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 Assess ment T y pe: 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 

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Health Effects Divison

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Environmental

# Fat**~~ fec** **~~ ~n** **~** Office of Pesticide Program

THR O UG H: Je ss Rowland

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## ~~~ Deputy Divi s ion Director
Health Effects Division
Office of Pe s ticide Programs

TO: Jolene Trujillo Chemical Review Manager
Pe s ticide Re-Evaluation Division Office of Pesticide Programs Ver .A pr. 2010

EPA has completed its Weight of Evidence (WoE) assessment eval u ating results of the
Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. The WoE documents for the 52 chemicals are li sted in Attac hm ent A a l ong with the chemical
and report identifiers

.

Page I of2Page 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

### EDSP: WEIGHT OF EVIDENCE ANALYSI S OF POTENTIAL
INTERACTION WITH THE ESTROG EN, ANDROGEN OR THYROID
PATHWAYS

CHEMICAL: ESFENVALERATE

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYESFENVALERATE 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 Rele vant 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-Estroge nic 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........................61ESFENVALERATE 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 Pathwa y A R Andro g en Rece p to r B max Bindin g at maximum BROD Benz y lox y resorufin-O-dealk y lase BUN Blood Urea Nitro g e n CA R Constitutive Androstane Rece p to r 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

DE R Data Evaluation Recor d DMSO Dimeth y l Sulfoxide DNA Deox y ribonucleic Aci d DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
E R Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase ) ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g enerationESFENVALERATE FINAL

iv Abbreviation Terminology

F2 Second filial g eneration
Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Intera g enc y Coordinatin g Committee on the Validation of Alternative K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

L Low dose LABC Levator Ani-Bulbocavernosus LAGDA Larval Am p hibian Growth and Develo p ment Assa y 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 Re p roduction Test
MH Medium hi g h
ML Medium low MoA Mode of Action MOE Mar g in of Ex p osure MRID Master Record Identifier

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated ConcentrationESFENVALERATE FINAL

v Abbreviation Terminology

MTD Maximum Tolerated Dose
N Ne g ative
NE Not examined/evaluate d
NF sta g e Nieuwkoo p and Faber’s Sta g in g Atlas NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level NS Not Statisticall y Si g nificant N R Not Re p orte d OCSPP Office of Chemical Safet y Pollution and Prevention OECD Organization for Economic Co-Operation and Development

OPP Office of Pesticide Programs

ORD Office of Research and Develo p ment OSCP Office of Science Coordination and Polic y OSRI Other Scientificall y Relevant Informatio n P Positive
P Parental g eneratio n PC Positive Control PC 10 Positive Control at 10% of response

PC 50 Positive Control at 50% of response

PND Post-Natal Da y POD Point of De p arture PPS Pre p utial Se p aration PROD Pentaox y resorufin-O-dealk y lase ( or de p ent y lase )
PX R Pre g nane X rece p to r QC Quality Control

RBA Relative Binding Affinity

RBC Red Blood Cells
RfD Reference Dose RPC max Relative to Positive Control at maximum

SAP Scientific Advisor y Panel SC Solvent Control

s.c Subcutaneous SDH Sorbitol deh y dro g enase SDWA Safe Drinking Water Act

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th SV Seminal Vesicles
T Th y roid ( hormonal p athwa y) T1WoERC EDSP Tier 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)ESFENVALERATE FINAL

vi Abbreviation Terminology

TP Testosterone Pro p ionate
T R Th y roid Rece p to r TSH Thyroid Stimulating Hormone

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

VP Ventral Prostate VTG Vitello g eni n WoE Wei g ht-of-EvidenceESFENVALERATE FINAL

Page 1 of 62 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen re ceptor, altered steroido genesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 data, other scientifically releva nt information (OSRI), including general toxicity data and open lite rature studies of sufficient qu ality, were considered in this weight of evidence (WoE) assessment.

