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: FOLPET 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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ii 

TABLE OF CONTENTS 

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

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

I. Introduction.......................................................................................3 

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

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

B. Other Scientifically Relevant Information (OSRI)......................................4 

III. Weight of Evidence (WoE) Evaluation.......................................................4 

A. EDSP Tier 1 Screening Assays.............................................................7 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................8 

1. Effects on Estrogen Pathway.....................................................8 

2. Effects on Androgen Pathway..................................................12 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................15 

IV. Committee’s Assessment of Weight of Evidence..........................................17 

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................17 

1. Tier 1 in vivo Assays.............................................................18 

2. OSRI...............................................................................18 

B. Estrogen Pathway...........................................................................20 

C. Androgen Pathway.........................................................................20 

D. Thyroid Pathway...........................................................................21 

E. Conclusions.................................................................................21 

V. EDSP Tier 2 Testing Recommendations....................................................21 

VI. References.......................................................................................22 

LIST OF TABLES: 

 Table 1. Tier 1 Assays for Folpet ......................................................................7 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Folpet ..........................................9 

 Table 3. Androgenic/Anti-Androgenic Pathway for Folpet ......................................13 

 Table 4. Thyroid Pathway for Folpet ...............................................................16 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................25 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................37 

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

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iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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iv 

Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

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v 

Abbreviation Terminology 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

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vi 

Abbreviation Terminology 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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Page 1 of 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 a pesticide chemical interacts with E, A, or T hormone pathways, the 

number and type of effects induced, the magnitude and pattern of responses observed across 

studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur 

were considered, in particular, whether or not endocrine-related responses occurred at dose(s) 

that also resulted in general systemic toxicity or overt toxicity. 

On December 3, 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 folpet 

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). 

There was no convincing evidence for potential interaction of folpet with the estrogen pathway 

in mammals. Folpet was negative in the estrogen receptor (ER), ER transactivation assay 

(ERTA) and steriodogenesis assays. In the aromatase assay, folpet was equivocal. No estrogenrelated effects were observed in the Tier 1 female pubertal assay or in any of the mammalian or 

wildlife Part 158 studies. However, in fish, folpet showed potential to interact with the estrogen 

pathway based on increased male plasma vitellogenin in the fish short-term reproduction assay 

(FSTRA). It is noted that there were no other significant effects observed in females and 

generally in males (a NS decrease in tubercle score is noted) in the FSTRA. 

There was no convincing evidence for potential interaction of folpet with the androgen pathway. 

Folpet was negative in the in vitro AR binding and steriodogenesis assays and in the in vivo 

Hershberger. No androgen-related effects were seen in the male pubertal assay in the absence of 

overt toxicity. Other than the effect on male vitellogenin (VTG), no effects were observed in 

males in the FSTRA [a non-significant decrease in tubercle score was noted)]. Additionally, no 

androgen-related effects were seen in any of the mammalian or wildlife Part 158 studies. 

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There was no convincing evidence for potential interaction of folpet with the thyroid pathway. In 

the male pubertal assay the only treatment-related finding was a decrease in serum T4 level at the 

low dose in the absence of overt toxicity. In the female pubertal assay, serum T4 levels were 

decreased at the low and high dose. Serum thyroid stimulating hormone (TSH) levels were 

unaffected and there were no alterations in thyroid weight or thyroid histopathology at any dose 

level in either sex. While growth (i.e. snout ventral length and body weight) was decreased in the 

amphibian metamorphosis assay (AMA), there were no effects on developmental stage or 

thyroid histopathology. 

Based on the weight of evidence evaluation, a mammalian EDSP Tier 2 testing is NOT 

recommended for folpet since there was no convincing evidence for potential interaction with the 

estrogen, androgen or thyroid pathways in mammals. 

In the FSTRA, male vitellogenin was increased at 8.6 μg a.i./L. Currently, the chronic fish risk 

assessments are based on a NOAEC of 8.8 μg a.i./L and a LOAEC of 17 μg a.i./L established in 

the fish early-life cycle toxicity study with the fathead minnow. Since the responses observed in 

the FSTRA occurred at similar concentrations as the current chronic endpoint, the EDSP Tier 2 

Medaka Extended One-Generation Reproduction Test (MEGORT) is recommended. 

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

duck are available for folpet. In both species, no effects on reproductive parameters were 

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

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

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

 

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I. Introduction

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

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

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

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

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In 

addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality were considered 

in this weight of evidence (WoE) assessment. 

In determining whether a pesticide chemical interacts with E, A, or T hormone pathways, the 

number and type of effects induced, the magnitude and pattern of responses observed across 

studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur 

were considered, in particular, whether or not endocrine-related responses occurred at dose(s) 

that also resulted in general systemic toxicity or overt toxicity. 

On December 3, 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 folpet 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). 

Folpet is a fungicide with a water solubility of 0.8 mg/L. Folpet degrades rapidly (half-life, t1/2, 

of 2.6 hours to 2 days) in aquatic and terrestrial environments. Bioaccumulation data suggest that 

folpet does not bioaccumulate in fish because of its rapid hydrolysis in water. Folpet has an 

intermediate, thiophosgene, which causes irritation to mucus membranes and is a skin irritant 

and sensitizer. 

The available information considered in determining the potential interaction of folpet 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 

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 Folpet (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 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 (AOP). Broadly, there are four main steps outlined in the guidance which provide the 

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

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

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

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

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

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

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

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

above. 

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the 

EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other 

scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays 

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consisting of in vitro and mammalian and wildlife in vivo assays. OSRI may include published 

literature studies as well as studies conducted under USEPA (often referred to as Part 158 data) 

or OECD guidelines submitted in support of pesticide registrations. Each study is evaluated for 

scientific quality and relevance for informing interactions with the E, A, or T 

pathway. Additionally, the consistency of the responses in the individual study is evaluated. For 

the Tier 1 in vivo assays, often multiple endpoints are measured in each assay. 

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the 

relative degree of diagnostic utility of a specific endpoint for discerning whether or not the 

chemical has interacted with the endocrine system, are considered. The collective response of the 

individual endpoints, as well as the conditions under which they were expressed, are considered 

when evaluating an overall indication of potential interaction as measured by the study. 

The second step in this WoE process is to formulate hypotheses and integrate the available data, 

along different levels of biological organization. Two keys elements in the integration of data as 

well as characterizing the extent to which the available data support a hypothesis that a chemical 

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

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

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

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

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

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

the whole organism level. The extent of expression of responses at higher levels of biological 

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

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

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

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

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

biological organization were the responses observed and whether organisms exhibit 

compensatory responses at higher levels of biological organization? Finally, under what 

conditions where responses occur including consideration of whether the responses were 

observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic 

toxicity introduces uncertainty in the ability to distinguish effects specifically related to an 

endocrine-related effect from a non-endocrine toxic response. 

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

the 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 

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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. 

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. Thus, the WoE approach involved 

consideration of data from the EDSP Tier 1 assays in reaching and supporting a conclusion to 

determine whether or not a substance has the potential to interact with the E, A, or T pathways. 

The lines of evidence indicating potential interaction with the E, A, or T pathway are depicted in 

Tables 2 - 4. These tables contain data that are considered scientifically and biologically 

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relevant with 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 folpet are shown below in 

Table 1. 

Table 1. Tier 1 Screening Assays for Folpet 

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement satisfied 

(MRID 48616903) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement satisfied 

(MRID 48616904) 

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement satisfied 

(MRID 48616901) 

Steroidogenesis Assay (Human cell line 

H295R) 

OCSPP 890.1550; 

OECD 456 

Requirement satisfied 

(MRID 48616906) 

Aromatase Assay (human recombinant 

microsomes) OCSPP 890.1200 Requirement satisfied (MRID 48616902) 

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement satisfied 

(MRID 48616907) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement satisfied 

(MRID 48616905) 

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement satisfied 

(MRID 48671201) 

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement satisfied 

(MRID 48671202) 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement satisfied 

(MRID 48684201) 

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; 

OECD 231 

Requirement satisfied 

(MRID 49140601) 

 . 

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

1. Effects on Estrogen Pathway 

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

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

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

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

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

considered further in the WOE assessment. 

Folpet was classified as negative in the Tier 1 in vitro estrogen receptor (ER) binding (rat uterine 

cytosol) assay, the ERα transcriptional assay, and steroidogenesis assay. In the Tier 1 aromatase 

assay, folpet was determined to be equivocal based on a 43% reduction in aromatase activity at 

10−5 M, the highest concentration tested. Folpet was also negative in an ERTA considered as 

OSRI conducted in Chinese hamster ovary (CHO) cells (Kojima et al., 2004). 

In the Tier 1 in vivo studies, folpet was classified as negative for estrogenic activity in the 

uterotrophic assay. In the female pubertal study, no estrogen-related effects were seen at the low 

dose (400 mg/kg/day). At the high dose (800 mg/kg/day), the number of antral ovarian follicles 

was increased (p<0.05) by 58% compared to the controls, which was likely related to an 

increased number of high dose animals in diestrus. Additionally, the number of other types of 

ovarian follicles (small, medium, and atretic), follicular cysts, and corpora lutea were not 

significantly different at this dose. While signs of toxicity (body weight loss, abnormal breathing, 

and gastrointestinal dilation) were seen 2 and 3 (out of 16) rats at the low and high dose 

respectively, the remaining animals survived with no treatment-related clinical signs of toxicity. 

In the FSTRA, there were no estrogen-related effects observed in the females. However, it is 

noted that in males a significant increase in male plasma vitellogenin was observed at 8.6 μg/L. 

No estrogen-related effects were seen in the mammalian Part 158 studies with folpet. In a twogeneration reproduction toxicity study in rats female fertility in the F1b generation was reduced 

(67%; not significant) at the mid dose (800 ppm) and 66% at the high dose (3600 ppm) when 

compared to controls (79%), and during the second mating of the F1b generation, fertility indices 

in females were also reduced at the low (200 ppm; 67%), mid (69%), and high (63%) dose 

groups as compared to controls (71%). However, overt toxicity, presenting as decreased maternal 

body weights and decreased pup body weights, were observed at the high dose. Additionally, 

these effects (i,e decreased fertility) were not replicated at doses up to 5000 ppm in another 

reproduction toxicity study in rats. 

There were no effects on reproductive parameters in Part 158 avian reproduction toxicity studies 

on mallard and quail. In a Part 158 early life stage study with fathead minnow, reduced growth 

was observed at 17.7 μg/L. 

Page 16 of 56
FOLPET FINAL 

Page 9 of 48 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Folpet. 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Folpet1

Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Vitlli

EDSP Tier 1 Assay 

ER Binding (MRID 48616903) N ERTA (MRID 48616904) N Aromatase (MRID 48616902) E Steroidogenesis (MRID 48616906) N Uterotrophic (MRID 48616907) N Female Pubertal Rat (MRID 48671201)

N N N N N N 

FSTRA (MRID 48684201) NE N N N N N

OSRI 

ERTA (Kojima et al., 

2004) 

 N 

Two-generation Reproduction (Rat, MRID 00151489) 

NE NE N NE NE N 
FOLPET FINAL 

Page 10 of 48 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Folpet. 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Folpet1

Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Vitlli

Two-generation Reproduction (Rat, MRID 40135901) 

NE N N NE NE N 

Developmental Toxicity (Rat, MRID 00132457) 

 N 

Chronic Toxicity (Rat, MRID 43640201) 

NE N N NE 

Chronic Toxicity/ Carcinogenicity (Rat, MRID 00151560) 

NE N N NE 

Chronic Toxicity (Dog, MRID 00161315) 

NE N N N 

Subchronic Toxicity (Rat, MRID 00115269) 

NR N N NR 

Avian Reproduction (Quail, MRID 00098004) 

 N 
FOLPET FINAL 

Page 11 of 48 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Folpet. 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Folpet1

Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Vitlli

Avian Reproduction (Duck, MRID 00098005) 

 N 

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

indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this aare absolute unless otherwise indicated. CS= Clinical signs; LW= Liver weight 

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

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 i

see Section IV. A 

4 Due to signs of toxicity (body weight loss, abnormal breathing, and gastrointestinal dilation, 2 and 3 (out of 16) rats were euthanized 

respectively, the remaining animals survived with no treatment-related clinical signs of toxicity. 

