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 56
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

Page 2 of 56
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: FLUTOLANIL 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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FLUTOLANIL FINAL 

ii 

TABLE OF CONTENTS 

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

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

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

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

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

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

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

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

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

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

2. Effects on Androgen Pathway..................................................13 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................17 

IV. Committee’s Assessment of Weight of Evidence..........................................21 

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................21 

1. Tier 1 in vivo Assays.............................................................22 

2. OSRI...............................................................................22 

B. Estrogen Pathway...........................................................................23 

C. Androgen Pathway.........................................................................24 

D. Thyroid Pathway...........................................................................24 

E. Conclusions.................................................................................24 

V. EDSP Tier 2 Testing Recommendations....................................................24 

VI. References.......................................................................................26 

LIST OF TABLES: 

 Table 1. Tier 1 Assays for Flutolanil..................................................................8 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Flutolanil ....................................10 

 Table 3. Androgenic/Anti-Androgenic Pathway for Flutolanil ..................................14 

 Table 4. Thyroid Pathway for Flutolanil.............................................................19 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................29 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................38 

APPENDIX 3. References Not Utilized in the Flutolanil WoE Analysis.............................47 

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FLUTOLANIL FINAL 

iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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iv 

Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

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v 

Abbreviation Terminology 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

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vi 

Abbreviation Terminology 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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Page 1 of 48 

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 flutolanil interacts with E, A, or T hormone pathways, the number and 

type of effects induced, the magnitude of responses, and the 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 August 13, 2014, the 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 of the potential interaction of flutolanil 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). 

The conclusion of the WoE evaluation is that flutolanil demonstrates no convincing evidence of 

potential interaction with the estrogen or androgen pathways in mammals. However, the 

complementary responses observed in both sexes in the fish short-term reproduction assay 

(FSTRA) suggest a potential for flutolanil to interact with the E and A pathways in fish. 

For the estrogen pathway, flutolanil was negative in the in vitro Tier 1 estrogen receptor (ER) 

binding, aromatase and steroidogenesis assays. In a published ERTA study (Kojima et al., 2004) 

considered as OSRI, flutolanil was reported to be negative for both ERα agonism and 

antagonism. Flutolanil was also negative for estrogenic activity in the in vivo Tier 1 uterotrophic 

assay and showed no evidence of an estrogenic response in the female pubertal rat assay or in the 

mammalian Part 158 toxicity studies. Overall flutolanil does not appear to have a potential to 

interact with the estrogen pathway in mammals. Conversely, in the FSTRA several 

complementary responses were observed that suggest that flutolanil may have the potential to 

interact with the estrogen pathway in fish. The FSTRA responses included decreased fecundity, 

decreased plasma VTG, increased female GSI and alterations in gonadal histopathology. In 

addition to the decrease in fecundity in fish, a 17% decrease in eggs laid was observed in the 

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FLUTOLANIL FINAL 

Page 2 of 48 

bobwhite quail reproduction (no other treatment-related effects observed); however, it is noted 

that this effect alone is limited in its diagnostic specificity for a potential estrogen-related effect. 

A 5% non-treatment responsive decrease in eggshell thickness was observed the mallard duck 

reproduction study, but no other effects were observed in this study (and not seen in the 

bobwhite quail study). Therefore, while there were largely negative or limited responses in the 

Tier 1 in vitro assays, in vivo mammalian dataset, and avian dataset, for the FSTRA there were 

complementary responses along several levels of biological organization for the females 

including alterations in enzyme activity (plasma VTG) up to the apical endpoint of fecundity. As 

such, significant effects of the reproductive capacity of fish were observed in the FSTRA 

suggests that flutolanil may potentially interact with the HPG axis.

For the androgen-pathway, although flutolanil showed an equivocal response in the Tier 1 AR 

binding assay, in a published ARTA study (Kojima et al., 2004) considered as OSRI, flutolanil 

was reported to be negative for AR transcriptional activation. Flutolanil did not alter in vitro

testosterone production in the Tier 1 steroidogenesis assay. In vivo, flutolanil was negative in 

both the Tier 1 Hershberger and in the male pubertal assay. There were no androgen-related 

responses in the mammalian Part 158 toxicity studies to support an interaction with the androgen 

pathway in mammals. No androgen-related effects were observed in the Part 158 avian 

reproduction studies. However, in the FSTRA, median male tubercle score was decreased by 

32% and multiple abnormalities in the testes were observed in the high treatment group (1.2 

mg/L). These findings suggest a potential effect on the androgen pathway in fish. As with the 

findings in the estrogen pathway, while the in vitro and mammalian/avian in vivo assays do not 

suggest a potential interaction, the results in the male fish, with complementary effects in the 

female fish, suggest a potential to interact with the A pathway in fish. 

There is no convincing evidence for the potential for flutolanil to interact with the T pathway in 

mammals or wildlife. There were no treatment-related effects on the thyroid parameters in the in 

vivo male or female pubertal rat assays or in the Part 158 mammalian toxicity studies. The 

effects on development, growth and histopathology observed in the amphibian metamorphosis 

assay (AMA) were not considered to be related to flutolanil treatment, but rather may be 

reflective of a response due to the presence of the solvent (similar effects observed in solvent 

control and treatment groups relative to negative control). Therefore, the results in the AMA do 

not suggest a potential for interaction with the thyroid pathway in amphibians. 

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

recommended for flutolanil since there was no convincing evidence of potential interaction with 

the estrogen, androgen or thyroid pathways. 

Based on the complementary responses observed in the FSTRA, including effects on the apical 

endpoint of fecundity, EDSP Tier 2 Medaka Extended One-Generation Reproduction Test is 

recommended. The results in the FSTRA indicate that there may be a potential for interaction 

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FLUTOLANIL FINAL 

Page 3 of 48 

with the estrogen and androgen pathway in fish. Currently, the chronic fish NOAEC value is 

0.233 mg a.i./L based on decreased length and wet weight in a fathead minnow early life-stage 

study. Effects on female GSI and gonadal histopathology were observed at 0.018 mg a.i./L in 

the FSTRA, with an increase in the number of effects observed in the males and females, 

including a cessation in fecundity, at 1.2 mg a.i./L. Therefore, as the concentration of the 

observed effects in the FSTRA are lower than the current regulatory NOAEC for fish, additional 

data are recommended to understand the dose-response relationship for potential reproductive 

effects. 

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

duck are available for flutolanil. 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 flutolanil 

exposure. Relative to the EDSP, additional testing is not recommended. As there was no 

convincing evidence of a potential thyroid interaction in amphibians, no additional testing is 

recommended. 

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Page 4 of 48 

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 flutolanil interacts with E, A, or T hormone pathways, the number and 

type of effects induced, the magnitude of responses, and the 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 August 13, 2014, the 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 flutolanil 

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). 

Flutolanil, is a systemic benzanilide fungicide. Flutolanil’s water solubility is 6.53 mg/L at 20°C 

with a vapor pressure of 4.88 x 10−8 mm Hg at 25°C. In the environment, flutolanil is 

anticipated to be stable and persistent. Flutolanil has a low propensity to volatilize, is resistant to 

hydrolysis and is stable to photolysis. Flutolanil is relatively resistant to aerobic and anaerobic 

degradation in soils and aquatic environments, with half-lives of more than 200 days in soils and 

greater than one year in water. Because of its reported water solubility and high soil binding 

capacity, there is potential for flutolanil sorbed to soil to be transported into surface water via 

erosion. Flutolanil has a low likelihood to bioaccumulate in fish and other aquatic organisms 

with an estimated bioconcentration factor of 100 with more than 95-99% depurated in 3 days. 

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

the E, A and/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 studies 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 flutolanil (USEPA, 2010). 

Since then, the Agency has also conducted a more recent search of available scientific literature 

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

Appendix 2. Additionally, literature/studies considered but not utilized for the WoE analysis are 

listed in Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

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

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

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

Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how 

the individual lines of evidence are assembled and integrated along two concepts (i.e., 

complementarity and redundancy) within the conceptual framework of an adverse outcome 

pathway). Broadly, there are four main steps outlined in the guidance which provide the 

foundation for WoE evaluations. The first step is to evaluate the individual studies for their 

scientific quality and relevance in evaluating potential endocrine interaction(s). The second step 

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

extent of complementarity (i.e., the concordance of endpoints within an assay that measures 

multiple endpoints) and redundancy (i.e., the concordance of endpoints/responses across assays) 

in the observed responses across these different levels of biological organization. The third step 

is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to 

evaluate whether additional testing is needed based on the evidence and conclusions described 

above. 

 

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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 

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 response s 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 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 

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Page 7 of 48 

introduces uncertainty in the ability to distinguish effects specifically related to an endocrinerelated effect from a non-endocrine toxic response. 

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

the 2013 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 non-endocrine organs or overall disruptions in homeostasis” (USEPA, 

2013). 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

forms of toxicity (e.g., 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 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. 

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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 in regard to a treatment-related effect which 

supports a conclusion of whether a substance has the potential to interact with the E, A, or T 

pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each 

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

A. EDSP Tier 1 Screening Assays 

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

in Table 1. 

Table 1. Tier 1 Screening Assays for Flutolanil 

Tier 1 Assays Test Guideline Test Order Status 

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

(MRID 48616805) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OSCPP 890.1300; 

OECD 455 

Requirement satisfied by 

OSRI 

(Kojima et al., 2004) 

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

(MRID 48616802) 

Steroidogenesis Assay (Human cell line 

H295R) 

OSCPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID 48616808) 

Aromatase Assay (human recombinant 

microsomes) OSCPP 890.1200 Requirement Satisfied (MRID 48616803) 

Uterotrophic Assay (Rat) OSCPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID 48616809) 

Hershberger Assay (Rat) OSCPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID 48616807) 

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied 

(MRID 48671101) 

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied 

(MRID 48671101) 

Fish Short-term Reproduction Assay OSCPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID 48616806) 

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID 48616801) 

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FLUTOLANIL FINAL 

Page 9 of 48 

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

1. Effects on Estrogen Pathway 

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

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

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

table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in 

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

considered further in the WOE assessment. 

In an evaluation of estrogenic/anti-estrogenic activity from Tier 1 testing results, flutolanil was 

classified as negative in the estrogen receptor (ER) binding, aromatase, ERα transcriptional 

activation (ERTA; Kojima et al., 2004) and steroidogenesis assays. 

Flutolanil was also classified as negative for estrogenic activity in the in vivo uterotrophic assay, 

and there were no estrogen-related effects observed in the female pubertal rat assay. 

