 

(MRID 48688302) N N N N N 

LW 

(M, 

H); 

KW 

(H) 

N 

AMA 

(MRID 48673701) P5

(H) 

Day 7: ↓1 NF 

stage (L,M,H);

 Day 21: ↓1 NF

stage (M); ↓5 

NF stage (H); 

↓56% HLL (H)

SVL: Day 7: 

↓16% (H); 

Day 21: 

↓6.3, 24% 

(L, H) 

BW: Day 7: 

↓33% (H); 

Day 21: 

↓12, 59% 

(L, H) 

 

P6

(M,H) N 

OSRI

Hershberger assay 

(Taxvig et al., 2008) N 

Developmental 

Toxicity (Taxvig et 

al., 2007) 

T3 ↓11% 

(H) 

X 

(H) N 

Subchronic Toxicity 

(Rat; MRID 

40700930) 

N X 

(L, M, H)

X 

(H) 

Subchronic Toxicity 

(Dog; MRID 

40700934) 

N N 

X 

(L, M, 

H) 

X 

(H) 

Chronic Toxicity 

(Dog; MRID 

40700940) 

N N X 

(M, H) N 

Chronic Toxicity 

(Dog; MRID 

42030601) 

N X

 (H) N 

Chronic toxicity/ 

carcinogenicity 

(Rat; MRID 

40700939) 

N X 

(L, M, H) N 

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Table 4. Thyroid Pathway for Tebuconazole.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Tebuconazole1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

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

Overt Toxicity Observed3

Oncogenicity 

(Mouse; MRID 

40700941) 

N X 

(L, M, H) N 

Oncogenicity 

(Mouse; MRID 

42175001) 

N X 

(L, H) N 

Thyroid Endocrine 

Disruption 

(Zebrafish; Yu et 

al., 2013) 

Whole 

body 

levels: 

T3 ↑28% 

T4 ↓10% 

(H) 

 N 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high 

treatment, H=High treatment Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates 

that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute 

unless otherwise indicated. 

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

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

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

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

4. These androgen-related effects occurred in the absence of overt toxicity but co-occurred with decreases in 

serum bilirubin (75% at low and not detected at high dose) and urea nitrogen (10% at high dose), decreased 

organ weights of kidneys (7-9%) and adrenals (18-13% not dose-dependent), and a 10% increase in liver 

weight (high dose, relative only). 

5. At the highest test concentration (0.341 mg/L) mild thyroid hypoplasia was observed in all tadpoles 

6. Histological findings in the liver were reported in the mid and high treatment. 

P Positive findings 

N Negative findings 

 

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IV. Committee’s Assessment of Weight of Evidence 

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

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

pathways) as well as recommendations regarding Tier 2 testing.. The results of the Tier 1 assays 

are considered, along with other scientifically relevant information (e.g., 40 CFR Part 158 test 

guidelines and published or publicly available peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence (i.e., specific Tier 1 assays and 

OSRI), then performing an integrated analysis of those lines of evidence. 

The WoE evaluation includes considerations of biological plausibility of the findings from the 

different lines of evidence by examining the consistency, coherence, and interrelationships 

among the measured endpoints within and across studies. The available findings from standard 

toxicology studies on the substance may contribute to the WoE evaluation in helping elucidate if 

effects seen in the Tier 1 assay are related to perturbations of the endocrine system per se or 

alternatively a sequelae of systemic effects. Endocrine modes of action may 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 Tire 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. Overt/Systemic Toxicity in the in vivo Tier 1 Assays and OSRI 

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

OSRI studies are described 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).

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1. Tier 1 in vivo Assays 

Rats were treated at doses of 75 and 150 mg/kg/day in all in vivo studies; in the Hershberger and 

uterotrophic assays a dose 35 mg/kg/day was also administered with no effects. In the 

Hershberger assay, overt toxicity was not observed in 58-days old male rats at any dose and 

other systemic toxicity was not evaluated. While mean overall body weight gain was decrease 

20% at the high dose in the androgen agonist portion of the Hershberger assay, this did not 

translate into a net decrease in mean body weight. Overt toxicity was also absent from the rat 

pubertal assays (22-day old females and 23-day old males at start of assay). A small decreased 

in mean body weight (8% compared to controls) was observed in pubertal males at the high dose 

but not in pubertal females. Liver weights were increased 8% at the low and 15-18% at the high 

dose in females, but in males only relative liver weight was increased 10% at the high dose. The 

mean liver weight changes seem to correlate with mean changes in blood urea nitrogen (BUN), 

total bilirubin and total cholesterol that are more significant in females (↓15-17%, not detected, 

and ↑17%, respectively) than in males (10%, 75%-not detected, no change). Kidney weights 

were decreased 7% at the low and 9% at the high dose in pubertal males but only relative kidney 

weight was increased at the high dose in pubertal females; creatinine was decreased 8% in 

females but not in males. Adrenal weights were significantly decreased in males at the low (15-

18%) and high (13%) doses, but not in pubertal females. The differences in systemic toxicity 

between pubertal males and females are most likely due to the different exposure durations (30 

days males vs. 20 days females). 

Conversely, overt toxicity was observed in 19-day old female rats in the uterotrophic assay at 

150 mg/kg/day tebuconazole as a mean body weight loss of 3.0 g (compared to a gain of 8.2 g in 

controls), which resulted in a 24% decrease in mean terminal body weight compared to controls 

(p<0.01). In addition, five of six females in the high dose group had reduced motor activity on 

the final day of the uterotrophic assay (Day 3). 

In the AMA, in the high concentration treatment group (0.341 mg/L), two tadpoles were reported 

as pale while two more tadpoles showed erratic behavior/loss of equilibrium. Additionally, 

effects on the liver were reported at the mid (0.067 mg/L) and high treatment [included 

decreased hepatocyte vacuolation, hepatocyte anisocylosis, hepatocyte anisokaryosis, increased 

multinucleate hepatocytes and individual hepatocyte necrosis (0.341 mg/L only)]. 

In the FSTRA, no signs of overt toxicity were observed in the treated groups. For the liver 

histopathology, for males, the occasional findings of microgranulomas (minimal) and individual 

hepatocyte necrosis (minimal) in the livers were likely treatment-related (based on female 

findings); there was also a dose-related decrease in hepatocyte vacuolation compared to control. 

In females, dose-dependent findings in the liver included individual hepatocyte necrosis which 

occurred in all three treatment groups. Additionally, in the mid and high treatments (0.011 and 

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0.0195 mg/L) increases in multinucleated hepatocytes, microgranulomas, hepatocyte 

megalocytosis and decreased hepatocellular vacuolation were reported. 

2. OSRI 

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

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

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

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

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

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

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

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

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

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

outcomes related to the liver and the endocrine system. The key events leading to these adverse 

effects have not been fully elucidated. However, mechanistic information related to toxicity 

pathways associated with the liver and endocrine system can be used to inform the EDSP WOE 

for tebuconazole for mammals. 

Lipid, sterol, and steroid homeostatic pathways are regulated by the constitutive androstane 

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

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

Development (ORD), relatively high doses of conazoles such as propiconazole appear to perturb 

fatty acid and steroid metabolism in the male rat liver predominantly through the CAR and PXR 

signaling pathways (Goetz and Dix; Goetz et al., 2006). Since both CAR and PXR regulate the 

expression of CYP genes and the associated enzyme activity of oxidative metabolism of steroid 

hormones as well as xenobiotics, it may be that CAR and PXR are also components of a 

regulatory network that regulates circulating hormone levels. 

As in the Tier 1 studies, the target organ for tebuconazole toxicity was generally the liver, with 

minor effects observed on the kidney and adrenal gland. dogs. In a published, multi-part study 

including a Hershberger assay by Taxvig et al. (2008), no signs of overt toxicity were observed; 

however, liver weights were increased at the high dose (150 mg/kg/day). 

In the rat subchronic toxicity study, overt toxicity was observed in the form of mortality of one 

high dose (1600 ppm) male and one high dose female. In the mid (400 ppm) dose males and 

high dose males and females, body weights were decreased, and overall body weight gains were 

also decreased at the high dose in both sexes. Effects on liver included increases in Ndemethylase activity in high-dose males and increases in activity of cytochrome P-450 in midand high-dose males, and in the low-, mid-, and high-dose females. Vacuole formation in the 

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zona fasciculata of the adrenal glands was noted in the high dose males and mid and high dose 

females. 

In the dog subchronic toxicity study, overt toxicity was noted, as one high dose (5000 ppm) 

female was found dead on Day 2. Body weights were decreased in the mid and high dose males 

and females, with an overall decrease in body weight gain in both sexes. Lens opacity of the 

eyes was observed in the high-dose group, such that all high-dose dogs were observed with this 

finding at Week 10. Increased platelet counts and signs of severe anisocytosis were noted at the 

high dose. Serum alkaline phosphatase activity was increased, and serum albumin was 

decreased in the high dose males. Liver N-demethylase activities were increased at the low (200 

ppm), mid and high dose in both sexes; cytochrome P-450 activities were increased to a lesser 

extent in the mid and high dose males and in the high dose females. Spleen weights were 

increased at the high dose in both sexes. Liver weights were slightly increased in the mid and 

high dose males. Microscopic findings in the high dose group included slight degeneration of 

the posterior wall of the lens of the eye, cataract-like eosinophilic plaques, bilateral cataracts and 

slight siderosis in the liver and spleen. In the females, increased vacuole formation was noted in 

the adrenals of one low dose, one mid dose and two high dose dogs. 

In a dog chronic toxicity study, there were no signs of overt toxicity. Lenticular lesions 

(opacities, lens stars) were noted in the eyes in two animals of the mid dose (400 ppm) as well as 

one dog at the high dose (1000 ppm). In addition, corneal opacities (cloudy, extensive) were 

elevated in one dog in each of the mid dose and high dose groups. Alkaline phosphatase levels 

were elevated over the course of treatment in the high dose males and females; liver Ndemethylase activity was significantly elevated at the high dose, as well as liver triglycerides. 

Absolute and relative liver weights were increased in the mid and high dose males, with a 

consistent change in the appearance of the liver in mid and high dose dogs, as well as a slight 

increase in the presence of siderin in mid and high-dose males and ORO-positive lipids in one 

dog in each of the mid and high-dose groups. Other potential target organs/tissues are the 

hematopoietic system, the adrenals, the kidneys and the spleen. 

In a second dog chronic toxicity study, no signs of overt toxicity were observed. Microscopic 

pathology demonstrated the adrenal gland to be a target organ at the high dose (150 ppm) in both 

sexes. Hypertrophy of adrenal zona fasciculata cells and fatty changes in the zona glomerulosa 

were observed in both sexes, and lipid hyperplasia in the cortex was noted in the males. 

In the rat chronic toxicity/carcinogenicity study, no signs of overt toxicity were noted. However, 

female body weights were decreased at the mid (300 ppm) and high (1000 ppm) dose throughout 

the study. The high-dose females also had slightly increased food consumption. In the highdose females, there were small decreases in hemoglobin, hematocrit and altered mean 

corpuscular concentrations and volumes which correlated with an increased deposition of splenic 

hemosiderin. Dose-related decreases in female absolute and relative adrenal weights were 

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associated with a dose-related decrease in the incidence of adrenal cortical hemorrhagic 

degeneration at the mid and high dose. There was also a dose-related increase in liver 

microsomal enzyme induction at all dose levels tested, based on histological examination. In the 

high dose males, gross pathology showed an increase in kidney cyst/cystic kidneys and reddened 

lymph nodes; histological examination of the lymph nodes revealed an elevation of blood-filled 

sinuses of the mesenteric lymph nodes. 

In a rat eight-week range-finding study,, there were no signs of overt toxicity, but body weights 

were consistently decreased by 1-2 g in the high dose (2000 ppm) females during treatment. In 

the males, total bilirubin was decreased at the high dose, indirect bilirubin was decreased at the 

low (500 ppm) and high dose, and serum iron was increased at the high dose. Absolute liver 

weights were increased at the low and high dose in both sexes. Absolute adrenal weights were 

decreased in the high dose females. Gross pathology revealed swollen or increased lobulation of 

the liver in the low and high dose males, and pale livers in the both sexes at the low and high 

dose. In the liver, there was an increase in the incidence and severity of cell degeneration, cell 

necrosis, focal necrosis, and the presence of vacuoles and lipid content at either or both doses. 

At the high dose, there was an increase in the incidence of iron-related pigment and round cell 

infiltrates of the kidneys. An increase in cortical cell lipids (mid and high dose) and sinus 

dilation (high dose) was observed in the male adrenals. N-demethylase activity was increased in 

females at the low (125 ppm, mid (500 ppm), and high dose (2000 ppm), and O-demethylase 

activity was increased in the high-dose males and in the mid-and high-dose females. 

Cytochrome P-450 activity was increased in the low-, mid-, and high-dose males and females. 

Finally, triglycerides were increased in the low dose males and in the mid and high dose males 

and females. 

In a mouse oncogenicity study, overt toxicity was not noted. A minimal decrease in male mean 

body weights was noted during the first third of the dosing period at the high dose (180 ppm). 

Bilirubin values were increased in the high dose females at Week 53 and in the low (20 ppm), 

mid (60 ppm) and high females at Week 92. Slight increases (NS) in absolute and relative liver 

weights were noted in high dose males and females at Weeks 53 and 92/93. Gross pathology 

demonstrated an increase in male liver enlargement and the presence of liver nodules. Nonneoplastic histopathology indicated the liver as a primary target organ at the mid and high dose 

in both sexes. In males, the most frequent liver lesions were vacuolation and lipid deposition; in 

females, hepatic vacuolation, minimal extramedullary hematopoiesis, sinusoidal cellularity and 

increased lipid deposition were noted. In males, the adrenals were affected at the mid and high 

dose with an increased enlargement and hyperplasia of cortical cells, and an increase in minimal 

stomach gastritis in the high dose group. Females also had elevations in the pancreatic 

interstitial edema and stomach gastritis at the mid or high dose levels. 

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In the second mouse oncogenicity study, there were no signs of overt toxicity, but distended 

abdomen was observed in the high dose (1500 ppm) males and females. In males, there were 

decreases in body weight at the low dose (500 ppm) during the initial half of the testing period, 

and at the high dose up to Week 84. In females, there were sporadic decreases in body weight at 

the low dose during the initial third of the study, and at the high dose, there were decreased body 

weights in 39 of the first 59 weeks and in 7 of the last 8 weeks. Mean food consumption was 

increased frequently in the mid dose males and generally throughout the study at the high dose in 

both sexes. At sacrifice, leucocytosis and changes consistent with anemia, (increased platelet 

counts and decreased thromboplastin times) were noted in the high dose males. In the high dose 

females, a reduced hematocrit and leucocytosis and an increased platelet count were noted. 

Plasma activities of liver enzymes (GPT, GOT, AP) were increased in the high dose females. In 

addition, increases were observed at the low dose for GPT in males and for GPT and GOT in 

females. Cholesterol and albumin were decreased in both sexes at the low and high dose. 

Absolute liver weights were increased in the low dose males, and at the high dose in both sexes. 

Absolute kidney weights were decreased in the low and high dose males. Gross observations 

included: enlarged liver at the high dose in both sexes; pale livers in both sexes at the low and 

high dose; areas of change in the liver in the high dose females; irregular surfaces in livers at the 

high dose in both sexes; and liver masses in the high dose females. Microscopically, there were 

increases in extramedullary hematopoiesis (males), focal hyperplasia of hepatocytes (both sexes), 

oval cell proliferation (both sexes), periacinar hepatocyte hypertrophy (females), eosinophilic 

foci of hepatocyte alteration (females) and pigment-laden Kupfer cells (females) at the high dose, 

and in hepatic panacinar fine fatty vacuolation the low dose males and high dose males and 

females. At the high dose, the incidence of hepatocellular adenomas and hepatocellular 

carcinomas was increased in males, and the incidence of hepatocellular carcinomas was 

increased in females. 

In a rat prenatal developmental toxicity study, overt toxicity was not observed. Mean body 

weights were slightly decreased in the high dose dams (120 mg/kg/day) on Day 21, and mean 

body weight gains were decreased during dosing. These decreases were accompanied by a 

reduction in food consumption during dosing. Mean maternal absolute and relative liver weights 

were increased at the mid (60 mg/kg/day) and high dose. Mean fetal weight was reduced at the 

high dose. 

In a rabbit prenatal developmental toxicity study, there were no signs of overt toxicity. Maternal 

mean body weight gains were decreased during dosing in the high dose (100 mg/kg/day) does. 

When GD 6-28 body weight gains were corrected for gravid weights, a slight loss was noted at 

the high dose. Food consumption was also decreased during dosing at the high dose. 

In a second rabbit prenatal developmental toxicity study, overt toxicity was not noted. Single 

cell necrosis of the liver was seen in all does in the low (10 mg/kg/day), mid (30 mg/kg/day) and 

high (100 mg/kg/day) dose groups. 

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In a published, multi-part study including a developmental toxicity study by Taxvig et al. (2008), 

no signs of overt toxicity were observed. However, the frequency of post-implantation loss was 

increased (11.4%) compared to controls (0%) at the only dose administered (50 mg/kg/day). 

In a published developmental toxicity study by Hass et al. (2012), overt or systemic toxicity 

were not observed at the high dose (50 mg/kg/day). 

In a published developmental toxicity study by Jacobsen et al. (2012), overt toxicity was not 

noted, however, liver weight was increase the high dose (50 mg/kg/day) males. 

In a two-generation reproduction toxicity study (1987), there were no signs of overt toxicity. 

Clinical signs of toxicity at the high dose (1000 ppm) included loss of hair in the P and F1b

females, inflammation/changes of the eye in the P males and ill/bad condition in the F1b males. 

Loss of hair was also increased in the mid dose (300 ppm) F1b females. Body weights were 

consistently decreased at the high dose in both sexes in both generations. Organ weight data 

presented for the high dose F1b parents indicated decreases in terminal body weights and absolute 

liver, kidney and adrenal weights in the males, and in terminal body weights and kidney weights 

in the females. 

