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 70
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 70
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: ESFENVALERATE 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

Page 3 of 70
ESFENVALERATE FINAL 

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..................................................13 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................18 

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

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

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

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

B. Estrogen Pathway...........................................................................25 

C. Androgen Pathway.........................................................................25 

D. Thyroid Pathway...........................................................................25 

E. Conclusions.................................................................................26 

V. EDSP Tier 2 Testing Recommendations....................................................26 

VI. References.......................................................................................27 

LIST OF TABLES: 

 Table 1. Tier 1 Assays for Esfenvalerate .............................................................7 

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

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

 Table 4. Thyroid Pathway for Esfenvalerate ......................................................19 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................31 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................40 

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

Page 4 of 70
ESFENVALERATE FINAL 

iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

Page 5 of 70
ESFENVALERATE FINAL 

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 

Page 6 of 70
ESFENVALERATE FINAL 

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) 

Page 7 of 70
ESFENVALERATE FINAL 

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

Page 8 of 70
ESFENVALERATE FINAL 

Page 1 of 62 

Executive Summary 

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

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

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

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

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

addition to the available Tier 1 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 esfenvalerate 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 July 2, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of 

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

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

esfenvalerate 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).

Esfenvalerate (enantiomer of fenvalerate) is a member of the synthetic pyrethroid class of 

insecticide having similar toxicity profiles that elicit neurotoxic behaviors in laboratory animals. 

This assessment includes studies conducted on esfenvalerate and/or fenvalerate. 

For the estrogen pathway, although Kojima et al., reported a positive response for fenvalerate in 

an ERTA conducted in CHO cells, Saito et al. reported that fenvalerate was negative for ER 

binding/ER activation in three different ER assay systems: a mammalian ER transactivation 

assay (ERTA) with HeLa cells, a yeast two-hybrid system, and a hER competitive ligandbinding (Saito et al. 2000). Additionally, esfenvalerate was negative in the Tier 1 ERTA, 

aromatase and steroidogenesis assays. The in vivo uterotrophic and female pubertal assays were 

negative, and no estrogen related effects were seen in the mammalian or avian Part 158 studies. 

While there was a 6% increase in fertilization success was reported in the FSTRA at the high 

dose there were no other treatment related effects in females. In the Part 158 studies with 

esfenvalerate, the only effect observed was a slight decrease in hatchling weights. Therefore 

there is no convincing evidence for a potential interaction with the estrogen pathway. 

Page 9 of 70
ESFENVALERATE FINAL 

Page 2 of 62 

For the androgen pathway, a generally consistent pattern of weak anti-androgenicity was 

observed for esfenvalerate/ fenvalerate in the in vitro Tier 1 and in vitro OSRI assays. However, 

esfenvalerate was negative in the in vivo Tier 1 Hershberger and male pubertal assays, and no 

androgen-related effects were seen in the mammalian or avian Part 158 toxicity studies. As 

reported above, the only effect observed in the FSTRA was an increase in fertility. In the Part 

158 studies with esfenvalerate, the only effect observed was a slight decrease in hatchling 

weights. Overall, there is no convincing evidence for an interaction with the androgen pathway. 

For the thyroid pathway, the available Tier 1 EDSP assays and OSRI for esfenvalerate do not 

indicate a potential for thyroid interaction in mammals. Although an increase in T3 was reported 

by Giray et al. (2010), thyroid weight, and concentrations of plasma TSH and total T4 were 

similar to the control animals. No thyroid related effects were observed in the Tier 1 male and 

female pubertal assays or in the Part 158 mammalian studies. However, an adequate amphibian 

metamorphosis assay (AMA) was not available for consideration in the weight-of-evidence, and 

given the observed high variability and apparent lack of concentration stability in the available 

study, esfenvalerate may not be amenable to testing in the current (AMA design (i.e., water 

column exposure). Therefore, the weight-of-evidence cannot conclude whether esfenvalerate has 

the potential for thyroid interaction in non-mammalian species. 

Based on weight of evidence conclusions, mammalian or wildlife EDSP Tier 2 testing is NOT 

recommended for esfenvalerate. 

A conclusion regarding potential interaction with the thyroid pathway in non-mammalian species 

could not be determined, at this time, and it is noted that there is a lack of a validated dietary 

exposure test design as well as the fact that the nature of esfenvalerate may make it difficult to 

test long-term in aquatic systems with amphibians. Except for an increase in fertility, no other 

treatment-related effects were observed in the FSTRA up to 0.23 μg a.i./L; it is noted that 

measured test concentrations were low and variable in this study. A fathead minnow life-cycle 

study was submitted to the agency (fish are used as surrogates for aquatic-phase amphibians), 

and based on a preliminary review, high variability in measured test concentrations within a 

treatment was observed. In this study, the most sensitive endpoints measured were decreased 

survival and fecundity in the parental generation and decreased survival in the second generation 

(MRID 49289201). The overall LOAEC for this study, based on survival in the second 

generation was 0.034 μg a.i./L, and the NOAEC was 0.017 μg a.i./L. The nominal test 

concentrations in the esfenvalerate AMA were 0.006, 0.02, and 0.05 μg/L which are similar to 

the concentrations tested in the fish life-cycle study. Given the lack of a validated dietary 

exposure test design with amphibians along with the observed difficulties in maintaining aquatic 

exposure from the available data, relevant to EDSP, additional amphibian testing is not 

recommended at this time. 

Page 10 of 70
ESFENVALERATE FINAL 

Page 3 of 62 

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 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 esfenvalerate 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 July 2, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of 

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

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

esfenvalerate 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).

This assessment includes studies conducted on esfenvalerate and/or fenvalerate. Esfenvalerate 

and fenvalerate are related synthetic Type II pyrethroid broad-spectrum acaricides/insecticides. 

Fenvalerate is a racemic mixture of four stereoisomers (Aα or S,S; Bα or R,S; Aβ or S,R; and Bβ

or R,R isomers) with equivalent abundances of approximately 23% each. Esfenvalerate is the 

S,S-enriched material (with an abundance of >75%). Esfenvalerate has a vapor pressure of 1.5 × 

10−9

 mm Hg at 25°C; esfenvalerate will exist as a particulate if released to the atmosphere. 

Esfenvalerate has a water solubility of <20 μg/L, and has an octanol/water partition coefficient 

(log Kow) of 6.2 to 6.5. Esfenvalerate is stable, and not subject to photolytic and hydrolytic 

degradation. Volatilization from water and soil is not expected to contribute to the dissipation of 

esfenvalerate in the environment. The estimated bioconcentration factor for esfenvalerate is 

12,000. 

The available information considered in determining the potential interaction of esfenvalerate 

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 

Page 11 of 70
ESFENVALERATE FINAL 

Page 4 of 62 

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. 

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 esfenvalerate (USEPA, 2010). Since then, 

the Agency has also conducted a more recent search of available scientific literature for any 

additional relevant information. Summaries of the available OSRI are presented in Appendix 2. 

Additionally, literature/studies considered but not utilized for the WoE analysis are listed in 

Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

above. 

Page 12 of 70
ESFENVALERATE FINAL 

Page 5 of 62 

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

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

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

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

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

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

effects on the HPG and HPT axes. OSRI may include published literature studies as well as 

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

submitted in support of pesticide registrations. Each study is evaluated for scientific quality and 

relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency 

of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple 

endpoints are measured in each assay. 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

the whole organism level. The extent of expression of 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 conditions did 

responses occur including consideration of whether the responses were observed in the presence 

of overt or systemic toxicity? The presence of overt and/or systemic toxicity introduces 

Page 13 of 70
ESFENVALERATE FINAL 

Page 6 of 62 

uncertainty in the ability to distinguish effects specifically related to an endocrine-related effect 

from a non-endocrine toxic response. 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

forms of toxicity (e.g., 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 discussed 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. 

Page 14 of 70
ESFENVALERATE FINAL 

Page 7 of 62 

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

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

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

which supports a conclusion of whether 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 and/or other scientifically relevant information (OSRI) submitted to satisfy the 

agency’s test order for esfenvalerate are shown below in Table 1. Executive Summaries are 

presented in Appendix 1. 

Table 1. Tier 1 Screening Assay for Esfenvalerate

Tier 1 Assays Test Guideline Test Order Status 

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

(Saito et al. 2001)

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement satisfied 

 (MRID 48670801) 

AR Binding Assay (Rat prostate 

cytosol) OCSPP 890.1150 Requirement satisfied by OSRI (Sumitomo et al., 2001) 

Steroidogenesis Assay (Human cell line 

H295R) 

OCSPP 890.1550; 

OECD 456 

Requirement satisfied 

(MRID 48670802) 

Aromatase Assay (human recombinant 

microsomes) OCSPP 890.1200 Requirement satisfied  (MRID 48616502) 

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement satisfied by OSRI 

(Kunimatsu et al., 2002) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement satisfied 

(MRID 48616501) 

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement satisfied 

(MRID 48683803) 

Pubertal Male Assay (Rat) OCSPP 890.1500 

Requirement satisfied by OSRI 

(Moniz et al., 1999; Zhang et al., 2009; 

and Kaul et al.,1996); however, a Tier 1 

assay was conducted (MRID 48683802) 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement satisfied 

(MRID 48995501) 

Amphibian Metamorphosis Assay 

(Frog) 

OCSPP 890.1100; 

OECD 231 

Requirement NOT satisfied (MRID 

49024501)1

1 This is based on an apparent lack of concentration stability in the available study, however, it is noted 

that esfenvalerate may not be amenable to testing in the current 890.1100 AMA study design (i.e., water 

column exposure) and another AMA study utilizing water column exposure is not recommended. 

Page 15 of 70
ESFENVALERATE FINAL 

Page 8 of 62 

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

1. Effects on Estrogen Pathway 

The potential for esfenvalerate 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. 

Esfenvalerate was negative in the Tier 1 ERTA, aromatase and steroidogenesis assays. The 

requirement for an ER binding study was satisfied by Saito et al. (2000) which showed that 

fenvalerate was negative in three different ER assay systems: a mammalian ERTA assay with 

HeLa cells, a yeast two-hybrid system and an hER competitive ligand-binding assay. In a 

literature study by Kojima et al. (2004), fenvalerate induced a positive response in an ERTA in 

Chinese hamster ovary (CHO) cells, with a 20% relative effective concentration (REC20) of 3.7 x 

10-6 M.

Esfenvalerate was negative in the in vivo uterotrophic assay (satisfied by Kunimatsu et al., 2002) 

and in the Tier 1 female pubertal rat assay no estrogen-related effects were observed. In the 

FSTRA, there were no treatment-related effects except for a significant increase of 6% in 

fertilization success in the high-dose group (0.23 μg a.i./L). 

There were no estrogen-related effects in the esfenvalerate or fenvalerate mammalian Part 158 

studies. In a Part 158 avian reproduction study with esfenvalerate, bobwhite quail hatchling 

weights were slightly, but significantly, decreased in the medium- and high-dose (504 and 10 

mg/kg-diet; 5 and 6 % reductions, respectively) groups compared to control, but no differences 

were observed in hatchling survival or 14-day offspring body weights. No other statistically 

significant treatment-related effects were noted. In the mallard duck reproduction study with 

esfenvalerate, there were no reproductive effects observed up to 1190 mg/kg-diet. Several avian 

reproduction studies were conducted with bobwhite quail and mallard duck for fenvalerate. In 

these studies, various study deficiencies (i.e., test levels below estimated environmental 

concentrations) were identified, however, these available supplemental studies indicate that an 

increase in cracked eggs may occur with exposure to fenvalerate. 

Page 16 of 70
ESFENVALERATE FINAL 

Page 9 of 62 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Esfenvalerate

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Esfenvalerate1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopat

hology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin 

EDSP Tier 1 Assay 

ER Binding Requirement satisfied by OSRI (Saito et al., 2000; see below) ERTA (MRID 48670801) N Aromatase (MRID 48616502) N Steroidogenesis (MRID 48670802) N Uterotrophic Requirement satisfied by OSRI (Sumitomo et al., 2001b and Kunimatsu et al., 2002; see bFemale Pubertal Rat (MRID 48683803) N N N N N N FSTRA (MRID 48995501) NE N N N Frt: ↑6% (H); N 

OSRI 

ER Binding/ERTA (Saito et al., 2000) N N ERTA (Kojima et al. ,2004) P Uterotrophic (Kunimatsu et al., 2002) 

N 
ESFENVALERATE FINAL 

Page 10 of 62 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Esfenvalerate

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Esfenvalerate1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopat

hology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin 

Subchronic toxicity (Rat; MRID 40215601) 

N N NE NE 

Subchronic toxicity (Mouse; MRID 41359701) 

NE N N N 

Subchronic dermal toxicity (Rat; MRID 45275401) 

N N N NE 

Subchronic dermal toxicity (Rabbit; MRID 42325101) 

NE NE N NE 

Developmental toxicity (Rat; MRID 43211504) 

NE N 

Developmental toxicity (Rabbit; MRID 43211503) 

NE N 

Two-generation reproduction (Rat; MRID 43489001) 

 NE NE NE NE N 

One-generation reproduction (Rat; MRID 48076104) 

 NE NE NE NE N 

Chronic toxicity/ carcinogenicity (Rat; MRIDs 00082244 and 00111888) 

NE NE N NE 
ESFENVALERATE FINAL 

Page 11 of 62 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Esfenvalerate

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Esfenvalerate1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopat

hology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin 

Chronic toxicity/ carcinogenicity (Rat; MRID 00079877) 

NE NE N NE 

Carcinogenicity (Mouse; MRID 00079876) 

NE NE N NE 

Carcinogenicity (Mouse; MRID 44260601) 

NE NE N NE 

Chronic toxicity (Dog; MRID 00163855) 

NE N N NE 

Avian reproduction (Quail; MRID 48027802) 

P6 

Avian reproduction (Duck; MRID 49008802) 

N 

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

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

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 OS

this column. For details see Section IV. A 

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

details see Section IV. A 

4. Clinical signs included tremors in all high-dose females (PND 22-36) and splayed hindlimbs or impairment of hindlimbs in high-d

5. Clinical signs (i.e., tremors) were observed in the high-dose fenvalerate (80 mg/kg/day) and esfenvalerate (20 mg/kg/day) females. 
ESFENVALERATE FINAL 

Page 12 of 62 

6. Hatchling weights were slightly decreased in the medium- and high-dose (504 and 10 mg/kg-diet; 5 and 6 % reductions, respective

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

Page 13 of 62 

2. Effects on Androgen Pathway 

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

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

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

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

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

table and not considered further in the WOE assessment. 

The Agency determined that the requirement for an AR binding study was satisfied based on the 

results of an AR transactivation (ARTA) study (Sumitomo, 2001), and that the requirement for a 

male pubertal assay was satisfied based on the results of three studies (Moniz et al., 1999; 

Zhang et al., 2009; and Kaul et al., 1996). However, the Test Order Recipient elected to conduct 

the male pubertal rat assay despite this decision. The results of additional OSRI that examined 

the effects of esfenvalerate in AR binding and ARTA assays were also evaluated in the 

determination of the androgenic potential of esfenvalerate/fenvalerate. 

Esfenvalerate was negative in the Tier 1 in vitro steroidogenesis assay. Fenvalerate 

demonstrated mixed responses in the OSRI in vitro AR binding/ARTA assays with negative or 

weak binding responses (androgenic/anti-androgenic). Fenvalerate was negative for agonist 

activity in two independent hAR reporter gene assays using HeLa (Sumitomo et al., 2001) and 

CV-1 (Xu et al., 2006) cell lines. However in the assay by Xu et al., (2006), antagonist activity 

was reported at the highest test concentration (10−5

 M). Kojima et al., (2004) also reported 

positive antagonist activity for fenvalerate in an ARTA in Chinese hamster Ovary (CHO) cells. 

Sumitomo et al. (2001) reported negative findings in a yeast two-hybrid assay evaluating 

potential interaction with the hAR ligand binding domain (LBD) and in a whole cell AR binding 

assay using MDA-MB-53 cells. In contrast, Eil and Nisula (1990) reported competitive to the 

AR of human skin fibroblast at a concentration of approximately 10−4

 M fenvalerate. 

The Tier 1 in vivo Hershberger assay was negative and no androgen-related effects were 

observed in the Tier 1 Male Pubertal assay. In the FSTRA, there were no esfenvalerate 

treatment-related effects in males. 

 

No androgen-related effects were observed in the mammalian Part 158 toxicity studies. As noted 

in the estrogen pathway section, slight, but significant reductions in hatchling weights were 

observed in the Part 158 bobwhite quail reproduction study; no reproductive effects were 

observed in the mallard duck study, but male adult body weight gains were significantly reduced 

at the highest treatment group. 