In the FSTRA, fecundity was decreased (↓100%) at the high concentration (1.2 mg/L) and 

fertilization success could not be assessed as no eggs were produced. Plasma vitellogenin (VTG) 

was decreased (↓72%) in high-concentration females compared to the negative control. Female 

GSI was increased (↑15-64%) relative to the negative control in all treatment groups (0.018-1.2 

mg/L). There was an increase in the number and severity (mild to severe) of oocyte atresia in 

high-concentration females and an increase in gonad size in each of the treatment groups; the 

latter observation was attributed to an increase in vitellogenic oocytes. Five fish were reported 

as lethargic on Day 1 in the high concentration, which progressively decreased to one fish by 

Day 4 which suggests the fish may have been stressed at test initiation but did not persist 

throughout the duration of the study. Additionally, there were no effects on survival or growth 

parameters.

There were no estrogen-related effect observed in the Part 158 mammalian toxicity studies. 

In a Part 158 reproduction study with bobwhite quail, there was a slight, but not statisticallysignificant, reduction in the number of eggs laid at the highest treatment concentration by 17% 

compared to the negative control and was considered biologically significant (4800 mg/kg-diet). 

In a Part 158 reproduction study with mallard ducks, there was a statistically significant, but nontreatment responsive, 5% reduction in eggshell thickness at 4800 mg/kg-diet; no other treatmentrelated effects were observed. In a fish early life stage study with fathead minnow, effects on 

survival and/or growth (length and wet weight) were observed at ≥0.486 mg/L. 

Page 17 of 56
FLUTOLANIL FINAL 

Page 10 of 48 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Flutolanil 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Flutolanil1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Uterine Weight 

Ovarian Weight/ 

GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Vitellogenin 

EDSP Tier 1 Assay 

ER Binding (MRID 48616805) N ERTA Requirement satisfied by OSRI (Kojima et al., 2004; MRID 48033008; see below) Aromatase (MRID 48616803) N Steroidogenesis (MRID 48616808) N Uterotrophic (MRID 48616809) N NE Female Pubertal Rat (MRID 48671101) N N N N N N FSTRA (MRID 48616806) NE 

↑15, 

30, 

64% 

(L, 

M, H) 

P 

(L, M, 

H)4

N 

Frt: N5; 

Fcd: 

↓100% 

(H) 

↓7

% 

(H

OSRI 

ERTA (Kojima et al., 2004) 

MRID 48033008 

N 

Two-generation reproduction (Rat; MRID 41850805) 

NE NE N NE NE N 
FLUTOLANIL FINAL 

Page 11 of 48 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Flutolanil 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Flutolanil1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Uterine Weight 

Ovarian Weight/ 

GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Vitellogenin 

Developmental toxicity (Rat; MRID 41850804) 

NE N 

Developmental toxicity (Rabbit; MRID 40342924) 

 NR N 

Chronic toxicity/ carcinogenicity (Rat; MRID 40342921) 

NE N N N 

Carcinogenicity (Mouse; MRID 41850803) 

NE NE N NE 

Chronic toxicity (Dog; MRID 40342922) NE N N N Subchronic Toxicity (Rat; MRID 4342919) NE N N NE Subchronic Toxicity (Dog; MRID 40342920) 

NE N N NE 

Avian reproduction (Quail; MRID 42932301) 

P (H)7 

Avian reproduction (Duck; MRID 42932302) 

P (H)8 
FLUTOLANIL FINAL 

Page 12 of 48 

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

↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable 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 O

in this column. For details see Section IV. A 

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

details see Section IV. A 

4. Ovaries displayed a mild to severe increase in atresia (15 of 16) at the high concentration (1.2 mg/L). Increases in gonad size wer

groups (8 to 14 of 16) concomitant with an increased presence of vitellogenic oocytes. 

5. Fertility could not be assessed at the high concentration as no eggs were produced. Fertility was not affected at the two lower con

6. Five fish were reported as lethargic on Day 1 which progressively decreased to one fish by Day 4 which suggests the fish may hav

initiation but did not persist throughout the duration of the study. Additionally, there were no effects on survival or growth param

7. Reduction in eggs laid at the highest treatment level by 17% compared to the negative control and was considered biologically sig8. Egg shell thickness was decreased 5% (effect not treatment-responsive) at the high dietary concentration. P Positive finding N Negative finding (in vitro)/No effect (in vivo) 

NE Not examined NR Not reported 
FLUTOLANIL FINAL 

Page 13 of 48 

2. Effects on Androgen Pathway 

The potential interactions of flutolanil with the androgen pathway are summarized in Table 3. 

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

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

This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that 

occurred in the presence of overt toxicity are discussed in the text but are not reported in the 

table and not considered further in the WOE assessment. 

In an evaluation of androgenic/anti-androgenic activity from Tier 1 testing results, flutolanil was 

classified as equivocal in the in vitro androgen receptor (AR) binding assay and negative in the 

steroidogenesis assay. The equivocal response in the AR binding assay resulted from negative 

responses in two of the three assay runs, and a variable response in a single assay run that 

yielded decreases in specific binding between 25 and 50%. In addition, flutolanil was negative 

for agonist and antagonist activity up to 10-5 M in an AR transactivation assay conducted by 

Kojima et al. (2004). 

Flutolanil was also classified as negative for androgenic and anti-androgenic activity in the in 

vivo Hershberger assay and there were no changes in the androgen-responsive endpoints in the 

male pubertal rat assay. 

In the FSTRA, median male tubercle score was decreased (21 vs. 27) in the high treatment group 

(1.2 mg/L) with a reported 36% decrease in dorsal nape pad (fatpad). In addition, three of the 

eight males in the high treatment group showed multiple, often related, abnormalities in the 

testes including increases in the number of spermatogonia (mild to moderate), increases in 

primary spermatocytes (moderate), decreases in secondary spermatocytes (mild to moderate) 

and/or decreases in spermatids (mild to moderate). These microscopic alterations interfered with 

staging of the testes in two of these males. 

There were no androgen-related effects seen in the Part 158 mammalian toxicity studies. There 

were no androgen-specific effects noted in the Part 158 avian reproduction studies. 

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FLUTOLANIL FINAL 

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Table 3: Androgenic/Anti-Androgenic Pathway for Flutolanil Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Flutolanil1Study Type/ Literature Citation 

AR Binding/ AR 

activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histo

pathology

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight 

at PPS 

Vitellogenin 

EDSP Tier 1 Assay 

AR Binding (MRID 48616802) E4Steroidogenesis (MRID 48616908) N Hershberger (MRID 48616807) NE N Male Pubertal Rat (MRID 48671101) N N N N N N N N FSTRA (MRID 48616806) NE N P (H)5

Tubercle 

score: 

(21 v 27) 

(H) 

fat pad 

↓36% (H) 

N6 N 

OSRI 

ARTA (Kojima et al., 

2004) 

N

Two-Generation Reproduction (Rat; MRID 41850805)

NE N NE N NE NE 

Chronic Toxicity / Carcinogenicity (Rat; MRID 40342921) 

N N NE N N NE 

Carcinogenicity (Mouse; MRID 41850803) 

N N NE N NE NE 
FLUTOLANIL FINAL 

Page 15 of 48 

Table 3: Androgenic/Anti-Androgenic Pathway for Flutolanil Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Flutolanil1Study Type/ Literature Citation 

AR Binding/ AR 

activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histo

pathology

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight 

at PPS 

Vitellogenin 

Chronic toxicity (Dog; MRID 40342922) 

NE N NE N N NE 

Subchronic Toxicity (Rat; MRID 40342919) 

N N NE N NE NE 

Subchronic Toxicity (Dog; MRID 40342920) 

N N N N NE NE 

Avian reproduction (Quail; MRID 42932301) 

N 

Avian reproduction (Duck; MRID 42932302) 

N 

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

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

absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsa(PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). 

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 

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 th

Section IV. A 

4. Mean specific-binding decreases in the presence of flutolanil were variable, and not dose specific, in Run 1 (>50% but <75% at several c

did not reduce binding <75% in Runs 2 and 3. 

5. Multiple, often related, abnormalities in the testes including increases in the number of spermatogonia (mild to moderate), increases in p

(moderate), decreases in secondary spermatocytes (mild to moderate), and/or decreases in spermatids (mild to moderate) in the three of etreatment group (1.2 mg/L). These microscopic alterations interfered with staging of the testes in two of these males.

6. Fertility could not be assessed at the high concentration as no eggs were produced. Fertility was not affected at the two lower concentrat
FLUTOLANIL FINAL 

Page 16 of 48 

7. Five fish were reported as lethargic on Day 1 which progressively decreased to one fish by Day 4 which suggests the fish may have been 

but did not persist throughout the duration of the study. Additionally, there were no effects on survival or growth parameters

E Equivocal assay response P Positive finding N Negative finding (in vitro)/No effect (in vivo) 

NE Not examined 
FLUTOLANIL FINAL 

Page 17 of 48 

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 flutolanil 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. 

No effects on the thyroid pathway (thyroid weight, histopathology, or hormone concentrations) 

were observed in the in vivo male and female pubertal rat assays. In the AMA, flutolanil 

significantly advanced median Nieuwkoop-Faber (NF) developmental stage by 2 stages at Day 

21 at all treatment concentrations (0.015, 0.13, and 1.1 mg/L) relative to the negative control, 

with no effect at Day 7. Increases in the severity and incidence of thyroid hypertrophy and 

increased colloid area were apparent in all treatment groups. Flutolanil significantly increased

Day 21 normalized hind-limb length (HLL) at all treatment concentrations in a nonconcentration-dependent manner (i.e., 28, 26, and 23% at the low, medium, and high treatment 

concentrations) relative to the negative control, with no effects noted at Day 7. Significant 

increases in snout-to-vent length (SVL) and body weight were observed on Days 7 and 21 in the 

low and medium treatment groups. Growth parameters in the high treatment group were not 

different compared to the negative control. With the exception of Day 7 body weight, for all 

endpoints in which there was a significant increase from the negative control, there was also a 

significant or marginally significant increase in response of the solvent control over the negative 

control. In addition, the severity of thyroid hypertrophy and increased colloid area was increased 

in the solvent control relative to the negative control. Therefore, these apparent solvent effects 

on both growth and developmental endpoints suggest that the observed changes in flutolaniltreated animals relative to the negative control were related to the presence of the solvent, and 

not a thyroid-related interaction. 

No thyroid effects were noted in any of the Part 158 mammalian chronic toxicity studies, but 

increased thyroid weights were observed in the subchronic rat and dog studies. Generally nonsignificant and/or non-dose-dependent increases in thyroid weights were noted in the high-dose 

female as well as the medium- and high-dose male rats. In the subchronic dog thyroid weighs 

were increased 25% at the high dose (2000 mg/kg/day) and 21 and 23% in females at the mid 

(400 mg/kg/day) and high (2000 mg/kg/day) dose, respectively. The thyroid weight increases in 

the dog were not statistically significant. Thyroxine (T4) and thyroid stimulating hormone (TSH) 

Page 25 of 56
FLUTOLANIL FINAL 

Page 18 of 48 

concentrations were not determined in any general toxicity studies. Histopathological 

examination of the pituitary did not reveal any treatment-related changes in any in vivo

mammalian studies. 