In a published reproductive toxicity study by Taxvig et al. (2007), no signs of overt toxicity were 

noted. Treatment-related effects observed at the high dose (100 mg/kg/day) included decreased 

body weight gain during gestation, increased gestation length, increased post implantation loss 

and body weight loss during lactation. 

In a developmental neurotoxicity study, there were no signs of overt toxicity. The following 

findings were noted at the high dose (1000 ppm); body weights were decreased in the P females 

during gestation and early lactation; body weight loss was noted during GD 6-9; body weight 

gains were decreased during GD 6-21 and 0-21; body weight loss was also noted during LD 1-4; 

body weight gains were decreased during LD 4-7. Both absolute and relative maternal food 

consumption were reduced beginning on GD 6, and continuing through early lactation. The 

number of live fetuses/dam was decreased while the number of dead fetuses/dam was increased. 

At the low (100 ppm), mid (300 ppm) and high dose, pup body weights and body weight gains 

were decreased in both sexes. Pinna unfolding was slightly delayed at the mid and high dose. 

In a published reproductive function study by Moser et al. (2001), no signs of overt toxicity were 

noted. At the high dose (60 mg/kg/day), maternal body weights were decreased on GD 21, with 

body weight gains during gestation decreased. Post-mating necropsy of F1 males showed 

reduced kidney weights at the high dose which was likely due to a decreased terminal body 

weight. The number of dead pups/litter was increased at the high dose. At the PND 46 

necropsy, relative (but not absolute) liver weight was increased in the high-dose females; the 

high-dose males had increased relative spleen and liver weights, and a decreased relative kidney 

weight. 

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Page 34 of 81 

In a Part 158 avian reproduction toxicity study on quail (MRID 40700910) and two avian 

reproductive studies on ducks (MRIDs 40700909 and 41818301), no overt or systemic signs of 

toxicity were noted at fed levels up to 74 ppm in quail and 611 ppm in ducks. In a second avian 

reproduction toxicity study on quail (MRID 41624201), there were no signs of overt toxicity, but 

treatment related lesions (liver) were observed in the birds at the high dose (290 ppm). 

In a Part 158 rainbow trout early life stage toxicity study, overt toxicity was noted at the four 

highest test concentrations (0.025, 0.061, 0.120, and 0.230 mg/L) based on reduced survival of 

larvae at test termination. Development of larvae in the mid-high (0.120 mg/L) and high (0.230 

mg/L) concentrations also appeared to be delayed by several days, and throughout the post-hatch 

exposure period larvae at the four highest test concentrations also exhibited abnormal appearance 

and behavior (i.e., dark coloration and lethargy). 

In the Part 158 sheepshead minnow early life stage toxicity study, no signs of overt toxicity were 

noted. However, mean body length at the high concentration (0.0993 mg/L) was reduced 

compared to the solvent controls. Mean wet weight at the mid-high (0.0475 mg/L) and high 

concentrations was reduced compared to the solvent controls. 

In a Part 158 fish full life cycle toxicity study with sheepshead minnow, overt toxicity was noted 

based on decreased parental survival on Days 58 and 177 at the high dose (0.197 mg/L). 

Parental body length was also reduced on Day 33 at the mid-high dose (0.0967 mg/L), and 

growth (i.e., length and weight) of the F1 generation was reduced at 28 days post-hatch at the 

mid-high dose. 

In a fathead minnow fish sexual development study, there was no effect on hatch but survival 

was significantly reduced at ≥0.045 mg/L with effects on female growth at 0.022 mg/L. Systemic 

toxicity was also reported including histopathological changes in the liver, pancreas and 

intestines (≥0.011 mg/L), kidneys (0.045 mg/L) and spinal deformities (≥0.022 mg/L). 

In a published thyroid endocrine disruption study by Yu et al. (2013) on zebrafish embryos, there 

were no signs of overt or systematic toxicity up to 4 mg/L. 

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Page 35 of 81 

B. Estrogen Pathway 

Although tebuconazole was negative in an OSRI ER binding assay and in the Tier 1 ERTA, it 

appears to alter estrogen production. In vitro studies showed tebuconazole can potentially alter 

steroidogenesis and inhibits aromatase activity. In the in vivo mammalian studies, a delay in VO 

was observed in the female pubertal assay; this finding was corroborated by other findings in 

mammalian OSRI such as decreased number of offspring and gestational/delivery alterations in 

the absence of overt toxicity. Additionally, complementary findings were observed in the 

FSTRA which included effects on plasma VTG, gonadal weight and histopathology. 

Furthermore, effects on gonadal histopathology were reported in a fish sexual development test. 

Therefore, the preponderance of the findings suggests that tebuconazole potentially interacts with 

the estrogen pathway both in vitro and in vivo. It is noted that generally, these in vivo findings 

co-occurred with liver effects that included increased weight and/or changes in histopathology. 

C. Androgen Pathway 

Effects on testosterone biosynthesis and AR interaction were consistently noted in the Tier 1 and 

OSRI in vitro studies. In the mammalian in vivo studies, anti-androgen effects were observed in 

young but not adult animals. Testosterone levels were decreased in the Tier 1 male pubertal 

assay, as well as in a reproductive toxicity in the published literature; this concurred with 

decreased weights of androgen-dependent tissues. While there were limited findings in male fish 

in the FSTRA which included a decrease in GSI, gonadal histopathological findings were 

reported in a fish sexual development test. The preponderance of the findings suggests that 

tebuconazole has anti-androgenic activity in vitro and in vivo only in the young, and can interact 

with the androgen receptor and interfere with steroidogenesis. Again, it is noted that these in vivo

findings co-occurred with liver effects that included increased weight and/or changes in 

histopathology. 

D. Thyroid Pathway 

Based on the available data, there is limited evidence for a direct effect of tebuconazole on the 

thyroid pathway or the HPT axis. Negative responses for the T pathway were seen for 

tebuconazole in the in vivo Tier 1 male and female pubertal assays and six of the seven pertinent 

general toxicity studies which showed no effect from tebuconazole treatment. In the published 

studies, effects were noted on dam thyroid hormone levels in developmental studies, but these 

occurred in the presence of systemic toxicity. Developmental delays (including NF stage and 

normalized HLL) along with thyroid hypoplasia were observed in the AMA study, however, this 

coincided with a significant reduction in growth (length and body weight) and liver effects. 

Although findings of thyroid gland hypoplasia are identified in the AMA guideline as diagnostic 

criteria, no effects were seen on the incidence of follicular cell hypertrophy or hyperplasia, which 

are expected to be more commonly affected in the presence of a thyroid-related developmental 

delay. Overall, the thyroid and developmental findings in the AMA with tebuconazole are 

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Page 36 of 81 

inconsistent with a direct, thyroid-related delay and may be more reflective of treatment-related 

growth inhibition. 

E. Conclusions 

Based on in vivo and in vitro results, tebuconazole showed potential to alter the steroidogenesis 

pathway which may result in endocrine-related effects in the estrogen and androgen pathways. In 

the androgen pathway, effects seem to have a greater impact on young than in adult mammals. 

It is noted that the effects observed in mammals and fish coincided with liver effects. 

Based on the available data, there was a lack of complementary and redundancy of evidence in 

the available Tier 1 and OSRI data to suggest a potential interaction with the thyroid pathway 

either in mammals or wildlife. 

V. EDSP Tier 2 Testing Recommendations 

Tebuconazole showed potential to alter the steroidogenesis pathway which may result in 

endocrine-related effects in the estrogen and androgen pathways 

For tebuconazole, the point of departure (POD) for risk assessment is based on developmental 

neurotoxicity endpoint observed in offspring in a developmental neurotoxicity study in rats. The 

basis for the POD is alterations in motor activity, decreases in body weight and brain weight, and 

changes in brain morphometrics observed in the offspring at 8.8 mg/kg/day, the lowest observed 

effect level (LOAEL). Since the study did not establish a no-observed-adverse-effect–level 

(NOAEL), the default 10X Uncertainty Factor (UFL) was applied (i.e., for the use of a LOAEL) 

resulting in an extrapolated NOAEL of 0.88 mg/kg/day. This value is used as the current POD 

for calculating the chronic Reference Dose (RfD) for dietary risk assessment and calculating the 

Margins of Exposure (MOEs) for non-dietary exposure risk assessments. This POD is 

approximately 75-fold lower than the lowest doses (75 mg/kg/day) that caused estrogen and 

androgen-related effects in the Tier 1 pubertal assays. Consequently, the chronic RfD and the 

MOEs are protective of the endocrine-related effects seen in the pubertal assays. 

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

recommended for tebuconazole since additional testing will not impact the current EPA 

established regulatory endpoints for human health risk assessments. 

For the wildlife the EDSP Medaka Extended One-Generation Reproduction Test is 

recommended. In the FSTRA, effects were observed at 19.5 μg/L. Gonadal histological effects 

in the fish sexual development test (FSDT) were observed at 0.045 and 0.022 μg/L for females 

and males, respectively, in the absence of effects on liver. The current chronic NOAEC for fish 

is 12 μg/L based on survival effects in the rainbow trout early-life cycle. It is noted that no 

effects on fecundity were observed in the sheepshead minnow life-cycle study at concentrations 

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Page 37 of 81 

up to 196 μg/L; effects on survival (F0) and growth (F1) were noted. Since the reproductive 

effects observed in the FSTRA and FSDT occur at concentrations similar or below the current 

NOAEC, additional information on potential reproductive effects is recommended. 

As there were no thyroid-related effects observed in the AMA or convincing evidence of a 

potential thyroid-related interaction for tebuconazole, no additional data are requested for 

amphibians. 

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard 

duck are available for tebuconazole. In both species, effects on reproductive parameters were 

observed. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) 

are considered sufficient for evaluating potential reproductive effects to birds from tebuconazole 

exposure. Relative to the EDSP, additional testing is not recommended. 

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Page 38 of 81 

VI. References 

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African Clawed-frog (Xenopus laevis) exposed to tebuconazole under flow-through conditions. 

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2012. MRID 48673701. Unpublished. 

Banman, C, Matlock, D, and Lam, C (2012) Short-term reproduction assay with fathead 

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Sterling, VA. Laboratory Report No. EBHWP002, July 2, 2012. MRID 48688301. 

Unpublished. 

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Umweltchemie AG, Itingen, Switzerland. Laboratory Project ID 96756, April 28, 1988. 

Sponsored by Bayer AG, Wuppertal, Federal Republic of Germany. MRID 40700943. 

Unpublished. 

Becker, H (1987) Embryotoxicity study (including teratogenicity) with HWG 1608 

TECHNICAL in the rabbit. RCC, Research and Consulting Company AG, and RCC, 

Umweltchemie AG, Itingen, Switzerland. Laboratory Project ID 96764, February 26, 1987. 

Sponsored by Bayer AG, Wuppertal, Federal Republic of Germany. MRID 40700945. 

Unpublished. 

Becker, H, and Biedermann, K (1995) Embryotoxicity study (including teratogenicity) and 

supplementary investigations on the maternal toxicity in pregnant rabbits. RCC, Research and 

Consulting Company Ltd., Itingen, Switzerland. Laboratory Project Nos. 319443 and 353294, 

May 31, 1995. MRID 43776201. Unpublished. 

Blanck, O (2011) Tebuconazole: evaluation in the in vitro (HeLa-9903) estrogen receptor 

transcriptional activation assay. Bayer S.A.S., Sophia Antipolis Cedex, France. Laboratory 

Report No. SA 11159, December 1, 2011. MRID 48619403. Unpublished. 

Blanck, M. (2012) Tebuconazole: evaluation in the Hershberger bioassay. Bayer S.A.S., Sophia 

Antipolis Cedex, France. Laboratory Report No. SA 11011, April 10, 2012. MRID 48673702. 

Unpublished. 

Blanck, M (2012) Tebuconazole: evaluation in the immature rat uterotrophic assay. Bayer 

S.A.S., Sophia Antipolis Cedex, France. Laboratory Report No. SA 11010, February 2, 2012. 

MRID 48673703. Unpublished. 

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Bomhard, E (1986) Subchronic toxicological study with rats; feeding for thirteen weeks. Bayer 

AG, Institute of Toxicology, Wuppertal, Federal Republic of Germany. Sponsored by Mobay 

Corporation, Stilwell, KS. Laboratory Study No. 94212, October 27, 1986. MRID 40700930. 

Unpublished. 

Bomhard, E, and Burnett, R (1991) HWG 1608: toxic dose range carcinogenicity study in 

NMRI mice (supplement to Study T 6018953-carcinogenicity in NMRI Mice with 

Administration in Diet over a 21-Month Period). Bayer AG, Institute of Toxicology, Wuppertal, 

Federal Republic of Germany. Laboratory Study No. 16376-A, December 12, 1991. MRID 

41275001. Unpublished. 

Bomhard, E, and Ramm, W (1988) HWG 1608: study for chronic toxicity and cancerogenicity 

in Wistar rats. Bayer AG, Institute of Toxicology, Wuppertal, Federal Republic of Germany. 

Laboratory Project ID 96711, January 25, 1988. MRID 40700939. Unpublished. 

Bomhard, E, and Ramm, W (1988) HWG 1608: study for cancerogenicity in NMRI mice 

(administration in diet for up to twenty-one months). Bayer AG, Institute of Toxicology, 

Wuppertal, Federal Republic of Germany. Sponsored by Mobay Corporation, Stilwell, KS. 

Laboratory Study No. 96709, January 25, 1988. MRID 40700941. Unpublished. 

Eiben, R (1987) HWG 1608: Two-generation study in rats. Bayer AG, Institute of Toxicology, 

Wuppertal, Federal Republic of Germany, and PATCO AG, Itingen, Switzerland. Laboratory 

Project ID 91064, November 12, 1987. Sponsored by Mobay Corporation, Kansas City, MO. 

MRID 40700946. Unpublished. 

Fletcher, D, and Pedersen, C (1990) Folicur Technical: toxicity and reproduction study in 

Mallard ducks. Bio-Life Associates, Ltd., Neillsville, WI. Laboratory Project ID not provided, 

completion date not provided. Sponsored by Mobay Corporation, Kansas City, MO. MRID 

41818301. Unpublished. 

Hass, U, Boberg, J, Christiansen, S, et al. (2012) Adverse effects on sexual development in rat 

offspring after low dose exposure to a mixture of endocrine disrupting pesticides. Reprod. 

Toxicol. 34: 261-274.

Jacobsen, P, Axelstad, M, Boberg, J, et al. (2012) Persistent developmental toxicity in rat 

offspring after low dose exposure to a mixture of endocrine disrupting pesticides. Reprod. 

Toxicol. 34: 237-250. 

Kjærstad, M, Taxvig, C, Nellemann, C, et al. (2010) Endocrine disrupting effects in vitro of 

conazole antifungals used as pesticides and pharmaceuticals. Reprod. Toxicol. 30: 573-582. 

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Lautraite, S (2012) Tebuconazole: assessment of pubertal development and thyroid function in 

juvenile/peripubertal male and female rats. Bayer S.A.S, Sophia Antipolis Cedex, France. 

Laboratory Report No. SA 11013, June 26, 2012. MRID 48688302. Unpublished. 

Laws, S, Yavanhxay, S, Cooper, R, et al. (2006) Nature of the binding interaction for 50 

structurally diverse chemicals with rat estrogen receptors. Toxicol. Sci. 94 (1): 46-56. 

Laws, S, Kariya, J, Wolf, M, et al. (2009) DSSTox EPA Estrogen Receptor Ki Binding Study 

(Laws et al.) Database - (KIERBL): SDF file and documentation, Launch version: 

KIERBL_v1a_278_17Feb2009, http://www.epa.gov/ncct/dsstox/sdf_kierbl.html. 

Moser, V, Barone, S, Smialowicz, R, et al. (2001) The effects of perinatal tebuconazole exposure 

on adult neurological, immunological, and reproductive function in rats. MRID 45486901. 

Published: Toxicol. Sci. 62: 339-352. 

Orton, F, Rosivatz, E, Scholze, M, et al. (2011) Widely used pesticides with previously unknown 

endocrine activity revealed as in vitro antiandrogens. Environ. Health Perspect. 119 (6): 794-

800. 

Overgaard, A, Holst, K, Mandrup, K, et al. (2013) The effect of perinatal exposure to ethinyl 

oestradiol or a mixture of endocrine disrupting pesticides on kisspeptin neurons in the rat 

hypothalamus. NeuroToxicology 37: 154-162. 

Parker, R (2000) Developmental neurotoxicity study of technical grade tebuconazole 

administered orally via diet to Crl:CD®BR VAF/Plus® presumed pregnant rats. Primedica 

Argus Research Laboratories, Inc., Horsham, PA. Laboratory Study No. 1702-004, March 1, 

2000. MRID 45074301. Unpublished. 

Porter, M, Jasty, V, Troup, C. M, et al. (1989) Safety evaluation of HWG 1608: chronic (1-year) 

feeding study in dogs. Miles Inc., Toxicology Department, Elkhart, IL. Laboratory Study No. 

99673, June 28, 1989. MRID 42030601. Unpublished. 

Ramm, W (1986) Range-finding toxicological study with NMRI mice to establish dosage for a 

chronic study (feeding for eight weeks) and for determinations of enzyme induction in the liver 

(feeding for five days). Bayer AG, Institute of Toxicology, Wuppertal, Federal Republic of 

Germany. Sponsored by Mobay Corporation, Stilwell, KS. Laboratory Project ID No. 94211, 

July 7, 1986. MRID 40700933. Unpublished. 

Rouquie, D (2011) Evaluation of tebuconazole in the androgen receptor binding assay. Bayer 

SAS, Sophia Antipolis Cedex, France. Laboratory Study No. SA 11212, December 8, 2011. 

MRID 48619401. Unpublished. 

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Scott Ward, G. (1991) Toxicity to embryos and larvae of the Sheepshead minnow (Cyprinodon 

variegatus) under flow-through test conditions. Toxikon Environmental Sciences, Jupiter, FL. 

Laboratory Study No. J9101002b, completion date not provided. Sponsored by Mobay 

Corporation, Agricultural Chemicals Division, Kansas City, MO. MRID 42038202. 

Unpublished. 

Surprenant, D (1988) The toxicity of HWG 1608 Technical to rainbow trout (Salmo gairdneri) 

embryos and larvae. Laboratory Report No. 87-11-2545, completion date not provided. 