Page 21 of 70
ESFENVALERATE FINAL 

Page 14 of 62 

Table 3. Androgenic/Anti-Androgenic Pathway for Esfenvalerate

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Esfenvalerate1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Ad Wih

EDSP Tier 1 Assay 

AR Binding Requirement satisfied by OSRI (Sumitomo et al., 2001; see below) Steroidogenesis (MRID 48670802) N Hershberger (MRID 48616501) NE N Male Pubertal Rat (MRID 48683802) N N N N N N N FSTRA (MRID 48995501) NE N N N 

Frt: ↑6% 

(H); 

 Fcd: N 

OSRI 

AR Binding/ARTA (Sumitomo et al., 2001) 

N N 

ARTA (Xu et al., 2006) P ARTA (Kojima et al., 2004) P AR Binding (Eil and Nisula, 1990) P Subchronic toxicity (Rat; MRID 40215601) 

N N NE NE NE NE 
ESFENVALERATE FINAL 

Page 15 of 62 

Table 3. Androgenic/Anti-Androgenic Pathway for Esfenvalerate

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Esfenvalerate1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Ad Wih

Subchronic toxicity (Mouse; MRID 41359701) 

N N NE N N NE 

Subchronic dermal toxicity (Rat; MRID 45275401) 

N N N N NE NE 

Subchronic dermal toxicity (Rabbit; MRID 42325101) 

N N NE N NE NE 

Two-generation reproduction (Rat; MRID 43489001) 

NE NE NE NE NE NE N 

One-generation reproduction (Rat; MRID 48076104) 

NE NE NE NE NE NE N 

Chronic toxicity/ carcinogenicity (Rat; MRIDs 00082244 and 00111888) 

N N NE NE NE NE 

Chronic toxicity/ carcinogenicity (Rat; MRID 00079877) 

N N NE NE NE NE 

Carcinogenicity (Mouse; MRID 00079876) 

 NE N NE N NE NE 
ESFENVALERATE FINAL 

Page 16 of 62 

Table 3. Androgenic/Anti-Androgenic Pathway for Esfenvalerate

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Esfenvalerate1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Ad Wih

Carcinogenicity (Mouse; MRID 44260601) 

N N NE N NE NE 

Chronic toxicity (Dog; MRID 00163855) 

N N NE N NE NE 

Avian reproduction (Quail; MRID 48027802) 

P6 

Avian reproduction (Duck; MRID 49008802) 

N 

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

(↓ or ↑) indicate the direction of the response. A shaded cell indicates that this parameter is not routinely evaluated or is not applica

in weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral proprostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). 

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

see Section IV. A 

4. Clinical signs included abnormal gait, ataxia, fast breathing, hyper-reactivity, paw shaking and licking, abnormal grooming, whole-b

altered posture, decreased muscle tone, and/or tremors in the medium- and high-dose groups.

5. Clinical signs included tremors, impaired use of left forelimb, impaired use of hind limb, and splayed hindlimb in high-dose animals 6. Hatchling weights were slightly decreased in the medium- and high-dose (504 and 10 mg/kg-diet; 5 and 6 % reductions, respectivelP Positive findings (in vitro)/No effect (in vivo) N Negative findings (in vitro)/No effect (in vivo) NE Not examined 
ESFENVALERATE FINAL 

Page 17 of 62 

NR Not reported 
ESFENVALERATE FINAL 

Page 18 of 62 

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

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

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

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

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

The potential for esfenvalerate to interact with thyroid regulation are summarized in Table 4. 

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

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

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. 

Changes in thyroid stimulating hormone (TSH) levels were seen at the high dose in the pubertals 

assays (↓31% TSH in females, ↑14% TSH in males); however, the changes were not statistically 

significant and occurred in the presence of overt toxicity (clinical signs, tremors). No changes in 

thyroid weight, histopathology or serum thyroxine (T4) concentration were observed in the male 

or female pubertal assays. The AMA conducted with esfenvalerate is considered unacceptable 

given the extremely high coefficient of variances (CVs >100%) for the measured esfenvalerate 

concentrations which resulted in overlapping measurements in all three treatment concentrations 

making the low, mid and high test groups indistinguishable in terms of exposure. Detections of 

the test substance in some of the negative and solvent control samples further compromise the 

test results. Therefore, this assay was not used to determine the potential for effects on the HPT 

axis. 

No thyroid effects were noted in any of the Part 158 mammalian studies. Increases in T4

concentrations were reported in a study by Kaul et al., 1996; however, both doses (100 and 200 

mg/kg/day) were considered to be overtly toxic (tremors, paralysis). Giray et al. (2010) reported 

an increase in total T3 concentration (approximately two-fold based on results presented 

graphically) in rats treated with 100 mg/kg/day fenvalerate (seven week study), but thyroid 

weight, concentrations of plasma TSH and total T4 were similar to the control animals. 

Page 26 of 70
ESFENVALERATE FINAL 

Page 19 of 62 

Table 4. Thyroid Pathway for Esfenvalerate

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Esfenvalerate1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH Levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, BL, SVL) Systemic Toxicity Observed 2

Overt Toxicity Observed 3 

EDSP Tier 1 Assay 

Female Pubertal Rat 

(MRID 48683803) N N N N N N X 

 (H) 4

Male Pubertal Rat 

(MRID 48683802 ) N N N N N N X 

(H) 5

AMA (Frog; MRID 

49024501) Unacceptable; does not satisfy requirement 

OSRI 

Thyroid Hormones 

(Rat; Giray et al., 

2010) 

N NE P6 N NE NR NR 

Subchronic toxicity 

(Rat; MRID 

40215601) 

NE NE NE X 

(H) N 

Subchronic toxicity 

(Mouse; MRID 

41359701) 

N N N X 

(H) N 

Subchronic dermal 

toxicity (Rat; MRID 

45275401) 

NE N NE 

X 

(ML, 

MH, H) 

N 

Subchronic dermal 

toxicity (Rabbit; 

MRID 42325101) 

NE N NE N N 

Two-generation 

reproduction (Rat; 

MRID 43489001) 

NE NE NE X 

(H) 

X 

(H) 

One-generation 

reproduction (Rat; 

MRID 48076104) 

NE NE NE N N 

Chronic toxicity/ 

carcinogenicity (Rat; 

MRIDs 00082244 and 

00111888) 

NE N NE N N 

Chronic toxicity/ 

carcinogenicity (Rat; 

MRID 00079877) 

NE N NE X 

(H) 

X 

(H) 

Carcinogenicity 

(Mouse; MRID 

00079876) 

NE N NE X 

(H) 

X 

(H) 

Page 27 of 70
ESFENVALERATE FINAL 

Page 20 of 62 

Table 4. Thyroid Pathway for Esfenvalerate

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Esfenvalerate1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH Levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, BL, SVL) Systemic Toxicity Observed 2

Overt Toxicity Observed 3 

Carcinogenicity 

(Mouse; MRID 

44260601) 

NE N NE X 

(M, H) 

X 

(H) 

Chronic toxicity (Dog; 

MRID 00163855) N N NE N N 

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

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

that this 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. Clinical signs included tremors in all high-dose females (PND 22-36) and splayed hindlimbs or impairment of 

hindlimb in high-dose females (n=4) on PND 24. 

5. Clinical signs included tremors, impaired use of left forelimb, impaired use of hindlimbs, and splayed hindlimbs 

in high-dose animals. 

6. Total T3 levels were increased (approximately 2-fold) at 100 mg/kg/day; however, T4 levels were similar to the 

controls. 

P Positive findings 

N Negative findings 

NE Not examined 

Page 28 of 70
ESFENVALERATE FINAL 

Page 21 of 62 

IV. Committee’s Assessment of Weight of Evidence 

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

potential of esfenvalerate 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 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 Tier 1 assays stated that “In 

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

disruption in the presence of overt toxicity. All effects in endocrine sensitive tissues should be 

evaluated in terms of primary interactions with the endocrine system vs. secondary effects 

related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 

2013). 

A. Systemic/Overt Toxicity in the 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) is sufficient for a dose/concentration to be considered overtly toxic. 

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

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

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

Page 29 of 70
ESFENVALERATE FINAL 

Page 22 of 62 

1. Tier 1 in vivo Assays 

Systemic or overt toxicity observed for esfenvalerate in the Tier 1 in vivo studies was typical of 

pyrethroid compounds, consisting generally of neurological effects. There were no consistent or 

significant effects noted on body weights and/or body weight gain in any in vivo Tier 1 studies. 

Clinical signs observed in the Hershberger assay (medium- and high-dose animals, 6 and 9 

mg/kg/day) included abnormal gait, ataxia, fast breathing, hyper-reactivity, paw shaking and 

licking, abnormal grooming, whole-body fasciculations, altered posture, decreased muscle tone 

and/or tremors. These effects were considered toxicologically relevant. In the female pubertal 

rat study, tremors were observed in all high-dose females (9 mg/kg/day) after dosing on PND 22-

36. On PND 24, four of the high-dose females were also observed with hindlimbs splayed or 

impairment of hindlimbs. In the male pubertal rat study, tremors were observed generally at ≥3 

hours after dosing in 12/15 high-dose animals (9 mg/kg/day) on PND 23-31. During the daily 

examinations, only two high-dose animals were observed with tremors. Impaired use of left 

forelimb (1 male), impaired use of hindlimbs (3 males), and splayed hindlimbs (4 males) were 

also observed at ≥3 hours after dosing on PND 24-42 in the high-dose group. Clear or red 

material around mouth was noted in both low- and high-dose animals during the treatment 

period. Finally, no treatment-related mortalities or clinical signs were present in the FSTRA. 

2. OSRI 

Systemic or overt toxicity in the OSRI in vivo studies also consisted primarily of neurological 

effects. In the uterotrophic assay (Kunimatsu et al., 2002), the highest doses of fenvalerate and 

esfenvalerate each exhibited typical clinical signs of pyrethroid exposure such as tremors; the 

daily incidence of tremor was 4-6/6 rats in the 80 mg/kg/day fenvalerate group and 2-4/6 rats in 

the 20 mg/kg/day esfenvalerate group. 

There was no mortality during a subchronic oral toxicity study in rats with esfenvalerate. 

Neurological signs were noted after Week 1, and hyperactivity was observed in females only 

between Weeks 10 and 13. During Weeks 11-13, jerky leg movements were observed in some 

high-dose (15 and 17.2 mg/kg/day) male and female rats. Males and females in the high-dose 

group had decreased mean body weight gains compared to control throughout the study. 

Numerous clinical signs were observed in the high-dose [106/113 mg/kg/day (M/F)] for both 

esfenvalerate and fenvalerate [(422/462 mg/kg/day (M/F)] in a subchronic oral toxicity study in 

mice: fibrillation, tremors, convulsions, hypersensitivity, abnormal gait and salivation during 

Week 1; scratching and licking after Week 3; and alopecia, scabs and sores after Week 4. No 

deaths occurred during the study. Decreased body weight gains were noted throughout the study 

in both sexes (males>females) in the high-dose esfenvalerate group and the fenvalerate group. 

Animals in both groups had decreased body weight gains during Days 0-28. Body weight gain 

Page 30 of 70
ESFENVALERATE FINAL 

Page 23 of 62 

decreases were lessened (<10%) in the females dosed with fenvalerate for the remainder of the 

study, and the fenvalerate males and high-dose esfenvalerate females by Days 63-90. 

Maternal toxicity was observed at all doses in the developmental toxicity in rats with 

esfenvalerate. Clinical signs in the low-dose group (2.5 mg/kg/day) included erratic jerking and 

extension of forelimbs, rapid side-to-side head movement and excessive grooming. Additional 

signs at the medium-low dose (5 mg/kg/day) consisted of hindlimb jerking and soft/mucoid 

stools, and in the medium-high dose group (10 mg/kg/day), further signs included 

hypersensitivity to touch and tremors. Finally, additional clinical signs in the high-dose dams 

(20 mg/kg/day) consisted of high carriage, goose-stepping ataxia, ataxia and convulsions. 

Incidence and frequency of these behaviors increased with increasing dose. Decreased body 

weight gains of 8% were observed in the medium-high and high-dose dams during GDs 6-15, but 

overall body weight gains were not affected. One high-dose rabbit (20 mg/kg/day) died on GD 

17 in the developmental toxicity in rabbits with esfenvalerate, but the death did not appear to be 

related to treatment. Maternal toxicity was also observed at all doses. Clinical signs in the lowdose group (3 mg/kg/day) included erratic jerking and extension of forelimbs, rapid side-to-side 

head movement, sneezing and excessive grooming. Additional signs at the medium-dose (10 

mg/kg/day) consisted of hindlimb jerking and hypersensitivity to touch, and further clinical signs 

in the high-dose dose animals consisted of tremors, ataxia and diarrhea. The incidence and 

frequency of these behaviors increased with increasing dose. Body weight gains were decreased 

during the dosing period (GDs 7-10 and 16-19) at the low, medium, and high doses, with 

compensatory increases during GDs 19-29. 

In a two-generation reproduction study in rats with esfenvalerate, P1 males in the high-dose 

group (18.8 mg/kg/day) exhibited abnormal gait/mobility, alopecia, scabs and sores. Two males 

in the high-dose group died, but only one of the deaths was believed to be due to treatmentrelated ulcerative dermatitis. High-dose P1 females (25.1 mg/kg/day) exhibited abnormal gait, 

and a P1 female in the medium-dose group was sacrificed in extremis (attributed to dystocia). F1

males and females in the high-dose group exhibited numerous clinical signs of neurotoxicity 

(abnormal gait/mobility, ataxia, barrel rolls, general spasms, hyperactivity, hypersensitivity, 

popcorn seizures, tremors and vocalization) and signs involving the skin (alopecia, swollen face, 

scabs and sores). The severity of these clinical signs led to early termination of the high-dose 

group. Clinical signs involving the skin were observed in low- and medium-dose animals 

[(5.10/5.47 and 6.7/7.27 mg/kg/day (M/F)]. 

Minimal effects on body weight gains occurred in the low- and medium-dose animals throughout 

the study. Body weight gains in the high-dose P1 animals during the premating period were 

generally decreased during the initial weeks, but overall body weight gains in high-dose P1 males 

and females were only decreased by 19 and 32%, respectively. Decreased body weights in the 

high-dose females during gestation were attributed to pre-existing conditions, but body weight 

gain was also decreased in the high-dose P1 females during lactation. Body weight gains in the 

Page 31 of 70
ESFENVALERATE FINAL 

Page 24 of 62 

high-dose F1 animals during the premating period were slightly decreased during the initial 

weeks, but increased mortality occurred in high-dose F1 animals by approximately Week 5. In a 

follow-up one-generation reproduction study in rats with esfenvalerate) at lower doses compared 

to the two-generation reproduction study, several animals were sacrificed in extremis or were 

found dead, but none of these deaths were considered to be treatment related. Additionally, there 

were no treatment-related effects on clinical signs of toxicity or body weight, and the overall 

body weight gain for the pre-mating period was not affected. 

There were no treatment-related differences in survival in a chronic toxicity/carcinogenicity 

study in rats with fenvalerate. In addition, there were no treatment-related clinical signs or 

weight decreases. In a follow-up chronic toxicity/ carcinogenicity study in rats with fenvalerate, 

there were no overall treatment-related differences in survival at a higher dose. Treated females 

began dying earlier compared to control and treated males, but control males began dying earlier 

compared to all other groups. Clinical signs included treatment-related transient hindlimb 

weakness (appearing by Week 3 or 4, and generally resolving within a week) and non-treatmentrelated alopecia. Body weights in males were decreased from Weeks 16-104; weights were 

decreased generally ≥10% by Week 72. Body weights in females were decreased from Weeks 

44-104, with weights generally decreased ≥10% by Week 60. 

In a carcinogenicity study in mice with fenvalerate, treatment-related decreases in survival were 

noted for the low-dose males and the high-dose males and females. Because decreases in 

survival were not continued in the medium-low and medium-high dose males, the decrease in the 

low-dose group is not considered to be related to treatment. The presence of clinical signs was 

not reported. There were no consistent significant effects on mean body weights in the low- and 

medium-low dose groups. Body weight decreases were generally consistent in the high-dose 

animals (187.5 mg/kg/day) throughout the study, with greater decreases observed during the 

second year through study termination (↓13-24%). In a second carcinogenicity study in mice 

with esfenvalerate, clinical signs and lesions observed in treated mice were attributable to selfmutilation or secondary effects of self-mutilation. Self-mutilation occurred due to the induction 

of dermal sensations that stimulate scratching by the mice (a well-known toxicological effect of 

pyrethroids). All treated mice exhibited increased alopecia, serosanguinous fluid from sores, 

skin sores, and swollen ears. There also was an increase in mortality in the high- and mediumdose groups due to skin inflammation/necrosis caused by self-trauma. Mortality was increased 

in the high-dose group by Study Day 56. This excessive mortality was attributed to skin 

inflammation/necrosis caused by self-trauma, and it was decided to remove the high-dose group 

from the study on Days 57-58. At 358 days, survival in the medium-dose group was 70-71% 

versus 95% in the low-dose and control groups. At termination (547 days), survival in the 

medium-dose group was 41-46% versus 66-75% in the other groups. Body weights and body 

weight gains in the low- and medium-dose groups generally were not affected by treatment. In 

Page 32 of 70
ESFENVALERATE FINAL 

Page 25 of 62 

the high-dose group, decreased mean body weight was reported for Study Day 56 (↓15% and 

11%, respectively, in males and females) prior to termination of the dose group. 