In determining whether esfenvalerate interacts w ith E, A or T hormone pathways, the number and type of effects induced, the magnitude and pattern of respons es observed across studies, taxa and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whethe r 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 th e Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) an alysis of the potential interaction of esfenvalerate with the E, A or T hormone pa thways. 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 fe nvalerate) 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 conducte d on esfenvalerate a nd/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 fenvalera te was negative for ER binding/ER activation in three different ER assay system s: a mammalian ER transactivationassay (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 e ffects were seen in the mammalian or avian Part 158 studies.
While there was a 6% increase in fertilizati on success was reported in the FSTRA at the high
dose there were no other treatment related effect s in females. In the Part 158 studies with esfenvalerate, the only effect obs erved was a slight decrease in hatchling weights. Therefore there is no convincing evidence for a potential interaction with the estrogen pathway.ESFENVALERATE FINAL

Page 2 of 62 For the androgen pathway, a generally consistent pattern of weak an ti-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 ma le pubertal assays, and no androgen-related effects were s een in the mammalian or avian Pa rt 158 toxicity studies. As reported above, the only effect observed in the FS TRA was an increase in fertility. In the Part 158 studies with esfenvalerate, th e 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 thyr oid interaction in mammals. Although an increase in T 3 was reported by Giray et al. (2010)

, thyroid weight, and concentrati ons of plasma TSH and total T 4 were similar to the control animals. No thyroid rela ted effects were observed in the Tier 1 male and female pubertal assays or in th e Part 158 mammalian studies. Ho wever, 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 concentr ation 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-ev idence cannot conclude whether esfenvalerate has the potential for thyroid intera ction in non-mammalian species.

Based on weight of evidence conclusions, mammalia n 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 not ed that there is a lack of a validated dietary exposure test design as well as th e fact that the nature of esfenvalerate may make it difficult to test long-term in aquatic systems with amphibian s. 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 vari able 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 end points 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 we re 0.006, 0.02, and 0.05 μg/L which are similar to
the concentrations tested in th e fish life-cycle study. Given th e 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.ESFENVALERATE FINAL

Page 3 of 62 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 data, other scientifically releva nt information (OSRI), including general toxicity data and open lite rature studies of sufficient qu ality, were considered in this weight of evidence (WoE) assessment.

In determining whether esfenvalerate interacts w ith E, A or T hormone pathways, the number and type of effects induced, the magnitude and pattern of respons es observed across studies, taxa and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whethe r 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 th e Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) an alysis of the potential interaction of esfenvalerate with the E, A or T hormone pa thways. 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 esfe nvalerate and/or fenva lerate. Esfenvalerate and fenvalerate are related synthetic Type II pyr ethroid broad-spectrum acaricides/insecticides. Fenvalerate is a racemic mixture of four stereoisomers (A α or S,S; B α or R,S; A β or S,R; and B β 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/wat er partition coefficient (log Kow) of 6.2 to 6.5. Esfenvalerate is stable , and not subject to pho tolytic and hydrolytic degradation. Volatilization from water and soil is not expected to contribu te to the dissipation of
esfenvalerate in the environment. The estimate d bioconcentration factor for esfenvalerate is 12,000.

The available information considered in determ ining the potential intera ction 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 st udies and other published
articles. These data are summarized in Sections III.A through III.C. An analysis of the dataESFENVALERATE FINAL

Page 4 of 62 submitted to the Agency, using the WoE appro ach outlined by the Agency (USEPA, 2011), is presented in Section IV. The ED SP 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 believe d 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 T eam on esfenvalerate (USEPA, 2010). Since then, the Agency has also conducted a more recent sear ch of available scientif ic literature for any
additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies c onsidered but not util ized 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 presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the indivi dual 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 endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to
evaluate whether additional te sting is needed based on the evidence and conclusions described
above.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 in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach 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 speci fic endpoint for discerni ng whether or not the chemical has interacted with the endocrine syst em, are considered. The co llective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indi cation of potential interaction as measured by the study.

The second step in this WoE process is to form ulate hypotheses and integr ate 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 suppor t a hypothesis that a chemical has the potential to interact E, A, or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, 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 ev ent, such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment 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 rele vant, robust and consistent evidence in terms of complementarity and redundancy, as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biol ogical organization. Finally, under conditions did responses occur including consideration of whethe r the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic toxicity introducesESFENVALERATE FINAL

Page 6 of 62 uncertainty in the ability to dist inguish effects specifically relate d to an endocrine-related effect from a non-endocrine toxic response.

This uncertainty in distinguishi ng whether the responses were endoc rine-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, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

For these WoE analyses, overt toxicity was gene rally 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 include d, but were not limited to: mortal ity; 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, es pecially 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/concentra tion 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 decr eases 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 ef fects were generally cons idered 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/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addres sing the three components discussed above is fundamental to the WoE process and in determ ining 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 eviden ce supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing th e need for additional data is based on a caseby-case analysis which took all availa ble toxicity data into account.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 bi ologically 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 s hown 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)

ER α 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 t esting in the current 890.1100 AMA study design ( i.e. , water column exposure) and another AMA study utilizin g water column exposure is not recommended.ESFENVALERATE FINAL

Page 8 of 62 B. Effects on Hypothalamic-P ituitary-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-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e 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 hE R competitive ligand-binding assay. In a literature study by Kojima et al. (2004), fenvalera te induced a positive response in an ERTA in Chinese hamster ovary (CHO) cells, with a 20% relative effective concentration (REC 20 ) of 3.7 x

10 -6 M.