Springborn Life Sciences, Inc., Wareham, MA. Sponsored by Mobay Corporation, Stilwell, KS. 

MRID 40700914. Unpublished. 

Toll, P.A. (1988) Effects of HWG 1608 (FOLICUR) on Mallard Duck Reproduction. Study No. 

87-675-01. Prepared by Mobay Corp., Biochemistry/Wildlife Effects Group. Research and 

Development Dept. Stilwell, KA. Submitted by MOBAY Corp., Agricultural Chemicals 

Division, Kansas City, MO. MRID 40700909. Unpublished. 

Taxvig, C, Hass, U, Axelstad, M, et al. (2007) Endocrine-disrupting activities in vivo of the 

fungicides tebuconazole and epoxiconazole. MRID 48277401. Published: Toxicol. Sci. 100 (2): 

464-473. 

Taxvig, C, Vinggaard, A, Hass, U, et al. (2008) Endocrine-disrupting properties in vivo of 

widely used azole fungicides. MRID 48264606. Published: Int. J. Androl. 31: 170-177. 

Tinwell, H (2011) Evaluation of tebuconazole in the aromatase assay. Bayer S.A.S. (Bayer 

CropScience, Sophia Antipolis Cedex, France. Laboratory Study ID SA 11064, Dec 9, 2011. 

MRID 48619402. Unpublished. 

Tinwell H (2011) Evaluation of tebuconazole in the H295R steroidogenesis assay. Bayer S.A.S., 

Bayer CropScience, Sophia Antipolis Cedex, France. Laboratory Study No. SA 11072, 

November 10, 2011. MRID 48619404. Unpublished. 

Toll, P (1988) Effects of HWG 1608 (FOLICURTM) on bobwhite quail reproduction. Mobay 

Corp., Biochemistry/Wildlife Effects Group, Research and Development Dept., Stilwell, KS. 

Laboratory Study No. 87-675-02, completion date not provided. MRID 40700910. 

Unpublished. 

Toll, P (1990) Technical HWG 1608 (FOLICUR): a one generation reproduction study with 

bobwhite quail. Mobay Corporation, Kansas City, MO. Laboratory Project ID not provided, 

completion date not provided. MIRD 41624201. Unpublished. 

USEPA. (2002) Development of estrogen binding data for approximately 300 chemicals: final 

data package submittal. Battelle, EPA Contract No. 68-W-99-033, Work Assignment 3-04. 

Unpublished.

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USEPA (2010) Tebuconazole: Report of the Endocrine Disruptor Review Team; Test Order#: 

EDSP-128997-142; -143; -144; -145; -147; -148; -149; -151; -153; -154; and -155. December 

20, 2010. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection 

Agency, Washington, DC. 

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

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

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

Protection Agency, Washington, DC. 

von Keutz, E (1987) HWG 1608: subchronic study of toxicity to dogs with oral administration 

(thirteen-week feeding study). Bayer AG, Institute for Toxicology, Wuppertal, Federal Republic 

of Germany. Sponsored by Mobay Corporation, Kansas City, MO. Laboratory Study No. 

94984, May 6, 1987. MRID 40700934. Unpublished. 

von Keutz, E (1987) HWG 1608: study of chronic toxicity to dogs after oral administration 

(twelve-month feeding study). Bayer AG, Institute for Toxicology, Wuppertal, Federal Republic 

of Germany. Sponsored by Mobay Corporation, Kansas City, MO. Laboratory Report No. 

956090, November 11, 1987. MRID 40700940. Unpublished. 

Wheat, J (1993) HWG 1608 (tebuconazole): life cycle chronic toxicity to the Sheepshead 

minnow (Cyprinodon variegatus) under flow-through conditions. Toxikon Environmental 

Sciences, Jupiter, FL. I.aboratory Study No. J9104001, completion date not provided. 

Sponsored by Miles Incorporated, Kansas City, MO. MRID 43009601. Unpublished. 

Yu, L, Chen, M, Liu, Y, et al. (2013) Thyroid endocrine disruption in zebrafish larvae following 

exposure to hexaconazole and tebuconazole. Aquat. Toxicol. 138: 35-42. 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100) 

The 21-day assay (MRID 48673701) of Tebuconazole (97.5% purity) on amphibian 

metamorphosis of South African clawed frogs (Xenopus laevis) was conducted under flowthrough conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80/group, 20 in each 

of four reps) were exposed to nominal concentrations of 0 (negative control), 0.0152, 0.076, and 

0.380 mg a.i./L; mean-measured concentrations were <0.0015 (<LOQ; control), 0.0170, 0.067, 

and 0.341 mg a.i./L. The test system was maintained at 21.4 to 22.7°C and a pH of 7.9 to 8.4. 

Unless otherwise indicated, all effects are reported based on comparison to the negative (clean 

water) control. The Day 21 survival averaged 100% in the control and low treatment group and 

98.3% in the mid and high treatment groups. Tadpoles with bent tails were observed in the 

control (16/80 tadpoles) and low (14) and mid (5) treatment groups. In the high treatment, two 

tadpoles were reported as pale while two more tadpoles showed erratic behavior/loss of 

equilibrium. 

Late-stage tadpoles made up more than 20% of the surviving tadpoles on Day 21 in the negative 

control (55%) and low treatment group (38%), whereas late-stage tadpoles accounted for 7 and 

0% of survivors in the mid and high treatment groups. All growth analyses were conducted 

excluding late-stage tadpoles and separate analyses were not conducted on the late-stage tadpoles 

because none were present in the highest dose. Tebuconazole had no effect on 7-day normalized 

hind-limb length (HLL), whereas the highest dose exhibited a significant 56% reduction relative 

to the control on Day 21. Significant delay of median NF developmental stage by one stage was 

observed at 7 days in all treatment groups and at 21 days in the mid and high treatment groups 

(by one stage at the mid and 5 stages at the high). No asynchronous development was observed 

in any treatment group. Given the large differences in NF stage between the control and high 

treatment group, additional control tadpoles were reared with the goal of selecting tadpoles at 

developmental stages similar to the high treatment (NF stage 56 and 57). Mild thyroid 

hypoplasia was observed at the high treatment group in all 20 tadpoles, whereas this finding was 

not observed in the concurrent control, low or mid treatment group but was observed in 11/20 

tadpoles in the stage-matched control. Mild follicular cell hypertrophy was observed in the 

concurrent control (6/20 tadpoles), staged-matched controls (2/20 tadpoles) and low and mid 

treatment groups (3/20 and 6/20 tadpoles respectively). The procedure of rearing additional 

control frogs to allow for stage-matching histopathological evaluation is not part of the guideline, 

and there is uncertainty in the ability to compare these frogs to the treatment groups given that 

they were not from the same batch of eggs and were not reared at the same time which may have 

resulted in slight differences in environmental conditions. 

Significant reductions in snout-vent length (SVL) and body weight (BW) were observed at the 

high concentration on both Day 7 (SVL: ⇘ 16%; BW: ⇘ 24%) and 21 (SVL: ⇘ 24%; BW: ⇘

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59%) and in the low treatment on Day 21 (SVL: ⇘ 6%; BW: ⇘ 12%). In the high treatment, two 

tadpoles were reported as pale while two more tadpoles showed erratic behavior/loss of 

equilibrium. Histological findings in the liver occurred in the mid and high treatment groups, and 

included decreased hepatocyte vacuolation, hepatocyte anisocylosis, hepatocyte anisokaryosis 

and increased multinucleate hepatocytes. An additional treatment-related finding in the high 

treatment group was individual hepatocyte necrosis. 

Androgen Receptor (AR) Binding Assay (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48619401), ventral prostate cytosol from 

male Sprague Dawley rats was used as the source of AR to conduct a saturation binding 

experiment and a competitive binding experiment. 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 androgen 

(R1881) prior to routinely conducting AR competitive binding experiments. The competitive 

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

H]-R1881 (1 

nM) in the presence of increasing concentrations of tebuconazole (logarithmic increase from 

10−10 to 10−3

 M). DMSO was used as the solvent at a final assay concentration of ≤3.3%. The 

assay included dexamethasone as a weak positive control, and R1881 as the ligand reference 

standard. 

In the saturation binding experiment, the mean maximum binding capacity (Bmax) was 14 

fmol/100 μg protein and the mean dissociation constant (Kd) was 1.01 nM. The Kd obtained 

from Saturation Assay #2 was low (0.59 nM), but the Kds from the other two runs were within 

the recommended range (0.685 to 1.57 nM) and the Scatchard plots for the three saturation 

assays were linear. The specific binding reached a plateau, and non-specific binding (NSB) was 

less than 20% of total binding (4.6-12.6%). The saturation binding experiment demonstrated that 

AR was present in a reasonable concentration and functioning with appropriate affinity for the 

R1881 ligand. 

In the competitive binding experiment, the estimated mean log IC50 was −4.3 M for tebuconazole 

compared to that of R1881 of −9.1 M and the weak positive control of −4.4 M. The mean RBA 

was 0.0014% for tebuconazole compared to 0.0021% for the weak positive control. No 

precipitation of the test compound was visually observed at any concentration. All performance 

criteria were met, with the exception that ligand depletion and NSB were not reported, but the 

IC50s for R1881 and the weak positive control were within the expected ranges demonstrating 

that the ligand was available at an appropriate level. These deviations were considered minor 

and performance criteria were found to be met. 

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Tebuconazole reduced the mean specific binding of the radiolabeled ligand in a concentration 

dependent manner in all three runs. The maximum binding reduction occurred at the highest 

concentration tested (10−3 M), with specific binding being reduced to 1.5-6.9%. Specific binding 

was also reduced at 10−4

 M (33-37%), but not at concentrations ≤10−5 M (82-111% binding). 

Specific [

3

H]-R1881 AR binding was reduced to <50% at the highest concentrations tested in all 

three runs. 

Based on the results from the three runs, tebuconazole is classified as a Binder in the Androgen 

Receptor Binding Assay. 

Aromatase Assay (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48619402), tebuconazole (97.5% a.i., batch# AE 

F069623-01-18) was incubated with human recombinant aromatase and tritiated androstenedione 

(1-β [3

H(N)]-androst-4-ene-3,17-dione; [3H]ASDN) in dimethyl sulfoxide (DMSO) at increasing 

logarithmic concentrations from 10–10 M to 10−3 M for 15 minutes to assess the potential of 

tebuconazole to inhibit aromatase activity. 

Aromatase activity was determined by measuring the amount of tritiated water produced at the 

end of a 15-minute incubation for each concentration of tebuconazole. Tritiated water was 

quantified using liquid scintillation counting (LSC). Three runs were conducted and each run 

included a full activity control, a background activity control, a positive control series (10−10 to 

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

tebuconazole series (10−10 to 10−3

) with three repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.250 to 0.365 nmol/mg-protein/min 

for the three test runs, with a mean and standard deviation of 0.306±0.038 nmol/mg-protein/min. 

Activity in the background controls ranged from 3.3 to 4.4% of the full control activity with an 

average of 3.6%. The response of the full activity controls and background controls was 

acceptable. 

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.2925±0.0202 

nmol/mg-protein/min at the lowest tested concentration (10−10 M) and 0.0028±0.0005 nmol/mgprotein/min at the highest tested concentration (10−5 M). These results were generally within the 

recommended ranges for the top of the curve, bottom curve, Hill slope, and log IC50. 

For tebuconazole, aromatase activity averaged 0.2706 ± 0.0069 nmol/mg-protein/min at the 

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

tested concentration (10−3

 M). The lowest portion of the response curve for tebuconazole across 

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runs was less than 50% activity. Tebuconazole reduced aromatase activity to 90% at 10−7 M, 

62% at 10−6 M, 19% at 10−5 M, 2.3% at 10−4 M, and 0.1% at 10−3 M. 

The estimated log IC50 ranged from −5.8 to −5.7 M for the test material and averaged −5.73 M 

compared to the positive control 4-OH ASDN, which averaged −7.20 M. The average slope of 

the concentration response curve was −0.99 for tebuconazole and −0.88 for 4-OH ASDN. 

Based on the data form the average response curve, tebuconazole is classified as an Inhibitor of 

aromatase activity in this assay. 

Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250) 

The requirements for a Tier 1 ER receptor binding assay were satisfied by the study by USEPA 

(2002). EPA tested tebuconazole in a rat uterine cytosol ER competitive binding assay as one of 

the chemicals used to evaluate a set of QSAR models in an unpublished report by Battelle (Work 

Assignment 3-04, EPA Contract number 68-W- 99-033). Tebuconazole was negative for ER 

binding. The procedures were described in Laws et al. (2006); however, this paper was a followup for the positive chemicals. A synopsis is presented in Appendix 2. 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48619403), hERα-HeLa-9903 

cells cultured in vitro were exposed to tebuconazole (97.5% a.i., Batch # K689052) at 

logarithmically increasing concentrations from 10−10 to 10−4

M in DMSO (0.1%). The 

experiments were performed using 96-well plates and each concentration was tested in triplicate 

(3 wells/plate). Cells were exposed to the test agent for 23 ±1 hour to induce reporter 

(luciferase) gene products. Luciferase expression in response to activation of the estrogen 

receptor was measured upon addition of a luciferase substrate and detection with a luminometer 

with acceptable sensitivity. 

Tebuconazole was tested up to the limit of solubility, 10−4 M. Performance criteria were met for 

all reference chemicals. The log PC10 for 17α-estradiol and 17α-methyltestosterone, and Hill 

Slope for 17α-estradiol were lower than the expected range in the first run, indicating increased 

sensitivity of the assay to weak agonists. The mean RPCMax for tebuconazole was -0.2% for the 

first run and -1.6% for the second run; the associated PCMax was 10−10 M for the first run and 

10−5

 M for the second run. Because the RPCMax < PC10 in two assay runs, tebuconazole was 

considered negative for estrogen receptor transcriptional activation in this test system. 

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Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350) 

The 21-day fish short-term reproduction assay (MRID 48688301) of tebuconazole (97.5% 

purity) with fathead minnows (Pimephales promelas) was conducted under flow-through 

conditions. Sixteen spawning groups of adult fish (2 males and 4 females per group; ca. 7 months 

of age) were exposed to tebuconazole at nominal concentrations of 0 (water control), 0.0065, 

0.013, and 0.026 mg a.i./L, which resulted in mean measured concentrations of <0.00065 

(<LOQ), 0.00497, 0.0113, and 0.0195 mg a.i./L, respectively. The mean measured recoveries 

ranged from 75 to 87% of nominal concentrations. The test system was maintained at 24.7 to 

25.6oC and a pH of 7.7 to 7.9. 

There were no significant differences in survival in the tebuconazole treatment groups compared 

to the control (overall survival was 100% in the control and 88-92% in the treatment groups). 

Additionally, there were no treatment-related clinical signs of toxicity observed in the treatment 

groups. No treatment-related effects were observed on body weight or length. 

Spawning occurred at least every 4 days in two of the four control replicates and mean fecundity 

in controls was 23.7 eggs/female/day/replicate (replicate range: 15.7-31.9 eggs/female/day); 

fertilization success in controls was 97.6%. There were no significant effects on fecundity or 

fertility (fertilization success) in the tebuconazole treatment groups. There were no significant 

effects on male tubercle score and there were no tubercles observed in females. At the high 

treatment concentration there was a significant (p<0.05; Jonckheere-Terpstra) decrease of 18.5% 

in female gonado-somatic index (GSI) and a marginally significant (p=0.056; JonckheereTerpstra) decrease of 19% in male GSI. There was a significant decrease of 35 and 55% in 

female plasma vitellogenin (VTG) at the mid and high treatment concentrations, respectively 

(p<0.05; Jonckheere-Terpstra); there were no significant differences in male VTG between the 

treatment groups and the control. 

According to the pathologist’s report, no histopathologic findings observed in the testes of the 

male fathead were clearly related to tebuconazole treatment. The mean testicular stage scores 

were decreased in the mid and high treatment groups compared to the controls, but the study 

authors report this decrease is unlikely to be biologically meaningful. In the ovary of the female 

fathead, the histopathologic finding of increased granulomatous inflammation (minimal to mild) 

in the high treatment group compared to the controls was most likely related to tebuconazole 

treatment. Additional histopathological findings for females included increased prevalence of 

post-ovulatory follicles and decreased prevalence of oocyte atresia in the tebuconazole-treated 

groups, neither of which were reported as potentially treatment-related by the pathologist; in the 

controls, 47% of the examined females were reported to have some level of increased oocyte 

atresia (2 fish with minimal and 5 fish with moderate increased atresia) whereas the low, mid and 

high treatment groups had 40, 20, and 20%, respectively. The mean ovarian stage scores were 

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decreased in the high treatment females compared to the controls (2.9 vs. 3.2), but the study 

authors report this decrease is unlikely to be biologically meaningful. Histological examination 

of the liver was also conducted with no substantially more prevalent findings for treated males 

compared to the control. However, for males, the occasional findings of microgranulomas 

(minimal; mid and high treatment groups) and individual hepatocyte necrosis (minimal; high 

treatment group) in the livers were likely treatment related; there was also a concentrationrelated decrease in hepatocyte vacuolation compared to control. In females, generally 

concentration-dependent findings in the liver included individual hepatocyte necrosis (occurred 

in all three treatment groups), multinucleated hepatocytes (mid and high treatment groups), 

microgranulomas (mid and high treatment groups), hepatocyte megalocytosis (mid and high 

treatment groups), and decreased hepatocellular vacuolation (all groups). Plasma sex steroids 

were not measured in this study. 

Hershberger Assay (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48673702) screening for androgenic activity, tebuconazole 

(97.5% a.i.; Batch # K689052) in 0.5% aqueous methylcellulose 400 + 0.4% Tween 80 was 

administered daily via oral gavage to groups of six 58-day old, castrated male Sprague Dawley 

rats at dose levels of 0 (vehicle), 75, or 150 mg/kg/day. An androgenic positive control group 

consisted of six castrated rats exposed to 0.4 mg/kg/day of testosterone propionate (TP) by 

subcutaneous (s.c.) injection. 