5 No effect was observed in female plasma VTG, however, male plasma VTG was significantly increased at the highest concentration 

values (11,000,000 ng/mL at 8.6 μg/L vs. 1600 ng/mL in control). 

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

E Equivocal response NE Not examined NR Not reported 
FOLPET FINAL 

Page 12 of 48 

2. Effects on Androgen Pathway 

The potential for folpet to interact with the androgen pathway is summarized in Table 3. The 

various targets of the estrogen 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. 

Folpet was classified as negative in both the AR binding and steroidogenesis assays. Folpet was 

also negative in an ARTA considered as OSRI conducted in Chinese hamster ovary (CHO) cells 

(Kojima et al., 2004). In the Tier 1 in vivo assays, folpet was negative in the Hershberger for 

both androgenic and anti-androgenic activity. 

In the male pubertal rat assay, no treatment-related effects were seen at the low dose (200 

mg/kg/day). At the mid (400 mg/kg/day) and high (800 mg/kg/day) dose groups decreases were 

seen in the weight of the epididymides (9-13%), pituitary (15-16%), and LABC (11-13%); 

however, at these doses, overt toxicity was manifested as morbidity, adverse clinical signs (e.g. 

abnormal breathing/rales, hunched posture and piloerection) and decreased body weights 

(>10%). Additionally, due to excess mortality (7/16 euthanized) at the mid dose, serum 

parameters (i.e. clinical chemistry and hormones) were not evaluated at this dose. No treatmentrelated effects were seen on age or weight at PPS and no histopathological changes in the testes 

or epididymides were seen at any dose. 

In the FSTRA, male tubercle score was decreased (NS; 30 in control vs. 26 in treatment) and 

male vitellogenin was significantly increased (p<0.05) at the highest concentration tested (8.6 

μg/L) compared with the negative control. Vitellogenin concentrations averaged 1600 ng/mL in 

the negative control and 11,000,000 ng/mL at 8.6 μg/L folpet. However, all other endpoints were 

unaffected by treatment. 

No androgen-related effects were seen in any of the mammalian Part 158 studies with folpet. As 

discussed in Section B 1 (Estrogen Pathway), the decrease in male fertility observed in a twogeneration reproduction study was not replicated at higher doses in a second two-generation 

reproduction study. In the wildlife Part 158 studies, there were no effects on reproductive 

parameters in avian reproduction toxicity studies on mallard and quail. 

Page 20 of 56
FOLPET FINAL 

Page 13 of 48 

Table 3: Androgenic/Anti-Androgenic Pathway for Folpet. 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Folpet1 Study Type/ Literature Citation 

AR Binding 

Steroidogenesis & 

Gene Expression 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 2° 

Sex Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

EDSP Tier 1 Assay 

AR Binding (MRID 48616901) N Steroidogenesis (MRID 48616906) N Hershberger (MRID 48616905) NE N Male Pubertal Rat (MRID 48671202) N N N N N N N N FSTRA (MRID 48684201) NE N N N N 

OSRI 

ARTA (Kojima et al., 2004) N Two-generation Reproduction (Rat, MRID 00151489) 

NE NE N NE N NE N NE 

Two-generation Reproduction (Rat, MRID 40135901) 

NE N N NE N NE N NE 

Chronic Toxicity (Rat, MRID 43640201) NE N N NE NE NE NE 
FOLPET FINAL 

Page 14 of 48 

Table 3: Androgenic/Anti-Androgenic Pathway for Folpet. 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Folpet1 Study Type/ Literature Citation 

AR Binding 

Steroidogenesis & 

Gene Expression 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 2° 

Sex Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

Chronic Toxicity/ Carcinogenicity (Rat, MRID 00151560) 

 NE N N N N NE NE 

Chronic Toxicity (Dog, MRID 00161315) NE N N N N N NE Avian Reproduction (Quail, MRID 00098004) 

N 

Avian Reproduction (Duck, MRID 00098005) 

N 

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

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

in weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral 

prostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP)Liver weight; m=male 

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

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 a

details see Section IV. A 

4 Male plasma VTG was significantly increased at the highest concentration tested compared to control values (11,000,000 ng/m

ng/mL in control). 

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

NE Not examined 
FOLPET FINAL 

Page 15 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 

female and male rat pubertal assays, and the amphibian metamorphosis assay (frog). 

The potential for folpet to interact with the thyroid pathway is summarized in Table 4. The 

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

facilitate determination of 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. 

As discussed before in Section III. A and B, overt toxicity characterized as morbidity, severe 

clinical signs, decreases in body weight, body weight and liver weight was seen at the two 

highest doses tested in the male pubertal assay and at both doses in the female pubertal assay. At 

the low dose in the male pubertal assay, the only treatment-related effect seen in the absence of 

overt toxicity was a decrease (p<0.01) in serum T4 level. In the female pubertal assay, serum T4 

level was decreased (p<0.05) 33% and 34% at 400 and 800 mg/kg/day, respectively. There were 

no changes in serum TSH levels, thyroid weight or thyroid histopathology in either sex. 

 In the AMA, decreased SVL (13%) and body weight (30%) was noted on Day 7 at the highest 

test concentration (9.6 μg/L) compared to the negative control. All other parameters were 

unaffected by treatment. 

No thyroid-related effects were seen in any of the mammalian Part 158 studies with folpet. 

Page 23 of 56
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Page 16 of 48 

Table 4. Thyroid Pathway for Folpet.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Folpet1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histo

pathology

Serum T4 Levels 

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

Overt Toxicity Observed3

EDSP Tier 1 Assay 

Male Pubertal Rat 

(MRID 48671202) N N ↓23% 

(L) N N 

LW 

↓11-18% 

(L, M, H) 

CS, ↓BW 

(M,H) 

Female Pubertal Rat 

(MRID 48671201) N N 

↓33, 

34% 

(L,H) 

N N N N4 

AMA 

(MRID 49140601) N N 

SVL: 

↓13%; 

BW: 

↓30% 

Day 7 

(H) 

 N 

OSRI

Two-generation 

Reproduction (Rat, 

MRID 00151489) 

NE N NE NE NE N X 

 (H) 

Chronic Toxicity (Rat, 

MRID 43640201) NE N NE NE NE X 

(L, M, H) 

X

 (H) 

Chronic Toxicity/ 

Carcinogenicity(Rat, 

MRID 00151560) 

NE N NE NE NE N X 

 (H) 

Chronic Toxicity 

(Dog, MRID 

00161315) 

N N NE NE N X X 

(H) 

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

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

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

otherwise indicated. 

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

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

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

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

4. Due to signs of toxicity (body weight loss, abnormal breathing, and gastrointestinal dilation, 2 and 3 (out of 16) 

rats were euthanized at the low and high dose respectively, the remaining animals survived with no treatmentrelated clinical signs of toxicity. 

P Positive findings 

N Negative findings 

NE Not examined 

Page 24 of 56
FOLPET FINAL 

Page 17 of 48 

IV. Committee’s Assessment of Weight of Evidence 

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

potential of folpet 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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Page 18 of 48 

1. Tier 1 in vivo Assays 

No overt or systemic toxicity was not observed in the uterotrophic assay at dose levels up to 

1000 mg/kg/day. 

In the Hershberger assay, at 800 mg/kg/day, 5/16 rats (3 were co-administered TP) were 

euthanized before study termination due to moribundity. Animals exhibited loss of body weight, 

abnormal breathing, and/or soft feces. Additionally, in the androgen agonist assay at 800 

mg/kg/day, a body weight loss of −3.3 g (p<0.05) was noted over Days 1-11 (compared to a gain 

of +38.2 g in the vehicle control). 

In the male pubertal assay which included three dose levels, no overt or systemic toxicity was seen 

at the low dose (200 mg/kg/day). At the mid (400 mg/kg/day) and high (800 mg/kg/day) dose 

groups, overt toxicity manifested as: mortality/moribundity (7/16 rats at 400 mg/kg/day and 5/16 at 

800 mg/kg/day (5/16 rats); severe clinical signs (abnormal breathing/rales, hunched posture, 

piloerection, distended abdomen and/or thick red nasal discharge); decreases in final body 

weights at 400 mg/kg/day (14%) and 800 mg/kg/day (11%); and decreases in liver weight !8% at 

400 mg/kg/day and 15% at 400 mg/kg/day. 

In female pubertal rat assay which tested only two dose levels, overt toxicity was observed at 

both doses and included: moribundity (2/26 at 400 mg/kg/day and 3/16 at 800 mg/kg/day); 

clinical signs (rales); decreases in body weight; and gastrointestinal dilatation in sacrificed 

animals. 

Overt toxicity was not observed in the FSTRA or AMA. 

2. OSRI 

In a rat two-generation reproduction toxicity study, mean body weights of the 3600 ppm F1

parental males were decreased (p<0.05) by 9-20% throughout the study. Body weights of the 

3600 ppm F1 parental females were decreased (p<0.05) by 18% on Day 1 of premating, but the 

decrease was only 5% (NS) on Day 176. Food consumption of the 3600 ppm P and F1 groups 

was reduced (p<0.05) frequently during the premating periods. During lactation, the 3600 ppm 

pup body weights were decreased (p<0.05) by 13-19% on PND 21 in the F1a, F1b, F2a, and F2b

litters. 

In a second rat two-generation reproduction toxicity study, parental body weight decreases were 

noted at 5000 ppm. During pre-mating, decreased (p<0.05) mean body weights were observed in 

the P and F1 males and females, resulting in decreased overall (Weeks 0-14) body weight gains. 

During gestation and lactation, body weights were decreased (p<0.05) in the P and F1 females, 

with decreased overall gestational body weight gains. Additionally at 5000 ppm, food 

consumption was decreased (p<0.05) throughout pre-mating in the P and F1 males, and during 

Page 26 of 56
FOLPET FINAL 

Page 19 of 48 

pre-mating, gestation and lactation in the P and F1 females. In the F1 pups, mean viable pup 

weights were decreased (p<0.05) at 1500 ppm on PND 21 and 25 and at 5000 ppm on PND 7, 

14, 21, and 25. In the F2 pups, mean viable pup weights were decreased (p<0.05) only at 5000 

ppm on PND 21. Microscopically, most or all of the 1500 and 5000 ppm males and females in 

both generations had slight, moderate or marked diffuse hyperkeratosis of the non-glandular 

epithelium of the stomach compared to only two control P males. Slight or moderate diffuse 

hyperkeratosis of the esophagus was reported for 1500 and 5000 ppm F1 females only. 

In a rat developmental reproduction toxicity study, maternal clinical signs of toxicity (rales, 

salivation, chromorrhinorhea, decreased motor activity, and soft or liquid feces) were noted at 

360 mg/kg/day. During GD 0-20, maternal body weight gains were decreased (p<0.05) by 15-

28% at 60 and 360 mg/kg in dams corrected for uterine weight, and by 16% at 360 mg/kg in 

dams with intact gravid uteri. Maternal feed consumption was also decreased (p<0.05) during 

GD 6-20 at 360 mg/kg. 