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FLUTOLANIL FINAL 

Page 19 of 48 

Table 4. Thyroid Pathway for Flutolanil.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Flutolanil1

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 Toxicity2 

Overt Toxicity Observed3

EDSP Tier 1 Assays 

Male Pubertal Rat 

(MRID 48671101) N N N N N N N 

Female Pubertal Rat 

(MRID 48671101) N N N N N N N 

AMA 

(MRID 48616801) N N4 

Day 21 NF 

stage: 

↑2 stages5 

Day 21 

HLL: 

↑28,26,23% 

(L, M, H)6

Day 7 BW: 

↑40, 54% 

Day 21 BW: 

↑20, 35% 

 Day 7 SVL: 

↑15, 18% 

Day 21 SVL: 

↑5, 9% 

 (L, M)7

 N 

OSRI

Chronic Toxicity/ 

Carcinogenicity (Rat; 

MRID 40342921) 

N N N X 

(H) N 

Carcinogenicity (Mouse; 

MRID 41850803) NE N NE N N 

Chronic toxicity (Dog; 

MRID 40342922) N N N N X 

(M, H) 

Subchronic Toxicity 

(Rat; MRID 40342919) N N NE X 

(M, H) N 

Subchronic Toxicity 

(Dog; MRID 40342920) N N NE X 

(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. 

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. Although not statistically-significant, increases in the severity and incidence of thyroid hypertrophy and 

increased colloid area were apparent in all treatment groups. However, the severity of thyroid hypertrophy and 

increased colloid area was significantly increased in the solvent control relative to the negative control, 

suggesting that the observed changes were related to the presence of the solvent. 

5. Increased median developmental stage (59 vs. 57 in the negative control). Median NF stage of the solvent 

control was also 59, suggesting that the observed changes were related to the presence of the solvent. 

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FLUTOLANIL FINAL 

Page 20 of 48 

6 Day 21 HLL of the solvent control was significantly increased by 47% compare to the negative control, 

suggesting that the observed changes were related to the presence of the solvent. 

7 Day 7 and 21 SVL of the solvent control was increased marginally (p = 0.053) by 11 and 5%, respectively, 

relative to the negative control. Day 7 and 21 BW of the solvent control were increased by 28% (NS) and 22% 

(p<0.05), respectively, relative to the negative control. Increased body weight on Day 7 (↑40 and 54%) and 

Day 21 (↑20 and 35%) in the low- and medium-dose groups, respectively. This suggests that the observed 

changes in growth were related to the presence of the solvent. 

P Positive finding 

N Negative finding 

NE Not examined 

 

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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 flutolanil 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 mediated through an endocrine pathway, therefore nonendocrine 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. Systemic/Overt 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. 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 

There was no evidence of overt toxicity in the Tier 1 in vivo studies. Systemic effects in the 

mammalian Tier 1 in vivo assays were confined to minimal changes in liver weights. Finally, no 

treatment-related mortalities or clinical signs were present in the FSTRA or the AMA. 

2. OSRI 

Systemic or overt toxicity in the OSRI in vivo studies was also minimal. The toxicology studies 

conducted on flutolanil demonstrate few biologically significant effects, mainly in the liver and 

gastrointestinal (GI) system. 

In a subchronic oral toxicity study in rats, no mortality or clinical signs were observed. Slight 

decreases in overall body weight gain were noted at the high dose. Absolute liver weights in 

males and females were increased by 9 and 19%, respectively, and relative liver weights were 

increased by 14 and 20%, respectively, in the high-dose group. Absolute and relative liver 

weights in females also were increased by 11 and 8%, respectively, in the medium-dose group. 

In a subchronic oral toxicity study in dogs, no mortality or clinical signs were observed, although 

swollen mammary areas were noted in the medium- and high-dose females. The absolute and 

relative weights for the liver were increased in both sexes in the high-dose animals. 

In a two-generation reproduction toxicity study in rats, no mortality or clinical signs were 

observed. Absolute and relative liver weights were increased in high-dose F0-generation animals 

and F1 females. There were no treatment-related effects on mortality or clinical signs in a 

prenatal developmental toxicity study in rats or rabbits. Reduced body weight gains during 

dosing were observed in all treatment groups in the rabbit developmental study, and lower 

overall body weight gain was observed in the high-dose group (↓12%). However, these 

differences were attributed to higher control group weights (↑11-15%) relative to the treated 

groups prior to dosing. 

In a combined chronic toxicity/carcinogenicity study in rats, no mortality or clinical signs were 

observed. Body weight gains were decreased up to 10% during Weeks 1-13 (with body weight 

decreases of 3-6%) in the high-dose male rats. Body weight gain differences decreased after 

Week 13. Relative liver weights were increased by 12 and 14%, respectively, at 3 and 24 

months in high-dose males. In the high-dose females, absolute liver weights were increased by 

15-18% at 3, 12, and 24 months, and relative liver weights were increased by 11 and 10%, 

respectively, at 3 and 12 months. In an oncogenicity study in mice, no mortality or clinical signs 

were observed. Body weight gains were decreased by 17 and 16%, respectively, in the high-dose 

males and females during Weeks 0-24. In medium-dose females, body weight gains also were 

decreased by 15% during Weeks 0-24. Absolute liver weight in the high-dose females was 

increased by 20%, and liver-to-body weight ratio in the high-dose females was increased by 

33%. No similar increases in liver weight were observed in high-dose males. In a chronic 

Page 30 of 56
FLUTOLANIL FINAL 

Page 23 of 48 

toxicity study in dogs, all dogs survived until study termination. There was a dose-related 

increase in the incidence of clinical signs (emesis, salivation, and soft stool) in both sexes in the 

medium- and high-dose groups from Week 65 until study termination. Decreased body weights 

were observed in high-dose males from Weeks 93-104 (↓4-8%) with decreased body weight 

gains (↓8-16%). Additionally, decreased body weights were observed in high-dose females from 

Weeks 87-104 (↓3-8%) with decreased body weight gains (↓6-17%).

In general, absolute and /or relative liver weight increases were considered treatment-related 

adaptive changes due to the lack of adverse effects during gross and microscopic evaluations. 

In a fish early life stage study with fathead minnow, effects on survival and/or growth (length 

and wet weight) were observed at ≥0.486 mg/L. 

No clinical signs of toxicity were observed in the avian reproduction studies. 

B. Estrogen Pathway 

Flutolanil was negative in the in vitro Tier 1 estrogen receptor (ER) binding, aromatase and 

steroidogenesis assays. In a published ERTA study (Kojima et al., 2004) considered as OSRI, 

flutolanil was reported to be negative for both ERα agonism and antagonism. Flutolanil was also 

negative for estrogenic activity in the in vivo Tier 1 uterotrophic assay and showed no evidence 

of an estrogenic response in the female pubertal rat assay or in the mammalian Part 158 toxicity 

studies. Overall flutolanil does not appear to have a potential to interact with the estrogen 

pathway in mammals. 

Conversely, in the FSTRA several complementary responses were observed that suggest that 

flutolanil may have the potential to interact with the estrogen pathway in fish. The FSTRA 

responses included decreased fecundity, decreased plasma VTG, increased female GSI and 

alterations in gonadal histopathology. In addition to the decrease in fecundity in fish, a 17% 

decrease in eggs laid was observed in the bobwhite quail reproduction (no other treatmentrelated effects observed); however, it is noted that this effect alone is limited in its diagnostic 

specificity for a potential estrogen-related effect. A 5% non-treatment responsive decrease in 

eggshell thickness was observed the mallard duck reproduction study, but no other effects were 

observed in this study (and not seen in the bobwhite quail study). Therefore, while there were 

largely negative or limited responses in the Tier 1 in vitro assays, in vivo mammalian dataset, and 

avian dataset, for the FSTRA there were complementary responses along several levels of 

biological organization for the females including alterations in enzyme activity (plasma VTG) up 

to the apical endpoint of fecundity. As such, significant effects of the reproductive capacity of 

fish were observed in the FSTRA suggests that flutolanil may potentially interact with the HPG 

axis.  

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FLUTOLANIL FINAL 

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C. Androgen Pathway 

Although flutolanil showed an equivocal response in the Tier 1 AR binding assay, in a published 

ARTA study (Kojima et al., 2004) considered as OSRI, flutolanil was reported to be negative for 

AR transcriptional activation. Flutolanil did not alter in vitro testosterone production in the Tier 

1 steroidogenesis assay. In vivo, flutolanil was negative in both the Tier 1 Hershberger and in the 

male pubertal assay. There were no androgen-related responses in the mammalian Part 158 

studies to support an interaction with the androgen pathway in mammals. No androgen-related 

effects were observed in the Part 158 avian reproduction studies. However, in the FSTRA, 

median male tubercle score was decreased by 32% and multiple abnormalities in the testes were 

observed in the high treatment group (1.2 mg/L). These findings suggest a potential effect on the 

androgen pathway in fish. As with the findings in the estrogen pathway, while the in vitro and 

mammalian/avian in vivo assays do not suggest a potential interaction, the results in the male 

fish, with complementary effects in the female fish, suggest a potential to interact with the A 

pathway in fish.

D. Thyroid Pathway 

There were no treatment-related effects on the thyroid parameters in the in vivo male or female 

pubertal rat assays or in the Part 158 mammalian toxicity studies. The effects on development, 

growth and histopathology observed in the AMA were not considered to be related to flutolanil 

treatment, but rather may be reflective of a response due to the presence of the solvent (similar 

effects observed in solvent control and treatment groups relative to negative control). Therefore, 

the results in the AMA do not suggest a potential for interaction with the thyroid pathway in 

amphibians. 

E. Conclusions 

The conclusion of the WoE evaluation is that flutolanil demonstrates no convincing evidence of 

potential interaction with the estrogen or androgen pathways in mammals. However, the 

complementary responses observed in both sexes in the FSTRA suggest a potential for flutolanil 

to interact with the E and A pathways in fish. There is no convincing evidence for the potential 

for flutolanil to interact with the T pathway in mammals or wildlife. 

V. EDSP Tier 2 Testing Recommendations 

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

recommended for flutolanil since there was no convincing evidence of potential interaction with 

the estrogen, androgen or thyroid pathways. 