To screen for potential anti-androgenic activity, tebuconazole in 0.5% aqueous methylcellulose 

400 + 0.4% Tween 80 was also administered daily via oral gavage to groups of six 58-day old, 

castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 35, 75, or 150 mg/kg/day in 

conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The 

anti-androgenic positive control group consisted of six castrated rats exposed to 0.4 mg/kg/day 

TP and 3 mg/kg/day flutamide (FT). TP alone was used as the 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 were collected and weighed. 

All animals survived until scheduled termination. No clinical signs of toxicity were observed in 

animals for any dose groups. 

In the androgen assay, body weights throughout the duration of the assay in the tebuconazoledosed groups and positive (vehicle + TP) control group were comparable to the vehicle controls. 

Overall (Days 1-10) body weight gains in the tebuconazole 150 mg/kg/day groups were 

decreased (p<0.05) by 20% compared to the vehicle controls. Overall body weight gains in the 

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positive control group were increased (p<0.05) by 29% compared to the vehicle controls. No 

treatment-related effects were observed in any of the accessory sex organs of tebuconazole-dosed 

animals. Rats in the positive control (vehicle + TP) group had accessory sex organ weight 

increases (p<0.05) as follows: 992% in seminal vesicles; 602% in ventral prostate; 160% in 

levator ani-bulbocavernosus (LABC); 449% in Cowper’s glands; and 65% in glans penis. 

In the anti-androgen assay, body weights throughout the study and body weight gain from Days 

1 to 10 were similar across all groups. At 75 mg/kg/day, mean cumulative body weight gains 

were decreased (p<0.05) from Days 1 to 3 by 24%. At 150 mg/kg/day, mean cumulative body 

weight gains were decreased (p<0.05) from Days 1 to 3 by 71%, Days 1 to 4 by 33%, and Days 1 

to 6 by 16% compared to the vehicle control; mean body weights were not affected by the end of 

the study. The accessory sex organ weights in all tebuconazole treatment groups were 

comparable to the negative controls. Rats in the positive control (FT + TP) group had accessory 

sex organ weight decreases (p<0.05) as follows: 71% in seminal vesicles; 69% in ventral 

prostate; 38% in LABC; 52% in Cowper’s glands; and 22% in glans penis. 

The dosing in this study was considered adequate based on the decreased overall body weight 

gains observed at the high-dose (150 mg/kg/day).

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

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

Hershberger assay. 

Female Pubertal Assay (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48688302), 15 female Crl(CD)SD rats/dose group were 

treated daily via oral gavage with tebuconazole (97.5% a.i., Lot # K689052) in 0.5% 

methylcellulose 400 + 0.4% Tween 80 at doses of 0 (vehicle), 75 or 150 mg/kg/day from postnatal day (PND) 22 to 42. Animals were examined for vaginal opening (VO) daily beginning on 

PND 22, and age and weight at day of attainment were recorded. Following sacrifice on PND 

42, blood was collected for clinical chemistry analyses, and total thyroxine (T4) and thyroid 

stimulating hormone (TSH) levels were determined using enzyme-linked immunoassay (ELISA) 

kits. Adrenal, liver, pituitary, thyroid, and urogenital organs were weighed, and microscopic 

examinations were performed on the ovaries, uterus, thyroid, and kidneys. 

No adverse, treatment-related effects were observed on mortality; estrous cyclicity; gross 

pathology in the liver, kidneys, or adrenal glands; or histopathology in the kidneys. No 

treatment-related effect was noted on serum T4 or TSH levels, or on thyroid weight or 

morphology. 

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All rats survived the 21-day test period. Mean daily body weight and body weight gain/day in 

the 150 mg/kg/day animals was decreased on several days through PND 33, but there were no 

treatment-related changes in final body weight or cumulative body weight gain (PND 22-42). 

Compared to the control, the mean age at initial VO was increased by 1.2 days (not significant, 

NS) and 2.7 days (p<0.01) at 75 and 150 mg/kg/day, respectively, and the mean age at complete 

VO was increased by 1.1 days (NS) and 2.0 days (p<0.05) at 75 and 150 mg/kg/day, 

respectively. However, there were no significant differences in body weights between groups at 

initial and complete VO. 

Administration of tebuconazole did not change the day of first estrus compared to the vehicle 

control group. Animals exhibited similar cycle lengths and regularity, and the majority of 

females in the 150 mg/kg/day group were in diestrus at necropsy. 

Liver weights and relative (to body) liver weights were increased (p<0.01) by 8% at75 

mg/kg/day and by 15-18% at 150 mg/kg/day, and there was an increase (p<0.01) of 6% in 

relative (to body) kidney weights at 150 mg/kg/day. No other treatment-related changes were 

observed in organ weights. 

Effects on clinical chemistry parameters included decreased mean creatinine (↓8%; p≤0.01) and 

increased total cholesterol (↑17%; p<0.01) concentrations in the 150 mg/kg/day animals, and 

decreased blood urea nitrogen (↓15-17%), total protein (↓6%), and total bilirubin (↓100%) 

concentrations (p<0.05 or 0.01) in females administered 75 or 150 mg/kg/day. 

There were no treatment-related changes in serum T4 and TSH levels or on thyroid weight or 

morphology associated with tebuconazole treatment. 

No overt toxicity was observed during treatment, but an earlier range-finding study noted 

increased salivation at 300 and 150 mg/kg/day, as well as a decrease in body weight gain. The 

doses tested were determined to be adequate since increased salivation, and decreased daily body 

weight and body weight gain were see at the high dose. 

 

Although increases in the age of initial and complete VO were observed in the tebuconazole 

treated rats, body weights at VO were similar between the control and treated groups, suggesting 

that the delay in VO for the tebuconazole treated groups resulted from the delayed growth in 

these groups during the early part of the study. 

Male Pubertal Assay (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48688302), 15 male Sprague Dawley [Crl:CD (SD)] rats/dose 

group were treated daily via oral gavage with tebuconazole (97.5% a.i., Batch #: K689052) in 

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aqueous 0.5% methylcellulose 400+0.4% Tween 80 at doses of 0, 75, or 150 mg/kg/day from 

post-natal day (PND) 23 to 53. Animals were examined for preputial separation (PPS) daily 

beginning on PND 30, and age and weight on the day of attainment was recorded. Following 

sacrifice on PND 53, blood was collected for clinical chemistry and hormone analysis. Total 

serum testosterone levels were determined by using an enzyme immunoassay (EIA), and 

thyroxine (T4) and thyroid stimulating hormone (TSH) levels were determined with enzymelinked immunosorbent assays (ELISA). Liver, pituitary, adrenals, thyroid, and urogenital organs 

were collected and weighed, and microscopic examinations were conducted on the testes, 

epididymides, thyroid, and kidneys. 

No adverse, treatment-related effects were observed on mortality; clinical chemistry parameters; 

gross pathology in the liver, kidneys, or adrenal glands; or histopathology in the kidneys. No 

treatment-related effect was noted on serum T4 or TSH levels, or on thyroid weight or 

morphology. 

Clinical signs of toxicity were limited to increased salivation in 9/15 males in the 150 mg/kg/day 

dose group compared to 0/15 in both the control and 75 mg/kg/day groups. At 150 mg/kg/day, 

mean body weight was decreased (p≤0.05 or 0.01) by 6-10% from PND 24 to 53, with a terminal 

body weight decrease (p≤0.05) of 8%. On PND 24, there was a mean body weight loss (p≤0.01) 

of 1.5 g compared to a mean body weight gain of 4.9 g in control animals. Differences in 

cumulative body gain at the high dose lessened throughout the study with an 8% decrease in the 

final cumulative body weight gain (PND 24-53). At 75 mg/kg/day, mean body weight was 

decreased (p≤0.05) on several occasions by 6 to 7%. Mean cumulative body weight gains were 

decreased by 53% (p≤0.05) on PND 24 and 18% on PND 25, but diminished to an 11% decrease 

by PND 33. Significant decreases in cumulative body weight gain in the low dose group did not 

continue past PND 47. 

Decreases (p≤0.05 or 0.01) were observed in kidney, pituitary and adrenal gland weights. These 

decreases in weight at 75 and 150 mg/kg/day were dose-dependent for kidneys (7 and 9%, 

respectively) and pituitary glands (9 and 12%), but not for adrenal glands (18 and 13%). 

Additionally, relative (to body) liver weight was increased (p≤0.01) by 10% at 150 mg/kg/day. 

Compared to the control, mean serum testosterone was decreased by 17% (not significant, NS) at 

75 mg/kg/day and by 53% (p≤0.05) at 150 mg/kg/day. The ages at initial PPS were delayed by 

ca. 2.5 days (p<0.01) in the high-dose group, but there was no difference in age at complete PPS 

between groups. The delay in initial PPS in the high-dose group was possibly related to delayed 

growth, as body weight gains were reduced in the high-dose group and body weights at initial 

and complete PPS were similar across groups. Although the age at complete PPS was similar 

between groups, there was a dose-related trend in the number of males in which the preputia 

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dose 7/15). Therefore, the lack of significant differences in age at complete PPS may be masked 

by the use of PND 54 (termination date, plus one) in calculating averages for complete PPS in 

the high-dose group. 

With the exception of the ventral prostate, there were dose-dependent decreases in the weights of 

androgen-dependent tissues. At 150 mg/kg/day, decreases (p≤0.05 or 0.01) were observed in the 

weights of the seminal vesicle and coagulating gland with and without fluid (↓23-33%), the 

dorsolateral prostate (↓21%), the LABC (↓24%), and the left and right epididymis (↓13-14%). In 

addition, the weights of the testes were decreased (NS) by 10-11% at the high dose. At 

75 mg/kg/day, dorsolateral prostate weights were decreased (p≤0.05) by 21%, and although not 

significant, substantial weight decreases were also noted in the seminal vesicle and coagulating 

gland with and without fluid (↓17-21%) and the LABC (↓11%). 

Atrophy was observed in the following androgen-dependent tissues (# affected/# examined): 

(i) testes (atrophic/small and/or soft) at 75 (2/15) and 150 (3/15) mg/kg/day compared to control 

(1/15); (ii) epididymis at 150 mg/kg/day (1/15) compared to control (0/15); (iii) seminal vesicle 

at 75 (5/15) and 150 (7/15) mg/kg/day compared to control (2/15); (iv) prostate at 75 (2/15) and 

150 (5/15) mg/kg/day compared to control (1); and (v) LABC at 75 (1/15) and 150 (3/15) 

mg/kg/day compared to control (0/15). Although the incidences of atrophy were small, it was a 

common finding in several androgen-dependent tissues, and there was additional evidence of 

effect on androgen-related systems. Additionally, a red focus was observed in the epididymis at 

150 mg/kg/day (2/15) compared to control (0/15). Tubular degeneration in the testes was 

increased at 350 mg/kg/day: unilateral (1/15 treated vs 0/15 control) and bilateral (3/15 treated 

vs 1/15 control). Bilateral oligospermia in the epididymis was observed at 75 (2/15) and 150 

(4/15) mg/kg/day compared to control (1/15). 

The high-dose used in the current study is considered adequate based on the effects observed on 

body weight and body weight gains, and the information from the available range-finding study. 

Steroidogenesis Assay (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48619404), H295R cells cultured in vitro in 24-well plates 

were incubated with tebuconazole (97.5% purity, Batch # AE F069623-01-18) at increasing 

logarithmic concentrations from 10−10 to 10−4

 M for 48 hours in triplicate for three independent 

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

0.1% (v/v). 

Testosterone and estradiol levels were measured using specific EIA kits. A Quality Control 

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

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

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

(forskolin) of estradiol and testosterone production. 

Guideline acceptability recommendations and requirements were generally met, including lack 

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

%CV), and appropriate induction and inhibition with positive controls, with the following 

exceptions. In the QC plates, the variability (CV) in the solvent control (SC) between the runs 

was >30% for estradiol (31%), and for Run 3, the forskolin control resulted in a 1.80–fold 

induction of testosterone, outside the ≥2-fold induction recommendation in the Guideline 

(average induction across runs was >2-fold). 

Precipitation of the test compound was not reported at any concentration. Cytotoxicity was not 

observed at any tested concentration. Decreases (p≤0.05) in testosterone concentrations (↓32-

98%) were observed at ≥10−6

 M, and estradiol production was completely inhibited at ≥10−5 M. 

An effect was not observed at other concentrations in these two assays. 

Based on the hormone responses in each of the three independent runs, tebuconazole treatment 

resulted in statistically significant and reproducible decreases in testosterone and estradiol 

production. 

Uterotrophic Assay (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48673703) conducted to screen for potential estrogenic activity, 

tebuconazole (97.5% a.i., Lot # K689052) in 0.5% aqueous methylcellulose 400 + 0.4% Tween 

80 was administered daily via oral gavage to groups of 6 immature female, Sprague-Dawley rats 

at dose levels of 0 (vehicle), 35, 75, or 150 mg/kg/day on post-natal days (PND) 19-21. A 

positive control group was treated with 17α-ethynyl estradiol (EE) in corn oil by gavage at a 

dose level of 1 μg/kg/day. All animals were terminated and necropsied on PND 22 

approximately 24 hours after the final dose to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. No clinical signs of toxicity were observed in 

any rats in the vehicle control, EE, or ≤75 mg/kg/day tebuconazole groups. Five of six animals 

in the 150 mg/kg/day group were observed to have reduced motor activity on Day 3. Body 

weights for the 35 and 75 mg/kg/day tebuconazole groups were comparable to the vehicle 

controls throughout the duration of the assay. Overall (Days 1-3) body weight gains were 

decreased (p<0.05) in the 75 mg/kg/day (↓38%) group compared to the vehicle controls. Final 

body weights were decreased (p<0.01) by 15-24% on Days 2-3 in the 150 mg/kg/day group 

compared to controls, resulting in an overall (Days 1-3) body weight loss (p<0.01) of 3.0 g 

(compared to a gain of 8.2 g in the controls). Uterine weights in the tebuconazole treated groups 

were comparable to the vehicle controls. Absolute wet and blotted uterus weights for the 

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positive control (EE) group were increased (p<0.05) as expected by 210% and 202%, 

respectively. 

Based on the observed decreases (15-24%) in body weights at 150 mg/kg/day, the dose levels 

used in this study were adequate. 

No statistically significant changes were seen in uterine weight in this assay. Tebuconazole is 

negative in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

ER Binding Assay 

In a published study by Laws et al. (2006), 50 structurally diverse chemicals were tested in the 

ER binding assay as a part of the then ongoing validation of this assay for the EDSP. EPA tested 

tebuconazole in the rat uterine cystol ER competitive binding assay as one of the chemicals used 

to evaluate a set of QSAR models in an unpublished report by Battelle (USEPA, 2002). 

Tebuconazole was negative for ER binding. The procedures were described in Laws et al., 

(2006); however, this paper was a follow-up for the positive chemicals. Information concerning 

this study is available at Laws et al. (2009) http://www.epa.gov/ncct/dsstox/sdf_kierbl.html. The 

concentration-response data are available from EPA. [Note: This study was not included in 

Table 2] 

Hershberger Assay 

In a published study by Taxvig et al. (MRID 48264606), groups of male Wistar rats (one group 

of intact rats and six groups of castrated rats; six rats/group) were used to investigate the antiandrogenic potential of tebuconazole. The group of intact rats and one group of castrated rats 

served as negative controls and were given peanut oil only. A second group of castrated rats was 

administered TP at 0.5 mg/kg/day s.c. only and served as the control group to which all other 

groups were compared. A third group of castrated rats received TP with flutamide (10 

mg/kg/day p.o.) and served as the anti-androgenic positive control group. The remaining three 

groups of castrated rats were given subcutaneous injection of TP as above and received 

tebuconazole (98.0% purity) in peanut oil via oral gavage at doses of 50, 100 or 150 mg/kg/day 

for seven consecutive days. After euthanasia, the testis (from intact groups), ventral prostate, 

seminal vesicles (with coagulating glands including fluids), LABC, paired bulbourethral glands, 

pituitary, liver and kidney were collected and weighed. Additionally, serum was collected and 

luteinizing hormone, rat follicle-stimulating hormone and T4 hormone levels were analyzed. 

No treatment-related effects were seen on the weights of the any of the secondary sex organs or 

in the hormone levels (LH, FSH, or T4). Body weights were unaffected by treatment. Liver 

weights were increased (p<0.05) by 23% in the 150 mg/kg/day group. 

In Vitro ER and AR Transactivation Assays 

In a multi-component study by Kjærstad et al. (2010), tebuconazole (98.0% purity) was tested 

for endocrine disrupting effects in three in vitro assays. In an MCF-7 cell proliferation assay, 

MCF-7 BUS cells were exposed at twenty concentrations of tebuconazole between 0.001 μM 

and 150 μM to test for estrogenic effects. Anti-estrogenic and estrogen potentiating effects were 

tested by co-treating the MCF-7 cells with l0 pM estradiol (which induces half of the maximum 

response). The combined effect on CYP19 activity (aromatase) and anti-estrogenic effect was 

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tested by co-treating the MCF-7 cells with 1 μM testosterone (which is presumed to be converted 

to estradiol by aromatase and thus induce about 65% of the maximum estradiol response). 

Effects on the production of estradiol, progesterone, and testosterone were tested in the human 

adrenocortical carcinoma cell line H295R. In an AR reporter gene assay, AR-transfected CHO 

cells were exposed at twelve concentrations between 0.025 μM and 50 μM in the presence and 

absence of R1881 (AR agonist) to test for AR agonism or antagonism. 

Tebuconazole inhibited the estradiol-induced proliferation of the MCF-7 cells with an IC50 value 

of 49 μM and inhibited the testosterone-induced proliferation with an IC50 value of 17 μM. 

While the effect on testosterone-treated cells may be related by inhibition of aromatase, this was 

not demonstrated in the study. Tebuconazole inhibited E2 production in a steroidogenesis assay 

with an IC50 of 1.2 μM. Tebuconazole was also an AR antagonist in the presence of R1881 with 

an IC50 value of 8.1 μM.