Esfenvalerate was negative in the in vivo uterotrophic assay (s atisfied 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 e ffects in the esfenvalerate or fenvalerate mammalian Part 158
studies. In a Part 158 avian reproduction study with esfenvale rate, bobwhite quail hatchling weights were slightly, but significantly, decr eased 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 re production study with esfenvalerate, there were no reproductive effect s 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 ava ilable supplemental studies indicate that an increase in cracked eggs may o ccur with exposure to fenvalerate.ESFENVALERATEFINALPage 9 of 62Table 2. Estrogenic/Anti-Estrogenic Pathway for EsfenvalerateLines of Evidence Indicating Potential Interaction withthe 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 Hi stopat h o l ogy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER BindingRequirement satisfied by OSRI (Saitoet al.,2000; see below)ERTA(MRID 48670801)NAromatase(MRID 48616502)NSteroidogenesis(MRID 48670802)NUterotrophicRequirement satisfied by OSRI (Sumitomoet al.,2001b and Kunimatsuet al.,2002; see below)Female Pubertal Rat(MRID 48683803)NNNN N NNX4(H)FSTRA(MRID 48995501)NENNNFrt:↑6% (H);NNOSRIER Binding/ERTA(Saitoet al.,2000)NNERTA(Kojimaet al. ,2004)PUterotrophic(Kunimatsuet al.,2002)NNX5(H)ESFENVALERATEFINALPage 10 of 62Table 2. Estrogenic/Anti-Estrogenic Pathway for EsfenvalerateLines of Evidence Indicating Potential Interaction withthe 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 Hi stopat h o l ogy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Subchronic toxicity(Rat; MRID40215601)NNNENEX(H)NSubchronic toxicity(Mouse; MRID41359701)NENNNX(H)NSubchronic dermaltoxicity (Rat; MRID45275401)NNNNEX(ML,MH, H)NSubchronic dermaltoxicity (Rabbit;MRID 42325101)NENENNENNDevelopmentaltoxicity (Rat; MRID43211504)NENX(L, ML,MH, H)NDevelopmentaltoxicity (Rabbit;MRID 43211503)NENX(L, M,H)NTwo-generationreproduction (Rat;MRID 43489001)NE NE NENENX(H)X(H)One-generationreproduction (Rat;MRID 48076104)NE NE NENENNNChronic toxicity/carcinogenicity (Rat;MRIDs 00082244and 00111888)NENENNENNESFENVALERATEFINALPage 11 of 62Table 2. Estrogenic/Anti-Estrogenic Pathway for EsfenvalerateLines of Evidence Indicating Potential Interaction withthe 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 Hi stopat h o l ogy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Chronic toxicity/carcinogenicity (Rat;MRID 00079877)NENENNEX(H)X(H)Carcinogenicity(Mouse; MRID00079876)NENENNENRX(H)Carcinogenicity(Mouse; MRID44260601)NENENNEX(M, H)X(H)Chronic toxicity(Dog; MRID00163855)NENNNEN NAvian reproduction(Quail; MRID48027802)P6N NAvian reproduction(Duck; MRID49008802)NN N1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatmentArrows (↓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 inweight 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 inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Clinical signs included tremors in all high-dose females (PND 22-36) and splayed hindlimbs or impairment of hindlimbs in high-dose females on PND 24.5. Clinical signs (i.e., tremors) were observed in the high-dose fenvalerate (80 mg/kg/day) and esfenvalerate (20 mg/kg/day) females.ESFENVALERATEFINALPage 12 of 626. Hatchling weights were slightly decreased in the medium- and high-dose (504 and 10 mg/kg-diet; 5 and 6 % reductions, respectively).P Positive findings (in vitro)/No effect (in vivo)N Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedESFENVALERATE 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 Ti er 1 assays are delineated so as to facilitate determination of potential for a ndrogenic, 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 di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