In a rat chronic toxicity study, systemic toxicity was observed at 5000 ppm. Body weights were 

decreased (p<0.05) by 7-12% in the males during Weeks 13-104. Food consumption was 

decreased (p<0.05) at almost all intervals in the males through Week 85 and in the females 

through Week 71. Water consumption was decreased for the majority of the study by 10% in the 

males and by 20% in the females, with both sexes having a 30% decrease during Week 1. At 

5000 ppm, slight to moderate diffuse hyperkeratosis of the esophagus was observed in 18/20 

males and 20/20 females compared to 0 controls. Slight to moderate diffuse hyperkeratosis of the 

non-glandular epithelium of the stomach was noted in 16/20 males and 20/20 females at 1500 

ppm and in 20/20 males and 19/20 females at 5000 ppm, with severity increasing with dose. 

Liver lesions, characterized as hepatic focal or areas of cellular alterations, occurred at all dose 

levels in both sexes including the controls. Also, hepatic nodular neoformation was observed in 

the liver of both sexes. 

In a combined chronic toxicity/carcinogenicity study, “thickened" stomach (no distinction was 

made between tissue of the glandular or non-glandular stomach) was noted in 1/23, 5/37, 5/32, 

and 6/31 control, low (200 ppm), mid (800 ppm), and high (3200 ppm) dose males, respectively, 

and in 3/31, 0/29, 0/31, and 5/30 of the respective female treatment groups at final sacrifice. 

Similar findings were also noted at the interim sacrifice in 1/10, 1/10, 2/10, and 3/10 control, 

low, mid and high dose males and in 0/10, 2/10, 2/10, and 3/10 of the respective female 

treatment groups. 

In a dog chronic toxicity study, body weights were decreased by 7-18% during Weeks 7-52 in 

the 60 mg/kg/day males, and by 8-24% during Weeks 3-52 in the 120 mg/kg/day males. Overall 

(Weeks 0-52) body weight gains were decreased by 50% at 60 mg/kg/day and by 62% at 120 

mg/kg/day. In the females, body weights were decreased by 7-10% during Weeks 3-52 at 60 

mg/kg/day, and by 7-11% during Weeks 3-52 at 120 mg/kg/day. Overall (Weeks 0-52) body 

Page 27 of 56
FOLPET FINAL 

Page 20 of 48 

weight gains were decreased by 35% at 60 mg/kg/day and by 42% at 120 mg/kg/day. Food 

consumption was decreased in a dose-related manner in the 60 and 120 mg/kg/day males for the 

first 13 weeks of the study and in the 60 and 120 mg/kg/day females during Weeks l and 2 and at 

four to five weekly intervals between weeks 9 and 16. Mean cholesterol, total protein, albumin, 

and globulin were decreased in the 60 and 120 mg/kg/day males and in the 120 mg/kg/day 

females at the 2-, 3-, 6-, 9-, and 12-month intervals. 

In a rat subchronic oral toxicity study, growth rates and body weights were decreased at 10,000 

ppm in males and females. Total serum protein was decreased in the 10,000 ppm males at Weeks 

6 and 13, and in the 10,000 ppm females at Week 13. Both albumin and globulin were decreased 

in the 10,000 ppm group at Week 13. LDH was increased in the 3000 and 10,000 ppm males. 

Both the 10,000 ppm males and females showed folpet-related effects in the non-glandular 

(upper) portions of the stomach such as acanthosis, hyperkeratosis, submucosal edema, and 

pleocellular infiltrate (inflammatory reaction), and an occasional instance of stomach focal 

erosion and ulceration. 

In avian reproduction toxicity studies in quail and in duck, overt toxicity was not observed at 

concentration levels up to 1000 mg/kg-diet. 

In a fish early-life stage study in fathead minnow (Pimephales promelas), no fish survived at 28 

days in the two highest treatment groups (34.9 and 65.3 μg/L) and growth was reduced at 17.7 

μg/L. 

B. Estrogen Pathway 

There was no convincing evidence for potential interaction of folpet with the estrogen pathway 

in mammals. Folpet was negative in the ER, ERTA and steriodogenesis assays. In the aromatase 

assay, folpet was equivocal. No estrogen-related effects were observed in the Tier 1 female 

pubertal assay or in any of the mammalian or wildlife Part 158 studies. However, in fish, folpet 

showed potential to interact with the estrogen pathway based on increased male plasma 

vitellogenin. However, in fish, folpet showed potential to interact with the estrogen pathway 

based on increased male plasma vitellogenin in the FSTRA. It is noted that there were no other 

significant effects observed in females and generally in males (a NS decrease in tubercle score is 

noted) in the FSTRA. 

C. Androgen Pathway 

There was no convincing evidence for potential interaction of folpet with the androgen pathway. 

Folpet was negative in the in vitro AR binding and steriodogenesis assays and in the in vivo 

Hershberger. No androgen-related effects were seen in the male pubertal assay in the absence of 

overt toxicity. Other than the effect on male VTG (a non-significant decrease in tubercle score 

Page 28 of 56
FOLPET FINAL 

Page 21 of 48 

was noted), no effects were observed in males in the FSTRA. Additionally, no androgen-related 

effects were seen in any of the mammalian or wildlife Part 158 studies. 

D. Thyroid Pathway 

There was no convincing evidence for potential interaction of folpet with the thyroid pathway. In 

the male pubertal assay the only treatment-related finding was a decrease in serum T4 level at the 

low dose in the absence of overt toxicity. In the female pubertal assay, serum T4 levels were 

decreased at the low and high dose. Serum TSH levels were unaffected and there were no 

alterations in thyroid weight or thyroid histopathology at any dose level in either sex. While 

growth (i.e. SVL and body weight) were decreased in the AMA, there were no effects on 

developmental stage or thyroid histopathology. 

 E. Conclusions 

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

potential interaction with the estrogen, androgen or thyroid pathways in mammals. In wildlife, 

there is evidence for potential interaction with the estrogen pathway based on the male 

vitellogenin increase in the FSTRA. Folpet did not interact with the androgen or the thyroid 

pathways in wildlife. 

V. EDSP Tier 2 Testing Recommendations 

Based on the weight of evidence evaluation, a mammalian EDSP Tier 2 testing is not 

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

estrogen, androgen or thyroid pathways. 

In the FSTRA, male vitellogenin was increased at 8.6 μg a.i./L. Currently, the chronic fish risk 

assessments are based on a NOAEC of 8.8 μg a.i./L and a LOAEC of 17 μg a.i./L established in 

the Fish Early Life Cycle toxicity study with the fathead minnow. Since the responses observed 

in the FSTRA occurred at similar concentrations as the current chronic endpoint, the EDSP Tier 

2 Medaka Extended One-Generation Reproduction Test (MEGORT) is recommended. 

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

duck are available for folpet. In both species, no effects on reproductive parameters were 

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

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

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

Page 29 of 56
FOLPET FINAL 

Page 22 of 48 

VI. References 

Beavers, J. and Fink, R. (1982) One-generation reproduction study in bobwhite quail: Phaltan 

Technical (SX-1111). Wildlife International, Ltd., St. Michael’s, MD. Sponsored by Chevron 

Chemical Co., Richmond, CA. Laboratory Report No. S-1889, June 16, 1982. Unpublished. 

(MRID 00098004) 

Beavers, J. and Fink, R. (1982). One generation reproduction study in mallard duck: Phaltan 

technical (SX-1111). Wildlife International, Ltd., St. Michael’s, MD. Sponsored by Chevron 

Chemical Co., Richmond, CA. Laboratory Report No. S-1888, June 23, 1982. Unpublished. 

(MRID 00098005) 

Cox, R. (1985) Combined chronic oral toxicity/oncogenicity study in rats with Chevron folpet 

technical (SX-2388). Hazelton Laboratories America, Inc., Vienna, VA. Sponsored by Chevron 

Chemical Co., Richmond, CA. Laboratory Project No. 2107-209, September 30, 1985. 

Unpublished. (MRID 00151560) 

Crown, S., Nyska, A., Waner, T., et al. (1989) Folpan: toxicity by dietary administration to rats 

for two years. Life Science Research Israel, Ltd., Israel. Sponsored by Makhteshim Chemical 

Works Ltd., Israel. Laboratory Report No. MAK/053/FOL, January 10, 1989. Unpublished. 

(MRID 43640201) 

Daly, I. and Knezevich, A. (1986) A one year subchronic oral toxicity study in dogs with folpet 

technical. Biodynamics Inc., East Millstone, NJ. Sponsored by Chevron Chemical Company, 

Richmond, CA. Laboratory Study No. 82-2677, April 7, 1986. Unpublished.( MRID 00161315) 

Davis, J. (2012). The Hershberger Bioassay (OPPTS 890.1400); Folpet. Integrated Laboratory 

Systems, Inc., Durham, NC. Laboratory Study No.: C200-200, January 4, 2012. Unpublished. 

(MRID 48616905) 

Davis, J. (2012) Pubertal development and thyroid function in intact juvenile/peripubertal female 

rats; Folpet. Integrated Laboratory Systems, Inc., Durham, NC. Sponsored by Makhteshim 

Chemical Works, Ltd., c/o Makhteshim Agan of North America, Inc., Raleigh, NC. Laboratory 

Project Study No.: C200-300, April 13, 2012. Unpublished. (MRID 48671201) 

Davis, J. (2012) Pubertal development and thyroid function in intact juvenile/peripubertal male 

rats; Folpet. Integrated Laboratory Systems, Inc., Durham, NC. Sponsored by Makhteshim 

Chemical Works, Ltd., c/o Makhteshim Agan of North America, Inc., Raleigh, NC. Laboratory 

Project Study No.: C200-301, April 18, 2012. Unpublished. (MRID 48671202) 

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Page 23 of 48 

Davis, J. (2012) The uterotrophic assay; Folpet. Integrated Laboratory Systems, Inc., Durham, 

NC. Sponsored by Makhteshim Chemical Works, Ltd., c/o Makhteshim Agan of North America, 

Inc., Raleigh, NC. Laboratory Project Study No.: C200-100, January 3, 2012. Unpublished. 

(MRID 48616907) 

Gordon, E (2013) Folpet: Weight of evidence evaluation of the U.S. EPA’s Endocrine Screening 

Program Tier 1 Screening Assay. Makhteshim Agan of North America, Inc., Raleigh, NC. 

Laboratory Project Study No. 839 13/2. 

Hardy, L. and Richter, W. (1985) Two-generation (two litter) reproduction study in rats with 

Chevron folpet technical. Chevron Environmental Health Center, Inc., Chevron Chemical 

Company, Richmond, CA, Laboratory Study No. SOCAL 2140, September 19, 1985.) 

Unpublished. (MRID 00151489) 

Hoberman, A. (1983) Teratology study in rats with folpet technical. Argus Research 

Laboratories Inc., Perkasie, PA. Sponsored by Chevron Chemical Co., Richmond, CA. 

Laboratory Project No. 303-001, August 23, 1983. Unpublished. (MRID 00132457) 

Kojima, H., Katsura, E., Takeuchi, S., Niiyami, 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): 524-531. 

Lee, M. (2013) Folpet - amphibian metamorphosis assay. Smithers Viscient, Wareham, MA. 