No mortality occurred during a chronic toxicity study in dogs with fenvalerate, and no consistent, 

treatment-related effects were observed regarding clinical signs of toxicity or body weight/body 

weight gain. 

There were no significant treatment-related mortalities or clinical signs of toxicity reported in the 

avian reproduction study with esfenvalerate in bobwhite quail. 

B. Estrogen Pathway 

Although Kojima et al., reported a positive response for fenvalerate in an ERTA conducted in 

CHO cells, Saito et al. reported that fenvalerate was negative for ER binding/ER activation in 

three different ER assay systems: a mammalian ERTA assay with HeLa cells, a yeast two-hybrid 

system and a hER competitive ligand-binding. Additionally, esfenvalerate was negative in the 

Tier 1 ERTA, aromatase and steroidogenesis assays. The in vivo uterotrophic and female 

pubertal assays were negative, and no estrogen related effects were seen in the mammalian Part 

158 studies. While there was a 6% increase in fertilization success was reported in the FSTRA at 

the high dose there were no other treatment related effects in females. In the Part 158 studies 

with esfenvalerate, the only effect observed was a slight decrease in hatchling weights. 

Therefore, there is no convincing evidence for a potential interaction with the estrogen pathway. 

C. Androgen Pathway 

A generally consistent pattern of weak anti-androgenicity was observed for 

esfenvalerate/fenvalerate in the in vitro Tier 1 and in vitro OSRI assays. However, esfenvalerate 

was negative in the in vivo Tier 1 Hershberger and male pubertal assays, and no androgen-related 

effects were seen in the mammalian or avian Part 158 toxicity studies. As reported above, the 

only effect observed in the FSTRA was an increase in fertility. In the Part 158 studies with 

esfenvalerate, the only effect observed was a slight decrease in hatchling weights Overall, there 

is no convincing evidence for an interaction with the androgen pathway. 

D. Thyroid Pathway 

The available Tier 1 EDSP data and OSRI for esfenvalerate do not indicate a potential for 

thyroid interaction in mammals. Although an increase in T3 was reported by Giray et al. (2010),

thyroid weight, concentrations of plasma TSH and total T4 were similar to the control animals. 

No thyroid related effects were observed in the Tier 1 male and female pubertal assays or in the 

Part 158 mammalian studies. However, an adequate AMA was not available for consideration in 

Page 33 of 70
ESFENVALERATE FINAL 

Page 26 of 62 

the weight-of-evidence, and given the observed high variability and apparent lack of 

concentration stability in the available study, esfenvalerate may not be amenable to testing in the 

current 890.1100 AMA study design (i.e., water column exposure). Therefore, the weight-ofevidence cannot conclude whether esfenvalerate has the potential for thyroid interaction in nonmammalian species. 

E. Conclusions 

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

of potential interaction with the estrogen or androgen pathway in mammalian and wildlife 

species. Additionally, the available information indicates no convincing evidence of potential 

interaction with the thyroid pathway for mammals, but a conclusion for this pathway could not 

be determined for non-mammalian species. 

V. EDSP Tier 2 Testing Recommendations 

Based on weight of evidence conclusions, mammalian or wildlife EDSP Tier 2 testing is NOT 

recommended for esfenvalerate. 

A conclusion regarding potential interaction with the thyroid pathway in non-mammalian species 

could not be determined, at this time, and it is noted that there is a lack of a validated dietary 

exposure test design as well as the fact that the nature of esfenvalerate may make it difficult to 

test long-term in aquatic systems with amphibians. Except for an increase in fertility, no other 

treatment-related effects were observed in the FSTRA up to 0.23 μg a.i./L; it is noted that 

measured test concentrations were low and variable in this study. A fathead minnow life-cycle 

study was submitted to the agency (fish are used as surrogates for aquatic-phase amphibians), 

and based on a preliminary review, high variability in measured test concentrations within a 

treatment was observed. In this study, the most sensitive endpoints measured were decreased 

survival and fecundity in the parental generation and decreased survival in the second generation 

(MRID 49289201). The overall LOAEC for this study, based on survival in the second 

generation was 0.034 μg a.i./L, and the NOAEC was 0.017 μg a.i./L. The nominal test 

concentrations in the esfenvalerate AMA were 0.006, 0.02, and 0.05 μg/L which are similar to 

the concentrations tested in the fish life-cycle study. Given the lack of a validated dietary 

exposure test design with amphibians along with the observed difficulties in maintaining aquatic 

exposure from the available data, relevant to EDSP, additional amphibian testing is not 

recommended at this time. 

 

Page 34 of 70
ESFENVALERATE FINAL 

Page 27 of 62 

VI. References 

Biegel, L.B. (1994) Reproductive and fertility effects with DPX-YB656-84 multigeneration 

reproduction study in rats. Unpublished study conducted by Dupont, Haskell Laboratory for 

Toxicology and Industrial Medicine, Newark, Delaware, Dupont HLR 331-94; Med. Res. Proj. 

No. 9657-001, December 1, 1994. (MRID 43489001) 

Delker, D.A. (2000) Esfenvalerate Technical: Repeated Dose Dermal Toxicity 21-Day Study in 

Rats. Unpublished study conducted by Haskell Laboratory for Toxicology and Industrial 

Medicine, Study No.: DuPont-4228, November 28, 2000. (MRID 45275401) 

Dinehart, S. (2012) Esfenvalerate Technical (DPX-YB656): Short-term Reproduction Assay 

with the Fathead Minnow, Pimephales promelas, Determined Under Flow-Through Conditions. 

Unpublished study conducted by ABC Laboratories, Inc., Columbia Missouri and Experimental 

Pathology Laboratories, Inc., Sterling, Virginia. Lab Study No.: 66821; Sponsor Project No.: 

DuPont 32258. Submitted by E.I. du Pont de Nemours and Company, Wilmington, Delaware. 

Completion date October 31, 2012. (MRID 48995501) 

DuPont (1987): D.M. Larson (1987) 13-Week dietary admix study of MO 70616 technical in 

rats. Unpublished study conducted by DuPont SR0-150-87. (MRID 40215601) 

DuPont (1992): MacKenzie, S.A (1992) Repeated dose dermal toxicity: 21-day study with DPXY4306-90 (Fenvalerate) in rabbits DuPont HLR 127-92. (MRID 42325101) 

Eil, C. and Nisula, B. C. (1990) The Binding Properties of Pyrethroids to Human Skin Fibroblast 

Androgen Receptors and to Sex Hormone Binding Globulin. J. Steroid Biochem. 35: 409-414. 

Fort, Douglas J. (2012) Esfenvalerate (DPX-YB656) Technical: 21-d Amphibian Metamorphosis 

Assay (AMA) with South African Clawed Frog, Xenopus laevis. Unpublished study conducted 

by Fort Environmental Laboratories, Inc., Stillwater, OK 74074. Laboratory project number 

DUPO01-00271. Study sponsored by E.I. du Pont de Nemours and Company (DuPont). (MRID 

49024501)

Giray, B., Cağlayan, A., Erkekoğlu, P., and Hıncal, F. (2010) Fenvalerate Exposure Alters 

Thyroid Hormone Status in Selenium-and/or Iodine-Deficient Rats. Biol. Trace Elem. Res.

Published online: September 2, 2009. 

Gordon, E.B., Wosu, N.J., and DePaoli, A. (1978) Lifetime Feeding Study in Rats: SD-43775 

Technical: LBI Project No. 2541. Final rept. (Unpublished study received May 9, 1978 under 

201-401; Unpublished study conducted by Litton Bionetics, Inc., submitted by Shell Chemical 

Co., Washington, D.C.; CDL:097075-A; 097076;097077;097078; 097079; 097080; 097081; 

097082) (MRIDs 00082244 and 00111888) 

Page 35 of 70
ESFENVALERATE FINAL 

Page 28 of 62 

Gordon, E.B. (1979) Lifetime Feeding Study in Rats, SD 43775 Unpublished study conducted by

Litton Bionetics Project No. 20733-01 (MRID 00079877) 

Higuchi, H. (1999) Reproduction study in rats with Esfenvalerate (in pelleted diet). Unpublished 

study conducted by Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd. 

Study No. 3356, October 12 1999. MRID 48076104. 

Johnston, C.D. (1979) Chronic Toxicity and Potential Carcinogenicity Study in the Mouse: 

Technical SD 43775: LBI Project No. 20738. Final rept. (Unpublished study received Oct 23, 

1979 under 201-401; Unpublished study conducted by Litton Bionetics, Inc., submitted by Shell 

Chemical Co., Washington, D.C.; CDL:241206-B; 241207) (MRID 00079876) 

Kaul, P.P., Rastogi, A., Hans, R.K., Seth, T.D., Seth, P.K., and Srimal, R.C. (1996) Fenva1erateinduced Alterations in Circulatory Thyroid Hormones and Calcium Stores in Rat Brain 

Toxicology Letters 89: 29-33. 

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. 

Kunimatsu, T., Yamada, T., Ose, K., Sunami, O., Kamita, Y., Okuno, Y., Seki, T., and 

Nakatsuka, I. (2002) Lack of (Anti-) Androgenic or Estrogenic Effects of Three Pyrethroids 

(Esfenvalerate, Fenvalerate, and Permethrin) in the Hershberger and Uterotrophic Assays. 

Regulatory Toxicology and Pharmacology 35, 227–237. 

Nabb, D.L. (2012) Esfenvalerate (DPX-YB656) Technical: H295R Steroidogenesis Assay. 

Unpublished study conducted by E.I. du Pont de Nemours and Company, DuPont Haskell Global 

Centers for Health & Environmental Sciences, Newark, DE. Laboratory Study No.: DuPont32096, February 8, 2012. (MRID 48670802) 

Ross, P.E. (1997) Carcinogenicity Study with DPX-YB656-84 Eighteen-Month Feeding Study in 

Mice. Unpublished study conducted by E. I. du Pont de Nemours and Company, Haskell 

Laboratory for Toxicology and Industrial Medicine, Newark, Delaware, April 3, 1997. 

Laboratory report number 853-95. (MRID 44260601) 

Saito, K., Tomigahara, Y., Ohe, N., Isobe, N., Nakatsuka, I., and Kaneko, H. (2000) Lack of 

significant estrogenic or antiestrogenic activity of pyrethroid insecticides in three in vitro assays 

based on classic estrogen receptor alpha-mediated mechanisms. Toxicol Sci. 57(1):54-60. 

Shell (1986): Dickie, B.C. (1986) One year oral study in dogs with MO 70616 technical. Shell 

Chemical Company Regulatory Information Record No. WRC RIR 467. Unpublished study 

conducted by Hazleton Laboratories America, Inc. (MRID 00163855) 

Page 36 of 70
ESFENVALERATE FINAL 

Page 29 of 62 

Snajdr, S.I. (2011) Esfenvalerate (DPX-YB656) Technical: In Vitro Aromatase Inhibition Using 

Human Recombinant Microsomes. Unpublished study conducted by E.I. du Pont de Nemours 

and Company, DuPont Haskell Global Centers for Health & Environmental Sciences, Newark, 

DE. Laboratory Project ID: DuPont-32091; November 16, 2011. (MRID 48616502) 

Snajdr, S.I. (2011) Esfenvalerate (DPX-YB656) Technical: 10-Day Hershberger BioAssay for 

Detecting Androgenic Activity. Unpublished study conducted by DuPont Haskell Global Centers 

for Health and Environmental Services, Newark, DE. Laboratory Project ID: DuPont-33441, 

November 8, 2011. (MRID 48616501) 

Stafford, J .M. (2009) Esfenvalerate: Reproductive toxicity test with northern bobwhite (Colinus 

virginianus) following OPPTS 850.2300 and OECD 206. Unpublished study conducted by 

Springborn Smithers Laboratories. Study sponsored by Sumitomo Chemical Company, Ltd., 

Chuo-ku, Tokyo, Japan. Report Nos. 13048.4110 and LLW-0137. (MRID 48027802) 

Sumitomo (1985): Y. Koyama (1985) Comparative subacute toxicity in B6C3Fl mice treated 

with S-1844 and S-5602 for 3 months. Unpublished study conducted by Sumitomo Chemical 

Co., Ltd. Unpublished report No.: LLT-50-0004 (MRID 41359701) 

Sumitomo (1990): M.D. Nemec (1990) A developmental toxicity study of S1844 in rabbits. 

Unpublished study conducted by Sumitomo Chemical Co., Ltd. Unpublished report No: LLT010135 (MRID 43211503)

Sumitomo (1991a): M.D. Nemec (1991a) A developmental toxicity study of S1844 in rats. 

Unpublished study conducted by Sumitomo Chemical Co., Ltd. Unpublished report No.: LLT110143 (MRID 43211504) 

Sumitomo (2001): Sumitomo Chemical Co., Ltd Report LLT-0217 Evaluation of hormonal 

activity (effects on human estrogen receptor alpha, androgen receptor and progesterone receptor) 

of fenvalerate using a suite of three in vitro assays. 

Toot, J.D. (2012) Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal 

Female Rats Following Oral Administration of Esfenvalerate (DPX-YB656) Technical. 

Unpublished study conducted by WIL Research Laboratories, LLC, Ashland, OH. Laboratory 

Project ID: WIL-189228, April 16, 2012. (MRID 48683803) 

Toot, J.D. (2012) Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal 

Male Rats Following Oral Administration of Esfenvalerate (DPX-YB656) Technical. 

Unpublished study conducted by WIL Research Laboratories, LLC, Ashland, OH. Laboratory 

Project ID: WIL-189231, April 16, 2012. (MRID 48683802) 

Page 37 of 70
ESFENVALERATE FINAL 

Page 30 of 62 

USEPA (2010) Esfenvalerate - Report of the Endocrine Disruptor Review Team; Test Order# 

EDSP-109303-80, 81, and 82. December 14, 2010. Office of Chemical Safety and Pollution 

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

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

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

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

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. 

Willoughby, J.A. (2012) Esfenvalerate (DPX-YB656): Estrogen Receptor Transcriptional 

Activation (Human Cell Line HeLa-9903)). Unpublished study conducted by CeeTox, 

Kalamazoo, MI. Laboratory Study No. 9144V-100359ERTA, February 8, 2012. (MRID 

48670801) 

Xu, L-C., Sun, H., Chen, J-F., Bian, Q., Song, L., and Wang, X-R. (2006) Androgen receptor 

activities of p,p'-DDE, fenvalerate and phoxim detected by androgen receptor reporter gene 

assay. Toxicology Letters 160: 151-157.

Page 38 of 70
ESFENVALERATE FINAL 

Page 31 of 62 

APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA): (OCSPP 890.1100) 

The 21-day assay (MRID 49024501) of esfenvalerate (DPX-YB656; 95.4% purity) on amphibian 

metamorphosis of African clawed frog (Xenopus laevis) was conducted under flow-through 

conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 per control and treatment 

group) were exposed to negative and solvent (0.1 mL/L triethylene glycol; TEG) controls, and 

nominal esfenvalerate concentrations of 0.0000060, 0.000020, and 0.00050 mg a.i./L. Time 

weighted average (TWA) measured concentrations calculated by the reviewer were <0.00000673 

(<LOD; control), 0.000193, 0.000111, and 0.000127 mg a.i./L. All analytical measurements at 

Days 0, 7, 14, and 21 were used in these calculations. The test system was maintained at 21.4 to 

21.8oC and a pH of 7.0 to 7.8. 

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

water) control. No mortalities or clinical signs (i.e., behavioral and other sublethal effects) were 

observed in any tadpoles at any treatment level. 