The Agency determined that the requirement fo r 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 Orde r 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 a nd ARTA assays were also evaluated in the determination of the androgenic potentia l 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-androge nic). Fenvalerate was negative for agonist activity in two independent hAR reporte r 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 highes t 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 a nd 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 secti on, slight, but signifi cant reductions in hatchling weights were observed in the Part 158 bobwhite quail repr oduction study; no reproductive effects were observed in the mallard duck study, but male adu lt body weight gains were significantly reduced at the highest treatment group.ESFENVALERATEFINALPage 14 of 62Table 3. Androgenic/Anti-Androgenic Pathway for EsfenvalerateLines of Evidence Indicating Potential Interaction withthe 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) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity 2 Overt Toxicity 3EDSP Tier 1 AssayAR BindingRequirement satisfied by OSRI (Sumitomoet al.,2001; see below)Steroidogenesis(MRID 48670802)NHershberger(MRID 48616501)NENNX(M, H)4Male Pubertal Rat(MRID 48683802)NNNNNNNNNX(H)5FSTRA(MRID 48995501)NENNNFrt:↑6%(H);Fcd: NNNOSRIAR Binding/ARTA(Sumitomoet al.,2001)NNARTA(Xuet al.,2006)PARTA(Kojimaet al., 2004)PAR Binding(Eil and Nisula, 1990)PSubchronic toxicity(Rat; MRID40215601)NNNENENENEX(H)NESFENVALERATEFINALPage 15 of 62Table 3. Androgenic/Anti-Androgenic Pathway for EsfenvalerateLines of Evidence Indicating Potential Interaction withthe 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) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity 2 Overt Toxicity 3Subchronic toxicity(Mouse; MRID41359701)NNNENNNEX(H)NSubchronic dermaltoxicity (Rat; MRID45275401)NNNNNENEX(ML,MH, H)NSubchronic dermaltoxicity (Rabbit;MRID 42325101)NNNENNENEN NTwo-generationreproduction (Rat;MRID 43489001)NENENENENENE NX(H)X(H)One-generationreproduction (Rat;MRID 48076104)NENENENENENE NN NChronic toxicity/carcinogenicity (Rat;MRIDs 00082244 and00111888)N N NENENE NEN NChronic toxicity/carcinogenicity (Rat;MRID 00079877)N N NENENE NEX(H)X(H)Carcinogenicity(Mouse; MRID00079876)NE N NE N NE NENRX(H)ESFENVALERATEFINALPage 16 of 62Table 3. Androgenic/Anti-Androgenic Pathway for EsfenvalerateLines of Evidence Indicating Potential Interaction withthe 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) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity 2 Overt Toxicity 3Carcinogenicity(Mouse; MRID44260601)N N NE NNE NEX(M, H)X(H)Chronic toxicity(Dog; MRID00163855)NNNENNENEN NAvian reproduction(Quail; MRID48027802)P6N NAvian reproduction(Duck; MRID49008802)NN N1. 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. Ashaded cell indicates that this parameter is not routinely evaluated or is not applicable in this assay. Changesin weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsalprostate (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 OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For detailssee Section IV. A4. Clinical signs 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 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 animals6. Hatchling weights were slightly decreased in the medium- and high-dose (504 and 10 mg/kg-diet; 5 and 6 % reductions, respectively)P Positive findings (in vitro)/No effect (in vivo)N Negative findings (in vitro)/No effect (in vivo)NE Not examinedESFENVALERATEFINALPage 17 of 62NR Not reportedESFENVALERATE FINAL

Page 18 of 62 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry 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 de tect 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 thyroi d 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 ar e not reported in the ta ble and not considered further in the WOE assessment.

Changes in thyroid stimulating hormone (TSH) leve ls 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 t oxicity (clinical signs, tr emors). No changes in thyroid weight, histopatho logy or serum thyroxine (T 4 ) concentration were observed in the male or female pubertal assays. The AMA conducted w ith esfenvalerate is considered unacceptable given the extremely high coeffi cient of variances (CVs >100%) for the measured esfenvalerate concentrations which resulted in overlapping measur ements in all three treatment concentrations making the low, mid and high test groups indistingui shable 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 a ssay 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 studi es. Increases in T 4 concentrations were repo rted in a study by Kaul et al. , 1996; however, both doses (100 and 200 mg/kg/day) were considered to be overtly toxic (t remors, paralysis). Giray et al. (2010) reported
an increase in total T 3 concentration (approximately two- fold based on results presented graphically) in rats treated with 100 mg/kg/da y fenvalerate (seven w eek study), but thyroid weight, concentrations of plasma TSH and total T 4 were similar to the control animals.ESFENVALERATE FINAL