Based on the complementary responses observed in the FSTRA, including effects on the apical 

endpoint of fecundity, EDSP Tier 2 Medaka Extended One-Generation Reproduction Test 

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FLUTOLANIL FINAL 

Page 25 of 48 

(MEGORT) is recommended. The results in the FSTRA indicate that there may be a potential for 

interaction with the estrogen and androgen pathway in fish. Currently, the chronic fish NOAEC 

value is 0.233 mg a.i./L, based on decreased length and wet weight in a fathead minnow early 

life-stage study. Effects on female GSI and gonadal histopathology were observed at 0.018 mg 

a.i./L in the FSTRA, with an increase in the number of effects observed in the males and 

females, including a cessation in fecundity, at 1.2 mg a.i./L. Therefore, as the concentration of 

the observed effects in the FSTRA are lower than the current regulatory NOAEC for fish, 

additional data are recommended to understand the dose-response relationship for potential 

reproductive effects. 

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

duck are available for flutolanil. 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 flutolanil 

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

As there was no convincing evidence of a potential thyroid interaction in amphibians, no 

additional testing is recommended. 

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Page 26 of 48 

VI. References 

Amanuma, T. (2011) Hershberger assay for flutolanil technical grade in rats. Unpublished study 

conducted by Research Center, Nihon Nohyaku Co., Ltd., Osaka, Japan. Final Report Number: 

LSRC-T10-117A, April 8, 2011. (MRID 48616807) 

Amanuma, T. (2012) Male and Female Pubertal Assay with Flutolanil Technical in Rats. 

Unpublished study conducted by Nihon Nohyaku Co., Ltd., Osaka, Japan. Laboratory Report 

No.: LSRC-T12-068A, June 18, 2012. (MRID 48671101) 

Amanuma, T. (2011) Uterotrophic Assay for Flutolanil Technical Grade in Ovariectomized Rats. 

Unpublished study conducted by Nihon Nohyaku Company, Ltd., Research Center, Osaka, 

Japan. Final Report No.: LSRC-T11-018A, April 15, 2011. (MRID 48616809) 

Atkinson, J. (1986) T21-Flutolanil: A three-month oral toxicity study of flutolanil in rats. 

Unpublished study conducted by Bio/dynamics, Inc. Project #85-2926. (T-3024). (MRID 

40342919) 

Atkinson, J. (1986) T22-Flutolanil: A three-month oral toxicity study of flutolanil in dogs. 

Unpublished study conducted by Bio/dynamics, Inc. Project #85-2927. (T-3025) (MRID 

40342920) 

Beavers, J.B., Hoxter, K.A., and Jaber, M. (1993) W64 flutolanil: Flutolanil technical: A one 

generation reproduction study with the bobwhite (Colinus virginianus). Wildlife International 

Ltd. Project No.: 244108 (W-3025). (MRID 42932301) 

Beavers, J.B., Hoxter, K.A., and Jaber, M. (1993) W65 flutolanil: Flutolanil technical: A one 

generation reproduction study with the mallard (Anas platyrhynchos). Wildlife International

Ltd. Project No.: 244109 (W-3026). (MRID 42932302) 

Cowlyn, I.E., Nicholls, I.M., Virgo, D.M., and Fowler, J.S.L. (1990) Flutolanil: Oncogenicity study 

by dietary administration to CD-1(1CR)BR mice for 78 weeks. Unpublished study conducted by 

Life Science Research, Ltd. LSR Report # 89/0043 (T-3041). (MRID 41850803) 

Hamasaka, Y. (2011) Steroidogenesis assay for Flutolanil in Human cell line- H295R. 

Unpublished study conducted by Research Center, Nihon Nohyaku Co., Ltd., Osaka, Japan. 

LSRCM11- 100A, November 22, 2011. (MRID 48616808) 

Ikuta, J. (2011) ER Binding Assay of Flutolanil. Unpublished study conducted by Research 

Center, Nihon Nohyaku Co. Ltd. Osaka, Japan. Laboratory Report No.: GA-27, 11-0063, 

November 24, 2011. (MRID 48616805) 

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Page 27 of 48 

Jäger, M. (2011) Human Androgen Receptor Binding Assay using Rat Prostate Cytosol (ARRPC) with Flutolanil. Unpublished study conducted by Harlan Cytotest Cell Research GmbH, 

Germany. Laboratory Study Number: 1423900, December 8, 2011. (MRID 48616802) 

Katoh, M., Suzuki, A., Ito, M., Chida, T., and Matsuzawa, I. (1982) Twenty-four month chronic 

toxicity study of NNF-136 in rats. Unpublished study conducted by Mitsubishi-Kasei Institute of 

Toxicological and Environmental Sciences. Report #54-039 (T-3013). (MRID 40342921) 

Kojima, H., Iida, M., Katsura, E., Kanetoshi, A., Hori, Y., and Kobayashi, K. (2003) Effects of a 

diphenyl ether-type herbicide, chlornitrofen, and its amino derivative on androgen and estrogen 

receptor activities. Environ Health Perspect. 111:497–502. 

Kojima, H., Katsura, E., Takeuchi, S., Niiyama, K., and Kobayashi, K. (2004) Screening for 

estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using 

Chinese hamster ovary cells. Environ Health Perspect. 112(5):524-31. 

Lambert, E.P. (1992). Flutolanil: Teratology study in the rat. Amendment to final report. 

Unpublished study conducted by Life Science Research Ltd. LSR Report #87 INHH023/554. 

First Amendment #92/NHH023/0490. (T3043). Original study completed 1987. (MRID 

41850804) 

Nagata, T., Sato, M., Nagata, T., Takeuchi, T., Murakami, U., and Enomoto, M. (1982) T3-

Flutolanil: Chronic toxicity study for NNF-136 administered orally to beagle dogs for 104 

weeks. Unpublished study conducted by Shin Nippon Biomedical Laboratories, Ltd. (T-3014). 

(MRID 40342922) 

Palmer, S.J., Kendall, T.Z., and Krueger, H.O. (2011) Flutolanil: Amphibian Metamorphosis 

Assay for the Detection of Thyroid Active Substances. Unpublished study conducted by Wildlife 

International, Ltd., Easton, Maryland 21601. Laboratory project number 397A-149. Study 

sponsored by Nihon Nohyaku Co. Ltd., Chuo-ku, Tokyo 103-8236 Japan. (MRID 48616801) 

Palmer, S.J., Zendall, T.Z., Martin, K.H., and Krueger, H.O. (2011) Flutolanil: Fish Short-term 

Reproduction Assay with the Fathead Minnow (Pimephales promelas). Unpublished study 

conducted by Wildlife International, Ltd., Easton, Maryland. Lab Study No.: 397A-148. Study 

sponsored by Nihon Nohyaku Co. Ltd., Chuo-ku, Tokyo, Japan. Study completed December 7, 

2011. (MRID 48616806) 

Rhodes, H., and Muckerman, M. (1995) Early life-stage toxicity of flutolanil to the fathead

minnow (Pimephales promelas) under flow-through conditions. Analytical Bio-Chemistry

Laboratories, Inc. Final Report #41685. Sponsor's Study #523AU (W-3030). (MRID 43552001) 

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Schroeder, R.E. (1991). A two-generation reproduction study in rats with flutolanil. Unpublished 

study conducted by Bio/dynamics, Inc. Project #89-3417. (T-3069). (MRID 41850805) 

Tauchi, K., Kawanishi, H., Takeshima, T., Toyohara, S., and Imai, S. (1987) Teratogenicity 

study of flutolanil in the rabbit. Unpublished study conducted by Imamichi Institute for Animal 

Reproduction. Study #173 (T-3042). (MRID 40342924) 

Tesh, J.M., McAnulty, P.A., Lambert, E.P., and Manning, T.J. (1987) Flutolanil: Preliminary 

teratology study in the rat. Life Science Research. Report #87 /NHH022/336. (T-3092) (MRID 

41850804) 

USEPA (2010) FLUTOLANIL - Report of the OPP Endocrine Disruptor Review Team- Test 

Order#: EDSP-128975-112. December 9, 2010. Office of Chemical Safety and Pollution 

Prevention. U.S. Environmental Protection Agency, Washington, D.C. 

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

Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. September 14, 2011. 

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

Washington, DC. 

USEPA (2011b) FLUTOLANIL - Response to Nichino America, Inc. request to evaluate

supplemental information provided to satisfy the Test Order requirement for the Estrogen

Receptor Transcriptional Activation Assay. October 13, 2011. Office of Chemical Safety and 

Pollution Prevention. U.S. Environmental Protection Agency, Washington, D.C. 

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

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

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

Yasunaga, R. (2011) Aromatase Study of Flutolanil. Unpublished study conducted by Research 

Center, Nihon Nohyaku Co., Ltd., Osaka, Japan. Laboratory Study No.: GA-27, 10-0122. June 

27, 2011. (MRID 48616803) 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100) 

The 21-day assay (MRID 48616801) of flutolanil (98.7% purity) on amphibian metamorphosis of 

African clawed frog (Xenopus laevis) was conducted under flow-through conditions. Amphibian 

larvae at developmental Nieuwkoop-Faber Stage (NF) 51 (80 per control and treatment group) 

were exposed to negative and solvent controls (0.02 mL/L dimethyl formamide, DMF) and 

nominal flutolanil concentrations of 0.015, 0.15, and 1.5 mg a.i./L. Mean-measured concentrations 

were <0.01 (<LOQ; control), 0.015, 0.13, and 1.1 mg a.i./L. The test system was maintained at 

21.2 to 22.2°C and a pH of 7.9 to 8.3. 

All effects, unless otherwise noted, are reported based on comparison to the negative control. 

There were no treatment-related effects on survival of tadpoles during the 21-day test; 1, 3, and 1 

deaths were observed in the negative control, low, and high treatment groups, respectively. 

Clinical signs of toxicity included one tadpole in the mid treatment group that was periodically 

noted as small with a misshapen head, and another was noted as weak. At test termination, one 

tadpole in the mid treatment group was noted to have an extra front leg. All other surviving 

tadpoles appeared normal throughout the test. Spinal curvature was observed in several tadpoles 

in the negative control (8) and solvent control (7) and low (1), and mid (1) treatment groups. 

By Day 21, several late stage tadpoles (>NF 60) were observed in the solvent control (12), low 

(6), mid (9), and high (5) treatment groups; none were observed in the negative control. Late 

stage tadpoles were excluded during analysis of growth parameters (e.g., SVL, BW, and HLL). 

7-day wet body weight (p≤0.016; ≥40%) and 7-day snout-vent length (SVL; p≤0.005; ≥15%) 

were significantly increased in the mean-measured low and mid treatment groups as compared to 

the negative control. No significant acceleration or delay of median NF developmental stage or 

normalized hind-limb length (HLL) was observed on Day 7. 

By Day 21, wet weight (p≤0.001; ≥20%) and SVL (p≤0.005; ≥5%) were significantly increased 

in the mean-measured low and mid treatment groups. Hind-limb length (HLL; p≤0.003; ≥23%), 

normalized (NHLL; p≤0.05; ≥23%), and NF stage (p=0.05) were significantly increased at all 

treatment levels as compared to the negative control. No asynchronous development was 

observed throughout the test. 