Sponsored by Makhetshim Agan NA, Raleigh, NC. Laboratory Report N. 11742.6177, May 22, 

2013. Unpublished. (MRID 49140601) 

Rhodes, J. and Stuerman, L. (1995) Early life-stage toxicity of folpet technical to the fathead 

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

Laboratories, Columbia, MO. Sponsored by Makheteshim-Agan of North America, Inc., New 

York, NY. Laboratory Report No. 42578, August 9, 1995. Unpublished. (MRID 43786301) 

Rubin, R. (1986) Folpan: two-generation reproduction study in the rat. Life Science Research 

Israel Ltd., Ness Ziona, Israel. Submitted by Makhteshim-Agan of North America, Inc., New 

York, NY. Laboratory Project ID. R-4347, December 29, 1986. Unpublished. (MRID 40135901) 

Snyder (1981). Subchronic toxicity study in rats: Phaltan. Hazleton Laboratories America, Inc., 

Vienna, VA. Sponsored by Chevron Chemical Co., Richmond, CA. Laboratory Project No. 

2107-100, October 22, 1981. Unpublished. (MRID 00115269) 

USEPA (2010) Folpet - Report of the Endocrine Disruptor Review Team - Test Order#: EDSP081601-175. Office of Chemical Safety and Pollution Prevention Science Coordination and 

Policy. U.S. Environmental Protection Agency, Washington, DC. 

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

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

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

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

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

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

Agency, Washington, DC. 

Wagner, H. (2012) Folpet: steroidogenesis (Human Cell Line – H295R). CeeTox, Inc., 

Kalamazoo, MI. Laboratory Study No. 9141V-100357STER, January 4, 2012. Unpublished. 

(MRID 48616906) 

Wilga, P. (2012) Folpet: human recombinant aromatase assay. CeeTox, Inc., Kalamazoo, MI. 

Laboratory Report No. 9141V-100357AROM, January 4, 2012. Unpublished. (MRID 48616902) 

Willoughby, J. (2012) Folpet: androgen receptor binding assay using rat prostate cytosol. 

CeeTox, Inc., Kalamazoo, MI. Laboratory Study No. 9141V-100357ARB, January 5, 2012. 

Unpublished. (MRID 48616901) 

Willoughby, J. (2012) Folpet: estrogen receptor binding assay using rat uterine cytosol. CeeTox, 

Inc., Kalamazoo, MI. Laboratory Study No. 9141V-100357ERB, January 5, 2012.. Unpublished. 

(MRID 48616903) 

Willoughby, J. (2012) Folpet: estrogen receptor transcriptional activation (human cell line 

(HeLa-9903)). CeeTox, Inc., Kalamazoo, MI. Laboratory Report No. 9141V-100357ERTA, 

January 5, 2012. Unpublished. (MRID 48616904) 

Willoughby, J.A. (2012). Supplemental information - laboratory proficiency data for ERTA 

assays and saturation binding data for AR and ER binding assays for assorted chemicals. 

CeeTox, Inc., Kalamazoo, MI. July 19, 2012. Unpublished. (MRID 48843501) 

York, D. (2012) Folpet: short-term reproduction assay with the fathead minnow (Pimephales 

promelas). Smithers Viscient Wareham, MA. Sponsored by Makhteshim Agan of North 

America, Inc., Raleigh, NC. Laboratory Study No.: 11742.6178, July 27, 2012. Unpublished. 

(MRID 48684201) 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100) 

The 21-day assay (MRID 49140601) of folpet on amphibian metamorphosis of African clawed 

frog (Xenopus laevis) was conducted under flow-through conditions. Amphibian larvae at 

Nieuwkoop-Faber (NF) stage 51 (80/control and treatment group; 20/replicate) were exposed to 

nominal concentrations of folpet (97.6% purity) at 0 (negative and solvent [0.004 mL/L 

dimethylformamide (DMF)] controls), 0.0002, 0.002, and 0.02 mg a.i./L; the reviewer-calculated 

time-weighted average (TWA) measured concentrations were <0.000014 (<LOQ; controls), 

0.000069, 0.00092, and 0.0096 mg a.i./L. The test system was maintained at 21 to 23°C and a pH 

of 6.8 to 7.9. 

There were no significant differences (p>0.05) between the negative control and solvent control 

for any of the endpoints. Unless otherwise indicated, all effects are reported based on 

comparison to the negative control. 

Survival rates in all treatment groups were similar to the negative control at test termination. 

Spinal deformities were observed in 3 to 18% of surviving tadpoles in both controls and all 

treatment groups, though this observation was not attributed to exposure to the test material. On 

Day 7, there were significant reductions (p<0.05) in snout-vent length of 13%, wet weight of 

30%, and hind leg length (HLL) of 9% at the high treatment level relative to the negative control; 

on Day 21, these endpoints were not significantly different from the negative control. 

Folpet did not significantly increase or decrease Day 7 or 21 normalized (for snout-vent length) 

HLL at any treatment level (p>0.05). No significant acceleration or delay of median NF 

developmental stage was observed after 7 or 21 days of exposure at any treatment level; no 

asynchronous development was observed. In the solvent control and the low and mid treatment 

groups, there were a total of 2, 1, and 1 late-stage tadpoles (NF>60), respectively; no late-stage 

tadpoles were observed in the negative control or high treatment group. 

Thyroid gland histopathology findings were observed in the controls and all treatment groups, 

though these effects did not appear to be treatment related. Histopathological findings included 

mild follicular hypertrophy and hyperplasia in the controls and all treatment groups and mild 

follicular lumen area increase in the controls and the low and mid treatment groups. 

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Androgen Receptor (AR) Binding Assay (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48616901), ventral prostate cytosol from 

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

which measured the binding of a single concentration of [3

H]-R1881 (1 nM) in the presence of 

increasing concentrations of folpet (94.5% purity, Lot# 00138518). Folpet was tested at 

concentrations from 10−10 to 10−3

 M in Run 1 or 10−11 to 10−4 M in Runs 2 and 3. DMSO was 

used as the solvent vehicle at a final assay concentration of approximately 3.2%. A total of three 

runs were performed, and each run included dexamethasone as a weak positive control, and 

R1881 as the ligand reference standard. 

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

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

radiolabeled reference androgen (R1881) prior to routinely conducting AR competitive binding 

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

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

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

H]-

R1881 was 0.613±0.041 nM and the estimated Bmax was 0.817±0.049 fmol/100 μg protein for 

the single batch of prostate cytosol that was used for this assay. The mean and individual Kd and 

Bmax values were below the expected ranges reported in the EPA validation program (Kd 0.685 to 

1.57 nM and Bmax 7 to 16 fmol/100 μg protein). Confidence in these numbers is high based on 

the goodness of fit (R2 = 0.957-0.984) and the small variation among runs.

In the competitive binding experiment, the estimated mean log IC50s for R1881 (−9.0 M) and the 

weak positive control, dexamethasone (−4.6 M), were within the expected ranges. The mean 

relative binding affinity (RBA) for the weak positive control was 0.004%. All performance 

criteria were generally met; however, potential drift in the study assay could not be assessed 

because all solvent control tubes were analyzed at the beginning of each run. 

Substantial precipitation of folpet was visually observed at a concentration of 10−3

 M in Run 1; 

therefore, the second and third runs were performed at a maximum test concentration of 10−4

 M. 

The specific [3

H]-R1881 binding was >75% at all soluble concentrations of folpet (10−11 to 10−4

M) in all runs. A log IC50 and RBA could not be calculated for folpet. 

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

Receptor Binding Assay. 

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Aromatase Assay (OCSPP 890.1200) 

In an in vitro aromatase (CYP19) assay (MRID 48616902), folpet (94.5% a.i. Batch # 00138518) 

was incubated with human recombinant aromatase and tritiated androstenedione ([1β3H(N)]-

androst-4-ene-3,17-dione; [3

H]ASDN) in dimethyl sulfoxide (DMSO) at final log concentrations 

of 10−11 to 10−5 M for 15 minutes to assess the potential of folpet to inhibit aromatase activity. In 

Run 1, the two highest concentrations (10−3 and 10−4 M) of folpet tested showed precipitation 

during the 15-minute incubation period and were not used in data interpretation. Therefore, the 

highest test concentration used for Runs 2 and 3 was 10−5

 M. 

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

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

using liquid scintillation counting (LSC). Three runs were conducted and each run included a 

full activity control, a background activity control, a positive control series (10−10 to 10−5

 M) 

using a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), and the folpet series (10−11

to 10−5 M) with three repetitions per concentration. 

Aromatase activity in the full activity controls was 0.608 ± 0.042 nmol·mg-protein−1

·min−1. The 

response of each full activity control within a run was between 90 to 110% of the average full 

activity. Activity in the background controls ranged 0.24 to 0.37% and averaged 0.30% of the 

full activity controls. 

For the positive control substance (4-OH ASDN), aromatase activity results were within the 

recommended ranges for the performance criteria. The estimated log IC50 for 4-OH ASDN 

averaged −7.27 M and the slope was −0.94. 

For folpet, aromatase activity at the lowest and highest tested concentrations evaluated, 10−11 and 

10−5 M, was 97.03% and 57.32% of the control, respectively. Since the average lowest portion of 

the response curve across runs was between 50 and 75% activity, the response was considered 

equivocal at soluble concentrations. 

Based on data from the average response curve, folpet is classified as Equivocal for aromatase 

inhibition in this assay. 

Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48616903) for folpet (94.5% purity, Lot#: 

00138518), uterine cytosol from Sprague Dawley rats was used as the source of ER for a 

competitive binding experiment, which measured the binding of a single concentration of [3

H]-

17β-estradiol (1 nM) in the presence of increasing concentrations (10−11 to 10−4

 M) of folpet. 

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DMSO was used as the solvent vehicle at a final concentration of 4%. A total of three valid runs 

were performed, and each run included 19-norethindrone as a weak positive control, 

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

material. 

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

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

submitted following a request by the Agency. The protein concentrations used in the saturation 

binding runs varied between each run, and were approximately 3- to 6-fold greater than 

recommended (160 to 320 μg versus 50 ±10 μg). The mean dissociation constant (Kd) for [3H]-

17β-estradiol was 0.331 ± 0.061 nM and the estimated Bmax was 74.55 ± 3.03 fmol/100 μg 

protein for the prepared rat uterine cytosol. The Kd for each run was within the expected 

Guideline range of 0.03 to 1.5 nM. Although the Scatchard plots fit straight lines to the data, the 

concavity observed in all of the data sets may indicate issues with ligand depletion. 

In the competitive binding experiment, the estimated mean log IC50 was −9.0 M for 17β-estradiol 

and −5.5 M for the weak positive control, 19-norethindrone. The mean relative binding affinity 

(RBA) was 0.0295% for the weak positive control. Performance criteria were generally met; 

however, octyltriethoxysilane displaced more than 25% of [3

H]-17β-estradiol from the ER at the 

highest concentration tested (10-3 M) in two of the three assay runs. One run was repeated. 

The specific [3

H]-ligand binding was >75% at all folpet concentrations tested in all three runs. A 

log IC50 and RBA could not be calculated for folpet. 

Based on the results from the three runs, folpet is classified as Not Interactive in the Estrogen 

Receptor Binding Assay. 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48616904), hERα-HeLa-9903 

cells cultured in vitro were exposed to folpet (94.5% a.i., Lot # 00138518) at logarithmically 

increasing concentrations from 10-12 M to 10-4M in DMSO (0.1%) in two independent runs. 

Experiments were performed using 96-well plates and each folpet concentration was tested in 6 

wells/plate in each run. Cells were exposed to test agents for approximately 24 hrs to induce 

reporter (luciferase) gene products. Luciferase expression in response to estrogen receptor 

activation was measured using a proprietary assay. 

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No cytotoxicity was observed at 10-4 M folpet in the range-finding test, however cytotoxicity was 

observed at that concentration in the main assays. The mean RPCMax was 3.5% for the first run 

and 3.9% for the second, and the associated PCMax was 10-7 M. 