In a published study by Orton et al. (2011), tebuconazole (>97% a.i.) was tested for endocrine 

disrupting effects in two in vitro ARTA assays. In MDA-kb2 cell assay (human breast cancer 

cells stably transfected with an androgen-response element-containing promoter with a luciferase 

reporter gene), cells were initially exposed eight serial dilutions of tebuconazole in the presence 

and absence of 0.25 nM DHT to test for AR agonism or antagonism. Cells were initially 

exposed at concentrations between 0.64 nM and 50 μM. In a yeast anti-androgen screen (YAS), 

AR-transfected yeast cells were exposed to tebuconazole at concentrations between 0.016 μM 

and 750 μM in the presence of DHT. 

In the MDA-kb2 cell assay, tebuconazole had an anti-androgen IC20 2.89 μM and a cytotoxic 

IC20 of 38.9 μM, and was negative as an androgen agonist. No results were provided for 

tebuconazole in the YAS, and no explanation was provided. 

Steroidogenesis Assays 

In a multi-component study, Kjærstad et al. (2010) examined the effect of tebuconazole on 

steroidogenesis in an in vitro assay using H295R cells exposed to six tebuconazole 

concentrations from 0.1 and 30 μM. The effects on E2, progesterone, and testosterone 

production were evaluated. Tebuconazole inhibited the production of both E2 (IC50 1.2 μM) and 

testosterone (IC50 0.5 μM), and increased progesterone production (data presented graphically). 

Puberty Onset and Kiss1 Expression in Female Rats 

In a published study by Overgaard et al. (2013), groups of time-mated female Wistar rats were 

administered tebuconazole in corn oil by daily oral gavage at dose levels of 0 (9 dams), 12.5 (6 

dams), or 50 (4 dams) mg/kg/day from GD 7-21, and then the day after delivery to PND 16. 

Puberty onset was examined in female offspring, as was mRNA expression of the Kiss1 gene 

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that encodes for kisspeptin, which is essential for activation of the HPG axis and the onset of 

puberty in rats. 

There were no effects of tebuconazole treatment on juvenile (PND 28) or adult body weights, or 

on onset of puberty. Kiss1 mRNA expression was not affected by tebuconazole exposure. 

Subchronic Toxicity (Rat, OCSPP 870.3100) 

In a subchronic toxicity study (MRID 40700930), groups of 10 Wistar (BOR:WISW[SPF Cpb]) 

rats/sex were fed diets containing tebuconazole (Batch # 16007/83; 93.4% purity) at 0, 100, 400, 

or 1600 ppm (equivalent to 0/0, 8.6/10.8, 34.8/46.5, and 171.7/235.2 mg/kg/day in 

males/females) for 13 weeks. In addition to the parameters normally examined, liver 

N-demethylase and cytochrome P-450 levels were determined. 

There were no treatment-related effects on clinical signs, ophthalmic examinations, hematology, 

urinalysis, or gross pathology. One 1600 ppm male died during Week 1 and one 1600 ppm 

female died during Week 4. It was stated that both deaths were probably due to hemorrhagic 

diathesis and were considered to be treatment-related. Additionally, two control males died; one 

during Week 4 and the second during Week 12. 

At ≥400 ppm, male body weights were decreased (p<0.05) by 5-12% at Week 13; overall (Week 

0-13) body weight gains were decreased (p<0.01) by 12% at 1600 ppm. In the 1600 ppm 

females, body weights were decreased (p<0.05) by 8% at Week 13; overall body weight gains 

were decreased (NS) by 12%. Food consumption was generally increased at 1600 ppm in both 

sexes throughout the study. Creatinine was decreased (p<0.01) by 22-33% in the 100 ppm and 

above males at Week 13; however, it was stated that the values of the treated groups were similar 

to historical control values, and were not considered adverse. N-demethylase activity was 

increased (p<0.01) in the 1600 ppm males; females were similar to controls. Cytochrome P-450 

activity was increased (p<0.05) by 26-107% at ≥400 ppm in males, and by 10-19% in females at 

≥100 ppm. Absolute liver weights were decreased (p<0.05) by 13-16% in the 400 ppm and 

above males, and by 11-13% in the 100 and 400 ppm females; absolute liver weights were 

increased (NS) by 2% in the 1600 ppm females. As there were no corroborating microscopic 

findings, the decreased liver weights were considered to be a reflection of the decreased terminal 

body weights. Absolute kidney weights were decreased (p<0.05) by 15% in the 1600 ppm males 

and by 8-9% in the 400 ppm and above females. As no corroborating microscopic findings were 

found in the kidney, these decreases were also considered to be a reflection of the decreased 

terminal body weights. Vacuole formation in the zona fasciculata of the adrenal glands was 

noted in the 1600 ppm males and at ≥400 ppm in females, and hemosiderosis was noted in a nondose-dependent manner in males at ≥100 ppm and females at 1600 ppm. No treatment-related 

changes were seen in absolute or relative weights of the testes and adrenal glands. No treatmentrelated histopathological lesions were seen in the testes, epididymides, seminal vesicles, prostate, 

ovaries, uterus, thyroid, adrenal, or pituitary glands. 

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The NOAEL is 34.8 mg/kg/day for the males and 10.8 mg/kg/day for the females. The LOAEL 

in males is 171.7 mg/kg/day based on decreased body weight, body weight gain, and 

histopathological findings, and the LOAEL in females is 46.5 mg/kg/day based on 

histopathological changes in the adrenal glands. 

Subchronic Toxicity (Dog, OCSPP 870.3150) 

In a subchronic toxicity study (MRID 40700934), groups of four beagle dogs/sex were fed diets 

containing tebuconazole (93.4% purity) at 0, 200, 1000, or 5000 ppm (equivalent to 0/0, 

73.7/73.4, 368.3/351.8, and 1749.1/1724.8 mg/kg/day in males/females) for 13 weeks. In 

addition to the parameters normally examined, serum triglycerides and liver N-demethylase and 

cytochrome P-450 levels were determined. 

Serum triglyceride levels, urinalysis parameters, and gross pathology were unaffected by 

treatment. One 5000 ppm female was found dead on Day 2, the cause of death was considered to 

be acute circulatory collapse and moderate pulmonary edema, and was considered possibly due 

to the test compound. Diarrhea, vomiting, and pasty stool were observed sporadically in both 

control and treated dogs throughout the study. A few 1000 ppm males and 5000 ppm males and 

females appeared thin compared to controls sporadically from Week 7 to study termination, and 

two 1000 ppm and five 5000 ppm dogs appeared thin or very thin at termination. This finding 

was considered compound related. Lens opacity of the eyes was observed in the 5000 ppm 

group with increasing frequency and severity, such that all 5000 ppm dogs were observed with 

this finding at Week 10. 

Body weights were decreased (not analyzed statistically) in the 1000 ppm and above males (↓5-

11%) and females (↓6-17%) at Week 13, resulting in overall decreases in body weight gain of 

25-46% and 19-48% in males and females, respectively. Week 13 body weights were also 

decreased by 7% in the 200 ppm females; however, the overall body weight gain for this group 

was similar to controls, and Week −1 body weights were 0.7 kg less than controls. Food 

consumption was slightly decreased throughout the study in the 5000 ppm males and 1000 ppm 

and above females. 

Platelet counts were increased in the 5000 ppm group, and signs of severe anisocytosis were 

noted in 6/8 dogs at 5000 ppm. Serum alkaline phosphatase activity was increased at 5000 ppm, 

and serum albumin was decreased in the 5000 ppm males. Liver N-demethylase activities were 

increased at ≥200 ppm in the males (↑32-255%) and females (↑46-208%); cytochrome P-450 

activities were increased to a lesser extent at ≥1000 ppm in males (↑25-73%) and at 5000 ppm in 

females (↑97%). Absolute spleen weights were increased by 44% and 19% in the 5000 ppm 

males and females, respectively. Absolute liver weights were slightly increased at ≥1000 ppm in 

males (↑8% both doses). No treatment-related changes were seen in absolute or relative weights 

of the testes, prostate, ovaries, thyroid and adrenal glands. 

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Microscopic findings in the 5000 ppm group included slight degeneration of the posterior wall of 

the lens of the eye, cataract-like eosinophilic plaques, bilateral cataracts, and slight siderosis in 

the liver and spleen. Increased vacuole formation was noted in the adrenals of one 200 ppm 

female, one 1000 ppm female, and two 5000 ppm females. No treatment-related 

histopathological lesions were seen in the testes, epididymides, prostate, ovaries, uterus, 

mammary, thyroid, and adrenal, or pituitary glands. 

The NOAEL is 74 mg/kg/day, and the LOAEL is 368.3/351.8 mg/kg/day (m/f) based on 

decreases in body weight gain, food consumption, and increased liver enzyme activities. 

Chronic Toxicity (Dog, OCSPP 870.4100) 

In a chronic toxicity study (MRID 40700940), groups of four beagle dogs/sex were fed diets 

containing tebuconazole (96.9% purity) at 0, 40, 200, or 1000 ppm (equivalent to 15.5, 77.3 and 

483 mg/kg/day) for 52 weeks. The high dose was increased to 2000 ppm at Week 40, and 

remained at this level until study termination. The reason for this increase was not provided. In 

addition to the parameters normally examined, serum triglycerides and liver N-demethylase and 

cytochrome P-450 levels were determined. 

No treatment-related effects were observed on mortality, clinical signs, body weights, body 

weight gains, food consumption, and urinalysis parameters. Several findings in the eye were 

observed in the mid- and high-dose groups. Lenticular lesions (opacities, lens stars) were noted 

in two animals of the mid dose as well as one dog at the high dose. In addition, corneal opacities 

(cloudy, extensive) were also apparently elevated in one dog in each of the mid dose and high 

dose groups. 

The liver was a target organ for both sexes, based upon several moderate findings: alkaline 

phosphatase levels were elevated over the course of treatment in the high-dose males and 

females; liver N-demethylase, but not cytochrome P-450 activity (both sexes) activity was 

significantly elevated at the high dose, as well as liver triglycerides; elevations in absolute and 

relative liver weights in the mid- and high-dose males; a consistent change in the appearance of 

the liver in 3/8 mid-dose and 5/8 high-dose dogs, respectively, as well as a slight increase in the 

presence of siderin in mid- (1/4) and high-dose (3/4) males and ORO-positive lipids in one dog 

in each of the mid and high-dose groups. 

Other potential target organs/tissues are the hematopoietic system (increased presence of 

moderate anisocytosis in mid and high dose animals), the adrenals (increased incidence of 

intracytoplasmic vacuoules of zona fasiculata) the kidneys (relative weights elevated in midand/or high-dose males and females) and the spleen (elevation in both absolute and relative 

weights of the high dose males and females). No treatment-related changes were seen in 

absolute or relative weights of the testes, prostate, ovaries, thyroid and adrenal glands. No 

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treatment-related histopathological lesions were seen in the testes, prostate, ovaries, uterus, 

mammary, thyroid, adrenal, or pituitary glands. 

The LOAEL was 77.3 mg/kg/day based on the findings of ocular lesions and changes in 

erythrocyte morphology; the NOAEL was 15.5 mg/kg/day. 

In a subsequent chronic toxicity study (MRID 42030601), groups of four beagle dogs/sex were 

fed diets containing tebuconazole (96% purity) at 0, 100, or 150 ppm (equivalent to 0/0, 

2.96/2.94, and 4.39/4.45 mg/kg/day in males/females) for 52 weeks. 

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

food consumption, ophthalmological examinations, hematology, clinical chemistry, urinalysis, 

organ weights, or gross pathology. One control female was euthanized on Day 70 in extremis. 

No treatment-related changes were seen in absolute or relative weights of the testes, ovaries, and 

adrenal glands. No treatment-related histopathological lesions were seen in the testes, 

epididymides, seminal vesicles, prostate, ovaries, uterus, and vagina, mammary, thyroid and 

pituitary glands. 

Microscopic pathology demonstrated the adrenal gland to be a target organ at 150 ppm in both 

sexes. In males there was hypertrophy of adrenal zona fasciculata cells (4/4 treated vs, 0/4 

controls). Other adrenal findings in males included fatty changes in the zona glomerulosa (3/4) 

and lipid hyperplasia in the cortex (2/4) at 150 ppm vs. 1/4 in controls. In females there was 

hypertrophy of zona fasciculata cells of the adrenal (4/4 treated vs. l/4 controls). Fatty changes 

in the zona glomerulosa of the adrenal was noted in 2/4 150 ppm females compared to l/4 

controls. 

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

sexes. 

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300) 

In a chronic toxicity/carcinogenicity study (MRID 40700939), groups of 50 Wistar 

(Bor:WISW[SPF Cpb]) rats/sex were fed diets containing tebuconazole (ca. 95% purity) at 0, 

100, 300, or 1000 ppm (equivalent to 0/0, 5.3/7.4, 15.9/22.8, and 55.0/86.3 mg/kg/day in 

males/females) for 24 months. Additionally, groups of 10 rats/sex were treated as above and 

terminated after 12 months. 

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

clinical chemistry, 

Male body weights were unaffected by treatment. Female body weights were decreased 

(p<0.05) by 3-5% at 300 ppm beginning at Week 15, and by 7-10% at 1000 ppm beginning at 

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Week 1. These decreases were observed consistently during the remainder of the study. The 

1000 ppm females were also noted to have slightly increased food consumption during dosing. 

In females, but not males, there was a small but consistent decreases in hemoglobin, hematocrit 

and altered mean corpuscular concentrations and volumes at 79 and 104 weeks of analyses which 

correlated with an increased deposition of splenic hemosiderin in the 1000 ppm females. Doserelated decreases in female absolute and relative adrenal weights (statistically significant at all 

dose levels) were associated with a dose-related decrease in the incidence of adrenal cortical 

hemorrhagic degeneration (statistically significant at 300 and 1000 ppm). There was also a doserelated increase in liver microsomal enzyme induction at all dose levels tested, based on 

histological examination. In the 1000 ppm males, gross pathology suggested an increase in the 

presence of kidney cyst/cystic kidneys and an increase in reddened lymph nodes. Histological 

examination of the lymph nodes indicated a possible elevation of blood-filled sinuses of the 

mesenteric lymph nodes in the 1000 ppm males. In males, but not females, C-cell thyroid 

adenoma and carcinoma were noted in treated but not control groups. These were non-doserelated findings and were not significant. The incidence of thyroid C-cell hyperplasia was 

somewhat elevated at 300 and 1000 ppm (1/50, control vs. 7/50, 300 ppm, 6/50, 1000 ppm) 

being statistically significant (p<0.05) at the mid but not high dose level. The pathology report, 

noted that thyroid C-cell neoplasia and hyperplasia can be combined since the differentiation is 

arbitrary depending mainly on size of the lesions. Combination of hyperplasia and neoplasia 

increased the statistical significance of the hyperplastic findings which resulted in the following 

respective incidences: 2%, 10%, 20%, and 18%. Historical control data from the Bayer 

laboratories for spontaneous tumors in Wistar TNO/W 70 rats from eleven studies initiated 

between 1973-1976 were submitted. The range of thyroid parafollicular tumors (which included 

interstitial or C-cell adenomas) was 0-19.3% with an average of 7.4%. Thus, the individual and 

combined C-cell tumors from the present study were essentially within the historical 

parafollicular control range. 

The NOAEL is 15.9 mg/kg/day for the males and 7.4 mg/kg/day for the females. The LOAEL in 

males is 55 mg/kg/day based on decreased testes weights, and the LOAEL in females is 22.8 

mg/kg/day based on decreases in body weight and weight gains, decreased erythrocyte 

parameters and increased incidence of splenic hemosiderosis (increased erythrocyte clearance). 

There was no evidence of carcinogenicity at the doses tested.

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

In an eight-week range-finding study (MRID 40700933, 1986), groups of 5 NMRI mice/sex 

were fed diets containing tebuconazole (purity information not provided) at 0, 500, or 2000 ppm 

for 8 weeks. Parameters examined included mortality, clinical signs, body weights, food and 

water consumption, clinical chemistry, and gross and microscopic pathology. A second study 

was performed using groups of 5 NMRI mice/sex that were fed diets containing tebuconazole at 

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0, 125, 500, or 2000 ppm for 5 days for determination of liver cytochrome P-450, triglycerides, 

and N- and O-demethylase levels. 

There were no effects of treatment observed on mortality, clinical signs, or food or water 

consumption. One control female and one 500 ppm male died during terminal blood collection, 

due to either overdose or hypovolemic shock. Body weights were consistently decreased by 1-2 

g in the 2000 ppm females during treatment; male body weights were unaffected by 

tebuconazole. In the males, total bilirubin was decreased (p<0.05) by 34% at 2000 ppm, indirect 

bilirubin was decreased (p<0.05) by 58-62% at 500 and 2000 ppm, and serum iron was increased 

(p<0.01) by 26% at 2000 ppm. 

Absolute liver weights were increased (p<0.05) by 34% and 37% in the 500 ppm males and 

females, respectively, and by 43% and 34% in the 2000 ppm males and females, respectively. 

Absolute adrenal weights were decreased (p<0.05) by 21% in the 2000 ppm females. There 

were no effects of treatment on testes or ovary weights, or on testes or ovary histopathology. 

Gross examinations revealed swollen or increased lobulation of the liver in the 500 and 2000 

ppm males, and pale livers in the 500 and 2000 ppm males and females. Microscopically, there 

was an increase in the incidence and severity (grade) of liver cell degeneration, liver cell 

necrosis, focal necrosis and presence of vacuoles and lipid content at either or both doses. At 

2000 ppm, there was an increase in the incidence of pigmentation (iron-related pigment) and 

round cell infiltrates of the kidneys. The adrenals were also significantly affected as evidenced 

by an increase in cortical cell lipids (500 and 2000 ppm) and sinus dilation (2000 ppm) of the 

males. 

In the 5-day enzyme induction study, N-demethylase was increased (p<0.01) by 68-156% in the 

125 ppm and above females; males were unaffected by treatment. O-demethylase was increased 

(p<0.01) by 80% in the 2000 ppm males and by 59-89% in the 500 ppm and above females. 

Cytochrome P-450 was increased by 52-259% (p<0.01) in the 125 ppm and above males and by 

12-242% (p,0.05) in the 125 ppm and above females. Finally, triglycerides were increased 

(p,0.01) by 196-454% in the 125 ppm and above males and by 105-500% in the 500 ppm and 

above females. 