No treatment-related effects were observed for thyroid gland histopathology, however there were 

significant differences between the negative and solvent control. 

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For all variables in which there was a significant increase (p<0.05) from the negative control, 

there was also a significant increase (p<0.05) in response of the solvent control over the negative 

control. . Both the solvent and the concentration used are within what is recommended in the 

guideline for this study. These effects however make it difficult to ascertain whether the effects 

observed were treatment related or mediated by the solvent. 

Androgen Receptor (AR) Binding Assay (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48616802), prostate cytosol from 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 radio-labeled 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 (logarithmic increase from 10−9

 to 10−4 M) of flutolanil (98.7% 

purity, Lot #: 9AE0011P). Ethanol was used as a vehicle at a final assay concentration of 2%. 

The assay included dexamethasone as a weak positive control, and R1881 as the ligand reference 

standard. 

In the saturation binding experiment, the dissociation constant (Kd) for [3

H]-R1881 was 3.02

(±0.38) nM and the estimated Bmax was reported as 9567 (±537) dpm. The Kd was not within the 

range reported in the EPA validation program (0.685 to 1.57 nM), but the study reports that the 

expected saturation kinetics were observed in the curve. Although the standard deviation for the 

average Kd (6% of mean) and Bmax (13% of mean) values were provided, the individual run Kd

and Bmax values were not provided, therefore the variation among runs and confidence in the 

reported Kd and Bmax numbers is uncertain. However, the data produced a linear Scatchard plot. 

In the competitive binding experiment, flutolanil was evaluated at a concentration range from 10-

9

 M to 10-4 M, not the 10-10 to 10-3 M (or up to the limit of solubility) range recommended in the 

test guideline. The mean specific binding of the radiolabeled ligand reduced in the presence of 

flutolanil was variable and not dose specific in the first run: 78-98% at 10−4, 10−5, and 10−6 M; 

67% at 10−7

 M; and 73-74% at 10−8 and 10−9 M. In the second and third runs, flutolanil did not 

reduce the mean specific binding at any concentration (92-105% and 81-98%, respectively). 

Flutolanil is classified as equivocal as specific radioligand binding was greater than 50% but less 

than 75% at several concentrations in the first run. As specific binding was not <50% at any 

flutolanil concentration, an IC50 and RBA could not be calculated for flutolanil. 

The estimated log IC50s for R1881 and the weak positive control (dexamethasone) were −8.8 and 

−4.5 M, respectively. The mean RBA for the weak positive control was 0.0046%. Confidence 

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in these numbers is high due to the small variation. No precipitation of the test compound was 

noted and the solvent control responses indicated no drift in the study assay. 

The performance criteria were not met for R1881 and dexamethasone indicating possible 

problems with the assay. For R1881, the top and bottom % binding was low in Run #1, and the 

Hill slope was high in Run #2. For dexamethasone, the top % binding was low in Runs #1 and 

#3, the bottom % binding was high in all Runs, and the Hill slope was low in Run #3. 

Examination of the curve for the reference material across the runs indicated variability in the 

top plateaus for R1881. The study reported that the maximal percent binding at the lowest 

concentration of R1881 was unexpectedly lower than 18% of the negative control but 

nonetheless the IC50 calculated were comparable to the results obtained for the other two runs 

showing the expected plateau level. Additionally, variability in the top % binding and Hill slope 

curves for the weak positive control, dexamethasone, was observed and the bottom of binding 

curves (14-33%) could not be achieved at the expected levels. 

Based on the results from the three assay runs, flutolanil is classified as Equivocal in the 

Androgen Receptor Binding Assay. 

Aromatase Assay (OCSPP 890.1200) 

In an in vitro aromatase (CYP19) assay (MRID 48616803), flutolanil (99.5% a.i., Lot #: 

8AE0010P) in ethanol (≤1%) was incubated with human recombinant aromatase and tritiated 

androstenedione ([1β3H(N)]-androst-4-ene-3,17-dione; [3H]-ASDN) for 15 minutes at 37°C to 

assess the potential of flutolanil to inhibit aromatase activity. Flutolanil was tested at logarithmic 

concentrations from 10–10 M to 10−3

 M (Run 1) and from 10–10 M to 10−4 M (Runs 2 and 3), due 

to precipitation observed at 10−3

 M and 10-3.5 M in Run 1. 

Aromatase activity was determined by measuring the amount of tritiated water produced at the 

end of a 15-minute incubation period for each concentration of chemical. Tritiated water was 

quantified using liquid scintillation counting (LSC). Three independent runs were conducted, 

and each run included a full activity control, a background activity control, a positive control 

series (10−10 M to 10−5

 M) using a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), 

and the test chemical series (10−10 M to 10−3

 M) with 3 repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.203 to 0.253 nmol·mgprotein−1

·min−1 for the three test runs, with a mean and standard deviation of 0.230 ± 0.017 

nmol·mg-protein−1·min−1. Activity in the background controls averaged 0.08% of the full 

activity. The response of the full activity controls and background controls was acceptable for 

each run. 

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Results for the positive control were generally within the recommended ranges for the top of the 

curve, bottom curve, Hill slope, log IC50, and coefficient of variation for replicates of each 

concentration within runs. Also, the coefficients of variations (%CVs) for replicates of each 

concentration of flutolanil within a run were within the 15% guideline. For 4-OH ASDN, the 

estimated log IC50 averaged –7.2 M, and the slope was -1.0. 

For flutolanil, aromatase activity averaged 0.230 ± 0.017 nmol·mg-protein−1

·min−1 at the lowest 

tested concentration of 10–10 M and 0.242 ± 0.011 nmol·mg-protein–1·min−1

 at the highest 

successfully tested concentration of 10−4 M (highest non-precipitating dose). Precipitation was 

observed in Run 1 at 10−3

 M and 10-3.5 M. The data for flutolanil were not suitable for modeling. 

The lowest portion of the response curve was greater than 75% activity at all concentrations of 

The mean aromatase activity of 10–4 M flutolanil was 105.56% of control activity, and the best 

fit mean inhibition response curve was a straight line (essentially no inhibition was observed at 

other doses). 

Based on the data from the average response curve, flutolanil is classified as a Non-inhibitor of 

aromatase activity in this assay. 

Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48616805), uterine cytosol from Sprague 

Dawley rats was used as the source of estrogen receptors (ER) to conduct saturation binding 

experiments and competitive binding experiments in this assay. The saturation binding 

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

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

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

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

H]-17βestradiol (1 nM) in the presence of increasing concentrations (logarithmic increase from 10−10 to 

10−4

 M) of flutolanil (99.5% a.i., Lot#: 8AE0010P). Ethanol was used as a vehicle at a final 

concentration of 2%. The assay included 19-norethindrone as a weak positive control, 

octyltriethoxysilane as a negative control, and 17-β-estradiol as the natural ligand reference 

material. 

In the saturation binding experiments, the maximum binding capacity (Bmax) was 41.3 ± 1.7 

fmol/100 μg protein and the dissociation constant (Kd) was 0.044 ± 0.004 nM. For the 

competitive binding experiments, performance criteria were met; the log PC50, log PC 10, log 

EC50, and Hill slope for the reference chemicals 17β-estradiol, 17α-estradiol, 19-norethindrone 

and octyltriethoxysilane all performed within the acceptable range. The mean RBA for 19-

norethindrone was 1.06 × 10−4

%. Flutolanil displaced less than 25% of the radiolabeled estradiol 

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from the ER over the range of soluble concentrations tested (10-10 to 10-4 M) in all competitive 

binding runs. 

Based on the results from the three competitive runs, flutolanil is classified as Not Interactive in 

the Estrogen Receptor Binding Assay. 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

Requirement satisfied by OSRI (Kojima et al., 2004; MRID 48033008); See Appendix 2. 

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350) 

The 21-day short-term reproduction assay (MRID 4861606) of flutolanil with fathead minnows 

(Pimephales promelas) was conducted under flow-through conditions. Adult fish, 20 spawning 

groups (2 males and 4 females in each group: ca. 6 months), were exposed to flutolanil (98.7% 

purity) at nominal concentrations of 0 (negative control), 0 (solvent control), 0.020, 0.20 and 2.0 

mg a.i./L; mean-measured concentrations were <LOQ (<0.010), <LOQ (<0.010), 0.018, 0.18 and 

1.2 mg a.i./L. The solvent control consisted of dimethylformamide (DMF) at a final 

concentration of 0.02 mL/L. The test system was maintained at 24.6 to 25.9o

C and a pH of 7.7 to 

8.2. 

Unless otherwise indicated, all effects are reported based on comparison to the negative (clean 

water) control. There were no significant effects on survival in any treatment group as compared 

to the negative control. At study termination, all surviving fish appeared normal. Female body 

weight and length were significantly increased (p<0.05; 22% and 4%, respectively of negative 

control) at the high treatment level; there were no significant effects on male body weight or 

length. 

Flutolanil significantly reduced fecundity (p<0.05) 100% (relative to control) at the high 

treatment level; the fertilization success in the high treatment group was not calculated because 

no eggs were produced in any replicate. Fertilization success in the remaining treatments was not 

significantly different from negative control (p>0.05). While not statistically significant, 

fecundity was reduced 17% from the negative control at the low and mid concentrations. 

In females in the high treatment group, plasma vitellogenin (VTG) was significantly reduced 

relative to negative control (p<0.01, 72%); while not statistically significant, female VTG at the 

mid treatment level was reduced 16% from the negative control. Female gonado-somatic index 

(GSI) levels were significantly promoted in a concentration-dependent manner, relative to the 

negative control, at all treatment levels (p<0.05; 15-64%). There were no significant differences 

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in male VTG or GSI compared to the negative control (p>0.05). Sex steroids (plasma 17βestradiol and testosterone) were not measured during this study. Nuptial tubercle scores 

were significantly reduced (p<0.05) in male fish at the high treatment level (21 vs. 27); no 

tubercles were observed for females. 

There was a statistically significant decrease in mean male fatpad score at the high treatment 

level compared to the pooled control (p<0.05; Wilcoxon Rank-Sum test; calculated by the study 

author). In males at the high concentration, there was an increased presence of spermatogonia 

and primary spermatocytes with a decreased presences of spermatocysts containing secondary 

spermatocytes and spermatids (p<0.05; Jonckheere-Terpstra test); the testes could not be 

normally staged in 2 of 8 fish. In females, ovaries showed a mild to marked increase in atresia at 

the high treatment level (p<0.05; Jonckheere-Terpstra test; 15 of 16 fish) and significant changes 

in gonad stage and size at all treatment levels (p<0.05; Jonckheere-Terpstra test). 