There was no significant acceleration or delay (p>0.05) of median NF developmental stage 

observed at Day 7 or 21 at any treatment level relative to the negative control. Further, no 

asynchronous development was observed. Effects on thyroid gland histopathology were 

observed at all treatment levels, including the control groups and there was no apparent pattern 

of response. Histopathological effects included mild follicular cell hypertrophy in three tadpoles 

in the solvent control, four tadpoles in the mid treatment group and one tadpole in the high 

treatment group. Mild follicular cell hyperplasia was observed in one tadpole in each of the 

negative control, solvent control, low treatment group and high treatment group. On Day 7, the 

reviewer’s analysis detected a significant decrease in hind-limb length (HLL) at the mid and high 

(nominal) treatment groups (Jonckheere-Terpstra, p<0.05), relative to the negative control, but 

no effects were detected on developmental endpoints at Day 7 or Day 21. The Day 21 no-late 

stage snout-to-vent length (SVL) was found to have a significant decrease at the low treatment 

group relative to the negative control (p<0.05). No other growth or developmental effects were 

noted in this study. 

The CV of measured test concentrations exceeded the recommended ≤20% in all test treatments 

at 159%, 180%, and 151% in the nominal low, mid, and high treatment groups, respectively. 

Given the very low concentrations used (<1 ppb), the tendency for pyrethroids to sorb onto 

glassware, CVs moderately higher than 20% are not unexpected. However, the extremely high 

CVs (>100%) and overlapping measurements in all three treatment concentrations indicate that 

the lack of stability in the test concentrations does interfere with the interpretation of test results. 

Detections of test substance in some of the negative and solvent control samples further 

compromise the test results. 

Page 39 of 70
ESFENVALERATE FINAL 

Page 32 of 62 

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

Requirement satisfied by OSRI (Sumitomo et al., 2001); see Appendix 2. 

Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48616502), esfenvalerate (95.4% total isomers 

with the active isomer accounting for 85.7%, Lot #0902-11) was incubated with human 

recombinant aromatase and tritiated androstenedione (1-β [3H(N)]-androst-4-ene-3,17-dione; 

[

3

H]-ASDN) at concentrations of 1 × 10−10 to 2.5 × 10−5 M for 15 minutes to assess the potential 

of esfenvalerate to inhibit aromatase activity. Acetone was selected as the vehicle solvent for the 

test substance because esfenvalerate was not soluble, or did not stay in solution, when EPArecommended solvents were used; dimethyl sulfoxide (DMSO) was used as the solvent vehicle 

for the reference compounds. The final concentration for each solvent vehicle was 1% in the 

assay. 

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). A total of four runs were conducted and each run 

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

10−5 M) using a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), and the 

esfenvalerate series (10−10 to 2.5 × 10−5 M), with three repetitions per concentration. Because the 

solvent vehicle was acetone for esfenvalerate and DMSO for 4-OH ASDN, separate sets of the 

background activity and full enzyme activity controls were run using only 1% acetone or 1% 

DMSO. In the first run, full activity controls had aromatase activity below the 0.1 nmol·mgprotein−1

·min−1 performance criteria specified in the Guideline. Therefore, the first run was 

excluded from the calculations and a fourth run was performed for a total of three valid runs. 

Aromatase activity in the full activity controls for the three successful test runs averaged 0.416 ± 

0.166 nmol·mg-protein−1

·min−1 in 1% DMSO, and 0.0.213 ± 0.083 nmol·mg-protein−1·min−1 in 

1% acetone. Activity in the background controls averaged 0.5% of the full activity control with 

DMSO, and 1.1% of the full activity control with acetone. 

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

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

ASDN averaged −7.2 M and the Hill slope was −0.9. 

For esfenvalerate, aromatase activity averaged 0.204 ± 0.101 nmol·mg-protein−1

·min−1 at the 

lowest tested concentration (10−10 M) and 0.178 ± 0.077 nmol·mg-protein−1·min−1 at the highest 

tested concentration (2.5 × 10−5 M). The lowest portion of the response curve was greater than 

Page 40 of 70
ESFENVALERATE FINAL 

Page 33 of 62 

75% activity at all soluble concentrations of esfenvalerate. Aromatase activity with esfenvalerate 

was ≥82.80% at concentrations from 10−10 to 2.5 × 10−5 M. 

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

of aromatase activity in this assay. 

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

Requirement satisfied by OSRI (Saito et al., 2000); see Appendix 2. 

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

In an estrogen receptor transcriptional activation assay (MRID 48670801), hERα-HeLa-9903 

cells cultured in vitro were exposed to esfenvalerate (95.4% a.i., Batch/Lot # 0902-11) at 

logarithmically increasing concentrations from 10−10.6 to 10−3.6 M in 0.1% acetonitrile for 24 

hours in two independent runs. The experiments were performed using 96-well plates and each 

esfenvalerate concentration was tested in 6 wells/plate in each run. Cells were exposed to the 

test agent for 24±2 hours at 37±2°C to induce reporter (luciferase) gene products. Luciferase 

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

luciferase assay. 

Esfenvalerate was tested up to 10−3.6 M as no cytotoxicity or precipitation occurred in either test 

run. At concentrations up to 10−4.6 M, the RTA for esfenvalerate was ≤4.1%, indicating that it is 

negative for estrogenic activity under the conditions of this assay. 

In the main assays, the responsiveness of the cells to the very weak positive control 17αmethyltestosterone was lower than the expected values, indicating a decreased sensitivity of the 

assay to very weak agonists. Although the conditions of this assay were not optimal to detect 

very weak activity, esfenvalerate responses were similar to those of the negative control 

corticosterone and not comparable to the responses of 17α-methyltestosterone, which was able to 

reach a maximum of 24.3-26.6% PC. Esfenvalerate was only able to reach a maximum of 4.0-

4.1% PC when tested up to the highest concentration possible based on cytotoxicity. Because 

the RPCMax < PC10 in both assay runs, esfenvalerate was considered negative for estrogen 

receptor transcriptional activation in this test system. 

Page 41 of 70
ESFENVALERATE FINAL 

Page 34 of 62 

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

The 21-day short-term reproduction assay (MRID 48995501) of esfenvalerate with fathead 

minnows (Pimephales promelas) was conducted under continuous flow-through conditions. 

Adult fish (16 spawning groups, 2 males and 4 females in each group; ca. 6 months old) were 

exposed to esfenvalerate (95.4% purity) at nominal concentrations of 0 (negative control), 0 

(solvent control) 0.000036, 0.00012, and 0.00040 mg a.i./L; mean-measured concentrations were 

below the limit of quantitation (LOQ; <0.00244 controls), 0.000027, 0.000051, and 0.000231 mg 

a.i./L. The test concentrations were less than the reported limit of detection (LOD; 

0.000732 mg/L) and LOQ (0.00244 mg/L). In the AMA conducted with esfenvalerate at the 

same laboratory (MRID 49024501), the LOD was 0.00000673 mg/L and LOQ was 0.0000244 

mg/L; therefore, it seems likely that the reported values for the LOD and LOQ in the present 

FSTRA study are in error. Due to the variability in measured recoveries, time-weighted averages 

(TWA) were calculated as 0.000030, 0.000050, and 0.00023 mg a.i./L. The solvent control 

consisted of triethylene glycol (TEG) at a final concentration of 0.02 mL/L. The test system was 

maintained at 24.7 to 25.4oC and a pH of 8.01 to 8.40. 

Adult survival was not affected by treatment with esfenvalerate. Male survival was 87.5, 100, 

100, 62.5, and 87.5% in the negative control, solvent control, low, mid and high treatment 

groups, respectively; female survival was 100% at all treatment levels. There were no notable 

observations with regards to behavior or changes in appearance (specifically color/banding, 

ovipositor appearance, or size of dorsal nape pad) in the control or treated groups. There was one 

male in the mid treatment group that displayed hemorrhaging and surfacing and several females 

displayed vertical banding in each control and treatment group. All other fish in the controls and 

treatment groups appeared normal and no overt signs of toxicity were observed during the test 

period. Body weight was not significantly affected by treatment; body length was not reported. 

In the negative control, spawning occurred at least once every 4 days and fecundity was 48 

eggs/female/day/replicate (range 43 to 53); fertilization success in the negative control group 

averaged 90% (range 86-97, CV=3.8%). The only endpoint affected was fertility, for which 

median values exhibited a concentration-dependent increasing trend; a significant increase of 

6.1% in fertility was detected at the high concentration (p<0.05). There were no other 

statistically significant differences in the treated groups compared to negative control for any 

other endpoint (p>0.05). Histopathology and gonadal stages were similar among the negative 

control and all treatment groups. Plasma sex steroids were not measured. 

The test material does not appear to be stable under the test conditions, and recoveries were poor 

and variable. Analytical verification of the test material from Days 0, 6, 12, and 21 at nominal 

concentrations of 0.000036, 0.00012 and 0.00040 mg a.i./L yielded mean recoveries of 76, 43 

and 58%, respectively; the %CVs were 13.7, 22.7 and 4.2%, respectively. The test solutions 

were clear and colorless with no visible precipitate, surface film, or undissolved test substance 

Page 42 of 70
ESFENVALERATE FINAL 

Page 35 of 62 

during the exposure period, and the low recoveries observed were similar to those obtained 

during the range-finding study. 

Hershberger Assay; (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48616501) screening for androgenic activity, esfenvalerate 

(95.4% total isomers with the active isomer accounting for 85.7%; Batch/lot # 0902-11) in corn 

oil was administered daily via oral gavage to groups of six 56-day old, castrated male Sprague 

Dawley rats at dose levels of 0 (vehicle), 3, 6, or 9 mg/kg/day. An androgenic positive control 

group consisted of six castrated rats exposed to the vehicle plus 0.4 mg/kg/day of testosterone 

propionate (TP) by subcutaneous (s.c.) injection. 

To screen for potential anti-androgenic activity, esfenvalerate in corn oil was administered daily 

via oral gavage to groups of six 56-day old, castrated male Sprague Dawley rats at dose levels of 

0 (vehicle), 3, 6, or 9 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 

mg/kg/day by s.c. injection. The anti-androgenic positive control group consisted of six 

castrated rats orally dosed with 3 mg/kg/day flutamide (FT, via oral gavage) and 0.4 mg/kg/day 

TP by s.c. injection. TP by s.c. injection plus corn oil by gavage was used as the anti-androgenic 

negative control. 

For both components of the assay, body weights were determined daily. Food consumption was 

determined over the entire dosing period. The animals were dosed for 10 consecutive days and 

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

androgen-dependent tissues and liver were collected and weighed. 

All animals survived until scheduled termination. Test substance-related clinical signs of 

toxicity were observed in rats administered esfenvalerate at 9 mg/kg/day in both the androgen 

and anti-androgen assays and in rats administered esfenvalerate at 6 mg/kg/day in the antiandrogen assay. Clinical signs observed included abnormal gait, ataxia, fast breathing, hyperreactivity, paw shaking, paw licking, abnormal grooming, whole-body fasciculations, altered 

posture, decreased muscle tone, and/or tremors. The clinical observations observed in the 6 and 

9 mg/kg/day treatment groups were consistent with previously reported clinical signs observed in 

other toxicity studies and were considered toxicologically significant. 

In the androgen assay, body weights throughout the duration of the assay and feed consumption 

over the dosing period in the esfenvalerate-dosed groups were comparable to the vehicle 

controls. Overall (Days 1-11) body weight gains in the esfenvalerate 6 and 9 mg/kg/day groups 

Page 43 of 70
ESFENVALERATE FINAL 

Page 36 of 62 

were decreased (not significant; NS) by 17% and 24%, respectively, compared to the vehicle 

controls. Overall body weight gains in the positive control (vehicle + TP) group were increased 

(NS) by 27% and feed consumption was increased (p<0.05) by 17% compared to the vehicle 

controls. No treatment-related effects were observed in any of the accessory sex organs of 

esfenvalerate-dosed animals. Rats in the positive control (vehicle + TP) group had accessory sex 

organ weight increases (p<0.05) as follows: 817% in seminal vesicles; 1296% in ventral 

prostate; 191% in levator ani-bulbocavernosus (LABC); 482% in Cowper’s glands; and 66% in 

glans penis. Liver weights in the esfenvalerate treatment groups and positive control group were 

comparable to the vehicle controls. 

 

In the anti-androgen assay, body weights throughout the duration of the assay, feed consumption 

over the dosing period, and overall body weight gains in the esfenvalerate-dosed groups and 

positive (FT + TP) control group were comparable to the negative controls. There were no 

treatment-related decreases in accessory sex organs weights for any treatment groups in the antiandrogen assay. Rats in the positive control (FT + TP) group had accessory sex organ weight 

decreases (p<0.05) as follows: 69% in seminal vesicles; 70% in ventral prostate; 49% in LABC; 

60% in Cowper’s glands; and 24% in glans penis. Liver weights in the esfenvalerate treatment 

groups and positive control group were comparable to the negative controls. 

The high-dose of esfenvalerate selected for this study (9 mg/kg/day) may have been excessive 

based on data from earlier studies showing clinical signs of toxicity at doses ≥12.5 mg/kg/day 

and the clinical signs of toxicity observed at 9 mg/kg/day in the current assay. However, the low 

and mid-dose levels were considered adequate. All performance criteria were met with the 

exception for the ventral prostate (49% vs. 40% maximum CV) in the esfenvalerate high dose 

group of the anti-androgen assay. 

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

issues. Esfenvalerate was negative for androgenicity and anti-androgenicity in the Hershberger 

assay. 

Female Pubertal Assay; (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48683803), 15 Crl:CD(SD) Sprague-Dawley rats/dose group 

were treated daily via oral gavage with esfenvalerate technical (95.4% total isomers; 85.7% 

active isomers; Lot #0902-11) in corn oil at doses of 0, 3.0 or 9.0 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 the age and weight at day of attainment was recorded. Following sacrifice on PND 

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

thyroid stimulating hormone (TSH), which were analyzed using an electrochemiluminescent 

immunoassay or a radioimmunoassay, respectively. Kidney, liver, adrenal gland, thyroid, 

Page 44 of 70
ESFENVALERATE FINAL 

Page 37 of 62 

pituitary, uterus, and ovary weights were recorded, and microscopic examinations were 

performed on the thyroid, kidney, ovary, and uterus. 

No treatment-related effect was observed on survival, body weight, body weight gain, age of 

attainment of VO, body weight at VO, or gross pathology. No treatment-related effect was noted 

on estrous cycle status at necropsy, mean age at first estrus, mean cycle length, or percent 

cycling. Serum T4 and TSH were not affected by treatment. No treatment-related microscopic 

lesions were observed on the thyroid, ovary, uterus, or kidney. No effects on organ weights were 

observed during the study. 

One animal in the control group was found dead on PND 35 due to possible intubation error. All 

other rats survived until scheduled sacrifice. At 9.0 mg/kg/day, tremors were observed in all 

females after dosing on PND 22-36. On PND 24, four of the 9.0 mg/kg/day females were also 

observed with hindlimbs splayed or impairment of hindlimbs. No other treatment-related 

clinical findings were observed. No effects were observed on body weights or body weight gain 

in the study. 

The high dose level was considered adequate based on the tremors, seen in all animals at this 

dose. 

Male Pubertal Assay; (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48683802), 15 Crl:CD(SD) Sprague-Dawley rats/dose group 

were treated daily via oral gavage with esfenvalerate (95.4% total isomers; 85.7% active 

isomers; Lot # 0902-11) in corn oil at doses of 0, 3.0 or 9.0 mg/kg/day from post-natal day 

(PND) 23 to PND 53 or 54. Animals were examined for preputial separation (PPS) daily 

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

PND 53 or 54, blood was collected for clinical chemistry analyses, including total thyroxine (T4), 

testosterone, thyroid stimulating hormone (TSH). Testosterone and T4 were analyzed using an 

electrochemiluminescent assay and TSH levels were analyzed using radioimmunoassay. Liver, 

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

were performed on the thyroid, kidney, testis and epididymis. 

No treatment-related effects were observed on mortality, body weights, body weight gain, organ 

weights, or gross pathology. Age and body weight at PPS in the treated groups were similar to 

controls. Thyroid hormone levels, colloid area and follicular cell height scores were similar in 

the treated groups to the control group. Serum testosterone levels were similar in the treated 

groups to the control group. No treatment-related microscopic findings were noted in the 

epididymis, testis, and kidney. 

Page 45 of 70
ESFENVALERATE FINAL 

Page 38 of 62 

At 9.0 mg/kg/day, tremors were observed almost exclusively at ≥3 hours after dosing for 12 

animals on PND 23-31. During the daily examinations, only two 9.0 mg/kg/day animals were 

observed with tremors. At 9.0 mg/kg/day, impaired use of left forelimb (1 male), impaired use 

of hindlimbs (3 males), and splayed hindlimbs (4 males) were observed at ≥3 hours after dosing 

on PND 24-42. During daily examinations, only one 9.0 mg/kg/day animal was observed with 

impaired use of left forelimb. The clinical sign of clear material around mouth was increased for 

9.0 mg/kg/day animals at up to 3 hours after dosing starting at PND 35 and continuing 

sporadically during the study. Red material around the mouth was observed at 3.0 mg/kg/day (6 

males) and 9.0 mg/kg/day (5 males) during the daily examinations on PND 26-53. 