Carcinogenicity (Mouse, OCSPP 870.4200) 

In a carcinogenicity study (MRID 40700941), groups of 50 NMRI (Bor:NMRI[SPF Han]) 

mice/sex were fed diets containing tebuconazole (ca. 95% purity) at 0, 20, 60, or 180 ppm 

(equivalent to 0/0, 5.9/9.0, 18.2/26.1, and 53.1/80.5 mg/kg/day in males/females) for 91/92 

weeks. Additionally, groups of 10 mice/sex were treated as above and terminated after 52 

weeks. 

No treatment-related effects were observed on mortality, clinical signs, food consumption, or 

hematology. A minimal depression (p<0.05) in male, but not female, mean body weights was 

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noted for several weeks during the first third of the dosing period (↓3-5%) at the high dose. 

Bilirubin values were increased (p<0.05) in the high dose females at Week 53 (↑37%) and in all 

treated females at Week 92 (↑18-64%). Slight increases (NS) in absolute and relative liver 

weights were noted in high dose group males (↑2-7%) and females (↑9-23%) at Weeks 53 and 

92/93, reflective of ability of tebuconazole to induce microsomal enzymes (cytochrome P-450, 

N- and O-demethylase). Gross pathology suggested a general increase in male liver enlargement 

and the presence of liver nodules. Interim and terminal sacrifice non-neoplastic histopathology 

indicated the liver as a primary target organ, usually at the mid and high dose levels in both 

sexes. In males the most frequent liver lesions were vacuolation (centrilobular, periportal) and 

lipid deposition (centrilobular, focal, periportal); in females, hepatic vacuolation (centrilobular, 

minimal diffuse), minimal extramedullary hemopoiesis, sinusoidal cellularity and increased lipid 

deposition were noted. In males, the adrenals were affected at the mid and high dose with an 

increased enlargement and hyperplasia of cortical cells and there was an increase in minimal 

stomach gastritis in the high dose group. Females also had apparent elevations in the pancreatic 

interstitial edema and stomach gastritis at the mid or high dose levels. Although these findings 

indicate that the high dose resulted in some toxicity in liver and other organs, the nature and 

severity of such toxicity suggested that the high dose was not high enough and thus the MTD 

was not achieved in this study. No treatment-related changes were seen in absolute or relative 

weights of the testes and adrenal glands. Ovaries were not weighed. No treatment-related 

histopathological lesions were seen in the testes, epididymides, seminal vesicles, prostate, 

ovaries, vagina, mammary glands, thyroid, adrenal, or pituitary glands. In males, there was an 

apparent slight elevation in benign but not malignant liver tumors at 60 and 180 ppm (4%/control 

vs 8% at the mid dose, 12% at the high dose), but comparison to combined benign plus 

malignant liver tumors to previous data indicated that the combined incidences were within the 

historical control range (6-18%). 

In a subsequent oncogenicity study (MRID 42175001, 1991), groups of 50 NMRI 

(Bor:NMRI[SPF Han]) mice/sex were fed diets containing tebuconazole (96.2% purity) at 0, 

500, or 1500 ppm (equivalent to 0/0, 84.9/103.1, and 279/356.5 mg/kg/day in males/females) for 

up to 91 weeks. Additionally, groups of 10 mice/sex were treated as above and terminated after 

52 weeks. 

There were no effects of treatment on mortality. Distended abdomen was observed in the 1500 

ppm males and females. In males, there were decreases (p<0.05) in body weight at 500 ppm 

during the first half of the testing period (↓3-5% from data presented) and at 1500 ppm up to 

Week 84 (↓5-8% from data presented). In females, there were sporadic decreases (p<0.05) in 

body weight at 500 ppm during the initial third of the study, and at 1500 ppm, there were 

decreases (p<0.05) in body weight in 39 of the first 59 weeks and in 7 of the last 8 weeks (↓5-6% 

from data presented). Mean food consumption was increased (p<0.05) frequently in males at 

500 ppm and generally throughout the study in both sexes at 1500 ppm. 

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Significant changes in blood parameters were observed at 1500 ppm in both sexes, though 

mainly in males. At sacrifice, in the high dose males, these changes included leucocytosis, and 

changes consistent with anemia (decreased erythrocytes, hemoglobin, MCHC, and hematocrit 

values). In addition, high-dose males had increased platelet counts and decreased thromboplastin 

times. In the case of high-dose females, a reduced hematocrit and leucocytosis were observed at 

51 weeks, and only an increased platelet count at terminal sacrifice. There were no consistent 

effects on differential blood counts. There was a dose-dependent increase in the plasma activity 

of liver enzymes (GPT, GOT, AP) at the interim and final sacrifices. Except for the interim 

sacrifice in high-dose females, the increase in plasma activity of liver enzymes reached 

significance for both the interim and final sacrifices in the 1500 ppm group. In addition, 

significance was observed at 500 ppm at both sacrifices for GPT in males and for GPT and GOT 

in females. Cholesterol was statistically significantly decreased vs controls in both sexes at 500 

and 1500 ppm for the interim sacrifice, and at 500 ppm for the final sacrifice. Albumin levels 

were transiently decreased at interim sacrifice at 500 ppm and 1500 ppm. 

Absolute liver weights were increased (p<0.05) at both the interim and terminal sacrifices in the 

500 (↑17-39%) and 1500 (↑112-254%) ppm males, and in the 1500 ppm females (↑103-273%). 

Absolute kidney weights were decreased (p<0.05) in the 500 and 1500 ppm males (↓12-15%). 

No treatment-related changes were seen in absolute or relative weights of the testes and adrenal 

glands. Ovaries were not weighed. 

At the interim sacrifice, the following increases (p<0.05) were noted grossly: i) liver appearing 

large at 1500 ppm in both sexes; ii) pale livers in both sexes at 500 and 1500 ppm; and iii) areas 

of change in the liver in the 1500 ppm females. At the terminal sacrifice, the following increases 

(p<0.05) were observed grossly: i) liver appearing large at 1500 ppm in both sexes; ii) irregular 

surfaces in livers at 1500 ppm in both sexes; and iii) liver masses in the 1500 ppm females. 

Microscopically, in the terminal sacrifice males, there were dose-dependent increases (p<0.05) in 

extramedullary hematopoiesis, focal hyperplasia of hepatocytes, and oval cell proliferation at 

1500 ppm, and in hepatic panacinar fine fatty vacuolation at 500 and 1500 ppm. In the 1500 

ppm females at terminal sacrifice, there were increases (p<0.05) in focal hyperplasia of 

hepatocytes, hepatic panacinar fine fatty vacuolation, periacinar hepatocyte hypertrophy, oval 

cell proliferation, eosinophilic foci of hepatocyte alteration, and pigment-laden Kupfer cells. 

There was also a dose-dependent increase (p<0.05) in ovarian atrophy at 500 ppm and 1500 

ppm. Also in the uterus, bilateral luteal cell hyperplasia was (p <0.05) increased at 1500 ppm. 

No treatment-related histopathological lesions were seen in the testes, epididymides, seminal 

vesicles, prostate, vagina, mammary glands, thyroid, adrenal or pituitary glands. 

At 1500 ppm, the incidence of hepatocellular adenomas and hepatocellular carcinomas was 

increased (p<0.05) in males, and the incidence of hepatocellular carcinomas was increased 

(p<0.05) in females. 

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Developmental Toxicity (Rat, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 40700943) groups of 25 pregnant Wistar rats 

were administered tebuconazole (98.3% purity) in distilled water + 0.5% Cremophor EL by oral 

gavage (10 mL/kg) at dose levels of 0, 30, 60, or 120 mg/kg/day during GD 6-15. The dams 

were euthanized on GD 21, and their fetuses were collected by cesarean section. 

There were no treatment-related effects on mortality, clinical signs, or gross pathology. Mean 

body weights were slightly decreased (p<0.05) in the high dose dams (↓5%) on Day 21, and 

mean body weight gains were decreased (↓28%) during dosing (GD 6-15). These decreases 

were accompanied by a reduction in food consumption (↓15%) during dosing. Mean maternal 

absolute and relative liver weights were increased (p<0.05) at the mid (↑8-9%) and high dose 

(↑9-15%). The maternal NOAEL is 30 mg/kg/day, and the LOAEL is 60 mg/kg/day based on 

increased absolute and relative liver weights. 

Pregnancy rates ranged from 88-96%. No abortions or premature deliveries were noted. Mean 

corpora lutea counts or mean implantations did not differ among the treated groups. Mean live 

fetuses/dam were decreased in the high dose group, due to an increase in both early (embryonic) 

and late (fetal) resorptions (total resorptions: 2.8/dam treated vs. 0.6/dam controls). Mean fetal 

weight was (NS) reduced by approximately 11% at the high dose, while sex ratios were 

unaffected by treatment. The fetal weight depression may be treatment-related as a decrease in 

mean fetal weight was still observed despite a reduction in litter size at the high dose. 

Developmental toxicity was evidenced at both the mid and high dose groups by delays in 

ossification of thoracic, cervical and sacral vertebrae, the sternum and fore- and hind limbs along 

with an increase in supernumerary ribs. Frank malformations were observed in two fetuses of 

two high dose dams as missing tail, agnatha, microstomia and anophthalmia. The developmental 

NOAEL is also 30 mg/kg/day, and the LOAEL is 60 mg/kg/day based on delayed ossification 

and increased number of fetuses with supernumerary ribs. 

Developmental Toxicity (Rabbit, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 40700945), groups of 16 pregnant Chinchilla 

(Kfm:CHIN, hybrids, SPF Quality) rabbits were administered tebuconazole (98.3% purity) in 

distilled water + 0.5% Cremophor EL by oral gavage (4 mL/kg) at dose levels of 0, 10, 30, or 

100 mg/kg/day during GD 6-18. The dams were euthanized on GD 28, and their fetuses were 

collected by cesarean section. 

There were no effects of treatment on mortality, clinical signs, liver weights, or gross pathology. 

One high dose doe died, but this was attributed to a gavage error. Maternal mean body weight 

gains were decreased by 38% during dosing at the high dose, and there was no evidence of a 

rebound during the post-dosing period. When GD 6-28 body weight gains were corrected for 

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gravid weights, a slight loss was noted at the high dose. Food consumption was also decreased 

during dosing (↓12%) at the high dose, but rebounded during the post-dosing period. The 

maternal NOAEL is 30 mg/kg/day, and the LOAEL is 100 mg/kg/day based on decreased body 

weight gain and food consumption. 

Pregnancy rates ranged from 88-94%. Mean numbers of corpora lutea/dam and 

implantations/dam were not different among the dose groups. There was a reduction in live 

fetuses/dam observed in the high dose group as compared to the controls (6.4 treated vs. 7.4 

controls), due to an increase in total (both early and late) resorptions/dam (2.4 treated vi. 0.7 

controls). Mean fetal weights and sex ratios were unaffected by treatment. Small decreases in 

the rate of ossification were noted in high dose fetuses in the right and left digits or toes of the 

fore- and hind-limb. Frank malformations in 8 fetuses of 5 litters (peromelia, palatoschisis, 

malrotation of right hind-limb, agenesis of claws) were noted at the high dose, but not in 

concurrent controls or at the same frequency in historical data. These effects were considered 

treatment-related. The developmental NOAEL is 30 mg/kg/day, and the LOAEL is 100 

mg/kg/day. 

Developmental Toxicity (Rabbit, OCSPP 870.3700) 

In a developmental toxicity study (MRID 43776201), 16 pregnant Chinchilla (CHbb:CH 

hybrids) rabbits/group were administered tebuconazole (96.3-96.8% purity) in distilled water + 

0.5% Cremophor EL by oral gavage at nominal doses of 0, 10, 30, or 100 mg/kg/day (analytical 

doses of 0, 8.8, 26.7, or 96.7 mg/kg/day) on GD 6-18. On GD 28, all surviving does were 

sacrificed and all fetuses were weighed and examined for external, visceral, and skeletal 

malformation/variations. In a supplementary study, blood was collected from 5 does/group on 

GD 6, 12, and 19 for hematology and clinical chemistry. On GD 19, these animals were 

necropsied following blood collection. The adrenals, kidneys, liver, and spleen were weighed 

and examined histologically. Liver samples were analyzed for cytochrome P-450 content, N- and 

O-demethylase activity, and triglyceride content. 

No effects of treatment were observed on mortality, clinical signs of toxicity, maternal body 

weights, body weight gains, food consumption, or at necropsy. In the supplementary study, 

single cell necrosis of the liver was seen in 5/5 does examined in all treatment groups, compared 

with 1/4 control animals. The maternal LOAEL was 8.8 mg/kg/day, based on histopathology of 

the liver (single cell necrosis) seen at all doses; the maternal NOAEL was not identified (<8.8 

mg/kg/day). 

No treatment-related differences were observed in the number of corpora lutea/doe, 

implantations/doe, post-implantation loss, resorptions/doe, fetal body weights, or fetal sex ratios. 

No treatment-related skeletal malformations/variations were observed in any fetuses. The 

combined incidence rate of litters containing fetuses with external and visceral malformations 

was 0/16, 2/15, 5/14 (p less than 0.05), and 5/14 (p less than 0.05), respectively. The 

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developmental LOAEL was 26.7 mg/kg/day based on external and visceral malformations/ 

variations; the developmental NOAEL was 8.8 mg/kg/day. 

Developmental Toxicity 

In a published study by Taxvig et al. (MRID 48277401, 2007), groups of pregnant Wistar 

(HanTac: WH) rats were administered tebuconazole (98% purity) in corn oil oral via oral gavage 

at dose levels of 0, 50, or 100 mg/kg/day from GD 7 to PND 16, and their offspring were 

assessed on GD 21 and PND 16 using histology and hormone measurements. Some dams were 

sacrificed on GD 21 for evaluation of the sexual differences in the fetuses. Anogenital distance, 

nipple retention, and hormone levels (testosterone and progesterone) were examined at birth, at 

PND 13 or PND 16, and semen quality was assessed in adults. Organ weights (testis, 

epididymides, seminal vesicle, ventral prostate, LABC, bulbourethral gland, ovaries, uterus, 

thyroid, adrenals, kidneys, and liver) were obtained on PND 16. Seminal vesicles, epididymides, 

ventral prostate thyroids and adrenal underwent histopathological examinations. 

Significant effects (p<0.05) observed at the high dose in dams included decreased body weight 

gain during gestation (↓29%), mean gestation length increased by 0.94 day, increased post 

implantation loss (27.3% treated vs. 6.6% controls), and body weight loss during the lactation 

period (−4.57 g treated vs. +7.53 g controls); concomitant changes at the low dose were not 

statistically significant but suggest a dose-response. The mean number of implantations and 

fetuses and the sex ratio were not affected. AGD was increased in both newborn female 

offspring (1.91 mm treated vs. 1.72 mm controls) and in female fetuses at GD 21 (2.02 mm 

treated vs. 1.65 mm controls). When expressed in mm, AGD in females was increased on GD 21 

at both doses and at birth at the high dose (p<0.05) but the AGD index, expressed per cubic root 

body weight, was statistically different only in the high dose group (p<0.01). An effect (p<0.01) 

on nipple retention at PND 13 was seen in male pups at both doses. 

In plasma from high dose dams at GD 21, there was a marked (7-fold) increase (p<0.05) in the 

progesterone level as well as decreases in both T3 (↓11%; p<0.05) and T4 (↓29%; NS) levels. 

Testis of GD 21 fetuses showed higher levels of 17α-hydroxyprogesterone (↑238-330%) and 

progesterone (↑127-178%) at both doses and a lower level of testosterone (↓50%) at the high 

dose. Progesterone levels were significantly increase (178%) in testis of fetuses treated at the 

low dose, but a similar increase (127%) at the high dose was not statistically significant. On PND 

16, no statistical differences were noted on testosterone levels in plasma for male pups or ovarian 

levels of estradiol, or in organ weights at either dose level. No effects were seen on semen 

quality of PND 22 pups at 50 mg/kg/day; semen was not evaluated at the high dose due to fetal 

toxicity. No treatment-related changes in organ weights or histopathological lesions were seen. 

In a second published study by Taxvig et al. (MRID 48264606), groups of ten female Wistar 

rats were administered tebuconazole (98.0% purity) in com oil by oral gavage at dose levels of 0 

or 50 mg/kg/day during GD 7-21. Dams were sacrificed on GD 21 and blood was collected from 

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dams and fetuses to measure 17α-hydroxyprogesterone, progesterone, testosterone and estradiol 

levels on GD 21. In addition, progesterone and testosterone were measured in fetal testis and 

estradiol in fetal ovaries. Ex-vivo production of testosterone and progesterone by fetal testis was 

measured. AGD were measured in all fetuses. 

Tebuconazole increased (p<0.05) the percent post-implantation loss to 11.4% compared to 0% in 

controls. AGD was not affected by treatment. In treated dams, estradiol levels were decreased 

(p<0.05) by 58%. In the fetuses born to treated dams, testicular progesterone levels were 

increased (p<0.05) by 238%. Tebuconazole had no effect on testicular testosterone 

concentration or steroid production ex-vivo. It was also reported (data not provided) that the fetal 

plasma testosterone or progesterone levels, or estradiol level in fetal ovaries did not change. 

Developmental Toxicity 

In a published study by Hass et al. (2012), groups of female Wistar rats were administered 

tebuconazole (98.5% purity) in com oil at 0 (22 dams), 12.5 (12 dams), or 50 (10 dams) 

mg/kg/day during GD 7 through PND 16. Maternal body weights were recorded daily, and dams 

were allowed to litter normally. Gestation length, AGD, nipple retention, and genital 

malformations in male pups were recorded. Pup weights were measured on PND 6, 13, and 22. 

Tebuconazole was also tested in an AR reporter gene assay at 0.03-30 μM, in a T-screen for 

thyroid receptor activity at 0.01-30 μM, and in a steroidogenesis assay using H295R cells. 