Hershberger Assay (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48619807) screening for androgenic activity, flutolanil (98.7% 

a.i., lot # 9AE0011P) in 0.5% (w/v) methyl cellulose 100 (MC) was administered daily via oral 

gavage (10 mL/kg) to groups of six 57-day old, castrated male Sprague Dawley rats at dose 

levels of 0 (vehicle), 500, or 1000 (limit dose) 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, flutolanil (98.7% a.i., lot # 9AE0011P) in MC 

was also administered daily via oral gavage (10 mL/kg) to groups of six 58-day old, castrated 

male Sprague Dawley rats at dose levels of 0 (vehicle), 500, or 1000 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 and food consumption 

was measured for Days 1-2, 4-5, and 8-9. The animals were dosed for 10 consecutive days and 

terminated approximately 24 hours after the final dose administration. At necropsy, the five 

androgen-dependent tissues and liver were collected and weighed.

All animals survived until scheduled termination. There were no clinical signs of toxicity and no 

treatment-related gross pathological findings. 

In the androgen agonist assay, body weights, overall (Days 1-11) body weight gains, and food 

consumption in the flutolanil treatment groups and the positive controls were comparable to the 

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vehicle controls. There were no treatment-related effects on weights of accessory sex organs or 

liver for any flutolanil treatment group. Compared to the vehicle control, animals in the TP 

group had increased (p≤0.05) accessory sex organ weights as follows: 1090% in seminal 

vesicles; 1280% in ventral prostate; 818% in levator ani-bulbocavernosus (LABC); 400% in 

Cowper’s gland; and 91% in glans penis. There were no dose-related effects on liver weights in 

the positive control group. The performance criteria indicated that this assay was performing as 

expected. 

In the anti-androgen assay, body weights, overall body weight gains, and food consumption in 

the flutolanil treatment groups and the positive controls were comparable to the TP control. 

There were no treatment-related effects in weights of accessory sex organs or liver for any 

flutolanil treatment group. Compared to the TP control group, animals dosed with TP + FT 

(positive control) had decreased (p≤0.05) accessory sex organ weights as follows: 78% in 

seminal vesicles; 67% in ventral prostate; 56% in LABC; 63% in Cowper’s gland; and 28% in 

glans penis. There were no dose-related effects on liver weights in the positive control group. 

The performance criteria indicated that this assay was performing as expected. 

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

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

Hershberger assay. 

Female Pubertal Assay (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48671101), 15 female Sprague-Dawley (Crl:CD (SD)) 

rats/dose group were treated daily via oral gavage with flutolanil (98.7% a.i., Lot # 9AE0011P) 

in 0.5% aqueous methylcellulose at doses of 0, 500, or 1000 mg/kg/day (limit dose) 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 and hormone analyses. Total thyroxine (T4) and 

thyroid stimulating hormone (TSH) levels were determined by ELISA and radioimmunoassay 

(RIA), respectively. Adrenals, kidneys, liver, thyroid, pituitary, and urogenital organs were 

weighed and microscopic examination of the uterus, ovaries, kidneys, and thyroid was 

conducted. 

No adverse, treatment-related related effects were observed on mortality, clinical signs, final 

body weights, overall body weight gain, age at VO, body weight at VO, organ weights, and 

clinical chemistry or hormone levels. There were no treatment-related histopathological findings 

in the thyroid, uterus, ovaries, or kidneys. 

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Relative (to body) liver weights were increased (p<0.05) by 6% at 500 and 1000 mg/kg/day 

compared to control values. The dose levels tested were adequate since the high dose was the 

Limit Dose (1000 mg/kg/day). 

Male Pubertal Assay (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48671101), 15 male Crl:CD(SD) Sprague Dawley rats/dose 

group were treated daily via oral gavage with flutolanil (98.7% a.i., Lot# 9AE0011P) in aqueous 

0.5% methylcellulose at doses of 0 (vehicle), 500, or 1000 mg/kg/day from post-natal day (PND) 

23 to 53. Animals were examined for preputial separation (PPS) daily beginning on PND 30, 

and age and weight at day of attainment were recorded. Following sacrifice on PND 53, total 

testosterone levels were determined by EIA, total T4 levels were determined with ELISA, and 

TSH levels were determined with a radioimmunoassay (RIA). Adrenals, liver, pituitary, thyroid, 

and urogenital organ weights were recorded and microscopic examinations of the testes, 

epididymides, thyroid, and kidneys were performed. 

No adverse, treatment-related related effects were observed on mortality, clinical signs, final 

body weights, overall body weight gain, age at PPS, body weight at PPS, organ weights (except 

liver), clinical chemistry, or hormone levels (serum T4, TSH, or testosterone). There were no 

treatment-related histopathological findings in the thyroid, testes, epididymides, or kidneys. 

At 1000 mg/kg/day, absolute, adjusted (for body weight on PND 23), and relative liver weights 

were increased by 9-10% compared to control values, and at 500 mg/kg/day, relative liver 

weights were increased by 6%. 

The dosing levels used in the study were acceptable, as the limit dose of 1000 mg/kg/day was 

used for high dose group. 

Steroidogenesis Assay (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48616908), H295R cells cultured in vitro in 24-well plates 

were incubated with flutolanil, (99.5%, Lot No. 8AE0010P) at concentrations of 0.0001, 0.001, 

0.01, 0.1, 0.3, 1, 3, 10, and 100 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was 

used as a vehicle, at a final concentration of 0.1%. 

Hormone concentrations in the assay were determined using an enzyme-linked immunosorbent 

assay (ELISA). Three independent experiments were performed. A Quality Control (QC) plate 

was run concurrently with each independent run of a test chemical plate to demonstrate that the 

assay responded properly to positive control agents at two concentration levels; positive controls 

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included a known inhibitor (prochloraz) and a known inducer (forskolin) of estradiol and 

testosterone production. 

Data at flutolanil concentrations with % live values ≤80% (≥3 μM) were discarded due to 

cytotoxicity. Precipitation of the test compound was not reported at any concentration. 

Guideline acceptability recommendations and requirements were generally met, including lack 

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

coefficients of variation, %CV), and appropriate induction and inhibition with positive controls. 

No effects on testosterone and17β-estradiol concentrations were observed at flutolanil 

concentrations of 10−10 M to 10−4 M. Based on hormone responses in three independent runs, 

flutolanil treatment did not result in statistically significant and reproducible alterations in 

testosterone or estradiol production. 

Uterotrophic Assay (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48616809) conducted to screen for potential estrogenic activity, 

flutolanil (98.7% purity, Lot #9AE0011P) in methyl cellulose was administered daily via oral 

gavage to groups of six ovariectomized Sprague-Dawley rats at dose levels of 0 (vehicle), 500 or 

1,000 (limit dose) mg/kg/day on post-natal days (PND) 67-69 for three consecutive days. A 

positive control group was treated with a daily dose of 17α-ethynyl estradiol (EE) in corn oil at 

0.5 μg/kg/day by subcutaneous (s.c.) injection. Body weights and food consumption were 

determined daily. All animals were terminated and necropsied on PND 70 approximately 24 

hours after the final dose administration to determine wet and blotted uterine weights. Liver 

weights were also determined at necropsy. 

All animals survived until scheduled termination. No clinical signs of toxicity were observed in 

animals for any flutolanil treated groups. Body weights and overall body weight gains (Days 1-

4) in the flutolanil treated groups were comparable to the vehicle controls throughout the study. 

Uterine and liver weights in the flutolanil treated groups were comparable to the vehicle controls. 

Body weights in the positive control (EE) group were comparable to the vehicle control group 

throughout the study, but overall body weight gains of the positive control group were 

significantly (p<0.01) lower (↓99%) than the vehicle control group. Absolute wet and blotted 

uterus weights for the EE group were increased (p<0.0001) by 426% and 195%, respectively, as 

expected. Liver weights were comparable to the vehicle controls. 

No statistically significant changes were seen in uterine weight in this assay. Flutolanil is 

negative in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

ER and AR Transcriptional Activation Assay (ERTA & ARTA) 

In a published, non-Guideline study (Kojima et al., 2004), the potential of flutolanil (>95% 

purity) to interact with two human ER subtypes (hERα and hERβ) and one human androgen 

receptor (hAR) in transactivation assays with Chinese hamster ovary cells (CHO-K1 cells) was 

investigated. The human ER (hERα) and AR (hAR) expression vectors (pcDNAERα and 

pZeoSV2AR, respectively) were constructed as described previously (Kojima et al., 2003). The 

hERβ expression vector was constructed by cloning the hERβ cDNA with reverse transcriptasepolymerase chain reaction from human placental RNA. The sequence was verified and inserted 

into the mammalian expression vector pcDNA3.1Zeo(-) creating pcDNAERβ. The construction 

of the estrogen or androgen responsive elements containing reporter plasmid (pGL3-tkERE and 

pIND-ARE, respectively) was described previously (Kojima et al., 2003). pRL-SV40 containing 

the Renilla luciferase gene was used as an internal control for transfection efficiency. 

For detection of hERα or hERβ activity, cells were transfected with pcDNAERα or pcDNAERβ, 

pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were transfected with 

pZeoSV2AR, pIND-ARE, and pRL-SV40. Flutolanil was tested over a concentration range 

from 10−8 to 10−5 M, and the responses were compared to the activity of 17β-estradiol (E2, >97% 

purity) or 5α-dihydrotestosterone (DHT, 95% purity) for the ER or AR assays, respectively. For 

measurement of antagonistic activity to hERα, hERβ and hAR, either 10−11 or 10−10 M E2 or 

10−10 M DHT was added to the cell cultures along with the test compound, respectively. 

Agonistic activities were evaluated by the relative activity expressed as 20% relative effective 

concentration (REC20), the concentration of the compound demonstrating 20% of the activity of 

10−10 M E2, 10−9 M E2, or 10−9 M DHT for ERα, ERβ, and AR, respectively. Antagonistic 

activities were evaluated similarly by the relative activity expressed as 20% relative inhibitory 

concentration (RIC20), the concentration of the compound showing 20% inhibition of the activity 

of E2 or DHT against ERα, ERβ, and AR, respectively. 

Based on the additional information provided to the Agency (USEPA, 2011b), no evidence of 

cytotoxicity was observed at any of the flutolanil concentrations evaluated, and the highest test 

concentration was close to the test material solubility limit (20.5 μM). The supplemental 

information also included raw data from each test concentration of the three independent 

experimental runs. 

The additional information allowed the Agency to confirm that flutolanil was negative for 

agonist or antagonist activity against ERα and ERβ, and negative for agonist or antagonist 

activity against AR in this transcriptional activation test system. 