The clinical sign of tremors observed at 9.0 mg/kg/day indicated that the high dose was 

adequate. 

Steroidogenesis Assay; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48670802), H295R cells cultured in vitro in 24-well plates 

were incubated with esfenvalerate (85.7% active isomer; 95.4% total isomers, Batch # 0902-11) 

at concentrations of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 μM for 48 hours in triplicate in three 

independent experiments. Acetonitrile was used as the vehicle for the test compound, at a final 

concentration of 0.05%, and dimethyl sulfoxide (DMSO) was used as the vehicle for the positive 

controls, at a final concentration of 0.05%. Precipitates were observed after the exposure period 

at 10 μM and 100 μM. Cytotoxicity greater than 20% was not observed. Samples with 

precipitation were not analyzed. 

Testosterone and estradiol levels were measured using HPLC-MS/MS with internal standards. 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. 

Guideline acceptability recommendations and requirements were generally met, including 

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

appropriate induction and inhibition with positive controls. The exceptions include: in Runs 2 

and 3, estradiol production in the blank and DMSO ranged from 29.6-36.2 pg/mL (but were 

greater than 2.5-fold compared to the MDL for estradiol, 5 pg/mL) and induction of testosterone 

with 10 μM forskolin resulted in an average increase of 1.7-fold rather than the 2-fold increase 

Guideline criterion. 

In the three assay runs comparing esfenvalerate treatment to the solvent control (SC), inhibition 

(p≤0.05) of testosterone production was noted at 0.0001, 0.01, and 1 μM, as well as inhibition of 

Page 46 of 70
ESFENVALERATE FINAL 

Page 39 of 62 

estradiol at 0.01 μM. However, these responses were not concentration-dependent; therefore, the 

apparent effects are not considered treatment-related. All other values of testosterone and 

estradiol in the treated cells were similar to the SC. 

Based on hormone responses in the three independent runs, esfenvalerate treatment did not result 

in statistically significant and reproducible alterations in testosterone or estradiol production. 

Uterotrophic Assay; (OCSPP 890.1600) 

Requirement satisfied by OSRI (Sumitomo et al., 2001b and Kunimatsu et al., 2002); see 

Appendix 2.

Page 47 of 70
ESFENVALERATE FINAL 

Page 40 of 62 

APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Published literature and Part 158 general toxicity studies considered relevant for determining the 

potential interaction of esfenvalerate (or fenvalerate) with the endocrine system are summarized 

below. Given the breadth of toxicity data available on fenvalerate and esfenvalerate, the only 

Part 158 studies discussed in this appendix are those that fulfill current guideline requirements 

and are directly relevant to assessing potential endocrine effects (i.e., multiple-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 fenvalerate; 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. In 

these assays, fenvalerate was negative for ERβ activity, but induced a positive ERα agonist 

response with a REC20 of 3.7 x 10-6 M and a positive AR antagonism response with an RIC20 of 

6.9 x 10-6 M. 

In an unpublished report, Sumitomo (2001) fenvalerate was tested in an in vitro hAR (human 

AR) ARTA assay. Construction of a hAR expression plasmid for a reporter gene assay with the 

same sequence as human prostate cDNA was completed (pRc/RSV- hAR Kozak), as well as a 

reporter plasmid for hAR to be used in a reporter gene assay with the androgen responsive 

element (ARE) and the firefly luciferase gene (pGL3-MMTV). A reporter plasmid for a control 

assay constitutively expressing a reporter gene under the transcriptional control of a TK 

promoter was also prepared (pGL3-TK). HeLa cells were maintained in cell culture in an 

atmosphere of 5% CO2:95% air at saturated humidity. 

HeLa cells were plated at a density of 2×l06 cells per 10-cm plate with the assay medium that 

contained 10% (v/v) twice-dextran:charcoal-treated PCS, and incubated overnight. Cells were 

Page 48 of 70
ESFENVALERATE FINAL 

Page 41 of 62 

transfected with 3.75 ng of receptor expression plasmid (pRc/RSV-hAR Kozak) and 3.25 μg of 

reporter plasmid (pGL3-MMTV) per dish. For control assays to evaluate plasmid transfection 

efficiency, cell toxicity of a chemical, non-specific luminescence without receptor, and other 

effects, cells in another plate were transfected with 7.0 μg of pGL3-TK/dish in serum-free 

medium. 

After transfection, cells were plated at a density of 2×104 cells per 100 μL/well in 96-well plates 

with fresh assay medium, which contained 10% (v/v) twice-dextran:charcoal-treated PCS, and 

incubated overnight. Dilutions of DHT (10−12 to 10−6

 M) and fenvalerate (10−5 M) in DMSO 

(final concentration 0.1%) were prepared fresh and added to the transfected cells for the agonist 

assay. The solubility of each chemical was visually confirmed under the light microscope to 

assess precipitation. The selected doses of fenvalerate were determined based on solubility and 

cell toxicity results from a control assay. For the antagonist assay, DHT (100 pM; 10−10 M) was 

incubated with fenvalerate (10−5 M) or the anti-androgen, hydroxyflutamide (10−6 M). Cells 

were cultured for an additional 40-hour period, followed by removal of medium, washing with 

phosphate-buffered saline, and preparation for evaluation by treatment with cell lysis buffer. 

After incubation for 30 minutes at room temperature, luciferase activity was assayed with a 

Lumat LB 9501 luminometer. 

To confirm the transcriptional activation of the reporter gene, luciferase activity was determined 

with DHT (1 pM to 1 μM; 10−12 to 10−6

 M). Luciferase activity was present at 10 pM DHT 

(3-fold induction) and saturated at 1 nM (approximately 34-fold induction). Based on these 

results, a DHT concentration of 100 pM was selected for evaluation of antagonistic activity. The 

relative fold-induction of luciferase by 10 μM fenvalerate was 0.9±0.3. Therefore, fenvalerate 

was negative in the reporter gene assay with HeLa cells for evaluation of agonistic activity. The 

antagonistic activity of hydroxyflutamide (1 μM) decreased DHT luciferase activity by 80%. 

There was no difference between vehicle control and 10 μM of fenvalerate, indicating that 

fenvalerate was also negative for antagonistic activity in this assay. 

In a published study, Xu et al. (2006) examined the AR activity of several pesticides (including 

fenvalerate) in a hAR reporter gene (ARTA) assay. A reporter plasmid mouse mammary tumor 

virus (pMMTV)-CAT based on the pCAT3-Basic vector was constructed by inserting the 

oligonucleotides of MMTV sequences containing four ARE sequences and a TATA promoter 

into the pCAT3-basic to construct the plasmid pMMTV-CAT. The plasmid pSV-β-galactosidase 

(internal control for transfection efficiency and cytotoxicity of the test substances) and the hAR 

expression plasmid AR/pcDNA3.1 containing the full open reading frame of hAR cDNA were 

also prepared. The CV-1 cell line (African monkey kidney cell) was maintained at 37°C in an 

atmosphere of 5% CO2:95% air at saturated humidity, and transfected by incubation for 24 hours 

with the three plasmids. 

Page 49 of 70
ESFENVALERATE FINAL 

Page 42 of 62 

The reported gene assay was conducted with various concentrations of DHT and fenvalerate 

(>99% purity) dissolved in medium were added for measurement of agonistic activity. Initial 

stock solutions were prepared in absolute ethanol; final ethanol concentrations in culture medium 

did not exceed 0.1% (v/v). In order to determine AR antagonistic activity, 0.5 nM DHT was 

added along with fenvalerate. Cells were harvested after incubation for 24 hours, lysed, 

centrifuged, and the cell lysates were analyzed. Chloramphenicol acetyltransferase (CAT) was 

measured with a commercial CAT-ELISA kit, and β-galactosidase (β-Gal) activity was 

determined with the β-galactosidase (β-Gal) Enzyme Assay System. The amount of CAT for 

each lysate was normalized by the β-Gal activity. 

DHT was assayed over a concentration range of 10−12 to 10−6

 M. DHT induced CAT activity in 

a concentration-dependent manner, with CAT increased at 10−10 M and a maximal induction of 

approximately 79-fold achieved at 10−8 M. The median effective concentration (EC50) value for 

DHT, determined from the dose–response curve, was 3.9×10−10 M. DHT was not able to induce 

a transactivation response in the absence of AR (CAT in non-transfected CV-1 cells was 

0.20±0.021 normalized CAT unit vs. 0.20±0.026 after DHT treatment with non-transfected CV-1 

cells). Pesticide treatments were conducted at ≤10−5

 M to avoid cytotoxicity in the assay. 

Fenvalerate treatment at 10−7 to 10−5 M did not demonstrate transcriptional activation in the 

assay. Fenvalerate was also tested for inhibition of CAT activity induced by 0.5 nM DHT. 

Fenvalerate did not affect DHT-induced CAT activity at a fenvalerate concentration of 10−7

 M, 

but CAT activity was decreased (p<0.01) at 10−5

 M, with an IC50 value of 7.10×10−5 M. 

AR Binding

Sumitomo (2001) also tested fenvalerate in in vitro hAR binding assays (a yeast two-hybrid 

assay and a hAR Competitive Binding assay). 

Construction of a plasmid for hAR in a yeast two-hybrid assay was conducted with cDNA 

containing the LBD of hAR (amino acids No. 623-919). The plasmid (pGBT9-hARLBD) 

expresses the fusion protein of the LBD of hAR and the DNA binding domain of GAL4. Yeast 

cells (Y190) were transformed with two plasmids (pGBT9-hARLBD and pGAD424-TlF2RID), 

and yeast transformants were grown overnight at 30°C. Dilutions of DHT (10 pM to 1 μM; 

10−11 to 10−6

 M) and fenvalerate (10−5 M) in DMSO were prepared fresh and added to the 

transformed cells in 96-well plates for the agonist assay. After incubation for 4 hours at 30°C, 

induction of β-galactosidase activity in the yeast cells was measured by chemiluminescent 

detection. Chemiluminescence by β-galactosidase was present at 1 nM DHT and activity was 

saturated at 10 nM (approximately 40-fold induction). Based on these results, a DHT 

concentration of 10 nM was selected for evaluation of antagonistic activity. 

The relative fold-induction of β-galactosidase by 10 μM fenvalerate was 1.0±0.1. Therefore, 

fenvalerate was negative in the yeast two-hybrid assay for evaluation of agonistic activity. The 

antagonistic activity of hydroxyflutamide (1 μM) decreased β-galactosidase activity by 70%. 

Page 50 of 70
ESFENVALERATE FINAL 

Page 43 of 62 

There was no difference between vehicle control and 10 μM of fenvalerate, indicating that 

fenvalerate was also negative for antagonistic activity in this assay. 

MDA-MB-453 cells (used for the hAR Competitive Binding assay) were maintained in culture at 

37°C in an atmosphere of 5% CO2:95% air at saturated humidity. Cells were grown to 70-80% 

confluence in tissue culture dishes. Competitive binding was determined by testing 

concentrations of fenvalerate (10−9

 to 10−5 M) and unlabeled DHT against [3H]-DHT (50 nM). 

Binding was determined by LSC after incubation for 1 hour at 4°C. DHT binding inhibition was 

assessed in the assay by the addition of non-labeled DHT and [3

H]-DHT (100 pM to 1 μM; 10−10

to 10−6 M). Binding inhibition of [3

H]-DHT was decreased by 60% at 1 nM (100% at 100 pM) 

and was decreased approximately 97% at 1 μM. Inhibition of [3H]-DHT binding was not 

observed over a fenvalerate concentration range of 10−9

 to 10−5 M; therefore, fenvalerate was 

negative in this Competitive Binding assay for hAR. 

In another published study, Eil and Nisula (1990) investigated pyrethroid (including fenvalerate) 

binding to human skin fibroblast AR and to sex hormone binding globulin (SHBG). Fibroblast 

cultures were established from genital skin specimens obtained from neonates at routine 

circumcision. Fenvalerate (80-100% purity) test concentrations were prepared in absolute 

ethanol; three concentrations between approximately 10−5 and 10−4 M were tested in the 

Competitive Binding assay, and a concentration of 10−4

 was tested in the SHBG assay. 

The binding of fenvalerate to the AR was determined by competitive displacement of [3H]R1881 

from receptor sites on cultured human fibroblasts with a dispersed, intact cell system. Binding 

studies were conducted at 22°C to minimize metabolism of the radioligand and competitors. The 

Competition Binding assays were conducted with 0.5-1.0 nM [3

H]R1881 and increasing amounts 

of the nonradioactive compounds. In some experiments, fixed amounts of competitor or vehicle 

(ethanol) were added to increasing amounts (0.1-1.0 nM) of [3

H]R1881. Non-specific binding 

was determined in the presence of 5×10−7 M R1881, and was subtracted from whole cell binding 

of [3

H]R1881 obtained in the absence of any inhibitor to assess binding to high affinity sites 

(specific binding). The amount of R1881 required for 50% displacement of [3H]R1881 was 

determined to be 0.50±0.08 nM. The RBA of fenvalerate was calculated as the ratio of the 

concentration of unlabeled R1881 to the concentration of fenvalerate required for 50% 

displacement of high affinity binding of [3

H]R1881. The association constant of pyrethroid 

binding to the androgen receptor was evaluated from this RBA; the previously determined 

association constant of R1881 for binding to the AR in this system is 2×109 M−1. 

The SHBG assay was conducted with pooled citrated plasma from which endogenous steroids 

had been removed by charcoal adsorption. Binding to SHBG was determined at 37°C with 

[

3

H]testosterone and a solid-phase method with Con-A-Sepharose. Cortisol (2 μg) was included 

in order to minimize the possible contribution of transcortin to total [3H]testosterone binding of 

Page 51 of 70
ESFENVALERATE FINAL 

Page 44 of 62 

the absorbed glycoprotein fraction. Non-specific or low affinity binding was determined in the 

presence of 10−5 M unlabeled testosterone. 

Fenvalerate demonstrated competition for AR binding with a similar slope to unlabeled R1881, 

but with an approximate 10−5

 lower potency. Fenvalerate had an intermediate potency with a Ki

of 1.8×10−4

 M and a RBA of 0.000027. Fenvalerate at a concentration of 10−4 M did not 

demonstrate any binding capacity for the SHBG in this assay. 

ERTA/ ER Binding/Cell Proliferation 

In a published study, Saito et al. (2000) investigated the activity of several pyrethroids 

(including fenvalerate, purity >93%) on the ERα pathway using three different assay systems: a 

mammalian ERTA assay with HeLa cells and a luciferase reporter, a yeast two-hybrid system 

(Nishikawa procedure), and hER competitive ligand-binding with fluorescence polarization (Pan 

Vera). Some of the assays with fenvalerate were conducted as part of an assessment of 

ER/AR/PR binding (Sumitomo et al., 2001). Construction of an hER expression plasmid for a 

reporter gene assay with the same sequence as human prostate cDNA was completed (pRc/RSVhERα Kozak), as well as a reporter plasmid for hER to be used in a reporter gene assay with the 

TATA vector and the firefly luciferase gene (pGL3-TATA-ERE×5). A reporter plasmid for a 

control assay constitutively expressing a reporter gene under the transcriptional control of a TK 

promoter was also prepared (pGL3-TK). HeLa cells were maintained in cell culture in an 

atmosphere of 5% CO2:95% air at saturated humidity. Construction of a plasmid for hER in a 

yeast two-hybrid assay was conducted with cDNA containing the LBD of hERα (amino acids 

No. 249-596). The plasmid (pGBT9-hERαLBD) expresses the fusion protein of the LBD of hER 

and the DNA binding domain of GAL4. Yeast cells (Y190) were transformed with two plasmids 

(pGBT9-hERαLBD and pGAD424-TlF2RID), and yeast transformants were grown overnight at 

30°C. 

Positive controls were run for agonism (17β-estradiol and 4-nonylphenol) and antagonism 

(4-hydroxytamoxifen). 17β-estradiol displayed the expected responses in all three assays, with 

dose-dependent induction in the ERTA and the yeast two-hybrid assay, and dose-dependent 

inhibition (displacement of fluorescence standard) in the competitive ligand-binding assay. 

Fenvalerate was tested at 10−7

, 10−6, and 10−5 M in the competitive binding assay, at 1 and 

10 μM in the HeLa cell assay, and 10 μM in the yeast two-hybrid assay; tested dose(s) were 

determined on the basis of solubility. The results were displayed graphically; negative results for 

estrogenic and anti-estrogenic activity with all tested pyrethroids (including fenvalerate) were 

observed in all assays. 