Page 19 of 62 Table 4. Thyroid Pathway for Esfenvalerate

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Esfenvalerate 1

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmentalstage (± orasynchronous, HLL)Growth (BW, BL,SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

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 P 6 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)ESFENVALERATE FINAL

Page 20 of 62 Table 4. Thyroid Pathway for Esfenvalerate

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Esfenvalerate 1

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmentalstage (± orasynchronous, HLL)Growth (BW, BL,SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

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 examinedESFENVALERATE FINAL

Page 21 of 62 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight 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 recommendati ons 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 pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (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 an alysis of those line s of evidence.

The WoE evaluation includes consid erations of biological plausibility of the findings from the different lines of evidence by examining the cons istency, coherence and in terrelationships among the measured endpoints within and across studi es. The available findings from standard toxicology studies on the substance may contribute to the WoE evaluati on in helping el ucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively sequelae of systemic effects. E ndocrine modes of action may elicit a number of phenotypic consequences other than thos e 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 (incl uding but not limited to mortalit y 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, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All effects in endocrine se nsitive 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 belo w. Generally, one parameter (i .e., death or >10% decrease in mammalian body weight) is sufficient for a dose/concen tration to be considered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were ge nerally less severe forms of toxicity (e.g., organ we ights or blood chemistry).ESFENVALERATE FINAL

Page 22 of 62 1. Tier 1 in vivo Assays

Systemic or overt toxicity observe d for esfenvalerate in the Tier 1 in vivo studies was typical of pyrethroid compounds, consisting gene rally of neurological effects. There were no consistent or significant effects noted on body weight s and/or body weight gain in any in vivo Tier 1 studies. Clinical signs observed in th e Hershberger assay (medium- and high-dose animals, 6 and 9 mg/kg/day) included abnormal ga it, ataxia, fast breathing, hy per-reactivity, paw shaking and licking, abnormal grooming, whole-body fasciculati ons, altered posture, d ecreased 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 we re 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 obser ved 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-relate d 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 sign s of pyrethroid exposure such as tremors; the daily incidence of tremor was 4-6/6 rats in th e 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 toxic ity study in rats with esfenvalerate. Neurological signs were noted after Week 1, and hyperactiv ity was observed in females only between Weeks 10 and 13. During Weeks 11-13, je rky 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 co mpared to control throughout the study. Numerous clinical signs were observed in th e high-dose [106/113 mg/kg/day (M/F)] for both esfenvalerate and fenvalerate [( 422/462 mg/kg/day (M/F)] in a subc hronic oral toxicity study in mice: fibrillation, tremors, convulsions, hypers ensitivity, abnormal gait and salivation during Week 1; scratching and licking af ter Week 3; and alopecia, scab s and sores after Week 4. No deaths occurred during the study. Decreased bo dy 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 decr eased body weight gains during Days 0-28. Body weight gainESFENVALERATE FINAL

Page 23 of 62 decreases were lessened (<10%) in the females dos ed 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 gr oup (2.5 mg/kg/day) incl uded 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), furt her 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, at axia and convulsions. Incidence and frequency of th ese behaviors increased with increasing dose. Decreased body weight gains of 8% were observed in the medi um-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. C linical 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 hypers ensitivity to touch, and further clinical signs in the high-dose dose animals consisted of trem ors, ataxia and diarrhea. The incidence and frequency of these behaviors increased with incr easing 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 stu dy in rats with esfenvalerate, P 1 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 P 1 females (25.1 mg/kg/day) exhibited abnormal gait,
and a P 1 female in the medium-dose group was sacrificed in extremis (attributed to dystocia). F 1 males and females in the high-dose group exhibite d numerous clinical si gns of neurotoxicity (abnormal gait/mobility, ataxia, barrel rolls, general spasms, hyperactivity, hypersensitivity, popcorn seizures, tremors and vocalization) and si gns 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 ga ins in the high-dose P 1 animals during the premating period were generally decreased during the in itial weeks, but overall body weight gains in high-dose P 1 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-existi ng conditions, but body weight gain was also decreased in the high-dose P 1 females during lactation. Body weight gains in theESFENVALERATE FINAL

Page 24 of 62 high-dose F 1 animals during the premating period were slightly decrease d during the initial weeks, but increased mortality occurred in high-dose F 1 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 effect s on clinical signs of toxicity or body wei ght, and the overall body weight gain for the pre-ma ting period was not affected.