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Subchronic Toxicity (Rat, OCSPP 870.3100) 

In a subchronic oral toxicity study in rats (MRID 40342919, 1986), flutolanil (97.5% purity) was 

administered in the diet to CD (SD-derived) rats (10/sex/dose group) at concentrations of 0, 500, 

4000, and 20,000 ppm for 90 days. The mean dietary concentrations were equivalent to 0, 37, 

299, and 1512 mg/kg/day, respectively, for males and 0, 44, 339, and 1743 mg/kg/day, 

respectively, for females. Organs that were weighed included the liver, kidneys, testes, ovaries, 

adrenals, and thyroid/parathyroids. Gross and microscopic pathological examinations were 

conducted on these tissues, as well as the prostate, epididymides, seminal vesicles, uterus, and 

mammary and pituitary glands. The GLP status of this study is unknown. 

No treatment-related effects were observed on mortality, clinical signs, or food consumption; one 

male control animal was sacrificed moribund on Day 86. Slight decreases (NS) in overall body 

weight gain were noted for the high-dose animals in the study. No treatment-related changes in 

ophthalmoscopic, hematological, clinical chemistry, gross pathology, or histopathology 

parameters were observed in either sex. Urinalysis examinations were not conducted. No 

treatment-related changes were observed for the absolute or relative weights of the kidneys, 

testes, ovaries, and adrenal glands. In males, absolute and relative thyroid weights were 

increased (p<0.01 or 0.05) at the mid (absolute, 28% and relative 23%) and high [absolute, 16% 

(NS) and relative, 20%] dose groups. In high-dose females, absolute and relative thyroid 

weights were increased (NS) by 13 and 14%, respectively. In the high-dose group, absolute liver 

weights in males and females were increased by 9 (NS) and 19% (p<0.01), respectively, and 

relative liver weights were increased (p<0.01) by 14 and 20%, respectively, compared to control. 

In the medium-dose group, the absolute and relative liver weights in females were increased 

(p<0.01 and 0.05) by 11 and 8%, respectively. Increased liver weight was considered to be an 

adaptive response to flutolanil treatment because these effects were not supported by clinical 

chemistry or histopathology findings. No treatment-related histopathological lesions were 

observed in any examined tissues. 

The reported NOAEL is 20,000 ppm (1512 mg/kg/day; HDT), and the LOAEL is not 

established. 

Subchronic Toxicity (Dog; OCSPP 870.3150) 

In a subchronic oral toxicity study in dogs (MRID 40342920, 1986), flutolanil (97.5% purity) 

was administered in gelatin capsules to Beagle dogs (4/sex/dose group) at concentrations of 0, 

80, 400, and 2000 mg/kg/day for 95-98 days. Organs that were weighed included the liver, 

kidneys, testes, ovaries, adrenals, and thyroid/parathyroids. Gross and microscopic pathological 

examinations were conducted on these tissues, as well as the prostate, epididymides, uterus, and 

mammary and pituitary glands. 

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No treatment-related effects were observed on mortality, clinical signs, body weights, or food 

consumption; swollen mammary areas were noted in the medium- and high-dose females. No 

treatment-related changes in the ophthalmoscopic parameters were observed. Alkaline 

phosphatase activity was increased (significance not reported) at 1.5 and 3 months in low- and 

high-dose males; observation in the low-dose males may be artifactual due to pretreatment 

elevations. Additionally in the high-dose males, cholesterol was slightly elevated (significance 

not reported) at 1.5 and 3 months, and SGOT and SGPT activities were decreased (significance 

not reported) at 3 months. Alkaline phosphatase activity also was increased (significance not 

reported) at 3 months in the high-dose females. In the high-dose group, the absolute and relative 

weights for the liver and thyroid were increased (magnitude and significance not reported) in 

both sexes. No treatment-related changes were observed in absolute or relative weights of other 

organs. Gross examination noted liver enlargement at the high dose and increased glycogen 

deposition (increased incidences from minimal/mild to moderate) were observed in the mediumand high-dose dogs. No other treatment-related histopathological lesions were observed. 

The systemic toxicity LOAEL is 400 mg/kg/day based on enlarged livers and increased glycogen 

deposition; the NOAEL is 80 mg/kg/day. 

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800) 

In a two-generation reproduction toxicity study in rats (MRID 41850805, 1991), flutolanil (97.6 

and 98.3% purity) was administered daily to SD rats (30/sex/dose) in the diet at nominal doses of 

0, 200, 2000, and 20,000 ppm (mean-calculated intakes of 0, 16, 159, or 1625 mg/kg/day for 

males and 0, 19, 190, or 1936 mg/kg/day for females). The F0 parental animals were treated for 

70 days prior to mating, and the F1 parental animals were treated for 98 days prior to mating. 

Rats were dosed during mating, gestation and lactation. The organ weights for liver and spleen 

were determined (no endocrine-sensitive organs were weighed). 

There were no treatment-related effects on mortality, clinical signs, body weight/body weight 

gain, or food consumption in either F0 or F1 animals. No treatment-related effects were observed 

for the F0, F1, or F2 adults in the reproductive parameters of mal40342924e or female mating, 

fertility and gestation indices, gestation length, live birth index, lactation index, parturition, or 

fetal sex ratio. No treatment-related histopathological lesions were seen in the parental animals, 

and no clinical or pathological findings were noted in pups from any litter or generation. 

Absolute and relative liver weights were increased (significance not reported) in high-dose F0

generation animals and F1 females. These weight increases were considered treatment-related 

adaptive changes due to the lack of adverse effects during gross and microscopic evaluation. 

The LOAEL is not established (>1000 mg/kg/day) due to the lack of parental (systemic), 

reproductive, and offspring toxicity; the NOAEL is >1000 mg/kg/day. 

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Developmental Toxicity (Rat, OCSPP 870.3700) 

In a prenatal developmental toxicity study in rats (MRID 41850804, 1987 and 1992), pregnant 

SD CD rats (22/dose group) were administered flutolanil (97.5% purity) in 0.5% aqueous 

methylcellulose by oral gavage at doses of 0, 40, 200, or 1000 mg/kg/day during GDs 6 through 

15. All dams were sacrificed on GD 20. All fetuses were weighed, sexed, and examined for 

gross external alterations. One-half of the fetuses in each litter were examined for visceral soft 

tissue alterations by the Wilson technique. The remaining fetuses in each litter were examined 

for skeletal alterations with a modified Dawson’s method. 

No mortalities or abortions occurred during the study. There were no treatment-related effects 

on clinical signs, body weight, or food consumption. No treatment-related changes were seen in 

pregnancy rate, number of corpora lutea, mean number of implantations/litter, pre-or post-natal 

implantations, early or late resorptions, number of live fetuses/litter, or fetal sex ratio. No 

developmental abnormalities (external, visceral, or skeletal) were noted at any dose. 

The maternal and developmental NOAELs are 1000 mg/kg/day (HTD). The LOAELs are not 

determined (>1000 mg/kg/day). 

Developmental Toxicity (Rabbit, OCSPP 870.3700) 

In a prenatal developmental toxicity study in rabbits (MRID 40342924, 1987), pregnant New 

Zealand White rabbits (16/dose group) were administered flutolanil (97.5% purity) in aqueous 

2% gum arabic by oral gavage at doses of 0, 40, 200, or 1000 mg/kg/day on GDs 6 through 18. 

On GD 28, all surviving does were sacrificed. Each fetus was weighed, examined for gross 

external observations, and sacrificed. All fetuses in each litter were examined for visceral soft 

tissue alterations by the method of Stuckhardt. Skeletal examinations were conducted by the 

method of Dawson. 

No treatment-related effects on mortality or clinical signs were observed. One low-dose dam 

died due to gavage error. Reduced body weight gains during dosing were observed in all 

treatment groups, and lower overall body weight gain was observed in the high-dose group 

(↓12%; not statistically analyzed). Control group weights were 11-15% higher than the treated 

groups prior to dosing, so body weight gain decreases of 15-20% during dosing may be 

artificially high. Food consumption was slightly less in all treatment groups during the dosing 

period, and lower overall food consumption was observed in the high-dose group. The 

differences observed in body weight gains and food consumption were not considered treatmentrelated. Additionally, no treatment-related effects were seen on any absolute or relative organ 

weights. 

The number of resorptions was increased in the mid- and high-dose groups, but not considered 

treatment-related based on historical control values. No treatment-related changes were seen in 

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pregnancy rate, number of corpora lutea, mean number of implantations/litter, pre-or postnatal 

implantations, or number of live fetuses/litter at any dose. There was an apparent shift in fetal 

sex ratio in high-dose litters (46:54, male:female) compared to the control, but only because the 

sex ratio in the control was skewed toward males (58:42). Sex the ratios in all treated groups 

were approximately 50/50 (low - 55:45; mid - 50:50; high – 46:54). No developmental 

abnormalities (external, visceral, or skeletal) were noted at any dose. 

The maternal and developmental NOAELs are 1000 mg/kg/day. The LOAELs are not 

determined (>1000 mg/kg/day). 

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300) 

In a combined chronic toxicity/carcinogenicity study in rats (MRID 40342921, 1982), Crj:CD 

(SD) SPF rats (50/sex/dose group) were fed diets containing flutolanil (97.5-99.8% purity; 

multiple batches) at nominal concentrations of 0, 40, 200, 2000, and 10,000 ppm (meanmeasured intakes of 0/0, 1.8/2.1, 8.7/10, 86.9/103.1, and 460.5/535.8 mg/kg/day in 

males/females) for 24 months. Additional rats were randomly allocated for 3-month 

(6 rats/sex/group) and 12-month (10 rats/sex/group) interim sacrifices. For clinical chemistry 

examinations, additional groups of 10 rats/sex were sacrificed at the 6- and 18-month intervals. 

Organs that were weighed included the liver, kidneys, testes, ovaries, pituitary, 

thyroid/parathyroids, and adrenals. Gross and microscopic pathological examinations were 

conducted on these tissues as well as the prostate, seminal vesicles, uterus, and mammary gland. 

No treatment-related clinical signs or differences in mortality were observed during the study. In 

the high-dose male rats, body weight gains were decreased (p<0.01) up to 10% compared to 

control for Weeks 1-13 (body weight differences were decreased 3-6%). The body weight gain 

differences between the high dose and control were less apparent after the initial 13 weeks. No 

specific treatment-related pattern was observed for occasional food consumption reductions in 

treatment groups during the study. 

No treatment-related effects were observed for the hematological, clinical chemistry, urinalysis, 

gross pathology, or histopathology parameters. Clinical chemistry results for liver-specific 

enzymes and compounds were variable with no clear treatment-related responses. In the highdose males, the relative liver weights were increased (p<0.05 and 0.01, respectively) by 12 and 

14%, respectively, at 3 and 24 months. In the high-dose females, absolute liver weights were 

increased (p<0.05 or 0.01) by 15-18% compared to control at 3, 12, and 24 months and relative 

liver weights were increased (p<0.05) by 11 and 10%, respectively, at 3 and 12 months. These 

increases in liver weights in high-dose male and female rats were considered to be an adaptive 

response to flutolanil because these increases were not supported by adverse clinical chemistry 

or histopathology findings. No treatment-related changes were seen in absolute or relative 

weights of the other examined tissues, and no treatment-related histopathological lesions were 

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observed in the testes, seminal vesicle, prostate, ovaries, uterus, or mammary, thyroid, adrenal, 

and pituitary glands.