There were no effects of treatment on maternal body weight gains from GD 7 to GD 21 or from 

GD 7 to PD 1. No clinical signs of toxicity were observed in the dams. Gestation length, the 

number of implantation scars in the uterus, post-implantation loss, and perinatal loss were 

unaffected by treatment. In the 50 mg/kg/day group, female AGD was increased (14.5 mm 

treated vs. 13.6 mm controls; p<0.05), and male nipple retention was increased (1.6 treated vs. 0 

controls; p<0.05). One 50 mg/kg/day male pup was observed with slight genital malformations 

(NS). Tebuconazole exhibited some degree of AR antagonism (data presented graphically). In 

the steroidogenesis assay, tebuconazole reduced testosterone and estradiol production and caused 

a slight increase in progesterone production (data presented graphically). Data for the T-screen 

assay were not presented. 

Developmental Toxicity 

In a published study by Jacobsen et al. (2012), groups of female Wistar rats were administered 

tebuconazole (98.5% purity) in com oil at 0 (22 dams), 12.5 (12 dams), or 50 (10 dams) 

mg/kg/day during GD 7 through PND 16. Maternal body weights were recorded daily, and dams 

were allowed to litter normally. Gestation length, AGD, nipple retention, and genital 

malformations in male pups were recorded. Pup weights were measured on PND 6, 13, and 22. 

On PND 16, 1-3 pups/sex/litter were weighed, decapitated, and trunk blood was collected for 

hormone analysis. Uterus, ovaries, thyroid, and liver were collected from one female pup/litter; 

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testes, male accessory sex organs, liver, and thyroid were collected from one male pup/litter. 

Testes, thyroids, and any organ showing a significant weight change were examined 

microscopically. On PND 22, 1-3 pups/sex/litter were weaned; the dams and any unselected 

pups were killed and blood collected for hormone analysis. The number of uterine implantations 

were also counted in the dams. The day of VO or PPS was determined in all weaned pups. 

Motor activity and habituation were measured in weaned pups on PND 28 and 59; learning and 

memory was examined at an age of 2-5 months; and male mating behavior was observed at an 

age of 2-9 months. Female offspring were necropsied at approximately 6 months of age, blood 

was collected for hormone analysis, and the thyroid was examined microscopically. Male 

offspring were necropsied at approximately 9 months of age. Semen motility was analyzed in 

spermatozoa were obtained from the distal cauda epididymis. The following organs were 

excised and weighed: right and left testis, ventral prostrate, seminal vesicles with seminal fluid, 

epididymis, bulbourethral glands, LABC, liver and thyroid. The right or left testis was fixed, 

processed, and stained with hematoxylin and eosin. The following organs were fixed and 

processed: caput epididymis, ventral prostrate, seminal vesicles and liver. In ventral prostate, 

the degree of epithelial atrophy, epithelial infolding and inflammation was scored. Additionally, 

prostates were evaluated for intraepithelial vacuolation, acini with concretions in the lumen, 

presence of areas with multilayering of epithelial cells, and focal acinar atrophy. 

There were no effects of treatment on maternal body weight gains from GD 7 to GD 21 or from 

GD 7 to PD 1. No clinical signs of toxicity were observed in the dams. Gestation length, the 

number of implantation scars in the uterus, post-implantation loss, and perinatal loss were 

unaffected by treatment. 50 mg/kg/day tebuconazole caused a liver weight increase (p<0.05) of 

7% in the males; females liver weights were unchanged. Male and female reproductive organs 

and thyroid were unaffected by treatment. Semen quality was unchanged by treatment. Age at 

VO or PPS was unaffected by treatment. There were no effects of treatment on motor activity, 

learning and memory, or male mating behavior. 

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800) 

In a two-generation reproduction toxicity study (MRID 40700946), tebuconazole (95.2% purity) 

was administered to 25 Wistar rats/sex/dose via the diet at dose levels of 0, 100, 300, or 1000 

ppm (equivalent to 0, 5, 15 and 50 mg/kg/day) during a pre-mating period of 120 days; with 

exposure continuing in dams through gestation, lactation and weaning. The P generation rats 

were mated twice to produce F1a and F1b litters. Twenty-five pups/sex were selected from the F1b

litters at PND 28, fed the same dietary concentrations as their parents for approximately 70 days, 

and then mated to produce F2a and F2b litters. 

There were no effects of treatment on mortality, food consumption, or gross pathology. Clinical 

signs of toxicity noted at the high dose included loss of hair in the P and F1b females, 

inflammation/changes of the eye in the P males, and ill/bad condition in the F1b males. Loss of 

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hair was also increased in the mid dose F1b females. Body weights were consistently decreased 

(p<0.05) at the high dose in both sexes in both generations (↓4-15% males; ↓5-13% females from 

data provided). F1b parents showed decreases (p<0.05) in terminal body weights (↓9%) and 

absolute liver (↓12%), kidney (↓10%), and adrenal (↓11%) weights in the F1b males, and in 

terminal body weights (↓5%) and kidney (↓7%) weights in the F1b females. The NOAEL for 

parental/systemic toxicity is 15 mg/kg/day, and the LOAEL is 50 mg/kg/day based on the 

increased incidence and severity of splenic hemosiderosis and decreased body weights. 

A significant (p <0.05) decrease in viability index (live pups at PND 5 ÷ PND 0 x 100) at the low 

(90.3%) and high (88.1%) doses was observed for F1a pups when compared to controls (98.5%), 

but not at the mid dose; similar changes in viability index observed in F1b litters were not 

statistically significant. The lactation index (live pups at 4 weeks ÷ PND 5 x 100) of F1a litters 

was decreased compared to control (86.3% vs. 95.2%) and that of F1b litters at the low and high 

doses (76.1-80.3% vs. 90.9%) although not in a dose-dependent manner, and was increased at 

the mid dose (98.2%). No such findings were reported for the F2 litters. Given the lack of dose 

response and consistency within generations, these effects on viability and lactation indices are 

not considered to be treatment-related. High dose F1a, F2a and F2b pup weights were decreased 

(p≤0.05) compared to controls from birth to week 4. No treatment-related changes were seen in 

the absolute or relative weights of the testes or ovaries of the F1b parents. No treatment-related 

histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, 

uterus, vagina, mammary glands, adrenal, or pituitary glands. 

The reproductive NOAEL is 50 mg/kg/day, and a LOAEL was not determined. For offspring 

toxicity, the NOAEL is 15 mg/kg/day and the LOAEL is 50 mg/kg/day based on decreased pup 

body weight. 

Developmental Neurotoxicity (Rat, OCSPP 870.6300) 

In a developmental neurotoxicity study (MRID 45074301), groups of 25 pregnant Sprague 

Dawley (Crl:CD®BR VAF/Plus®) rats were administered tebuconazole (96-96.9% purity) in 

corn oil via the diet from GD 6 to LD 11 at dose levels of 0, 100, 300 or 1000 ppm (equivalent to 

0/0, 8.8/16.3, 22.0/41.3, and 65.0/125.4 mg/kg/day [GD/LD]). P dams were allowed to deliver 

naturally. On PND 5, litters were standardized to a maximum of 10 pups/litter. Pups were 

assigned to one of 5 Subsets (20 pups/sex/dose/subset). Physical development landmarks were 

evaluated for all subsets (including surface righting, eye opening, pinna unfolding, acoustic 

startle response, and pupil constriction); sexual maturation was evaluated in subsets 2-4. Subset 

1 pups were sacrificed on PND 12, all brains were weighed, and histopathological evaluations 

and morphometric analyses were performed. Subset 2 pups were evaluated for learning and 

memory (passive avoidance and Water M-maze). Subset 3 pups were evaluated for motor 

activity and for auditory startle habituation. Subset 4 pups received detailed weekly clinical 

evaluations. In addition, Subset 4 pups were sacrificed on PND 83, brains were weighed, and 

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histopathological evaluations and morphometric analyses were performed. Subset 5 pups were 

sacrificed and necropsied on PND 22. 

At 1000 ppm, two P females died as a result of prolonged gestation. Body weights were slightly 

decreased (p<0.01) in the P females during gestation (↓4-8%) and early lactation (↓6-12%). A 

body weight loss was noted during GD 6-9 (−9.3 g); body weight gains were decreased (p<0.05) 

during GD 6-21 (↓22%) and 0-21 (↓16%). A body weight loss was also noted during LD 1-4 

(−3.6 g); body weight gains were decreased (p<0.05) during LD 4-7 (↓55%). Both absolute 

(g/animal/day) and relative (g/kg/day) maternal food consumption were reduced (p<0.05) 

beginning on GD 6, and continuing through early lactation (up to LD 12). There was also an 

increase in alopecia in high dose dams. The number of live fetuses/dam was decreased (p<0.01); 

while the number of dead fetuses/dam was increased (p<0.01). No treatment-related findings 

were observed in dams at 300 or 100 ppm. The maternal LOAEL was 1000 ppm (equivalent to 

65.0/125.4 mg/kg/day [GD/LD]) based on decreased body weights, body weight gains, and food 

consumption, prolonged gestation with mortality, and an increased number of dead fetuses. The 

maternal NOAEL was 300 ppm (equivalent to 22.0/41.3 mg/kg/day [GD/LD]). 

There was a slight increase in mean gestation length at the high dose (23.0 days) compared to the 

controls (22.5 days). A slight delay in VO was noted at 1000 ppm (PND 33.2) compared to 

controls (PND 31.6). No treatment-related changes were seen in the age at PPS. At 1000 ppm, 

the stillborn index was increased (p<0.01) and the number of pup deaths was increased during 

PND 1-5. Body weights were decreased (p<0.01) in the males during PND 5-86 (↓7-23%) and 

in the females during PND 5-72 (↓5-24%). Pinna unfolding was slightly delayed (p<0.01) at 

1000 ppm (PND 3.8) compared to controls (PND 3.2). There were decreases in several 

morphometric measurements of the brain, including decreased (p<0.01) thickness of the 

cerebellum in the males and females on PND 12 and 83, and an increased thickness of the 

germinal layer of the cerebellar cortex in the PND 12 males (p<0.01). Absolute brain weights 

were decreased in the PND 12 and 83 animals (p<0.05). Relative (to body) brain weights were 

increased (p<0.05) in the PND 12 males and females. There were also changes in motor activity 

on PND 14 and 22, and changes in auditory startle amplitude on PND 23 and 63. 

At 300 ppm, there were also decreases in body weight (↓3-7%) and body weight gain (↓4-16%, 

PND 5-23 and 72-86 in males, PND 5-51 in females). Pinna unfolding was slightly delayed 

(p<0.01) at 300 ppm (PND 3.7) compared to controls (PND 3.2). There were changes in 

auditory startle amplitude in both sexes: a dose-related decrease in females on PND 23, and a 

dose-related increase in males on PND 63. In addition, there was a decrease in absolute brain 

weight in both sexes on PND 12, and in anterior/posterior cerebrum measurements. 

At 100 ppm, there were decreases in body weight (↓3-7%) and body weight gain (↓5-13%) (PND 

5-37 in males, PND 5-51 in females). There were decreases in motor activity on PND 14 and 18 

in males, and changes in auditory startle amplitude on PND 14 and 63. There was also a decrease 

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in absolute brain weight in both sexes on PND 12, and in anterior/posterior cerebrum 

measurements. 

The LOAEL for offspring toxicity was 100 ppm based on decreases in body weights, decreases 

in absolute brain weights and measurements, and decreases in motor activity. The NOAEL was 

not determined. 

Reproductive Function Study 

In a published study by Moser et al. (MRID 45486901), groups of pregnant Sprague-Dawley 

(Tac:N[SD]fBR) rats (P generation) were administered tebuconazole (97.4% purity) in 0.7% 

methylcellulose via oral gavage at dose levels of 0, 6, 20, or 60 mg/kg/day from GD 14 to PND 

7. Beginning on PND 7, F1 pups were dosed daily by oral gavage at the same dose levels as their 

dams until PND 42. In a subset of these pups, PPS and VO were evaluated beginning on PND 

35 and PND 25, respectively. As adults, these offspring were mated with untreated animals to 

produce F2 litters that were reared to PND 10 to assess normal maturation. Another second F1

subset (11-12 pups/sex/dose group) was sacrificed on PND 45/46 for assessment of organ 

weights and histopathology (testes, epididymides, uterus, liver, and adrenal). Blood was 

collected for hematology and clinical chemistry parameters. Testes and epididymides were 

processed for sperm count and motility. (Note: in this study, a second cohort of rats were used 

for neurotoxicity and immunotoxicity studies. As endpoints from these studies are not useful in 

determining endocrine systems disruption, they are not discussed in this summary.) 

At 60 mg/kg/day, maternal body weights were decreased by 4% on GD 21, with body weight 

gains during gestation decreased by 16%. No treatment-related effect was observed on AGD for 

males or females on PND 1, or on age of PPS or VO. The acceleration (p<0.05) of VO for the 

mid-dose group was considered unrelated to treatment, due to the lack of a difference for the 

high-dose group. For F1 females mated with untreated males, no overall effects on reproductive 

systems were observed. Post-mating necropsy of F1 males showed reduced (p<0.05) absolute 

epididymides (↓17%) and kidney (↓16%) weights at 60 mg/kg/day. These decreases were likely 

due to a decreased (p<0.05) terminal body weight (↓10%); however, relative kidney and 

epididymides weights were not reported. Sperm counts for the testis and epididymis were 

unaffected in F1 males. No effects on estrous cycle length, the number of female becoming 

pregnant or delivering a litter, or litter size were observed. The number of dead pups/litter was 

increased (p<0.05; 2.2 treated vs. 0.4 controls) at 60 mg/kg/day. No effect was observed on 

relative uterine weight. At the PND 46 necropsy, female terminal body weight was unchanged 

(157.1 vs. 155.7 g for control), and relative (but not absolute) liver weight was increased by 12% 

in the high-dose females; tissue histology was unchanged. The PND 46 male necropsy data 

showed few differences. Male body weight was unchanged. The high-dose group had a 26% 

increase in relative spleen weight, a 10% increase in relative liver weight, and an 8% decrease in 

relative kidney weight. 

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Avian Reproduction Toxicity (Bobwhite Quail, OCSPP 850.2300) 

In an avian reproduction toxicity study (MRID 40700910), groups of 18 bobwhite quail/sex 

(Colinus virginianus) were fed diets containing tebuconazole (97.4% purity) at 0, 5, 20, or 80 

ppm (mean measured concentrations 0, 4.96, 18.4, and 73.5 ppm). The birds were fed the diets 

prior to the onset of egg laying (13 weeks), and then during egg laying (10 weeks) for a total of 

24 weeks. The birds were housed with one male and female per pen, with 18 pens per dose 

group. 

Incidental mortality occurred in all groups due to cage mate aggression (2 females in each of the 

control and 18.4 ppm group and one female in the 4.96 and 73.5 ppm). There was one female in 

each of the 4.96 and 18.4 ppm groups and one male in the 73.5 ppm group that had a nervous 

disorder affecting the carriage of the head and at times affected the birds’ general coordination. 

This effect was not considered compound-related since this same observation had been noted in 

previously untreated birds. These birds appeared to have normal reproduction tendencies and 

there were no observable treatment-related lesions noted in any of the birds at necropsy. The 

73.5 ppm dietary level male and female birds showed a slight weight reduction (NS) at the time 

of the adult sacrifice. There were no differences in feed consumption. There were no 

differences in any of the reproductive parameters examined. There was a slightly lower 

percentage of 21-day embryo survivors and slightly lower hatch percentage at the 75.8 ppm 

dietary level. This difference, however, was not statistically different from controls. There were 

no significant differences between the control group and treated groups in mean hatch weights, 

14-day survivor weights or percent survival. There were no significant treatment-related 

differences shown by any of the treatment levels in either shell strength or shell thickness. Based 

on the results of this study, the NOAEC for tebuconazole on bobwhite quail reproduction is 73.5 

ppm, the highest level tested. 

In a second avian reproduction toxicity study (MRID 41624201), groups of 18 bobwhite 

quail/sex (Colinus virginianus) were fed diets containing tebuconazole (97% purity) at 0, 170, or 

340 ppm (mean measured concentrations 0, 156, and 290 ppm). The birds were fed the diets 

prior to the onset of egg laying (duration of treatment not provided), and then during egg laying 

(duration of treatment not provided) for a total of 21 weeks. The birds were housed with one 

male and female per pen, with 18 pens per dose group. 

This study showed no effects on adult body weight or feed consumption; however, when 

necropsy examinations were performed, treatment related lesions (abnormal livers) were 

observed in the birds of the 290 ppm concentration. Effects on reproduction were also observed 

at 290 ppm. These effects included reduction in: i) the number of eggs laid (33%); ii) normal 

hatchlings of live 3-wk embryos (11%); iii) hatchling (7%) and 14-d survivor (11%) body 

weights; iv) 14-day survival of the offspring (37%); and v) increased eggshell strength. The only 

effect seen at 156 ppm was a significant reduction in hatchling (4%) and 14-d offspring survivor 

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(6%) body weight; however, there was no difference in survivorship of the offspring. Based on 

the results of this study, the NOAEC was <156 ppm based on depressed hatchling and 14-day 

survivor body weights. 

Avian Reproduction Toxicity (Mallard duck, OCSPP 850.2300) 

In an avian reproduction toxicity study (MRID 40700909), groups of 15 Mallard ducks/sex 

(Anas platyrhyncos) were fed diets containing tebuconazole (97.4% purity) at 0, 5, 20, or 80 ppm 

(mean measured concentrations 0, 4.6, 18.8, and 75.8 ppm). The birds were fed the diets prior to 

the onset of egg laying (10 weeks), and then during egg laying (10 weeks). The total duration of 

the study was 20 weeks. The birds were housed with one male and one female per pen, with 15 

pens per dose group. 

There were no treatment-related mortalities over the course of the study. One 18.8 ppm pair and 

two 75.8 ppm pairs were euthanized because the females began producing eggs prior to being 

dosed for the required 10 weeks. No overt signs of toxicity were noted during the study. There 

were no grossly observable treatment-related lesions noted in any of the birds. There were no 

differences in body weights or feed consumption. There were no differences in any of the 

reproductive parameters examined. There was a slight dose-related trend toward a lower hatch 

percentage and eggs cracked of eggs laid, with the greatest difference in the 75.8 ppm group; 

however, these differences were not significantly different from controls. There were no 

differences in mean hatch weights, 14-day survivor weights or percent survival, and there were 

no differences in mean eggshell strength or thickness. Based on the results of this study, the 

NOAEC on mallard duck reproduction is 75.8 ppm, the highest level tested. 