In the main assays, the responsiveness of the cells to the very weak positive control 

17α-methyltestosterone was lower than the expected values, indicating a decreased sensitivity of 

the assay to very weak agonists. Furthermore, 17α-methyltestosterone responses reached only 

6.3-7.9% PC and were very similar to those of the negative control corticosterone, which 

averaged 2.6-3.7% PC. Therefore, under the conditions of this study, the reviewer cannot 

determine if folpet is a very weak estrogen. Nevertheless, the responses of folpet (3.5-3.9% PC) 

were not comparable to the response of the weak estrogen control, 17α-estradiol (117-124% PC) 

and the RPCMax < PC10 in both assay runs. 

Fish Short Term Reproduction Assay (FSTRA; OCSPP 890.1350) 

The 21-day short-term reproduction assay (MRID 48684201) of folpet with fathead minnows 

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

spawning groups; 2 males and 4 females in each group; 4 groups per treatment; ca. 6 months 

old), were exposed to folpet (94.5% purity) at nominal concentrations of 0 (negative and solvent 

[0.010 mL/L dimethylformamide, DMF] controls), 0.00054, 0.0036, and 0.024 mg a.i./L; meanmeasured concentrations were <0.000031 (<LOQ, controls), 0.00018, 0.0010, and 0.0086 mg 

a.i./L. The test system was maintained at 24 to 25o

C and a pH of 6.8 to 7.4. 

There were no significant differences (p>0.05) between the negative and solvent controls for any 

of the endpoints. Unless otherwise indicated in this DER, all effects are reported based on 

comparison to the negative control. 

Adult survival was unaffected by treatment with folpet; survival was 100% for all test levels and 

controls, except for females in the solvent control which showed 94% survival. No abnormal 

observations of secondary sexual characteristics (e.g., body color, coloration patterns, body 

shape, size of dorsal nape pad in males and ovipositor size in females) or clinical signs of 

toxicity were observed during the exposure period or at study termination. There were no effects 

on body weight or length at any treatment level compared to the negative control. 

In the negative control group, spawning occurred at least every four days in three of the four 

replicates; mean fecundity was 14 eggs/female/day/replicate; and fertilization success averaged 

98%. Replicate B in the negative control averaged 12 eggs/female/reproductive day while 

another replicate C averaged 7.5 eggs/female/reproductive day and did not spawn at least every 

four days. In the solvent control group, spawning occurred at least every four days during the 

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exposure period; mean fecundity was 16 eggs/female/day/replicate; and fertilization success 

averaged 98%. Replicate B in the solvent control averaged 14 eggs/female/reproductive day. 

There were no significant differences (p>0.05) between the treatment groups and the negative 

control for any endpoint except male vitellogenin (VTG), which showed a significant increase 

(p=0.0049; Jonckheere-Terpstra test) at the high treatment level. The mid and high treatment 

groups had only three replicates available for male VTG analysis because there were no data 

available for one replicate; no explanation was provided by the study authors for the missing 

data. 

Treatment-related effects were not observed for histopathology severity scores or gonadal stage. 

However, there was a slight increase in granulomatous inflammation and increased oocyte 

atresia (females) with increasing folpet concentration. For four, six, and five cases from the low, 

mid, and high treatment groups, respectively, the oocyte atresia and/or granulomatous 

inflammation was attributed to Microsporidia infection. 

 

Hershberger Assay (OCSPP 890.1400) 

In a Hershberger assay (MRID 48616905) screening for androgenic activity, folpet (97.6%, 

batch no. 00138518) in aqueous 1% carboxymethylcellulose was administered daily via oral 

gavage to groups of eight 61/62-day old castrated male Sprague Dawley rats at dose levels of 0 

(vehicle), 250, or 800 mg/kg/day for 10 consecutive days. An androgenic positive control group 

consisted of eight castrated male rats exposed to carboxymethylcellulose by gavage plus 0.4 

mg/kg/day of testosterone propionate (TP) in corn oil by subcutaneous (s.c.) injection. 

To screen for potential anti-androgenic activity, folpet in aqueous 1% carboxymethylcellulose 

was administered daily via oral gavage to groups of eight 61/62-day old castrated male Sprague 

Dawley rats at dose levels of 0 (vehicle), 100, 250, or 800 mg/kg/day in conjunction with a daily 

dose of reference androgen TP in corn oil at 0.4 mg/kg/day by s.c. injection. The 

anti-androgenic positive control group consisted of eight castrated rats exposed to 0.4 mg/kg/day 

TP and 3 mg/kg/day flutamide (FT) in corn oil. The negative control group consisted of the 

same animals that served as positive control for the androgenic portion of the assay. 

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

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

androgen-dependent tissues [seminal vesicles with coagulating glands, ventral prostate, levator 

ani-bulbocavernosus (LABC), Cowper’s glands, and glans penis] were excised, examined 

macroscopically, and weighed. 

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Measurements of food consumption, serum hormone concentrations, and liver, kidney, and 

adrenal weights are optional according to the Guideline and were not performed in this study. 

No treatment-related effect was noted on mortality due to the chemical (rather than the 

administration route) or on clinical signs. No gross effect was reported on the sex-related 

tissues; although, gastrointestinal dilatation was common in the treated groups. 

At 800 mg/kg/day, 5/16 rats (3 were co-administered TP) were euthanized before study 

termination due to moribundity; no signs of dosing error were observed, but animals exhibited 

loss of body weight, abnormal breathing, and/or soft feces. Additionally, 2/8 rats treated at 800 

mg/kg/day folpet (without TP) and 1/8 rats treated with 250 mg/kg/day folpet with TP died 

before study termination due to gavage error. All other animals survived to the scheduled 

sacrifice. No explanation was provided why similar moribundity was not observed in the dose 

range finding study. The oral LD50 in rat is >2000 mg/kg and the administration of 

approximately 1000 mg/kg/day in the diet for three weeks is tolerated. The study author stated 

that the increase in moribundity may have been due to gavage-related reflux. 

In the androgen agonist assay at 800 mg/kg/day, body weights were decreased (not statistically 

significant [NS]) by 10% at termination, and a body weight loss of 3.3 g (p≤0.05) was noted over 

Days 1–11 (compared to a gain of 38.2 g in the vehicle control). Terminal body weights and 

body weight gains at 250 mg/kg/day were similar to controls. In the anti-androgen assay, 

terminal body weights and body weight gains in the treated groups were similar to the TP 

negative control group. 

Organ weights in the folpet treated groups were similar to the controls in both the androgen 

agonist and anti-androgen assays. 

There was no effect of flutamide on body weights or body weight gains. TP administration 

produced a 49% increase in overall (Days 1–11) body weight gain. These body weight gain 

increases were consistent with an androgenic response in the test animals. For the androgenic 

portion of the study, the TP positive control caused increases (p≤0.05) in the weights of the 

seminal vesicles with coagulating glands (↑1007%), ventral prostate (↑923%), LABC (↑168%), 

Cowper’s glands (↑593%), and glans penis (↑40%), indicating that the test system was sensitive 

to an androgenic response. For the anti-androgenic portion of the study, the flutamide positive 

control caused decreases (p≤0.05) in weights of the seminal vesicles with coagulating glands 

(↓79%), ventral prostate (↓72%), LABC (↓49%), Cowper’s glands (↓64%), and glans penis 

(↓18%) compared to weights in the negative control group. These data indicate that the test 

system was sensitive to an anti-androgenic response. 

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The dose levels for this study were considered adequate based on the moribundity, clinical signs 

of toxicity, and body weight decreases observed. The Guideline criteria for %CV were met in all 

cases for the negative control and high dose groups. 

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

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

assay. 

Female Pubertal Assay (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48671201), 16 Sprague Dawley rats/dose group were treated 

daily via oral gavage with folpet (97.6% a.i., Batch/lot # 00138518) in aqueous 1% 

carboxymethylcellulose (CMC) at doses of 0 (vehicle), 400, or 800 mg/kg/day from post-natal 

day (PND) 22 to 42 or 43. 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 

or 43, blood was collected for clinical chemistry analyses; total thyroxine (T4) and thyroid 

stimulating hormone (TSH) levels were determined using radioimmunoassays. Adrenal, liver, 

pituitary, thyroid, and urogenital organs were weighed, and microscopic examinations were 

performed on the ovaries, uterus, thyroid, and kidneys. 

There were no treatment-related clinical signs of toxicity, and no dose-related effects were noted 

on final body weights, body weight gain, age and body weight at VO, mean age at first vaginal 

estrus, mean cycle length, percent cycling, or percent regularly cycling. 

Two rats in the 400 mg/kg/day group and three rats in the 800 mg/kg/day group were euthanized 

prior to study termination due to body weight loss and abnormal breathing. No signs of dosing 

error were observed at necropsy; however, gastrointestinal dilatation was observed in all five 

animals. All other rats survived until scheduled sacrifice. 

At 400 mg/kg/day, relative liver weights were decreased (p<0.05) by 5% compared to the vehicle 

controls; no changes in liver weights were observed in rats in the 800 mg/kg/day group. There 

were no dose-related effects on the weights of kidneys, pituitary, adrenals, ovaries, uterus (wet 

and blotted), or thyroid. 

Serum T4 levels were decreased (p<0.01) by 33-34% in rats dosed at 400 and 800 mg/kg/day. 

Sodium and chloride were increased (p<0.01) by 2% and 4%, respectively, in rats dosed at 800 

mg/kg/day; chloride was also increased (p<0.05) in rats dosed at 400 mg/kg/day (↑2%). Alanine 

aminotransferase and alkaline phosphatase were decreased (p<0.05) in rats dosed at 400 and 800 

mg/kg/day (↓30% and ↓52%, respectively, for alanine aminotransferase, and ↓20% and ↓18%, 

respectively, for alkaline phosphatase). There were no dose-related effects on levels of serum 

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TSH, potassium, calcium, phosphorus, aspartate aminotransferase, gamma glutamyl transferase, 

blood urea nitrogen, creatinine, total bilirubin, sorbitol dehydrogenase, total protein, or albumin. 

There were no dose-related histopathological changes in the uterus, thyroid glands, or kidneys. 

At 800 mg/kg/day, the number of antral ovarian follicles was increased (p<0.05) by 58% 

compared to the controls, which the study author concluded was likely due to the stage of the 

estrous cycle. The number of other types of ovarian follicles (small, medium, and atretic), 

follicular cysts, and corpora lutea were not significantly changed in rats from the 400 or 800 

mg/kg/day folpet groups compared to vehicle control. 

While signs of toxicity (body weight loss, abnormal breathing, and gastrointestinal dilation) were 

seen 2 and 3 (out of 16) rats at the low and high dose respectively, the remaining animals 

survived with no treatment-related clinical signs of toxicity. 

Male Pubertal Assay (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48671202), 16 Sprague Dawley rats/dose group were treated 

daily via oral gavage with folpet (97.6% a.i., Batch/lot # 00138518) in aqueous 1% 

carboxymethylcellulose (CMC) at doses of 0 (vehicle), 200, 400, or 800 mg/kg/day from postnatal day (PND) 23 to 53 or 54. Animals were examined for preputial separation (PPS) daily 

beginning on PND 30 and age and weight at day of attainment was recorded. Following 

sacrifice on PND 53 or 54, blood was collected for clinical chemistry analyses; total serum 

testosterone, thyroxine (T4), and thyroid stimulating hormone (TSH) levels were determined 

using radioimmunoassays. Adrenal, liver, pituitary, thyroid, and urogenital organs were weighed 

and microscopic examinations of the testes, epididymides, thyroid, and kidneys were performed. 