The NOAEL is 460 mg/kg/day (10,000 ppm), and the LOAEL is not established (>10,000 ppm). 

Carcinogenicity (Mouse, OCSPP 870.4200) 

In a carcinogenicity study in mice (MRID 41850803, 1990), CD-1(ICR)BR mice (52/sex/dose 

group) were fed diets containing flutolanil (97.6% purity) at nominal concentrations of 0, 300, 

1500, 7000, or 30,000 ppm (mean-measured intakes of 0/0, 32/34, 162/168, 735/839, and 

3333/3676 mg/kg/day in males/females) for 78 weeks. Organs that were weighed included the 

liver, kidneys, and testes. The tissues subjected to histological examination included these 

organs, as well as epididymides, prostate, seminal vesicles, ovaries, uterus, and mammary, 

pituitary, and thyroid/parathyroids glands. 

No treatment-related effects were observed on mortality, clinical signs, food consumption, 

hematology, gross pathology, or histopathology. Urinalysis and clinical chemistry evaluations 

were not conducted. Body weight gains were decreased (p<0.01) by 17 and 16%, respectively, 

in the high-dose males and females during Weeks 0-24. In medium-dose females, body weight 

gains also were decreased (p<0.05) by 15% during Weeks 0-24. Body weights during the study 

were not affected by treatment. 

No treatment-related changes were observed in organ weights, including the absolute or relative 

testicular weights. Absolute liver weight in the high-dose females was increased (NS) compared 

to control (↑20%), and liver-to-body weight ratio in the high-dose females was increased 

(p<0.0l) by 33%. No similar increase in relative liver weight was observed in high-dose males. 

The increase in liver weights may be attributable to fatty vacuolation, which is not considered 

adverse. No treatment-related histopathological lesions were observed in the testes, 

epididymides, seminal vesicle, prostate, ovaries, uterus, or mammary, thyroid, adrenal, and 

pituitary glands. Flutolanil was not considered oncogenic under the conditions of this study. 

Based on decreased body weight gains, the LOAEL is 3333 mg/kg/day for males (30,000 ppm) 

and 839 mg/kg/day (7000 ppm) for females. The NOAEL is 735 mg/kg/day (7000 ppm) for 

males and 168 mg/kg/day (1500 ppm) for females. 

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Chronic Toxicity (Dog, OCSPP 870.4100) 

In a chronic toxicity study in dogs (MRID 40342922, 1982), Beagle dogs (6/sex/dose group) 

were administered flutolanil (97.5-99.8% purity; multiple batches) in gelatin capsules at doses of 

0, 50, 250, or 1250 mg/kg/day for two years. Organs that were weighed included the liver, 

kidneys, ovaries, and pituitary, adrenals, and thyroid glands. Gross and microscopic pathological 

examinations were conducted on these tissues as well as the testes, prostate, and uterus. 

All dogs survived until study termination. There was a dose-related increase in the incidence of 

clinical signs (emesis, salivation, and soft stool) in both sexes in the medium- and high-dose 

groups from Week 65 until study termination. Decreased (p<0.01 or 0.001) body weights were 

observed in high-dose males from Weeks 93-104 (↓4-8%; unknown significant decrease at Week 

90) with decreased body weight gains (NS, ↓8-16%). Additionally, decreased (p<0.05 or 0.001) 

body weights were observed in high-dose females from Weeks 87-104 (↓3-8%) with decreased 

body weight gains (NS, ↓6-17%). Decreased food consumption (from Week 79 in males and 

from Week 74 in females; significance not reported) also was observed. No treatment-related 

changes in ophthalmoscopic, hematological, or clinical chemistry parameters were observed. No 

treatment-related changes were observed in organ weights or histopathological lesions at any 

dose. 

The systemic toxicity LOAEL is 250 mg/kg/day based on increased incidence of clinical signs 

(emesis, salivation, and soft stool). The NOAEL is 50 mg/kg/day. 

Avian Reproduction (OCSPP 850.2300) 

In an avian reproduction study (MRID 42932301, 1993), Bobwhite quail (Colinus virginianus; 

19-weeks old) were administered flutolanil (98.9% purity) in the feed at nominal concentrations 

of 0 (0.2% acetone/0.08% corn oil), 400, 960, 1920, or 4800 ppm (not adjusted for purity) for 

20 weeks (10 weeks prior to the start of egg production and during 10 weeks of egg production). 

Each treatment group was comprised of sixteen mating pairs, with one pair per pen. A 

progressive photo period was employed in this study: 8 hours/day through Week 7 and 

17 hours/day until the end of the study. Mean temperature was maintained at 19.6±1.2°C 

throughout the study and mean humidity was maintained at 33±13%. Individual adult body 

weights were recorded at study initiation, biweekly through Week 8, and at study termination. 

Food consumption in each replicate was measured biweekly throughout the pre-treatment and 

treatment periods of the adult phase. Individual chick body weights were recorded after hatching 

and on Day 14 after hatching; egg shell thickness was measured weekly during egg production. 

Adult birds and chicks were observed routinely for mortality and clinical signs. 

There were no overt signs of toxicity or treatment-related reductions in food consumption or 

body weight at any test concentration compared to control. Two incidental deaths occurred in 

the low-medium concentration group. Although there were no significant effects on any 

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reproductive parameter, there was a slight reduction in the number of eggs laid at the high 

concentration compared to control. This reduction was related to an increase in the number of 

hens that laid a smaller number of eggs (≤20) during the study. This reduction in egg production 

was also reflected in the number of hatchlings and 14-day survivors as percentages of the 

maximum number of eggs set. However, the proportion of eggs set/eggs laid indicated that 

fertility was not necessarily affected at the high dose. The LOAEC is 4800 ppm based on the 

reduction in egg production, and the NOAEC is 1920 ppm. 

In another avian reproduction study (MRID 42932302, 1993), Mallard ducks (Anas 

platyrhynchos; 21-weeks old) were administered flutolanil (98.9% purity) in the feed at nominal 

concentrations of 0 (0.2% acetone/0.08% corn oil), 400, 960, 1920, or 4800 ppm (not adjusted 

for purity) for 20 weeks (10 weeks prior to the start of egg production and during 10 weeks of 

egg production). Each treatment group was comprised of sixteen mating pairs, with one pair per 

pen. A progressive photo period was employed in this study: ≤8 hours/day through Week 7 and 

17 hours/day until the end of the study. Mean temperature was maintained at 17.8±1.8°C 

throughout the study and mean humidity was maintained at 47±15%. Individual adult body 

weights were recorded at study initiation, biweekly through Week 8, and at study termination. 

Food consumption in each replicate was measured biweekly throughout the pre-treatment and 

treatment periods of the adult phase. Individual chick body weights were recorded after hatching 

and on Day 14 after hatching; egg shell thickness was measured weekly during egg production. 

Adult birds and chicks were observed routinely for mortality and clinical signs. 

There were no overt signs of toxicity or treatment-related mortalities at any test concentration, 

and there were no reductions in adult body weight or feed consumption. At the high 

concentration, there was a 5% decrease (p<0.05) in egg shell thickness compared to control. 

However, the mean shell thickness was not treatment-responsive and was comparable to the lowconcentration group mean value; the mid-low and mid-high mean eggshell thickness values were 

comparable to control. The reduction in egg shell thickness at the high concentration was 

considered to biologically (toxicologically) relevant. There were no other treatment-related 

effects on any study parameters determined at any test concentrations. The LOAEC is 4800 ppm 

based on decreased egg shell thickness, and the NOAEC is 1920 ppm. 

Fish Early Life-Cycle Toxicity (Fathead minnow, OCSPP 850.1400) 

In a fish early life-cycle toxicity study (MRID 43552001, 1995), groups of Fathead minnow 

(Pimephales promelas) embryos (30 embryos/cup; 4 replicates/treatment concentration) were 

exposed to flutolanil (98.9% purity) at nominal concentrations of 0 [negative and solvent (DMF; 

0.025 mL/L) controls], 0.13, 0.25, 0.50, 1.0, and 2.0 mg/L. Mean-measured concentrations were 

0.116, 0.233, 0.486, 0.933, and 1.94 mg/L (89-97% of nominal). After hatching, >30 fry per 

treatment group were impartially selected to begin the 35-day exposure period. Fry were 

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counted on Days 4, 5, 6, 7, 8, 11, 17, 21, and 35 post-hatch. At Day 35 post-hatch, percent 

survival, mean total length, and average wet weight were determined. 

There were no effects on the percentage hatch of eggs, but Day 35 survival was decreased 

(p<0.05) by 96-100% at the high concentration. Mean total length and wet weight of fry were 

decreased (significance not reported) at the medium and medium-high concentrations (not 

determined for the high-concentration group due to ≥96% mortality). The LOAEC is 0.486 

mg/L due to decreased length and wet weight, and the NOAEC is 0.233 mg/L. 

 

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APPENDIX 3: References Not Utilized in the Flutolanil 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 Flutolanil but were not utilized due to one or more of the following reasons1) 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. 

Auletta, C.S. (1990) A 21-day dermal toxicity study in rats with flutolanil. Bio/dynamics, Inc. 

Project#89-3497 (T -3070). (MRID 41841205) 

Martin, M.T., Mendez, E., Corum, D.G., Judson, R.S., Kavlock, R. J., Rotroff, D.M., and Dix, D. 

J. (2009) Profiling the reproductive toxicity of chemicals from multigeneration studies in the 

toxicity reference database. Toxicological Sci. 110(1), 181–190. 

Matsumoto, K., Hosokawa, M., Kuroda, K., et al. (2009) Toxicity of Agrichemicals in Daphnia 

magna. Osaka City Med. J.: 55: 89-97. 

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FLUTOLANIL FINAL 

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Rhodes, J.E and Gormley, M. (1991) Early life-stage toxicity of flutolanil technical to the 

fathead minnow (Pimephales promelas) under flow-through conditions. Analytical 

BioChemistry Laboratories, Inc. Final Report #38436. NOR-AM Study #E642.06.89 (W-3012). 

(MRID 41841221) 

Tsujimura, S., Igarashi, N., Kawanishi, H., and Takeshima, T. (1982) Three generation 

reproduction and teratogenicity study on flutolanil in the rat. Imamichi Institute for Animal 

Reproduction. Study #144 (T-3015). (MRID 40342923) 

 

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