There were no treatment-related differences in su rvival in a chronic t oxicity/carcinogenicity study in rats with fenvalerate. In addition, ther e were no treatment-rela ted 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 treat ed 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 w ithin 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 fe males 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 signifi cant 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 increas ed 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 inflamma tion/necrosis caused by self-trauma. Mortality was increased in the high-dose group by Study Day 56. This ex cessive mortality was attributed to skin inflammation/necrosis caused by self-trauma, a nd it was decided to remove the high-dose group from the study on Days 57-58. At 358 days, su rvival 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 me dium-dose groups generally were not affected by treatment. InESFENVALERATE 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) pr ior to termination of the dose group.

No mortality occurred during a ch ronic toxicity study in dogs with fenvalerate, and no consistent,
treatment-related effects were observed regarding clinical sign s of toxicity or body weight/body weight gain.

There were no significant treatment-related mortalities or clinical si gns of toxicity reported in the avian reproduction study with esfe nvalerate 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 ne gative for ER binding/ER activation in three different ER assay systems: a mammalian ER TA assay with HeLa cells, a yeast two-hybrid system and a hER competitive ligand-binding. A dditionally, esfenvalerate was negative in the Tier 1 ERTA, aromatase and steroidogenesis assays. The in vivo uterotrophic and female pubertal assays were negative, a nd no estrogen related effects were seen in the mammalian Part
158 studies. While there was a 6% increase in fer tilization success was reported in the FSTRA at the high dose there were no other treatment related effects in fe males. In the Part 158 studies with esfenvalerate, the only effect observed wa s a slight decrease in hatchling weights. Therefore, there is no convincing evidence for a potential interaction wi th the estrogen pathway.

C. Androgen Pathway

A generally consistent pattern of w eak 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 pubert al assays, and no androgen-related
effects were seen in the mammalian or avian Pa rt 158 toxicity studies. As reported above, the only effect observed in the FSTRA was an increa se in fertility. In the Part 158 studies with esfenvalerate, the only effect obser ved was a slight decrease in hatchling weights Overall, there is no convincing evidence for an inte raction 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 T 3 was reported by Giray et al. (2010)

, thyroid weight, concentrations of plasma TSH and total T 4 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 adequa te AMA was not available for consideration inESFENVALERATE FINAL

Page 26 of 62 the weight-of-evidence, and given the observe d high variability and apparent lack of concentration stability in the av ailable 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 ha s the potential for thyr oid interaction in nonmammalian species.

E

. Conclusions

The conclusion of the WoE evaluation is that es fenvalerate 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 pa thway for mammals, but a conclu sion for this pathway could not be determined for non-mammalian species.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence conclusions, mammalia n 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 not ed that there is a lack of a validated dietary exposure test design as well as th e fact that the nature of esfenvalerate may make it difficult to test long-term in aquatic systems with amphibian s. 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 vari able 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 end points 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 we re 0.006, 0.02, and 0.05 μg/L which are similar to
the concentrations tested in th e fish life-cycle study. Given th e 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.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 Medici ne, Newark, Delaware, Dupont HLR 331-94; Med. Res. Proj. No. 9657-001, December 1, 1994. (MRID 43489001)

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

Rats. Unpublished study conducted by Haskell Laboratory fo r Toxicology and Industrial Medicine, Study No.: DuPont- 4228, November 28, 2000. (MRID 45275401)

Dinehart, S. (2012) Esfenvale rate Technical (DPX-YB656): Short-term Reproduction Assay with the Fathead Minnow, Pimephales promelas , Determined Under Flow-Through Conditions. Unpublished study conductedby ABC Laboratories, Inc., Colu mbia Missouri and Experimental Pathology Laboratories, Inc., St erling, Virginia. Lab Study No.: 66821; Sponsor Project No.: DuPont 32258. Submitted by E.I. du Pont de Ne mours and Company, Wilmington, Delaware. Completion date October 31, 2012. (MRID 48995501)

DuPont (1987): D.M. Larson (1987) 13-Week di etary 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 Pyrethro ids to Human Skin Fibroblast Androgen Receptors and to Sex Hormone Binding Globulin. J. Steroid Biochem. 35: 409-414.