One 200 mg/kg/day male was found dead on Day 25 as a result of a gavage error. At 400 

mg/kg/day, 6 males were euthanized before scheduled termination due to moribundity, and one 

additional rat was found dead on Day 25. At 800 mg/kg/day, 5 males were euthanized prior to 

scheduled termination due to moribundity. All other rats survived until scheduled sacrifice. 

One control and one 200 mg/kg/day rat were observed with a rough coat on Day 2; no abnormal 

findings were noted in the remaining animals in these groups. Abnormal breathing/rales, 

hunched posture, and/or thick red nasal discharge was observed in all six 400 mg/kg/day rats 

euthanized prior to study termination and two of the surviving rats. No abnormal findings were 

noted in the remaining eight 400 mg/kg/day rats. In the 800 mg/kg/day group, clinical 

observations noted in animals 24 hours after dosing included abnormal breathing, distended 

abdomen, and piloerection in three of five rats euthanized prior to study termination and four of 

the surviving rats. No abnormal observations were noted in the remaining nine 800 mg/kg/day 

rats. 

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Final body weight was decreased (p<0.05) at 200, 400, and 800 mg/kg/day, by 8%, 14%, and 

11%, respectively. Overall body weight gains were also decreased (p<0.05) at 200, 400, and 800 

mg/kg/day, by 10%, 17%, and 14%, respectively. There were no treatment-related effects on age 

or weight at attainment of PPS. 

There were no effects of treatment on the weights of adrenal glands, seminal vesicle plus 

coagulating gland, ventral prostate, dorso-lateral prostate, testes, or thyroid glands. At 800 

mg/kg/day, absolute and adjusted (for body weight on PND 21) liver weights were decreased 

(p<0.01) by 15%, and absolute and adjusted levator ani-bulbocavernosus (LABC) and 

epididymis (right only) weights were decreased (p<0.05) by 12% (LABC) and 9% (epididymis). 

Absolute pituitary weights were decreased (p<0.05) by 15% and relative kidney weights were 

increased (p<0.05) by 6%. 

At 400 mg/kg/day, absolute and adjusted weights for the following organs were decreased 

(p<0.05): liver (↓18%), kidney (↓10-11%), epididymides (↓13%), and LABC (↓11-13%). 

Absolute pituitary weights were decreased (p<0.05) by 16%. 

At 200 mg/kg/day, absolute and adjusted liver weights were decreased (p<0.05) by 11%. The 

weights of kidneys, pituitary, LABC, and epididymides were comparable to the controls. 

Serum T4 was decreased (p<0.01) in rats dosed at 200 (↓23%) and 800 (↓33%) mg/kg/day. At 

200 mg/kg/day, sodium, chloride, and sorbitol dehydrogenase were increased (p<0.05) by 3%, 

5%, and 20%, respectively. At 800 mg/kg/day, chloride was increased (p<0.01; ↑3%), and the 

following parameters were decreased (p<0.05): alanine aminotransferase (↓42%), alkaline 

phosphatase (↓27%), total protein (↓5%), and albumin (↓5%). There were no dose-related 

effects on serum TSH, testosterone, potassium, calcium, phosphorus, aspartate aminotransferase, 

gamma glutamyl transferase, blood urea nitrogen, creatinine, or total bilirubin. Serum from rats 

in the 400 mg/kg/day group was not evaluated due to the low survival of rats in this group and 

the Guideline requirement of a minimum of two treatment levels. 

There were no dose-related histopathological changes in the testes, epididymides, thyroid glands, 

or kidneys. No changes in follicular cell height or colloid area were observed in the thyroid 

glands of rats dosed at 200 or 800 mg/kg/day compared to the controls. Histopathological 

analyses were not conducted for rats in the 400 mg/kg/day group. 

The most common clinical observations noted 24 hours post dosing in rats at 400 and 800 

mg/kg/day were abnormal breathing/rales, distended abdomen, piloerection, hunched posture 

and/or thick red nasal discharge occurring coincident with decreased body weight (14% at 400 

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mg/kg/day and 11% at 800 mg/kg/day) when compared to controls. Necropsy of the dead and 

moribund animals did not show evidence for dosing error. 

The agency does not concur with the study author’s rationale for the mortality. The clinical signs 

such as abnormal breathing/rales, gasping, and hunched posture indicate that the cause of these 

signs is likely dosing errors. The mortalities cannot clearly be attributed to gavage-reflux, as 

rationalized by the study author, since in the range finding study, except for one rat at 800 

mg/kg/day, all rats survived comparable doses, and there were no dosing errors. Additionally, it 

is also possible that the doses tested were excessive based on significant decreases (11-14%) in 

body weight in rats at the 400 and 800 mg/kg/day groups. Overt toxicity was not seen at the low 

dose (200 mg/kg/day). 

Steroidogenesis Assay (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48616906), H295R cells cultured in vitro in 24-well plates 

were incubated with folpet, (94.5% purity, Batch # 00138518) at log concentrations from 0.0001 

to 100 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a 

final concentration of 0.05%. 

Testosterone and estradiol concentrations were measured by HPLC/MS-MS with positive ion 

multiple reaction monitoring. Four 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 included the known inhibitor (prochloraz) and inducer (forskolin) of 

estradiol and testosterone production. 

Laboratory proficiency testing was not conducted in the current study, and details of a previous 

proficiency determination were not included in the study report. The data from Run #1 were not 

analyzed due to an error in one of the reference chemical concentrations on the QC plate. 

Because of precipitation, the highest suitable concentrations of folpet in Runs #2 and #4 were 0.1 

μM and 1 μM. Cytotoxicity at the 100 μM concentration, and decreased cell viability (81.9%) at 

the 10 μM concentration, was noted in Run #3. 

Guideline acceptability recommendations and requirements were met, including adequate 

production of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate 

induction and inhibition with positive controls. 

The results of the steroidogenesis assay with folpet indicate that folpet is neither an inducer nor 

an inhibitor of testosterone synthesis in this assay. The results of this assay also indicate that, 

although folpet is not an inducer of estradiol synthesis, it may be an inhibitor of estradiol 

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production at high concentrations (≥10 μM). This effect could not be confirmed due to 

precipitation in the test wells after incubation in two of the three runs at concentrations of 10 and 

100 μM. 

Based on the hormone responses in the three independent runs, folpet 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 48616907) conducted to screen for potential estrogenic activity, 

folpet (97.6% a.i., batch/lot # 00138518) in 1% carboxymethylcellulose was administered daily 

via oral gavage (5 mL/kg) to groups of eight ovariectomized female Sprague Dawley rats at dose 

levels of 0 (vehicle), 313, or 1,000 (limit dose) mg/kg/day on post-natal days (PND) 56-58. A 

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

0.05 mg/kg/day by oral gavage. Body weights were determined daily. All animals were 

terminated and necropsied on PND 59 approximately 24 hours after the final dose administration 

to determine wet and blotted uterine weights. 

All animals survived until scheduled termination and no treatment-related clinical findings were 

observed in folpet treated animals. Final body weights, overall body weight gains, and uterine 

weights in the folpet treated groups were comparable to the vehicle controls. 

Comparative statistical analyses were not conducted for the final body weights and body weight 

gain of animals in the positive control (EE) group. However, final body weights were slightly 

decreased (↓6%) in the EE group compared to the vehicle controls, with a larger decrease in 

overall body weight gain (↓94%). Absolute wet and blotted uterus weights for the EE group 

were increased (p<0.05) by 77% and 66%, respectively, as expected. 

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

in the uterotrophic assay. 

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APPENDIX 2: Other Scientifically Relevant Information (OSRI) 

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

interaction of folpet with the endocrine system are summarized below. The only Part 158 studies 

and/or published literature articles discussed in this appendix are those that fulfill current 

guideline requirements or are directly relevant to assessing potential endocrine effects (i.e. two 

generation rodent reproductive studies, development studies, chronic toxicity studies, etc.). 

ER/AR Transcriptional Activation 

In a published, non-Guideline study (Kojima et al., 2004), the potential of 200 chemicals 

(including folpet; purity 95-100%) 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. 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; purity >97%) or 5α-dihydrotestosterone (DHT; purity 95%) 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. 

Folpet did not show agonist or antagonist activity for ERα and ERβ, or AR in this study. 

Two-Generation Reproduction in Rodents (Rat; OCSPP 870.3800)

In a two-generation reproduction toxicity study (MRID 00151489), groups of 30 SpragueDawley (Crl:COBS/CD) rats/sex were administered folpet (89.5% purity) in the diet at dose 

levels of 0, 200, 800, or 3600 ppm (equivalent to corrected concentrations of 0, 7.5, 34.5, and 

160 mg/kg/day) in the diet for two generations. The P rats were fed the test diets for 62 days, and 

then mated to produce the F1a offspring. The F1a pups were sacrificed at weaning (PND 21-23). 

The P rats were re-mated (different pairs) after a 2-week rest period to produce the F1b pups. 

After weaning, 30/sex/dose level F1b pups were fed the test diet for 12 weeks and then mated to 

produce the F2a pups. After weaning of pups, F1b rats were re-mated (after a 1-week rest period) 

to produce F2b litters, which were sacrificed at weaning. Pups were evaluated for abnormalities 

shortly after birth and were weighed on LD 0, 4, 7, 14, and 21. Litters were evaluated for size, 

pup sex, number of stillborn, number of live births, survival, and pup observations. All animals 

were subjected to a gross necropsy. No organ weights were recorded. Selected tissues from 

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reproductive organs of parental animals from the control and high-dose groups were examined 

histologically. Abnormal tissues from animals in all groups were examined microscopically. 

No compound-related mortalities or clinical observations were seen in the P or F1 adults. Mean 

body weights of the 3600 ppm P males and females were less (↓5%; p<0.05) than the controls at 

study initiation due to randomization procedures, and no treatment-related effects were noted on 

body weights in this group. Body weights of the 3600 ppm F1 parental males were decreased 

(p<0.05) by 9-20% throughout the study. Body weights of the 3600 ppm F1 parental females 

were decreased (p<0.05) by 18% on Day 1 of premating, but the decrease was only 5% (NS) on 

Day 176. Body weights of the P and F1 parents dosed with 200 and 800 ppm were generally 

comparable to the controls at all times. No compound-related effects were noted for P or F1

maternal body weight changes during gestation and lactation. 

The food consumption of the 3600 ppm P and F1 groups was reduced (p<0.05) frequently during 

the premating periods. Food consumption during the gestation and lactation periods of F1

females were generally comparable for all groups at all times. 

No treatment-related effects were observed in the reproductive performances (male or female 

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

fetal sex ratio) of the P males or females. However, in the F1b generation, there was a dosedependent decrease in the male fertility index to produce the F2a litter at the low (80%), mid 

(69%) and high (64%; p<0.05) dose groups when compared to controls (88%). Female fertility in 

the F1b generation was also reduced at the mid (67%) and high (66%) dose groups when 

compared to controls (79%), but the differences did not attain statistical significance. During the 

second mating of the F1b generation males and females, fertility indices in males were reduced at 

the low (69%), mid (63%) and high (65%) dose groups when compared to controls (78%). 

Fertility indices in females were also reduced at the low (67%), mid (69%) and high (63%) dose 

groups as compared to controls (71%). It was concluded that the F1 male and female fertility 

indices were adversely affected during both litter intervals. 

During lactation, the 3600 ppm pup body weights were decreased (p,0.05) by 13-19% on PND 

21 in the F1a, F1b, F2a, and F2b litters. No compound-related adverse effects were reported for 

litter size or pup survival during the lactation periods of any generation in this study. 

No treatment-related histopathological lesions were seen in the testes, epididymides, seminal 

vesicle, prostate, ovaries, uterus, thyroid or pituitary glands in the parental animals or in the 

offspring. 