Uterotrophic Assay 

In a published study examining the potential estrogenic effects of several pyrethroid pesticides 

(Kunimatsu et al., 2002), permethrin, fenvalerate (97.3% purity), and esfenvalerate (94.2% 

Page 52 of 70
ESFENVALERATE FINAL 

Page 45 of 62 

purity; active and total isomers not reported) were assessed in an in vivo Uterotrophic assay. 

Only the results for fenvalerate and esfenvalerate will be discussed. All test substances were 

administered in corn oil. 

Five-week old female Crl:CD (SD) IGS rats were maintained under standard laboratory 

conditions and provided with standard laboratory diet and water ad libitum. After an acclimation 

period of seven days, the rats were ovariectomized on PND 42 and allowed to recover for two 

weeks prior to initiation of dosing on PND 56. After the recovery period, rats were monitored 

for estrus cycling (only rats in diestrus or metestrus were selected), weighed, and randomly 

assigned to dose groups (n=6). Test and reference substances were suspended or dissolved daily 

in vehicles (corn oil including 10% ethanol for ethynyl estradiol; corn oil only for other 

compounds). Clinical signs, body weight, food consumption, and weights of liver and kidneys 

were assessed as indices of systemic toxicity. Clinical signs including any abnormal appearance 

of behavior were recorded twice a day in each animal. Food consumption for each cage was 

measured over 2 to 3 consecutive days. 

Fenvalerate, esfenvalerate, ethynyl estradiol, or methoxychlor were administered to 

ovariectomized rats by oral gavage for 3 days. The doses of fenvalerate and esfenvalerate were 

selected on the basis of a dose-finding study. Ethynyl estradiol (0.03 mg/kg/day) and 

methoxychlor (125 mg/kg/day) were administered as reference chemicals for estrogenicity; doses 

of fenvalerate and esfenvalerate were 20, 40, and 80 or 5, 10, and 20 mg/kg/day, respectively. 

One day after the final administration, rats were euthanized by blood withdrawal from the 

abdominal aorta under light ether anesthesia. The uterus, liver, and kidneys were dissected and 

weighed after careful trimming, and the wet (with luminal contents) and blotted (without luminal 

contents) uterine weights were determined. Each uterus was individually processed to carefully 

blot the excess fluid as follows: both uterine horns were pierced, placed on filter paper, and 

gently pressed to absorb the luminal fluid. 

Ethynyl estradiol did not demonstrate systemic toxicity based on clinical signs, body weight, 

food consumption, and liver and kidney weights. Methoxychlor also did not show any clinical 

signs, but decreased (p<0.01) final body weight (↓10%), food consumption (↓37%), and absolute 

liver weight (↓16%). Treatments with ethynyl estradiol and methoxychlor increased (p<0.01) 

both wet and blotted uterine weights as expected (↑74 and 73%, and ↑185 and 140, respectively). 

The highest doses of fenvalerate and esfenvalerate each exhibited typical clinical signs of 

pyrethroid exposure such as tremor; the daily incidence of tremor was 4-6/6 rats in the 

80 mg/kg/day fenvalerate group and 2-4/6 rats in the 20 mg/kg/day esfenvalerate group. 

However, no changes were observed in final body weight, food consumption, or liver, kidney, 

and uterine weights in all treated groups. One minor exception was a slight, non-dose-dependent 

decrease (p<0.05) in food consumption at the medium dose of fenvalerate. Under these 

Page 53 of 70
ESFENVALERATE FINAL 

Page 46 of 62 

experimental conditions, fenvalerate and esfenvalerate had no effects on uterine weights at any 

tested doses. 

Subchronic Oral Toxicity in Rodents (Rat; OCSPP 870.3050) 

In a subchronic oral toxicity study in rats (MRID 40215601), esfenvalerate (98.6% purity; 

82.9% Aα) was administered for seven weeks (interim sacrifice; 10/sex/dose) or thirteen weeks 

(15/sex/dose) in the diet to SD rats at nominal concentrations of 0, 75, 100, 125, and 300 ppm 

(equivalent to 0, 0.88, 3.8, 7.5 and 15 mg/kg/day for males; 0, 1.04, 4.3, 8.5, and 17.2 mg/kg/day 

for females). After seven or thirteen weeks, all rats were weighed, and sacrificed for gross and 

histopathological evaluation. Organs that were weighed included the liver, kidneys, testes, 

ovaries, and uterus; gross and microscopic pathological examinations were conducted on these 

organs as well. 

There was no mortality during the study. Neurological signs were noted after Week 10; 

hyperactivity was observed in females only (n=5) between Weeks 10 and 13. During Weeks 11, 

12, and 13, jerky leg movements were observed in 6, 5, and 12 high-dose rats, respectively (both 

sexes). Males and females in the high-dose group had decreased mean body weight gains 

compared to control throughout the study; decreases were only significant (significance not 

reported, with only selected data reported) for males at Weeks 1 and 2 and for females at Weeks 

3, 4, 5, 7, and 12. Slight decreases in food consumption were considered negligible. No 

treatment-related effects were noted for hematology, clinical chemistry, or urinalysis parameters. 

Changes in relative liver weights in males were not considered to be related to treatment. 

Treatment-related increases (significance not reported) in absolute kidney weights were seen in 

high-dose females (↑8 and 9%), and relative kidney weights were increased in high-dose males 

and females. Gross pathology was negative, and microscopic histopathology was not conducted. 

The LOAEL is 300 ppm (7.5 mg/kg/day for males; 8.5 mg/kg/day for females) based on the 

neurological clinical signs in both sexes. The NOAEL is 125 ppm (3.8 mg/kg/day for males; 

4.3 mg/kg/day for females). 

Subchronic Oral Toxicity in Rodents (Mouse; OCSPP 870.3050) 

In a subchronic oral toxicity study in mice (MRID 41359701), fenvalerate (95.5% purity; 

Aα=24.2%) or esfenvalerate (94.5% purity; Aα=87.2%) were administered to B6C3F1 mice 

(12/sex/group) at concentrations of 0 (vehicle; corn oil); 50, 150, or 500 (esfenvalerate); or 

2000 ppm fenvalerate in the diet (achieved doses of esfenvalerate were 10.5/12.6, 30.5/36.8, and 

106/113 mg/kg/day; achieved doses of fenvalerate were 422/462 mg/kg/day in males and 

females, respectively) for three months. Organs that were weighed included the liver, kidneys, 

testes, ovaries, pituitary, adrenal, and thyroid. Gross and microscopic pathological examinations 

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

mammary gland. 

Page 54 of 70
ESFENVALERATE FINAL 

Page 47 of 62 

No deaths occurred during the study. Clinical signs were observed in the high-dose 

esfenvalerate group and the fenvalerate group: fibrillation, tremors, convulsions, 

hypersensitivity, abnormal gait, and salivation (Week 1); scratching and licking (after Week 3); 

and alopecia, scabs, and sores (after Week 4). Hypersensitivity decreased after Week 4. 

Decreased body weight gains were noted throughout the study in both sexes (males>females) in 

the high-dose esfenvalerate group and the fenvalerate group. Animals in both groups had 

decreased body weight gains during Days 0-28 (esfenvalerate ↓43.5-52.8%; fenvalerate ↓24.1-

27.6%), with diminished decreases (<10%) in the females dosed with fenvalerate for the 

remainder of the study, and the fenvalerate males and high-dose esfenvalerate females by 

Days 63-90. No treatment-related differences in food consumption were observed. Decreases in 

water intake were noted for the first few weeks, but increased by Week 5. 

Absolute liver weights were decreased in the high-dose esfenvalerate males and females (↓13 

and 9%, respectively), but were increased in the fenvalerate males and females (↑9 and 13%, 

respectively). Relative liver weights were also increased in the fenvalerate-treated animals. 

Some increases were also observed in absolute and relative spleen weights, and absolute/relative 

salivary gland weights in the high-dose esfenvalerate and fenvalerate males and females. Gross 

pathology findings were primarily associated with the long-term effects of scratching (alopecia, 

scabs, and sores), as were many non-neoplastic lesions (hyperkeratosis, dermatitis, ulceration, 

and hair follicular cysts). An increased incidence of stomach lesions in the high-dose 

esfenvalerate animals was also noted, but no neoplastic tissue was observed. Effects seen at the 

high-dose of esfenvalerate were similar to those present due to the 2000 ppm dose of fenvalerate. 

The LOAEL is 500 ppm for esfenvalerate (equivalent to 106/113 mg/kg/day, males/females), 

based on clinical signs of toxicity in both sexes, increased water intake, skin lesions, and 

stomach lesions. The NOAEL is 150 ppm for esfenvalerate (equivalent to 30.5/36.8 mg/kg/day, 

males/females). 

Subchronic Dermal Toxicity in Rodents (Rat; OCSPP 870.3200) 

In a 21-day dermal toxicity study (MRID 45275401) esfenvalerate (98.6% purity; % Aα not 

reported) was applied to the shaved skin of Crl:CD®(SD)IGS BR rats (10/sex/dose group) at 

nominal doses (range) of 0, 25 (20 to 30), 125 (105 to 155), 500 (403 to 642), or 1000 (821 to 

1205) mg/kg/day, 6 hours/day, over a 21-day period. The rats were assessed for clinical signs, 

dermal effects, and FOB and motor activity were assessed at pretest and just prior to sacrifice. 

Organs that were weighed included the liver, kidneys, testes, epididymides, ovaries, uterus, and 

adrenal. Gross and microscopic pathological examinations were conducted on these tissues as 

well as the pituitary, thyroid, prostate, seminal vesicles, and mammary gland. 

All rats survived the 21-day dosing period. Abnormal hind limb gait was noted during the daily 

assessment for clinical signs and was noted in 0, 1, 10, 10 and 10 females and 1, 0, 5, 10 and 10 

males in the control, low-, medium-low, medium-high, and high-dose groups, respectively. 

Page 55 of 70
ESFENVALERATE FINAL 

Page 48 of 62 

Abnormal gait generally was seen on Day 0 to about Day 6 or 7 with only occasional 

occurrences on later days, and was not noted in the FOB assessment prior to sacrifice. The 

single incident in the low-dose females was noted on Day 2 when the rats treated at higher doses 

also displayed this condition. However, the single incident in the control male group was noted 

on Day 10 after most of the males stopped showing the condition. Since abnormal gait is 

consistent with esfenvalerate toxicity and all of the females at the next higher dose developed 

this condition, the single incident at 25 mg/kg/day is considered related to treatment. Other 

clinical signs noted included hyperactivity/hyperreactivity and vocalization in the medium-high 

and high-dose females. Motor activity data were confounded by the pretest baseline data being 

elevated in the higher dose groups, but even after accounting for the higher baseline there was an 

increased mean number of movements and duration of movements in the female medium-high 

and high-dose groups. However, it could not be determined whether the property of 

esfenvalerate to produce a dermal sensory stimulation may have been related to certain reactions 

in females such as hyperactivity and reactivity, corneal opacity, and increased motor activity at 

doses of 500 mg/kg/day and above, or if these were the result of systemic esfenvalerate after 

dermal absorption. No deleterious, treatment-related effects on body weight, body weight gain, 

food consumption, organ weights, or microscopic pathology were noted. 

The systemic NOAEL is 25 mg/kg/day, and the systemic LOAEL is 125 mg/kg/day based on 

abnormal hind limb gait. Note: It should be noted that 25 mg/kg/day is regarded as a LOEL as 

one female was affected. However, because the effect was no cumulative, this single incident of 

abnormal gait in one low-dose animal was not considered an adverse effect. The LOAEL for 

local site of application for dermal irritation is >1000 mg/kg/day. However, no NOAEL and 

LOAEL is being assigned for sensory stimulation following dermal application since HED does 

not have established criteria for evaluating this endpoint. 

Subchronic Dermal Toxicity in Non-Rodents (Rabbit; OCSPP 870.3200) 

In a 21-day dermal toxicity study (MRID 42325101) fenvalerate (95.4% purity) was applied to 

the shaved skin of New Zealand White rabbits (5/sex/dose group) at nominal doses of 0, 100, 

300, or 1000 mg/kg/day, 6 hours/day, over a 21-day period. Organs that were weighed included 

the liver, kidneys, adrenal, and testes. Gross and microscopic pathological examinations were 

conducted on these tissues as well as the pituitary, thyroid, prostate, seminal vesicles, 

epididymides, ovaries, and uterus. 

Four animals died on study, one male and one female each in the control and medium-dose 

groups (un-related to treatment). Moderate erythema was present in all animals except the 

control and low-dose males, with no dose response. Mild edema, superficial necrosis, and scar 

tissue were noted in medium-high and high-dose females. No deleterious, treatment-related 

effects on body weight, body weight gain, food consumption, organ weights, or microscopic 

pathology were noted. 

Page 56 of 70
ESFENVALERATE FINAL 

Page 49 of 62 

The systemic NOAEL is >1000 mg/kg/day; the LOAEL is not determined. The NOAEL for 

local site of application for dermal irritation is 100 mg/kg/day, and the LOAEL is 

300 mg/kg/day. 

Prenatal Development in Rodents (Rat; OSCPP 870.3700) 

In a developmental toxicity study in rats (MRID 43211504), esfenvalerate (97.1% purity; % Aα

not reported) was administered by gavage to mated SD (Crl:CD®BR) rats (25/dose group) at 

doses of 0 (corn oil), 2.5, 5, 10, or 20 mg/kg/day on GDs 6-15, inclusive. The rats were 

sacrificed at GD 21 of gestation and subjected to gross necropsy. All fetuses were weighed, 

sexed, crown-rump measurement taken, and examined for gross external alterations. One-half of 

the fetuses in each litter were examined for visceral soft tissue alterations by the Wilson 

technique. The remaining fetuses in each litter were examined for skeletal alterations. 

Maternal toxicity was observed at all doses. Behavioral/CNS incidences in the low-dose group 

included erratic jerking and extension of forelimbs (22/25), rapid side-to-side head movement 

(19/25), and excessive grooming (22/25). Additional signs at the medium-low dose consisted of 

hindlimb jerking and soft/mucoid stools. In the medium-high dose group, further signs included 

hypersensitivity to touch and tremors. Finally, additional clinical signs in the high-dose dams 

consisted of high carriage, goose-stepping ataxia, ataxia, and convulsions. Incidence and 

frequency of these behaviors increased with increasing dose. Dams in the pilot study displayed 

similar behaviors at esfenvalerate concentrations of ≥4 mg/kg/day, but not ≤3 mg/kg/day. 

Decreased (p<0.05) body weight gains of 8% were observed in the medium-high and high-dose 

dams during GDs 6-15. Overall body weight gains were not affected, and no treatment-related 

changes in food consumption were reported. There were no treatment-related findings at 

necropsy. 

Reproductive (cesarean section) parameters were not affected by treatment with esfenvalerate; 

there were no abortions or early deliveries. No treatment-related effects were noted during 

external, visceral, or skeletal examinations. The maternal LOEL is 2.5 mg/kg/day, based on 

behavioral/CNS clinical signs, and the maternal NOEL is 2.0 mg/kg/day (developed from the 

pilot study results). The developmental LOEL is not determined (>20 mg/kg/day), and the 

developmental NOEL is ≥20 mg/kg/day. 

Prenatal Development in Non-Rodents (Rabbit; OCSPP 870.3700) 

In a developmental toxicity study in rabbits (MRID 43211503), esfenvalerate (97.1% purity; % 

Aα not reported) was administered to artificially-inseminated, female New Zealand White 

rabbits (20/dose group) by gavage at doses of 0 (corn oil), 3, 10 or 20 mg/kg/day from GDs 7-19, 

inclusive. On GD 29, all surviving does were sacrificed, and all fetuses were weighed, sexed, 

crown-rump measurements were taken, and external examinations were conducted. Heads from 

Page 57 of 70
ESFENVALERATE FINAL 

Page 50 of 62 

one-half of the fetuses in each litter were examined for visceral soft tissue alterations by the 

Wilson technique. All carcasses were eviscerated and processed for skeletal examination. 

One high-dose rabbit died on GD 17, but the death did not appear to be related to treatment. 

Maternal toxicity was observed at all doses. Behavioral/CNS incidences in the low-dose group 

included erratic jerking and extension of forelimbs (11/20), rapid side-to-side head movement 

(2/20), sneezing (3/20), and excessive grooming (11/20). Additional signs at the medium-dose 

consisted of hindlimb jerking (6/20) and hypersensitivity to touch (1/20). Further clinical signs 

in the high-dose animals consisted of tremors (10/20), ataxia (9/20), and diarrhea (2/20). The 

incidence and frequency of these behaviors increased with increasing dose. Dams in the pilot 

study displayed similar behaviors at esfenvalerate concentrations of ≥3 mg/kg/day, but not 

2 mg/kg/day. Body weight gain was decreased during the dosing period (GDs 7-10 and 16-19) 

at the low (NS; ↓120 and 21%), medium (p<0.01 and NS; ↓720 and 70%) and high (p<0.0 and 

NS; ↓1380 and 81%) doses, with compensatory increases during GDs 19-29 (overall gains 

ranged from ↓14 to ↑28%). Decreases (p<0.01 or 0.05) in food consumption were also noted in 

the medium- and high-dose groups during GDs 7-13, but overall food consumption was within 

±10% of control values. There were no treatment-related findings at necropsy. 