Fort, Douglas J. (2012) Esfenvalerate (DPX-YB6 56) 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. Laborat ory project number DUPO01-00271. Study sponsored by E.I. du Pont de Nemours and Company (DuPont). (MRID 49024501)

Giray, B., Ca ğ layan, A., Erkeko ğ lu, P., and H ı ncal, F. (2010) Fenvalerate Exposure Alters Thyroid Hormone Status in Seleni um-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 r eceived 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)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 Labor atory, 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. Fina l 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 activi ties in 200 pesticides by in vitr o 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-) Androgeni c 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. Laborator y 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 antiestroge nic activity of pyrethroid insectic ides in three in vitro assays based on classic estrogen recepto r alpha-mediated mechanisms. Toxicol Sci. 57(1):54-60.

Shell (1986): Dickie, B.C. (1986) One year or al study in dogs with MO 70616 technical. Shell Chemical Company Regulatory Info rmation Record No. WRC RIR 467. Unpublished study conducted by Hazleton Laboratories Am erica, Inc. (MRID 00163855)ESFENVALERATE FINAL

Page 29 of 62 Snajdr, S.I. (2011) Esfenvale rate (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 fo r 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 t oxicity test with nort hern bobwhite (Colinus virginianus) following OPPTS 850.2300 and OECD 206. Unpublished study conducted by Springborn Smithers Laboratories. Study sponsor ed by Sumitomo Chemical Company, Ltd., Chuo-ku, Tokyo, Japan. Report Nos. 13048.4110 and LLW-0137. (MRID 48027802)

Sumitomo (1985): Y. Koyama (1985) Comparativ e 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 develo pmental 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 Chem ical Co., Ltd. Unpublished report No.: LLT110143 (MRID 43211504)

Sumitomo (2001): Sumitomo Chemical C o., Ltd Report LLT-0217 Evaluation of hormonal activity (effects on human estrogen receptor alph a, androgen receptor and progesterone receptor) of fenvalerate using a suite of three in vitro assays.

Toot, J.D. (2012) Pubertal Development and Thyr oid 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 Thyr oid 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)ESFENVALERATE FINAL

Page 30 of 62 USEPA (2010) Esfenvalerate - Report of the En docrine Disruptor Review Team; Test Order# EDSP-109303-80, 81, and 82. December 14, 2010. Offi ce of Chemical Safety and Pollution
Prevention. U.S. Environmental Pr otection Agency, Washington, D.C.

USEPA. (2011) Endocrine Disruptor Screeni ng Program – Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.

USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
held July 30 - August 1, 2013 on Weight-of

- Evidence: Evaluati ng Results of EDS P Tier 1 Screening. Office of Chemical Safety and Pollution Pr evention. U.S. Environmental Protection Agency, Washington, DC.

Willoughby, J.A. (2012) Esfenvalerate (DPX-YB 656): 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., Ch en, 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

.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 (N F) 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 concentra tions 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 calculati ons. The test system was maintained at 21.4 to

21.8 o C and a pH of 7.0 to 7.8.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. No mortal ities or clinical signs ( i.e. , behavioral and other s ublethal 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 tr eatment level relative to the negative control. Further, no asynchronous development was observed. Eff ects on thyroid gland histopathology were observed at all treatment levels, including the control groups and there was no apparent pattern of response. Histopathological e ffects 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, solven t control, low treatment group and high treatment group. On Day 7, the reviewer’s analysis detected a significant decr ease in hind-limb length (H LL) 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 e ndpoints 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 concen trations 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% ar e 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 do es interfere with the interpretation of test results.
Detections of test substance in some of th e negative and solvent control samples further compromise the test results.ESFENVALERATE FINAL

Page 32 of 62 Androgen Receptor (AR) Bindi ng 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 tr itiated androstenedione (1- β [ 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]-ASDN) at concen trations of 1 × 10

− 10 to 2.5 × 10

− 5 M for 15 minutes to assess the potential of esfenvalerate to inhibit aromatase activity. Ac etone was selected as the vehicle solvent for the
test substance because esfenvalerate was not so luble, or did not stay in solution, when EPArecommended solvents were used; dimethyl sulf oxide (DMSO) was used as the solvent vehicle for the reference compounds. The final concentr ation 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. Tritia ted 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 inhib itor (4-hydroxyandrostenedione ; 4-OH ASDN), and the esfenvalerate series (10

− 10 to 2.5 × 10

− 5 M), with three repetitions pe r concentration. Because the
solvent vehicle was acetone for esfenvalerate an d 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 aromatas e activity below the 0.1 nmol·mgprotein

− 1 ·min

− 1 performance criteria specified in the Gu ideline. 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 activ ity 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 back ground controls averaged 0.5% of the full activity control with DMSO, and 1.1% of the full act ivity control with acetone.