Two-Generation Reproduction in Rodents (Rat; OCSPP 870.3800)

In a two-generation reproduction toxicity study (MRID 40135901), groups of 25 SpragueDawley rats/sex were administered folpet (91% purity) in the diet at dose levels of 0, 250, 1500 

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or 5000 ppm (equivalent to 0/0, 19.1/22.5, 112/134, and 370/436 mg/kg/day for P males/females, 

and 25.1/28.4, 150/168, and 520/565 mg/kg/day for F1 males/females; both during pre-mating). 

In addition to the pre-mating period, the females received the appropriate dose concentrations 

during gestation and lactation periods. Treatment began for the P parents 14 weeks prior to 

mating until the F1 litters were weaned. The F1 pups were treated at least 14 weeks during 

weaning (F1 litters), as well as during mating (F1 adult males), gestation (F2 litters) and lactation 

(weaning of the F1 litters) until the birth of the F2 litters. The F1 adult males were similarly dosed 

until the birth of the F2 litters when they were sacrificed. 

No treatment-related mortalities or clinical signs were observed in the P or F1 generations. 

During pre-mating, decreased (p<0.05) mean body weights were observed in the 5000 ppm P 

males at Weeks 1, 7, and 14 (↓8% all points) resulting in decreased overall (Weeks 0-14) body 

weight gains (↓15%). Decreased (p<0.05) mean body weights were also observed during 

pre-mating in the 5000 ppm F1 males at Weeks 0, 1, 7, and 14 (↓14-25%), resulting in decreased 

overall (Week 0-14) body weight gains (↓13%). At 5000 ppm, decreased (p<0.05) body weights 

were noted in the P females at Weeks 7 (↓6%) and 14 (↓5%) of pre-mating and in the F1 females 

at Weeks 0, 1, 7 and 14 (↓8-20%) of premating, with associated decreased overall body weight 

gains (↓10% P females; ↓5% F1). 

At 5000 ppm during gestation, body weights were decreased (p<0.05) by 4-7% on GD 0, 6 and 

20 in the P females, and by 7-10% on GD 0, 6, 14, and 20 in the F1 females. Overall gestational 

body weight gains were decreased by 11% in the P females, and by 8% in the F1 females. 

At 5000 ppm during lactation, body weights were decreased (p<0.05) by 6-7% on LD 1, 4, 7 and 

14 in the P females, and by 5-12% on LD 1, 4, 7, 14, and 21 in the F1 females. However, overall 

lactational body weight gains were greater than controls in both the P and F1 groups. 

Food consumption was decreased (p<0.05) throughout pre-mating in the 5000 ppm P and F1

males. In the 5000 ppm P females, food consumption was decreased (p<0.05) during pre-mating, 

during GD 12-20, and during LD 15-25. In the 5000 ppm F1 females, food consumption was 

decreased (p<0.05) during pre-mating, during GD 18-20, and during LD 15-21. 

No reproductive effects were observed in the P or F1 generation. In the F1 pups, mean viable pup 

weights were decreased (p<0.05) at 1500 ppm on PND 21 and 25 (↓8%) and at 5000 ppm on 

PND 7, 14, 21, and 25 (↓8-23%). In the F2 pups, mean viable pup weights were decreased 

(p<0.05) only at 5000 ppm on PND 21 (↓9%). 

No treatment-related changes were seen in absolute or relative testes or ovaries in any 

generation. No treatment-related histopathological lesions were seen in the testes, epididymides, 

seminal vesicle, prostate, ovaries, uterus or vagina in the parental animals or in the offspring. 

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There were no macroscopic findings. Microscopically, most or all of the 1500 and 5000 ppm 

males and females in both generations had slight, moderate or marked diffuse hyperkeratosis of 

the non-glandular epithelium of the stomach compared to only two control P males. Slight or 

moderate diffuse hyperkeratosis of the esophagus was reported for F1 females only (15/24 at 

1500 ppm and 24/25 at 5000 ppm vs. 0 controls). 

Developmental Toxicity (Rat; OCSPP 870.3700) 

In a developmental toxicity study (MRID 00132457), groups of 25 pregnant Sprague-Dawley 

(Crl:COBS CD (SD) BR) rats were administered folpet (89.5% purity) in aqueous Tween 80 and 

CMC by daily oral gavage at dose levels of 0, 10, 60 or 360 mg/kg/day on GD 6-19. All females 

were sacrificed on GD 20, the uterus and ovaries were exposed, and the number and location of 

live and dead fetuses, early and later resorptions, implantations; number of corpora lutea; and 

uterine weight were recorded. Fetuses were individually weighed and examined for gross 

external anomalies. All live fetuses were killed, and for each litter, approximately half of the 

fetuses were examined for soft tissue anomalies, and half of the fetuses were examined for 

skeletal defects. 

Three 360 mg/kg/day females died, one each on GDs 12, 17 and 20. Two of these females died 

due to intubation errors; the third female died from unknown causes, and was not pregnant. 

Maternal clinical signs of toxicity were noted at 360 mg/kg/day, and included rales, salivation, 

chromorrhinorhea, decreased motor activity, and soft or liquid feces. During GD 0-20, maternal 

body weight gains were decreased (p<0.05) by 15-28% at 60 and 360 mg/kg in dams corrected 

for uterine weight, and by 16% at 360 mg/kg in dams with intact gravid uteri. Maternal feed 

consumption was also decreased (p<0.05) during GD 6-13 (↓11%) and GD 13-20 (↓16%) at 360 

mg/kg. 

No compound-related postmortem maternal gross lesions were observed. 

There were no effects of treatment on % pregnant animals, number of corpora lutea, number of 

implantations, number of resorptions (early or late), mean litter size or number of live fetuses (no 

fetuses were dead), live fetal body weights/litter (males and females combined or each sex 

separately), % dead or resorbed implantations/litter, or fetal sex ratios. 

No treatment-related effects were noted during the external or visceral examinations. One 60 

mg/kg fetus was observed externally with agnathia, presenting as a shortened, fused mandible at 

skeletal examination. One 360 mg/kg/day fetus was observed externally with a thread-like tail, 

but no caudal vertebrae were ossified upon skeletal examination. One 60 mg/kg fetus presented 

with malpositioned cardiac vessels plus ectopic accessory liver lobes surrounding the stomach, 

and one 360 mg/kg fetus was observed to have a slight dilation of the brain lateral ventricles. 

These findings were not considered compound related. 

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Skeletal examinations of the 360 mg/kg/day fetuses revealed one shortened rib, maligned 

thoracic vertebrae, and nonossified caudal vertebrae were observed in one fetus., Incomplete 

ossification of one or both pubes and/or eschia was observed in in one fetus in each of three high 

dose litters compared to three fetuses in one control litter. These incidences were not significant 

but could be considered suggestive of a mild fetotoxic effect. No other compound related 

changes in fetal ossification site averages were apparent. 

Chronic Toxicity (Rat; OCSPP 870.4100)

In a chronic toxicity study (MRID 43640201), groups of 20 Fischer 344 rats/sex were 

administered folpet (91.1% purity) in the diet at dose levels of 0, 250, 1500 or 5000 ppm 

(equivalent to 0/0, 12/15, 81/100 and 291/351 mg/kg/day in males/females) for 24 months. 

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

hematology, urinalysis, clinical chemistry, organ weights (including testes, ovaries or adrenal 

glands), gross pathology or neoplastic microscopic pathology. No treatment-related 

histopathological lesions were seen in the testes, prostate, ovaries, uterus, mammary, thyroid, 

adrenal or pituitary glands at any dose level. 

Systemic toxicity was observed at 5000 ppm. Body weights were decreased (p<0.05) by 7-12% 

in the males and by 5-8% in the females during Weeks 13-104, resulting in overall (Weeks 0-

104) body weight gain decreases of 9% in the males and 13% in the females. Food consumption 

was decreased (p<0.05) at almost all intervals in the males through Week 85 and in the females 

through Week 71. Water consumption was decreased for the majority of the study by 10% in the 

males and by 20% in the females, with both sexes having a 30% decrease during Week 1. 

At 5000 ppm, slight to moderate diffuse hyperkeratosis of the esophagus was observed in 18/20 

males and 20/20 females compared to 0 controls. Slight to moderate diffuse hyperkeratosis of the 

non-glandular epithelium of the stomach was noted in 16/20 males and 20/20 females at 1500 

ppm and in 20/20 males and 19/20 females at 5000 ppm, with severity increasing with dose. 

Liver lesions, characterized as hepatic focal or areas of cellular alterations, occurred at all dose 

levels in both sexes including the controls. Significance (p<0.05) was observed in males as well 

as in combined sexes and exhibited a nonsignificant (trend) increase in females. Also, hepatic 

nodular neoformation in the liver of both sexes was observed with statistical significance 

reported in females and for the combined sexes. Additionally, the study report indicated an 

increase (p<0.05) in the incidence of males only with hyperplasia of thyroidal follicular cells at 

5000 ppm (6/20 treated vs. 4/20 controls). 

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Chronic Toxicity/Carcinogenicity (Rat; OCSPP 870.4300) 

In a combined chronic toxicity/carcinogenicity study (MRID 00151560), groups of 60 SpragueDawley rats/sex were administered folpet (89.5% purity) in the diet at dose levels of 0, 200, 800, 

or 3200 ppm (equivalent to 0/0, 9/11, 35/45, and 145/180 mg/kg/day in males/females) for up to 

104 weeks. After 52 weeks of treatment, 10 rats/sex/dose were sacrificed and examined tor gross 

and microscopic changes. Remaining animals were sacrificed at study termination (Week 105). 

No effects of treatment were observed on mortality, clinical signs, body weights, overall (Weeks 

0-104) body weight gains, food consumption, hematology, clinical chemistry, urinalysis, 

ophthalmoscopic examinations, or organ weights (including ovary and testes with epididymidis). 

At final sacrifice, “thickened" stomach (no distinction was made between tissue of the glandular 

or non-glandular stomach) was noted in 1/23, 5/37, 5/32, and 6/31 control, low, mid, and high 

dose males, respectively, and in 3/31, 0/29, 0/31, and 5/30 of the respective female treatment 

groups. Similar findings were also noted at the interim sacrifice in 1/10, 1/10, 2/10, and 3/10 

control, low, mid and high dose males and in 0/10, 2/10, 2/10, and 3/10 of the respective female 

treatment groups. A similar effect of treatment on the stomach was not apparent in animals that 

died on test. Other potentially treatment-related non-neoplastic findings included "medullary 

tubule hyperplasia” of the ovaries at all dose levels, and increased "foci of vacuolated 

hepatocytes" in the liver at all doses in the males. 

Non-neoplastic lesions were observed in the testes and thyroid glands. In the testes, interstitial 

cell hyperplasia was increased at the low (10/60; 16%) mid (11/60; 18%) and high (9/60; 15%) 

dose groups when compared to controls (1160; 1 %). In the thyroid, observed incidences of 

C-cell hyperplasia in the males were as follows: control (3/60; 5% ), low (6/60; 10%}, mid (4/60; 

7%) and high (8/60; 13%) dose groups; in the females, the incidences were: control (14/60; 

23%}, low (12/60; 20%), mid (8/60; 13%) and high (6/60; 10%) dose groups. 

Neoplastic lesions were observed in the testes and thyroid glands. In the testes, interstitial cell 

tumors were increased at the low (5/60; 8%) mid (4/60; 6%) and high (8/60; 13%) dose groups 

when compared to controls (1160; 1 %). However, the incidences were within the range of 

historical controls (2-23%; mean, 10.1 %). In the thyroid, the incidences of C-adenomas in males 

were control (0/60; 0%), low (8/60; 13%), mid (2/60; 3%) and high (2/60; 3%) dose groups. In 

females, the incidences were control (4/60; 6%), low (4/60; 6%), mid (3/60; 5%) and high (2/60; 

3%) dose groups. 