Reproductive (cesarean section) parameters were generally not affected by treatment with 

esfenvalerate. Three does aborted during the study (one each in the control, low-, and high-dose 

groups), but a relation to treatment was not established. No treatment-related effects were noted 

during external, visceral, or skeletal examinations. The maternal LOAEL is 3.0 mg/kg/day, 

based on behavioral/CNS clinical signs, and the maternal NOAEL is 2.0 mg/kg/day (developed 

from the pilot study results). The developmental LOAEL is not determined (>20 mg/kg/day), 

and the developmental NOAEL is ≥20 mg/kg/day. 

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

In a two-generation reproduction in rats (MRID 43489001, 1994), esfenvalerate (98.8% purity; 

% Aα not reported) was administered to Crl:CD (SD) BR rats (30/sex/dose) at nominal 

concentrations of 0, 75, 100, or 350/150 ppm (dietary concentration reduced to 150 ppm after 

approximately four months of dosing). Mean compound intake for males was 0, 5.10, 6.70, and 

18.8 mg/kg/day and for females was 0, 5.47, 7.27, and 25.1 mg/kg/day in the 0, 75, 100 and 

350 ppm groups respectively (intake was not calculated for the 150 ppm, high-dose, F1

generation). Two successive generations were produced, each consisting of one breeding (two 

sets of litters). 

Seventy-three days after initiation of the study, each P1 female was mated with a randomly 

selected male in the same dose group until a copulatory plug was observed or until three weeks 

had passed. F1 rats were mated in a similar manner after they had been administered their diets 

for 105 days. Siblings were not mated. 

Page 58 of 70
ESFENVALERATE FINAL 

Page 51 of 62 

P1 males in the high-dose group exhibited abnormal gait/mobility, alopecia, scabs and sores. 

Two males in the high-dose group died; one death was due to genitourinary tract inflammation, 

but the second death was believed to be due to treatment-related ulcerative dermatitis. Highdose P1 females exhibited abnormal gait. One P1 female in the medium-dose group was 

sacrificed in extremis (attributed to dystocia). F1 males and females in the high-dose group 

exhibited various clinical signs of neurotoxicity (abnormal gait/mobility, ataxia, barrel rolls, 

general spasms, hyperactivity, hypersensitivity, popcorn seizures, tremors and vocalization) and 

signs involving the skin (alopecia, swollen face, scabs and sores). These clinical signs were 

observed in males in the high-dose group (all dead or sacrificed in extremis by Day 34); death 

was attributed to treatment. These signs were so severe in females that all but nine females in 

the high-dose group were dead or were sacrificed in extremis by Day 33 (all dead or sacrificed in 

extremis by Day 43). Clinical signs involving the skin were observed in low- and medium-dose 

animals. 

Body weight data was not available for review, but body weight gain data for selected periods, 

and overall body weight gain data, for the P1 and F1 animals were available. Minimal effects 

occurred in the low- and medium-dose animals throughout the study. Body weight gains in the 

high-dose (350 ppm) P1 animals during the premating period were generally decreased during 

the initial weeks, but overall body weight gains in high-dose P1 males and females were only 

decreased (p<0.05) by 19 and 32%, respectively. Decreased body weights in the high-dose 

females during gestation were attributed to pre-existing conditions. Body weight gain was 

decreased (p<0.05) in the high-dose P1 females during lactation. Body weight gains in the highdose (150 ppm) F1 animals during the premating period were slightly decreased during the initial 

weeks, but increased mortality occurred in high-dose F1 animals by approximately Week 5. 

Food consumption was generally decreased in the high-dose animals. 

There were no differences in the mating indices, fertility indices, or gestation length of the P1 or 

F1 groups. The mean number of F1 pups/litter on PNDs 4 (preculling), 7, 14, and 21 was 

decreased at the high dose (350 ppm). In addition, the viability (↓22%), lactation (↓44%) and 

litter survival indices (↓22%) were decreased (p<0.05) compared to control. F1 pup weights were 

decreased (p<0.05) in the high-dose group at each time point (↓14-15% on PNDs 0-4 and ↓28-

30% on PNDs 7-21). F1 pup weights were also decreased in the medium-dose group on PNDs 

14 and 21 by approximately 6%. The mean number of F2 pups/litter born alive and alive on 

PND 4 pre- and postculling was decreased (p<0.05; ↓12-13%) in the medium-dose group (data 

for the high-dose group was not available due to mortality). F2 pup weights were decreased 

(p<0.05) in the medium-dose group at PNDs 4 (post-culling), 7, 14, and 21, by up to 5% on 

PND 21. Some clinical observations were noted in the high-dose F1 pups, including abnormal 

gait/mobility, tremors, and weakness. 

Gross pathology of the parental animals showed skin ulcerations (and related microscopic 

pathology) in a few P1 animals and a dose-dependent response in a greater number of F1 parents. 

Page 59 of 70
ESFENVALERATE FINAL 

Page 52 of 62 

No differences were noted in gross necropsy findings of offspring. The LOAEL for parental 

toxicity is 75 ppm (5.10/5.47 mg/kg/day, [males/females]) based on decreases in mean body 

weights of F1 females and an increased incidence of skin lesions; the NOAEL was not 

determined. The LOAEL for reproductive toxicity is 100 ppm (6.70/7.27 mg/kg/day, 

[males/females]) based on decreases in F1 pup weights on PND 21 of lactation, decreases in litter 

size and F2 pup weights, and an increased incidence of subcutaneous hemorrhage. The NOAEL 

is 75 ppm (5.10/5.47 mg/kg/day, [males/females]). 

In a supplemental non-guideline, one-generation (two-litter) reproduction toxicity study (MRID 

48076104), esfenvalerate [97.3% (total isomer); 86.0% (active isomer)] was administered in the 

diet to Crj:CD(SD)IGS SPF rats (24/sex/dose group) by pellets at concentrations of 0, 20, 40, or 

100 ppm (equivalent to 0/0, 0.98/1.29, 1.89/2.48, and 4.74/6.34 mg/kg/day in males/females). 

The parental males were fed the test diets continuously until termination (approximately 

25 weeks). The parental females were fed the test diets continuously during pre-mating 

(10 weeks for the F1a litters, 7 weeks for the F1b litters), mating, gestation, and lactation. On 

PND 21, pups (24/sex/dose group) were selected from the F1a and F1b litters to continue as 

F1 adults. The F1a adults were fed the test diets for 203-205 days; the F1b adults were fed the test 

diets for 105 days. This supplemental study was conducted because in the two-generation 

reproduction toxicity study with esfenvalerate (MRID 43489001), body weights were decreased 

in the F1 generation at 75 ppm (the lowest dose tested), and a NOAEL was not determined. The 

effect at the low dose was thought to have been due to reduced food consumption due to skin 

paresthesia caused by esfenvalerate in pulverized diet. This study was conducted using 

pelletized diet to reduce dermal exposure to the test material and thus determine a NOAEL. 

Two P males (low- and medium-dose groups) were sacrificed in extremis. One P female each in 

the control and low-dose groups was found dead on GD 23 of production of F1b pups, and 

another low-dose P female was euthanized on GD 23 of production of the F1b pups due to the 

duration of delivery (>24 hours). None of these deaths were considered to be treatment-related. 

Additionally, there were no treatment-related effects on clinical signs of toxicity, food 

consumption, body weight, or macroscopic pathology. In the high-dose males, body weight gain 

was decreased (p<0.05) during the pre-mating period (↓10%). In the high-dose females, body 

weight gains were decreased (p<0.05) for Days 1-15 (↓10%) and 1-22 (↓10%) of pre-mating for 

the F1a litters, but the overall body weight gain for the pre-mating period was not affected. The 

LOAEL for parental toxicity is 100 ppm (equivalent to 4.74/6.34 mg/kg/day in males/females) 

based on reduced body weight gain in both sexes. The NOAEL is 40 ppm (equivalent to 

1.89/2.48 mg/kg/day in males/females). 

There were generally no treatment-related effects on reproductive performance. In the 

production of the F1b litters, the fertility indices were lower (non-dose-dependent) in the control 

and all treated groups (27-72%; p<0.01 at the medium dose, ↓63%). The fertility index for the 

F1a litters was also low (76-87%). This effect was considered to be due to the advanced age of 

Page 60 of 70
ESFENVALERATE FINAL 

Page 53 of 62 

the parental animals at the time of mating for the production of the F1b litters. The LOAEL for 

reproductive toxicity was not observed. The NOAEL for reproductive toxicity is 100 ppm 

(equivalent to 4.74/6.34 mg/kg/day in males/females). 

F1a offspring body weights were decreased in males and females on PNDs 0 and 4 in the highdose group, but weights recovered through PND 21. After weaning, body weights (PNDs 21, 

112, and 244) and body weight gain (PNDs 21-244) in females selected as F1a adults were 

decreased (p<0.05 or NS) in the high-dose group. Non-dose-dependent decreases (p<0.05 or 

NS) were observed in the low- and medium-dose males and females selected as F1b adults, but 

decreases were <10%. The LOAEL for offspring toxicity is 100 ppm (equivalent to 4.74/6.34 

mg/kg/day in males/females) based on decreased body weight and body weight gain in females 

post-weaning. The offspring NOAEL is 40 ppm (equivalent to 1.89/2.48 mg/kg/day in 

males/females). 

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat; OCSPP 870.4300) 

In a chronic toxicity/carcinogenicity study in rats (MRIDs 00082244 and 00111888), 

fenvalerate (98% purity) was administered in the diet to SD rats at nominal concentrations of 0 

(vehicle control, hexane; 183/sex), 1, 5, 25, or 250 ppm (93/sex/dose group; estimated intakes of 

0, 0.05, 0.25, 1.25, or 12.5 mg/kg/day) for approximately two years. Satellite control and highdose groups administered 0 or 500 ppm (22/sex/dose group) were sacrificed after three (interim) 

and six months of dosing. At the interim sacrifices or at study termination, all selected or 

surviving rats were examined for gross and histopathological evaluation. Interim sacrifices at 3, 

6, 12, and 18 months were conducted on the main study animals in the control, 25, and 250 ppm 

dose groups. Select organs weighed at necropsy included the liver, kidneys, testes, adrenals, and 

brain. Gross and microscopic pathological examinations were conducted on these tissues, as 

well as the pituitary, thyroid, prostate, ovaries, uterus, and mammary gland. 

There were no treatment-related differences in survival. Transient clinical signs were attributed 

to aging or injury. Body weight determinations at 3, 6, 12, and 18 months and study termination 

showed occasional significant differences (p<0.05) compared to control (increases or decreases). 

Body weight changes in males were ≤6%; females in the low-dose group exhibited a 7% 

decrease at Week 77 and the 500 ppm group had a 10% decrease after three months. Food 

consumption was considered unremarkable. No treatment-related effects on urinalysis, 

hematology, or clinical chemistry parameters were observed, and organ weights were generally 

unremarkable. 

No treatment-related effects on gross and microscopic pathology were observed throughout the 

study. Pituitary tumors were noted in all test animals, and mammary gland tumors were 

attributed to aging. The NOAEL is 250 ppm (7.5 mg/kg/day). No remarkable toxicological 

findings were determined in the concurrent 26-week study at 500 ppm fenvalerate. 

Page 61 of 70
ESFENVALERATE FINAL 

Page 54 of 62 

In a complementary chronic toxicity/carcinogenicity study in rats (MRID 00079877), 

fenvalerate (98% purity) was administered in the diet to Crl:CD (SD) BR rats (50/sex/dose 

group) at nominal concentrations of 0 (vehicle control, acetone), or 1000 ppm (estimated intakes 

of 0 or 50 mg/kg/day) for approximately two years. Interim sacrifices were not reported; at 

study termination, all selected or surviving rats were examined for gross and histopathological 

evaluation. Select organs weighed at necropsy included the liver, kidneys, testes, adrenals, and 

brain. Gross and microscopic pathological examinations were conducted on these tissues, as 

well as the pituitary, thyroid, prostate, ovaries, uterus, and mammary gland. 

There were no overall treatment-related differences in survival; treated females began dying 

earlier compared to control and treated males, but control males began dying earlier compared to 

all other groups. Clinical signs included treatment-related transient hind limb weakness 

(appearing by Week 3 or 4, and generally resolving within a week) and non-treatment-related 

alopecia. Body weights in males were decreased from Weeks 16-104; weights were decreased 

generally ≥10% by Week 72. Body weights in females were decreased from Weeks 44-104, 

with weights generally decreased ≥10% by Week 60. Food consumption was considered 

unremarkable. In general, no treatment-related effects on urinalysis, hematology, or clinical 

chemistry parameters were observed, and organ weights were generally unremarkable. 

No treatment-related effects on gross and microscopic pathology were observed throughout the 

study related to endocrine endpoints. Anterior pituitary tumors were noted in all test animals, 

but a treatment-related increase in spindle cell sarcomas was noted. A NAOEL is not determined 

in this study. The single dose level (1000 ppm; approximately 50 mg/kg/day) demonstrated 

treatment-related effects on body weight, hind limb weakness, and spindle cell sarcomas. 

Carcinogenicity in Rodents (Mouse; OCSPP 870.4200) 

In a carcinogenicity study in mice (MRID 00079876), fenvalerate (98% purity) was 

administered to B6C3F1 mice (50/sex/dose group) at concentrations of 0 (two vehicle control 

groups), 10, 50, 250, and 1250 ppm (estimated intakes of 0, 0, 1.5, 7.5, 38, or 187.5 mg/kg/day) 

for 104 weeks. Interim sacrifices were not reported. At study termination, all surviving mice in 

all groups were sacrificed for gross and histopathological evaluation. Select organs weighed at 

termination included the liver, kidneys, adrenal, and brain. Gross and microscopic pathological 

examinations were conducted on these tissues as well as the pituitary, thyroid, prostate, 

testes/ovaries, epididymides, uterus, and mammary gland. 

Treatment-related decreases (p<0.05) in survival were noted for the low-dose males and the 

high-dose males and females. Because decreases in survival were not continued in the mediumlow and medium-high dose males, the decrease in the low-dose group is not considered to be 

related to treatment. The presence of clinical signs was not reported. There were no consistent 

significant effects on mean body weights in the low- and medium-low dose groups; decreases 

(p<0.05) were noted at occasional time intervals, as well as increases (p<0.05), with decreases of 

Page 62 of 70
ESFENVALERATE FINAL 

Page 55 of 62 

10% only observed in males and females at Weeks 56 and 0, respectively. Single instances of 

weight decreases >10% were noted for selected measurement intervals in the medium-high dose 

animals (↓12% in males at Week 56 and ↓11% in females at Week 0). Body weight decreases 

were generally consistent in the high-dose animals throughout the study, with greater decreases 

(p<0.05) observed during the second year through study termination (↓13-24%). No treatmentrelated effects on food consumption were noted. 

Absolute kidney weights were decreased (p<0.05) in high-dose males and relative kidney 

weights were increased (p<0.05) in high-dose females; both findings were related to terminal 

body weight decreases (p<0.05) described previously. Carcinogenicity was not indicated in this 

study; the only non-neoplastic pathology observed was multifocal granulomata that were 

primarily present in the medium-high and high-dose animals. 

The LOAELs are 50 ppm for males (7.5 mg/kg/day) and 250 ppm for females (38 mg/kg/day) 

based on the incidences of granulomata (with decreased body weight and increased mortality at 

the high dose); the NOAELs are 10 and 50 ppm for males and females (1.5 and 7.5 mg/kg/day, 

respectively). 

In another chronic carcinogenicity study in mice (MRID 44260601, 1997), Crl:CD-1 BR mice 

(80/sex/dose) were administered diets containing esfenvalerate [98.8% (total isomer); 84.8% 

(active isomer)]at concentrations of 0, 35, or 150 ppm [mean-measured doses of 0, 4.29/5.74, or 

18.3/24.7 mg/kg/day (males/females)] for 18 months. In addition, a 350-ppm group was started 

concurrently, but was terminated after 57-58 days due to excessive morbidity and mortality. At 

study termination, all surviving mice in all groups were sacrificed for gross and histopathological 

evaluation. Select organs weighed at termination included the liver, kidneys, testes, adrenal, and 

brain. Gross and microscopic pathological examinations were conducted on these tissues as well 

as the pituitary, thyroid, prostate, ovaries, epididymides, seminal vesicles, uterus, vagina, and 

mammary gland. 