In a second avian reproduction toxicity study (MRID 41818301), groups of 16 Mallard ducks/sex 

(Anas platyrhyncos) were fed diets containing tebuconazole (96.9% purity) at 0, 170, 340, or 680 

ppm (mean measured concentrations 0, 157, 320, and 611 ppm). The birds were fed the diets 

prior to the onset of egg laying (10 weeks), and then during egg laying (9 weeks). The total 

duration of the study was 19 weeks. The birds were housed with one male and one female per 

pen, with 16 pens per dose group. 

One female in the 611 ppm group died during Week 15 after appearing listless and anorexic the 

previous day. Gross pathological examination revealed petechial hemorrhaging in the skin, a 

very hard liver, a dark-colored spleen, and an enlarged heart. It was concluded these 

observations were incidental to treatment. No clinical signs were noted in any of the survivors 

during the study. No abnormal findings were noted at necropsy of the surviving ducks. No 

differences in body weights were observed at test initiation, prior to egg laying (test week 7), or 

at termination. No differences in food consumption were recorded. 

In the reviewer’s analysis, reported a significant (p<0.05) reduction in the number of eggs 

hatched per hen in the 611 ppm group. No other statistical differences, with regard to other egg 

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parameters, were noted during the study. Although some statistically significant differences in 

mean body weights were recorded for certain groups within hatches, these intergroup differences 

were not considered to be consistent or dose related. The NOAEC is 320 ppm and the LOAEC 

is 611 ppm. 

Fish Early Life Stage Toxicity (Rainbow Trout, OCSPP 850.1400) 

In a fish early life stage toxicity study (MRID 40700914), groups of rainbow trout (Salmo 

gairdneri) were exposed to tebuconazole at nominal concentrations of 0 (negative and solvent 

[100 μL/L acetone] controls), 0.015, 0.030, 0.060, 0.120, or 0.240 mg/L (mean measured 

concentrations 0, 0.012, 0.025, 0.061, 0.120, and 0.230 mg/L). At study initiation, unfertilized 

eggs and sperm were mixed, left undisturbed for 45 minutes, and then randomly selected and 

distributed to 28 embryo incubation cups (50 eggs/cup; 4 cups/concentration). On Day 15, 

twenty viable embryos/replicate/group were selected and further incubated to produce larvae. 

Following completion of hatching on Day 23, twenty larvae/replicate/group were released into 

their respective aquaria for the 60-day post-hatch exposure. Exposure was continuous for a total 

of 83 days. Behavior and appearance of larvae were observed daily and the number of surviving 

larvae was counted twice weekly. At 60 days post-hatch exposure (test termination), the 

percentage larval survival was determined. The larvae were measured and weighed individually 

to calculate the mean and standard deviation of total length and wet weight. 

Exposure to all concentrations tested did not adversely affect the embryo viability or survival of 

organisms at hatch. By Day 42, (19 days post-hatch) nearly all exposed larvae in treatment 

levels of ≤0.061 mg/L and the controls completed their development to the swim-up stage. 

Development of larvae in concentrations of 0.120 and 0.230 mg/L appeared to be delayed by 

several days as compared to the larvae in the controls. Throughout the post-hatch exposure 

period, larvae at the four highest test concentrations exhibited abnormal appearance (dark 

coloration) and behavior (lethargic, partial or complete loss of equilibrium). At test termination, 

larval survival in the four highest test concentrations (i.e., 0.025, 0.061, 0.120, and 0.230 mg/L) 

was significantly affected compared to pooled control. Because larval survival was significantly 

reduced at the four highest concentrations, the growth data at these levels was not statistically 

compared to the growth of the control organisms. However, growth at these levels appeared to 

be adversely affected. Larval growth in the remaining treatment (0.012 mg/L) was not 

significantly different from that of the pooled controls. Based on these results, the NOAEC for 

rainbow trout survival and growth is 0.012 mg/L. 

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

In a fish early life stage toxicity study (MRID 42038202), groups of sheepshead minnow 

(Cyprinodon variegatus) were exposed to tebuconazole at nominal concentrations of 0 (negative 

and solvent [25 μL/L dimethylformamide] controls), 0.00625, 0.0125, 0.025, 0.050, or 0.100 

mg/L (mean measured concentrations 0, 0.00504, 0.0920, 0.0219, 0.0475, and 0.0993 mg/L). 

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Unfertilized eggs were obtained by stripping adult females and fertilized with a sperm 

suspension. Twenty embryos were used per concentration. Exposure was continuous for at least 

36 days. 

Viability of embryos was verified 3 days after fertilization. Viability of negative and solvent 

control embryos was 91%; viability of treated embryos was 90-96%. Hatching in the negative 

controls was completed in four days, while the solvent controls was completed in nine days. 

Hatching was 85% in the negative controls, 66% in the solvent controls (p<0.05 compared to 

negative controls), and 71-85% in the treated groups. Survival of juvenile fish was 72-93% in 

the treated groups, and 88% and 87% in the negative and solvent controls, respectively. 

Abnormalities were few with no apparent concentration relationship and included two-headed 

fry and spinal deformations. Mean lengths of fish ranged from 12.7-14.1 mm. Mean wet 

weights of fish ranged from 57.3-85.0 mg. Growth, as measured by length and wet weight, was 

significantly reduced in the negative controls compared to the solvent controls. Mean length at 

0.0993 mg/L was significantly reduced compared to the solvent controls. Mean weight at 0.0475 

and 0.0993 mg/L was significantly reduced compared to the solvent controls. 

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

In a fish full life cycle toxicity study (MRID 43009601), groups of 200 sheepshead minnow 

(Cyprinodon variegatus) embryos (50 embryos/cup; 4 cups/dose level) were exposed to 

tebuconazole at nominal concentrations of 0 (negative and solvent [20.55 μL/L triethylene 

glycol] controls), 0.0125, 0.025, 0.050, 0.100, or 0.200 mg/L under flow-through conditions for 

203 days. Mean measured concentrations were 0.0092, 0.019, 0.0436, 0.0967, and 0.1969 mg 

ai/L for the F0 phase; 0.0092, 0.0219, 0.0490, 0.0986, and 0.196 mg ai/L for the reproductive 

phase, and 0.0096, 0.0184, 0.0469, 0.0978, and 0.192 mg ai/L for the F1 phase. At study 

initiation, the embryos were <24 h post-fertilization. On Day 58, 25 fish/dose level were 

randomly selected and maintained until Day 113 for reproduction. Mature adult fish (generally 2 

males and 5 females) were selected and placed in a spawning chamber. Four spawning session, 

each lasting 14 days, were conducted. At the end of the spawning sessions, the adult fish were 

sacrificed for length and weight measurements and submitted for residue analysis. 

The F1 phase of the study was initiated on Day 163. Where possible, 25 embryos, obtained 

during the period between spawning sessions 3 and 4, were distributed to each of two embryo 

incubation chambers per replicate tank (i.e., 50 embryos/replicate,100 embryos/treatment). 

Survival was recorded until hatching was complete. Approximately two weeks after hatching, 

all fish were transferred into screen retention chambers within the same test chamber. Survival 

and sublethal effects were monitored daily. On Day 28, all fish were sacrificed for length and 

weight measurements and then submitted for residue analysis. 

Hatching success (%) of treated F0 embryos was not significantly different from the pooled 

controls (negative controls and solvent controls were 86.5% and 89.0%, respectively). F0 

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survival on Day 58 was reduced (p<0.05) at 0.1969 mg/L compared to the pooled controls 

(negative controls and solvent controls were 93.6% and 89.3%, respectively). F0 survival on 

Day 177 was reduced (p<0.05) at 0.1969 mg/L compared to the pooled controls (negative 

controls and solvent controls were 76% and 56%, respectively). Due to the significant survival 

effects, the 0.1969 mg/L exposure group was not included in the growth analyses. F0 length on 

Day 33 was reduced (p<0.05) at 0.0967 mg/L compared to the pooled controls. F0 growth (i.e., 

length and weight) on Days 58 and 177 was not significantly reduced at any exposure 

concentration compared to the pooled controls or to the solvent controls. The residue in tissues 

of the F0 sheepshead minnows generally increased with concentrations of test material in water. 

The mean tissue residue ranged from 0.12 mg/kg for fish in the lowest test level to 12.87 mg/kg 

for fish in the highest test level. The exposure solutions showed no significant effect on the 

reproductive parameters when compared to the control data. For spawning sessions 1 and 2, 

reproductive data at 0.0092 mg/L was substantially lower than the pooled control. However, this 

difference was not statistically significant and was not confirmed by the results at higher 

exposure concentrations. 

Hatching success (%) of F1 embryos exposed to 0.0184 mg/L was significantly reduced when 

compared to that of the pooled controls and the solvent controls; however, this difference was 

regarded as spurious. There were no significant differences at any other exposure concentration. 

Hatching success of the negative controls and solvent controls were 93.7% and 100%, 

respectively. F1 survival of the treated groups were higher than the solvent controls at 28 days 

post-hatch. Survival of the negative controls and solvent controls on Day 28 post-hatch was 

95.5% and 86.0%, respectively. F1 growth (i.e., length and weight) at 28 days post-hatch was 

reduced (p<0.05) at 0.0978 mg/L when compared to the pooled controls. F1 length at 28 days 

post-hatch was also reduced (p<0.05) at 0.0436 mg/L when compared to the pooled controls The 

residue in F1 sheepshead minnow tissues generally increased with concentrations of the test 

material in water. The mean tissue residue ranged from 0.31 mg/kg for fish in the lowest test 

level to 2.96 mg/kg for fish in the highest test level. 

Fish Sexual Developmental Test (FSDT) (Fathead Minnow, OECD 234) 

A fish sexual developmental test (FSDT) (MRID 48109802, 2007) with Tebuconazole using 

fathead minnows (Pimephales promelas) was studied under flow-through conditions. The study 

was conducted on freshly fertilized eggs (<24hrs old) for 122-125 days. Test organisms were 

exposed to tebuconazole at nominal concentrations of 0 (control), 0.00313, 0.00625, 0.0125, 

0.0250, 0.050 and 0.100 mg a.i./L; mean-measured concentrations were <LOQ (<0.00035), 

0.00265, 0.00583, 0.0112, 0.0221, 0.045 and 0.0896 mg a.i./L. The test system was maintained 

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

In this study, early life stage survival, growth and sexual development were examined. At the 

juvenile fish (study days 30 to 52) and young adult fish (study days 53 to test termination) stage, 

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very few clinical signs (e.g, underdeveloped fish) were reported for control fish and fish at test 

levels up to 0.0112 mg a.i./L, with the exception of one fish in the 0.0112 mg a.i./L level which 

had a deformed spine and other symptoms starting on day 55 until test termination. However, 

multiple sublethal effects, including spinal deformations, were observed in fish at the 0.0221, 

0.045 and 0.0896 mg a.i./L levels during both the juvenile and young adult stages of the study. 

The percent of egg survival at study days 0-5 (post hatch day 1) was 91, 86, 87, 90, 91, 90 and 

88% in the mean-measured 0 (control), 0.00265, 0.00583, 0.0112, 0.0221, 0.045 and 0.0896 mg 

a.i./L treatment levels, respectively. There were no significant differences between treated and 

control for egg survival, and time to hatch/hatching success. On study day 54, all individuals at 

the highest test concentration (0.0896 mg a.i./L) had died. Mean survival at test termination 

was 85, 85, 83, 88, 87, 31 and 0% in the mean-measured 0 (control), 0.00265, 0.00583, 0.0112, 

0.0221, 0.045 and 0.0896 mg a.i./L treatment levels, respectively. The mean overall survival at 

test termination for the 0.045 and 0.0896 mg a.i./L treatment levels was significantly decreased 

compared to the negative control (p<0.05). As survival was significantly affected in the 0.045 

,mg a.i./L treatment group which may have affected loading rates and environmental conditions, 

data from this treatment were excluded from analysis of growth parameters. 

There was a statistically significant decrease in female body weight and length at the 0.0221 mg 

a.i./L treatment level (p<0.01; Jonckheere). There were no significant differences in male body 

weight and body length or the sex ratio of male/female fish (p>0.05). 

According to the study report, changes in the liver were observed at 0.00583 mg a.i./L and above 

in males and at 0.0112 mg a.i./L and above in females and consisted of hepatocellular 

hypertrophy, minimal to slight pleomorphism of liver cells and nuclei; the study author regarded 

these findings as adaptive and not adverse. However, at 0.0112 mg a.i./L and above for both 

males and females, liver cell degeneration was observed and at 0.0221 mg a.i./L and above 

significant inflammations in the liver was also observed. 

In males, maturation of the testes was more advanced (more adult and less juvenile fishes) at 

0.0221 and 0.045 mg a.i./L. Also at 0.045 mg a.i./L, observations of increased spermatozoa in 

the sperm ducts and significant increases in spermatogonia in the testes were reported. 

In females, reduced yolk accumulation in oocytes was recorded in some fishes at 0.0112 mg 

a.i./L and above. Also at 0.045 mg a.i./L reduced maturity of follicles (less late vitellogenic 

oocytes) as indicated by staging of the ovary were reported. Additionally, degeneration of early- 

/previtellogenic follicles was significantly increased at 0.045 mg a.i./L. While at 0.00583 ug 

a.s./L and above a significantly reduced incidence of degeneration of late and mid vitellogenic 

follicles was observed but it was not dose-responsive. 

Additionally, findings were reported in the pancreas (0.0112 mg a.i./L and above), the kidneys 

(0.045 mg a.i./L) and spinal column/cord deformations (0.0221 mg a.i./L and above). Fungal 

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growth was reported in the lumen in the intestines in fish at 0.0112 and 0.0221 mg a.i./L without 

any associated histopathological findings, whereas a decrease in the incidence of inflammation in 

the caudal intestine was reduced at 0.0221 mg a.i./L and above. 

In regards to performance criteria, criteria relating to dissolved oxygen were met; data was not 

available to determine if temperature differed by no more than 1.5°C for all chambers at one 

time. The variation of the measured test concentrations were greater than ±20% of the mean 

measured value for several test concentrations; the ratio of the maximum/minimum measured 

concentrations ranged from 1.30 to 1.54 except for the second lowest treatment which was 1.91. 

However, this study was conducted using a fish species that does not have specific performance 

criteria outlined in the OECD 234 guideline. However, the survival and growth of the control 

fish appear to be appropriate. 

This study was conducted in accordance with OECD FSDT Draft Proposal (version as of July 

26, 2006 and adopted July 28, 2011) and therefore is different than the guidance as outlined in 

U.S. EPA OCSPP 890.1350: “Fish Short-Term Reproductive Assay” and OECD 229 (2009). 

This study was designed to assess the toxicity of the test item during the early, juvenile and 

sexual maturation life stages. The two primary endpoints analyzed were phenotypic sex 

expression via gonad tissue and gonad staging. This study does not evaluate reproductive 

success measured by fecundity and fertilization success. Additionally, this study did not 

measure plasma vitellogenin levels. 

Thyroid Endocrine Disruption in Fish 

In a published study by Yu et al. (2013), zebrafish embryos were exposed to tebuconazole at 

concentrations of 0, 1, 2, or 4 mg/L from shortly after spawning to 120 h post-fertilization. T3

and T4 levels were measured in extracts from 300 larvae/ concentration using commercially 

available ELISA kits. Additionally mRNA was isolated and gene expression in the HPT axis 

was examined. 

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

T4 levels were decreased (p<0.05) by 10%, and T3 levels were increased (p<0.05) by 28%. The 

following HPT axis genes appeared to be upregulated by tebuconazole treatment: 

corticotrophin-releasing hormone, thyroid stimulating hormone, sodium/iodide symporter, 

transthyretin, uridine diphosphate glucuronosyltransferase, thyronine deiodinase 2, and thyroid 

hormone receptor β. Thyronine deiodinase 1, thyroid hormone receptor α, and thyroglobulin did 

not change significantly. 

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APPENDIX 3. References Not Utilized in the Tebuconazole 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 tebuconazole but were not utilized due to one or more of the following reasons: 

1) the article was not available in English; 2) the compound of interest 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. 

Andersen, H, Andersson, A-M, Arnold, S, et al. (1999) Comparison of short-term estrogenicity 

tests for identification of hormone-disrupting chemicals. Published: Environ. Health Perspect.

107 (Suppl 1): 89-108. 

Desai, Y (2006) Acute toxicity study of tebuconazole technical in rainbow trout, Onchorhynchus 

mykiss. Jai Research Foundation, Gujrat, India. Laboratory Project Number: 5744, January 31, 

2006. Sponsored by Punjab Chemicals & Crop Protection Ltd., Mumbai, India. MRID 

46919204. Unpublished. 

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Horwitz, K, Costlow, M, and McGuire, W (1975) MCF-7: a human breast cancer cell line with 

estrogen, androgen, progesterone, and glucocorticoid receptors. Published: Steroids: 26 (6): 

785-795. 

Jacobsen, P, Christiansen, S, Boberg, J, et al. (2010) Combined exposure to endocrine disrupting 

pesticides impairs parturition, causes pup mortality, and affects sexual differentiation in rats. Int. 

J. Androl. 33: 434-442. 

Jensen, B, Petersen, A, Christiansen, S, et al. (2013) Probabilistic assessment of the cumulative 

dietary exposure of the population of Denmark to endocrine disrupting pesticides. Food. Chem. 

Toxicol. 55: 113-120. 

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

compounds in vitro. Int. J. Androl. 33: 425-433. 

Orton, F, Rosivatz, E, Scholze, M, et al. (2012) Competitive androgen receptor antagonism as a 

factor determining the predictability of cumulative antiandrogenic effects of widely used 

pesticides. Environ. Health Perspect. 120 (11): 1578-1584. 

Surprenant, D (1987) Acute toxicity of HWG- 1608 (Technical grade) to rainbow trout (Salmo 

sairdneri) under flow-through conditions. Springborn Life Sciences, Inc., Wareham, MA. 

Laboratory Report No. 87-5-2394, completion date not provided. Sponsored by Mobay 

Corporation, Stilwell, KS. MRID 40700911. Unpublished. 

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