There was a dose-dependent increase in C-cell carcinomas in males; the incidences were: low 

(1/60; 2%), mid (2/60; 3%) and high (4/60; 6%) dose groups when compared to control males 

(1160; 2%). The incidences, however, were within the historical control range (0-14%); mean, 

5.8%. In females, the incidences were as follows in the low (1160; 2%}, mid (3/60; 5%) and high 

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(0/60; 0%) when compared to control females (1160; 2%). The combined incidences of 

adenomas/carcinomas were comparable between the treated and control groups. 

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

In a chronic toxicity study (MRID 00161315), groups of 6 beagle dogs/sex were administered 

folpet (89.5% purity) via gelatin capsules at 0, 10, 60 or 120 mg/kg/day for one year. 

There were no treatment-related effects on mortality, clinical signs, ophthalmologic 

examinations, hematology or urinalysis. 

In the males, body weights were decreased by 7-18% during Weeks 7-52 at 60 mg/kg/day, and 

by 8-24% during Weeks 3-52 at 120 mg/kg/day. Overall (Weeks 0-52) body weight gains were 

decreased by 50% at 60 mg/kg/day and by 62% at 120 mg/kg/day. In the females, body weights 

were decreased by 7-10% during Weeks 3-52 at 60 mg/kg/day, and by 7-11% during Weeks 3-52 

at 120 mg/kg/day. Overall (Weeks 0-52) body weight gains were decreased by 35% at 60 

mg/kg/day and by 42% at 120 mg/kg/day. 

Food consumption was decreased in a dose-related manner in the 60 and 120 mg/kg/day males 

for the first 13 weeks of the study. This decrease was significant (p <0.05) during Weeks 1-3 and 

at five weekly intervals between Weeks 5 and 13 for the 120 mg/kg/day males, and during 

Weeks 1, 2, and 13 for the 60 mg/kg/day males. Food consumption was decreased in a similar 

manner in 60 and 120 mg/kg/day females during Weeks l and 2 and at four to five weekly 

intervals between weeks 9 and 16. 

Mean cholesterol, total protein, albumin, and globulin were decreased in the 60 and 120 

mg/kg/day males and in the 120 mg/kg/day females at the 2-, 3-, 6-, 9-, and 12-month intervals; 

the values of cholesterol and total protein were also found to be slightly decreased in high-dose 

females at the end of the first month. These decreases were found to be significant (p<0.05) in 

males at 3, 6, 9, and 12 months for total protein, albumin, and globulin. Albumin was 

significantly decreased in high-dose females at 2 months and cholesterol was significantly 

decreased at 2 and 3 months. The decrease in total protein was significant at 2, 3, and 12 months. 

No treatment-related changes were seen in absolute or relative weights of the testes, ovaries, 

thyroid, adrenal or pituitary glands at any dose level. No treatment-related histopathological 

lesions were seen in the testes, epididymides, prostate, ovaries, uterus, mammary, thyroid, 

adrenal or pituitary glands at any dose level. Slight decreases (p <0.05) were found in the 

absolute left adrenal and brain weights of the 120 mg/kg/day males. Left adrenal weights in 

control males were found to be unusually high when compared to the contralateral organ weights 

of these same animals; therefore, the decreased adrenal weights of high-dose males were not 

considered treatment-related. When left and right adrenal weights were combined and adrenal-tobrain weight ratios were calculated, the same decreased trend was found in the adrenal weights 

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of high-dose males when compared to the unusually high control weights. Adrenal and brain 

weights in females were comparable to controls. Kidney and liver weights in high-dose males 

and kidney weights in low- and high-dose females were also found to be slightly decreased (NS). 

These decreases were reported to be consistent with reduced body weights found in the highdose group. Histopathological examination of the organs did not reveal any alterations that 

would correlate with these organ weight changes. 

Subchronic Oral Toxicity in Rodents (OCSPP 870.3100) 

In a subchronic oral toxicity study (MRID 001152692), groups of Sprague-Dawley rats (# of 

rats/sex not provided) were administered folpet (purity not provided) in the diet at dose levels of 

0, 300, 1000, 3000, and 10,000 ppm (equivalent to 0, 15, 50, 150 and 500 mg/kg/day) for 13 

weeks. 

There were no effects of treatment on mortality or food consumption. 

Growth rates and body weights were retarded at 10000 ppm in males and females, and only in 

females after two weeks of recovery with no further folpet feeding. Total protein was decreased 

in the 10,000 ppm males at Weeks 6 and 13, and in the 10,000 ppm females at Week 13. Both 

albumin and globulin were decreased in the 10,000 ppm group at Week 13. LDH was increased 

in the 3000 and 10000 ppm males. 

Relative brain weights decreased in the 10000 ppm group whereas kidney weights increased; 

gross pathology showed no treatment-related effects. No treatment-related changes were seen in 

absolute or relative weights of any other organs (including the testes, epididymides or ovaries). 

Only the stomach showed remarkable histological effects. Both the 10,000 ppm males and 

females showed folpet-related effects in the non-glandular (upper) portions of the stomach such 

as acanthosis, hyperkeratosis, submucosal edema, and pleocellular infiltrate (inflammatory 

reaction), and an occasional instance of stomach focal erosion and ulceration. These effects are 

likely due to irritation to the stomach by folpet. No treatment-related histopathological lesions 

were seen in the testes, epididymides, prostate, ovaries, uterus, thyroid, adrenal or pituitary 

glands. 

Freshwater Fish Early Life Stage Study (OPPTS 850.1400) 

In a freshwater fish early life-stage toxicity test (MRID 43786301, 1995), Fathead minnow 

(Pimephales promelas) embryos and larvae were exposed to folpet (93.2% purity) at nominal 

concentrations of water (negative) control, solvent control, 23, 45, 90, 180, and 360 μg/L (meanmeasured concentrations of negative control, solvent control, 4.1, 8.8, 17.7, 34.9, and 65.3 μg 

a.i./L in a flow-through system for 28 days. Observations were made on embryo hatching (%), 

and larval survival and growth (length and weight). Data were analyzed by analysis of variance 

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(ANOVA), parameters examined were percentage of hatch and the survival, length, and weight 

of larvae after 28 days of exposure. If treatment effects were indicated, the means of the test 

parameters were compared to those of the controls using Dunnett’s procedure. 

There was 100% mortality in the two highest treatment groups (mean measured concentrations 

of 34.9 and 65.3 μg a.i/L and therefore growth endpoints were not analyzed for these 

concentrations. Percent hatch was also significantly (p<0.05) reduced from the negative control 

at these concentrations. Mean wet weight was significantly (p<0.05) reduced at the middle 

treatment concentration (mean measured 17.7 μg a.i/L. The NOAEC for fathead minnow 

exposed to folpet was 8.8 μg a.i/L, based on wet weight. 

Avian Reproduction Toxicity (OCSPP 850.2300) 

In an avian reproduction toxicity study (MRID 00098004), groups of male and female Bobwhite 

quail (age not reported) were administered folpet (90.4% purity) in corn oil in the diet at dose 

levels of 0, 100, 300, and 1000 ppm in diet. Quail were randomly assigned to treatment groups 

with one quail/sex/pen, 12 pens/group. 

No mortalities occurred in the study. Lacerated scalps appeared in one cock and one hen during 

weeks 8 and 9; lethargy was associated with these observations (the test group of these animals 

was not identified). One hen at 300 ppm had some loss of coordination during week 17, but 

recovered after one day, and at 1000 ppm one cock with lacerated scalp exhibited depression, 

reduced reaction to stimuli, and had a ruffled appearance, but subsequently recovered and was 

asymptomatic. 

Slight reductions (p<0.05) in adult body weights occurred at 100 ppm during Weeks 2, 4, and 6, 

and at 1000 ppm during Week 2; no effects on body weight were seen in the 300 ppm group. 

Additionally, average body weights of normal hatchlings of all parents receiving treated diets 

were slightly (p<0.05) reduced, as compared to controls. There were no effects, at any level, on 

the eggs laid, eggs cracked, viable embryos, live 3-week embryos, normal hatchlings on 14-day 

old survivors. No statistically significant effect were observed on body weights of 14-day old 

survivors, egg weights, or egg shell thickness. 

In another avian reproduction toxicity study (MRID 00098005), groups of male and female 

Mallard ducks (age not reported) were administered folpet (90.4% purity) in corn oil in the diet 

at 0, 100, 300, and 1000 ppm for 10 weeks. Ducks were randomly assigned to treatment groups 

with one duck/sex/pen, 12 pens/group. 

No mortalities occurred in the study. In the control group, one drake had a lame leg for four days 

during Week 8, and in the treatment groups: at 100 ppm one hen was observed prone with legs 

extended behind her for one day during Week 15; at 300 ppm one hen was observed prone with 

legs extended behind her for one day during Week 13, and another hen exhibited the same 

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symptoms for seven days during Weeks 14 and 15; and at 1000 ppm one hen was observed with 

lower limb rigidity for one day during Week 13 and continued to exhibit loss of coordination 

through Week 14. Lacerated scalps appeared in one cock and one hen during Weeks 8 and 9; 

lethargy was associated with these observations (the test group of these animals was not 

identified). One hen at 300 ppm had some loss of coordination during Week 17, but recovered 

after one day, and at 1000 ppm one cock with lacerated scalp exhibited depression, reduced 

reaction to stimuli, and had a ruffled appearance, but subsequently recovered and was 

asymptomatic. 

No statistically significant effects on adult body weights were observed at any level. Slight, 

though statistically significant (p<0.05) reduction in food consumption occurred at all levels 

tested, however, only during certain intervals. 

There was a slight, though statistically significant (p<0.01) reduction in average body weights of 

normal hatchlings of parents receiving 100 ppm folpet in their diets. However, this reduction was 

not observed for controls, or for birds receiving 300 and 1000 ppm folpet in diets. 

No effect, at any level was observed for the following reproductive parameters: eggs laid, eggs 

cracked, viable embryos, live 3-week embryos, normal hatchlings, or 14-day old survivors. 

Additionally there were no significant effects on body weights of 14-day old survivors, egg 

weights, or eggshell thickness. 

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APPENDIX 3: References Not Utilized in the Folpet 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 Folpet but were not utilized due to one or more of the following reasons: 1) the 

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

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

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

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

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

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

results; 10) the 40 CFR Part 158 guideline study was classified as unacceptable/inadequate; 11) 

the study dealt only with non-EDSP assay development; 12) no specific endocrine-related 

endpoints were assessed in the study; and 13) the study contained only data on invertebrates. 

Burgess, P. (1989). Chronic toxicity of Folpet to Daphnia magna under flow-through conditions. 

Project ID 37035. Analytical Biochemistry Laboratories. (MRID 42122013) 

Calandra, J.C. (1961). Chronic oral toxicity of Phaltan in albino rats. Industrial Biotest 

Laboratories for California Chemical Company. (MRID 00064323) 

Calandra, J.C. (1967). Three-generation reproduction study in albino rats - Phaltan. IBT No. 

B3566. Industrial Biotest Laboratories for Chevron Chemical Company. (MRID 00065143) 

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Cohle, P.J. (1989). Early life stage toxicity of Folpet technical to Fathead minnows (Pimephales 

promeles) in a flow-through system. Project ID ABC 37034. Analytical Bio-chemistry 

Laboratories. (MRID 42122012) 

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