Clinical signs and lesions observed in treated mice (medium- and high-dose) were attributable to 

self-mutilation or secondary effects of self-mutilation. Self-mutilation occurred due to the 

induction by esfenvalerate of dermal sensations that stimulate scratching by the mice (a wellknown toxicological effect of pyrethroids). All treated mice exhibited increased (p<0.05) 

alopecia, serosanguinous fluid from sores, skin sores, and swollen ears. There was an increase 

(p<0.05) in mortality in the high- and medium-dose groups due to skin inflammation/necrosis 

caused by self-trauma. Excessive morbidity and mortality in the high-dose group continued, and 

this group was removed from the study on Study Days 57-58. The animals were examined at 

necropsy and tissues were collected, but data from these animals were not used to evaluate 

esfenvalerate toxicity or carcinogenicity. 

Mortality was increased (p<0.05) in the high-dose group (6/80 or 23/80 treated vs 0/80 or 0/80 

control, respectively, in males and females) by Study Day 56. This excessive mortality was 

Page 63 of 70
ESFENVALERATE FINAL 

Page 56 of 62 

attributed to skin inflammation/necrosis caused by self-trauma, and it was decided to remove the 

high-dose group from the study on Days 57-58. At 358 days, survival in the medium-dose group 

was 70-71% versus 95% in the low-dose and control groups. At termination (547 days), survival 

in the medium-dose group was 41-46% versus 66-75% (p<0.05) in the other groups. The 

survival rates exceeded the Guideline requirement (not less than 25%) for this interval. Body 

weights for selected study days in the low- and medium-dose animals were slightly decreased 

(p<0.05 or NS; <10%). Body weight gains for Study Days 0-547 were decreased in low- and 

medium-dose males (↓13 and 19%, NS) and females (↓7 and 22%, NS and p<0.05). In the highdose group, decreased mean body weight was reported for Study Day 56 (↓15% and 11%, 

respectively, in males and females) prior to termination of the dose group. Food consumption, 

overall food efficiency, hematology, and clinical chemistry parameters were unchanged or not of 

toxicological concern. 

At termination, increased absolute (↑25%, p<0.05) and relative (↑52%, p<0.01) spleen weights 

were detected in the 150 ppm females. The increased spleen weights are believed to be due to the 

increased extramedullary hematopoiesis in females afflicted with skin erosion and/or ulcers. No 

organ weight effects were noted in males. Gross pathological examination of the medium-dose 

male and female mice exhibited enlarged spleens, increased skin ulceration/erosion, and 

alopecia. The medium-dose females also exhibited enlarged mandibular lymph nodes. 

Microscopically, the medium-dose group had increased skin erosions/ulcers and eye lesions 

(erosions/ulcers/keratopathy). These lesions are considered secondary physiologic responses to 

the chronic inflammation due to self-mutilation. Additionally, spleen extramedullary 

hematopoiesis, bone marrow granulocytic hyperplasia, and mandibular lymph node 

lymphocytic/plasmacytic hyperplasia were observed in the medium-dose animals. In conclusion, 

the treatment levels used in this study were the highest possible because of self-mutilation. 

There was no indication of carcinogenicity in either sex of mouse. 

Based on the observations of skin lesions due to scratching in all treated mice, as a response to 

the dermal sensations induced by esfenvalerate, the systemic LOAEL in this study is 35 ppm 

(4.29/5.74 mg/kg/day; males and females); a NOAEL is not established. No treatment-related 

increase in neoplasia was observed up to 150 ppm (18.3/24.7 mg/kg/day; males and females) 

esfenvalerate in Crl:CD-1 BR mice. 

Chronic Oral Toxicity/Carcinogenicity in Non-Rodents (Dog; OCSPP 870.4100) 

In a chronic toxicity/carcinogenicity study in dogs (MRID 00163855), fenvalerate (98.7% 

purity) was administered to Beagle dogs (6/sex/dose group) in the diet at nominal concentrations 

of 0 (acetone), 25, 50, 100, or 200 ppm for 52 weeks. The mean-measured doses of fenvalerate 

were 0, 0.68, 1.36, or 5.29 mg/kg/day. Organ weights determined included adrenals, kidneys, 

liver, ovaries, testes, mammary gland, and thyroid; histopathological examinations were 

conducted on these tissues as well as the pituitary, epididymides, prostate, and uterus. 

Page 64 of 70
ESFENVALERATE FINAL 

Page 57 of 62 

No mortality occurred during the study. Control and treated animals displayed clinical signs 

including soft stools, mucoid stools, and food-like vomitus; no treatment-related clinical signs 

were observed. Body weight gains were generally similar to control in treated animals although 

decreases (significance not reported) were noted in medium-low and high-dose males at Week 6 

and all males except the low-dose group at Week 7. Food consumption was generally similar to 

control. There were no treatment-related effects on organ weights, or on gross or microscopic 

pathology, and no evidence for esfenvalerate being oncogenic in the dog. A LOAEL was not 

observed, and the NOAEL is greater than 200 ppm (5.29 mg/kg/day), the HDT.

Mechanistic Studies: Thyroid Hormones (Rat) 

In a published study (Kaul et al., 1996), the effect of fenvalerate treatment on male rat 

circulating thyroid hormones was determined. 

Male ITRC-bred Wistar rats (20/dose group; further divided into subgroups of 5/subgroup) were 

administered fenvalerate (98.9% pure, technical grade) in corn oil by i.p. at 0 (vehicle control), 

100, or 200 mg/kg/day for a period of 45 days. For all the estimations, serum/brain tissues from 

five animals (each subgroup) were pooled and taken to represent one sample. Blood was 

withdrawn from all the animals by orbital sinus puncture under aseptic conditions at 15-day 

intervals. Serum was collected and stored at −80°C prior to analysis; tri-iodothyronine (T3) and 

T4 concentrations were determined by radioimmunoassay. After Day 45, animals were sacrificed 

and the brains were removed for determination of total and protein- bound calcium 

concentrations (not discussed further). 

Rats administered fenvalerate at 100 and 200 mg/kg/day exhibited hyperexcitability and mild 

tremors by Day 7. There was a gradual increase in the intensity of the tremors throughout the 

study period (through Day 45). Paralysis was first observed on Day 12 in high-dose animals, 

with the first case of paralysis in the low-dose group recorded on Day 17. All fenvalerate-treated 

animals developed paralysis by the end of the study (Day 45), with the degree of paralytic 

severity dependent on dose. At Day 0, the concentrations of T3 and T4 were similar in all study 

animals (T3 = 0.35±0.027 ng/mL, T4 = 33.0±0.009 μg/dL). Control animals administered corn 

oil alone for 15, 30 or 45 days did not exhibit any changes in the concentrations of T3 or T4. 

Administration of fenvalerate increased concentrations of both T3 and T4, with a dose-dependent 

relation in both dose and time (duration of dosing). Over the 45-day treatment period, T3

concentrations increased by 54, 119, and 138% in the low-dose group and increased by 144, 210, 

and 278% in the high-dose group, respectively. Similarly, T4 concentrations increased by 22, 42, 

and 49% in the low-dose group and increased by 40, 96, and 121% in the high-dose group, 

respectively. 

It was postulated that the increases in circulatory concentrations of T3 and T4 could be due to a 

direct effect of fenvalerate on the mechanism of thyroid activity or due to some other 

perturbation of the HPT axis. 

Page 65 of 70
ESFENVALERATE FINAL 

Page 58 of 62 

In another published study (Giray et al., 2010), the effect of fenvalerate treatment on male rat 

circulating thyroid hormones and thyroid weight was determined as part of a study examining the 

effects of selenium and iodine deficiency in rats. Male Wistar rats (8/dose group) were 

administered fenvalerate (92% purity, technical grade) by i.p. at 0 (vehicle not specified) or 

100 mg/kg/day during the last week of a 49-day feeding period (seven weeks total). Thyroid 

weight, and concentrations of plasma TSH and total T4 were not different compared to control at 

study termination. Total T3 concentration was increased (p<0.05) compared to control 

(approximately two-fold), but the data were only presented graphically. These data differ from 

the previous study (Kaul et al., 1996) that observed increased concentrations of both T3 and T4 at 

an identical dose after a longer dosing period.

Avian Reproduction (OCSPP 850.2300) 

In an avian reproduction study (MRID 48027802), 23-week old Bobwhite quail (Colinus 

virginianus) were administered esfenvalerate (87.3% purity; percentages of total isomer and 

active isomer are not reported) in the feed at nominal concentrations of 0 (acetone, 0.35% v/w), 

250, 500, or 1000 mg/kg diet for 25 weeks (fifteen weeks prior to the start of egg production and 

ten weeks of egg production). Calculated mean dietary doses were 257, 504 and 1056 mg/kgdiet (19.1, 38.7, and 78.5 mg/kg/day), respectively for the low-, medium-, and high-dose birds. 

Each treatment group was comprised of eighteen mating pairs, with one pair per pen. A 

progressive photo period was employed in this study: 7 hours/day through Week 10 and 

17 hours/day until the end of the study. Temperature was maintained at 16-27°C throughout the 

study and humidity was maintained at 42-70%. Individual adult body weights were recorded at 

Week 0, 2, 4, 42, 6, 8, 10, and 25, and body weight changes were determined for each interim 

period. Food consumption in each replicate was measured weekly throughout the pre-treatment 

and treatment periods of the adult phase. Individual chick body weights were recorded after 

hatching and on Day 14 after hatching; egg shell thickness was measured every two weeks 

during egg production. Adult birds and chicks were observed routinely for mortality and clinical 

signs. 

There was no reported treatment-related effect on adult mortality; there were 0, 1, 2, and 2 deaths 

during the study in the control, low-, medium-, and high-dose groups, respectively. Several of 

the deaths were attributed to pen-mate aggression (cages in the present study provided only 13% 

of the recommended floor space, and overcrowding was cited as potential confounder). Adult 

body weight changes and food consumption during the treatment period were generally similar 

in all groups, and there was no evidence of a treatment-related effect. A negative body weight 

change was observed for the Weeks 0-2 period in male birds, with a 107% greater decrease 

(p=0.125) in the high-dose group, and low- and medium-dose females during the Weeks 2-4 

period had decreased (p=0.0261) weight gains of 51 and 46%, respectively. However, overall 

male and female weight gain were not statistically significant. Also, food consumption was 

decreased (p=0.057) by 7% in the low-dose group. 

Page 66 of 70
ESFENVALERATE FINAL 

Page 59 of 62 

A decrease (p=0.0483) in the proportion of 14-day old survivors of hatchlings in the low-dose 

group compared to control (89 vs. 95% control) was observed. It was reported that the reduction 

was the result of poor hatchling survival in pens, where survival was only 78 and 58%, 

respectively; no difference was noted if the data from those two pens were excluded. 

Furthermore, there was no difference from control at the two higher treatment levels. Hatchling 

weights were decreased (p=0.055) in the medium- and high-dose groups compared to control, 

but no differences were observed in hatchling survival or 14-day offspring body weights. No 

other treatment-related effects were noted on any study parameters. The reproductive NOAEC 

for bobwhite quail exposed to esfenvalerate in the diet for 25 weeks was 19.1 mg/kg/day (257 

ppm). The LOAEC is 38.7 mg/kg/day (504 ppm) based on decreased hatchling weights. All 

other parameters had a NOAEC of 78.5 mg/kg/day (1000 ppm). 

The one-generation reproductive toxicity of Esfenvalerate to 16 pairs per level of 25-week old 

mallards (Anas platyrhynchos) was assessed over 20 weeks (MRID 49008802, 2012). 

Esfenvalerate was administered to the birds in the diet at nominal concentrations of 0 (negative 

control), 250, 500, and 1000 mg ai/kg diet. Mean-measured concentrations were <20 (<LOQ, 

control), 297, 608, and 1190 mg ai/kg diet. 

There were no treatment-related mortalities, overt signs of toxicity, or effects on food 

consumption at any dietary level. Additionally, there were no treatment-related effects on any 

reproduction parameters measured at any concentration. No statistically significant differences in 

egg shell thickness were observed between the control group and any of the treatment groups. 

However, mean adult female weight gain was significantly reduced at the highest treatment 

level. The NOAEC was 608 mg ai/kg diet (mean-measured concentration). 

This study is scientifically sound and classified as supplemental, due to the presence of 3% 

acetone in the food, adding to uncertainty. Though not fully acceptable due to the guideline 

deviation, it satisfies the guideline requirement for a Mallard (Anas platyrhynchos) reproductive 

toxicity study and the study does not need to be repeated. Results may be used quantitatively to 

calculate risk. 

Several one-generation avian reproduction studies were conducted using fenvalerate with both 

bobwhite quail and mallard duck. In one set of studies, bobwhite quail and mallard ducks were 

administered fenvalerate in the diet at concentrations of 25, 75 and 125 mg/kg-diet (MRID 

00037111 and 00037112, 1980). In these studies, no treatment-related clinical signs of toxicity 

or mortality were reported. In the bobwhite quail study, increases in the percentage of eggs 

cracked was reported at all test concentrations and was reported in the top two concentrations in 

the mallard duck study. The reviewer noted that there were some questions regarding the top 

concentration (125 mg/kg-diet) as measured samples reported concentrations of 60-85 mg/kgdiet. In two other reproduction studies with bobwhite quail and mallard (MRID 00109832 and 

Page 67 of 70
ESFENVALERATE FINAL 

Page 60 of 62 

00121827, 1975), birds were administered fenvalerate in the diet at 0.2 and 2.0 mg/kg-diet, 

which was regarded by the reviewer as lower than estimated environmental concentrations. In 

each study it was reported that food consumption and weight gain were comparable across 

control and treatment groups and there were no reported significant effects on any reproductive 

parameter. In the bobwhite quail study, the reviewer indicated that mean eggshell thickness was 

slightly below the normal range and percent of cracked eggs was higher than normal for control 

and treatment groups which suggested the birds may have been subjected to a deficiency in their 

diet. Overall, all four studies were classified as supplemental with data only to be used 

qualitatively in a risk assessment. 

Page 68 of 70
ESFENVALERATE FINAL 

Page 61 of 62 

APPENDIX 3: References Not Utilized in the Esfenvalerate 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 Esfenvalerate 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. 

Garey, J. and Wolff, M.S. (1998) Estrogenic and Antiprogestagenic Activities of Pyrethroid 

Insecticides. Biochemical and Biophysical Research Communications 251, 855–859. 

Ghodageri, M.G. and Katti, P. (2013) In vitro induction/inhibition of germinal vesicle 

breakdown (GVBD) in frog (Euphlyctis cyanophlyctis) oocytes by endocrine active compounds. 

Drug and Chem. Toxicol. 36(2), 217-223. 

Hagino, S. (2007) A state of the art of testing methods for endocrine disrupting chemicals in fish 

and daphnids. Pesticide Chemistry: Crop Protection, Public Health, Environmental Safety 

(2007), eds. Okawa, H.,Miyagawa, H. and Lee, P., 415-424. 

Page 69 of 70
ESFENVALERATE FINAL 

Page 62 of 62 

Kelce, W.R., Stone, C.R., Laws, S.C., Gray, L.E., Kemppainen, J.A., and Wilson, E.M. (1995) 

Persistent DDT metabolite p, p'-DDE is a potent androgen receptor antagonist. Nature 375, 581-

585. 

Kim, I.Y., Han S.Y., Kang, T.S., Lee B.M., Choi B.S., Moon H.J., Kim T.S., Kang I.H., Kwack 

S.J., Moon A., Ahn M.Y., and Kim H.S. (2005) Pyrethroid Insecticides Fenvalerate and 

Permethrin Inhibit Progesterone Induced Alkaline Phosphatase Activity in T47D Human Breast 

Cancer Cells. J. Toxicol. Environ. Health 68(23-24): 2175-2186. 

Mani U., Islam F., Prasak A.K., Kumar P., Kumar V.S., Maji B.K., and Dutta K.K. (2002) 

Steroidogenic Alterations in Testes and Sera of Rats Exposed to Formulated Fenvalerate by 

Inhalation. Human & Experimental Toxicology 21: 593-597. 

Materna, E.J., Rabeni, C.F., and LaPoint, T.W. (1995) Effects of the synthetic pyrethroid 

esfenvalerate, on larval leopard frogs (Rana, spp.). Environmental Toxicology and Chemistry

14 (4): 613-622. 

Sumida, K., Ooe, N., Saito, K., and Kaneko, H. (2003) Limited species differences in estrogen 

receptor alpha-medicated reporter gene transactivation by xenoestrogens. J. Steroid Biochem. 

Mol. Biol. 84, 33-40. 

Page 70 of 70