For the positive control substance (4-OH ASDN) , aromatase activity results were generally within the recommended ranges for the perf ormance criteria. The estimated log IC 50 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 thanESFENVALERATE FINAL

Page 33 of 62 75% activity at all soluble concentrations of esfe nvalerate. 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 classifi ed as a Non-inhibitor of aromatase activity in this assay.

Estrogen Receptor (ER) Bindi ng Assay; (OCSPP 890.1250)

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

ER α Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48670801), hER α -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 (luciferas e) gene products. Luciferase expression in response to activation of the estr ogen 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 th e cells to the very weak positive control 17 α

methyltestosterone was lower than the expected values, indicating a decrea sed sensitivity of the assay to very weak agonists. Although the conditi ons of this assay were not optimal to detect very weak activity, esfenvalerate responses we re similar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to
reach a maximum of 24.3-26.6% PC. Esfenvalera te was only able to reach a maximum of 4.0- 4.1% PC when tested up to the highest concentr ation possible based on cytotoxicity. Because
the RPC Max < PC 10 in both assay runs, esfenvalerate wa s considered negative for estrogen receptor transcriptional activa tion in this test system.ESFENVALERATE FINAL

Page 34 of 62 Fish Short Term Reproduction Assay (FSTRA); (OCSPP 890.1350)

The 21-day short-term reproduction assay (M RID 48995501) of esfenvalerate with fathead minnows ( Pimephales promelas) was conducted under contin uous 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 lim it of detection (LOD; 0.000732 mg/L) and LOQ (0.00244 mg/L). In the AM A conducted with esfenvalerate at the same laboratory (MRID 49024501), the L OD 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 25.4 o C 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, solv ent 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 chan ges 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 tr eatment group. All other fish in the controls and treatment groups appeared normal and no overt signs of toxicity were ob served during the test period. Body weight was not si gnificantly affected by treatm ent; body length was not reported.

In the negative control, spawning occurred atleast once every 4 days and fecundity was 48 eggs/female/day/replicate (rang e 43 to 53); fertilization succe ss 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-depend ent 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 differen ces in the treated groups compar ed to negative control for any other endpoint (p>0.05). Histopathology and gonada l 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 verifica tion 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 precipit ate, surface film, or undissolved test substanceESFENVALERATE 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) screen ing for androgenic activity, esfenvalerate (95.4% total isomers with the active isomer acc ounting 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 subcutan eous (s.c.) injection.

To screen for potential anti-androge nic activity, esfenvalerate in co rn 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-andr ogenic positive control group consisted of six castrated rats orally dosed with 3 mg/kg/day fl utamide (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 assa y, body weights were determined daily. Food consumption was determined over the entire dosing period. The an imals were dosed for 10 consecutive days and terminated approximately 24 hours after the fina l dose administration. At necropsy, the five androgen-dependent tissues and liver were collected and weighed.

All animals survived until sche duled termination. Test substa nce-related clin ical signs of toxicity were observed in rats administered es fenvalerate at 9 mg/kg/ day in both the androgen and anti-androgen assays and in rats administ ered esfenvalerate at 6 mg/kg/day in the antiandrogen assay. Clinical signs observed includ ed abnormal gait, ataxia, fast breathing, hyperreactivity, paw shaking, paw licking, abnormal grooming, whole-body fasc iculations, 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 repor ted clinical signs observed in other toxicity studies and were considered toxicologically significant.

In the androgen assay, body wei ghts throughout the duration of th e assay and feed consumption over the dosing period in the esfenvalerate-dos ed groups were comparable to the vehicle controls. Overall (Days 1-11) body weight gains in the esfenvale rate 6 and 9 mg/kg/day groupsESFENVALERATE FINAL

Page 36 of 62 were decreased (not significant; NS) by 17% and 24%, respectively, compared to the vehicle controls. Overall body weight ga ins in the positive control (veh icle + TP) group were increased (NS) by 27% and feed consumption was increas ed (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 posi tive control (vehicle + TP) group had accessory sex organ weight increases (p<0.05) as follows: 817% in seminal vesicl es; 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 we ights throughout the duration of the assay, feed consumption over the dosing period, and overall body weight gains in the es fenvalerate-dosed groups and positive (FT + TP) control group were comparable to the negative controls. There were notreatment-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 comp arable 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 androgeni city 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 esfe nvalerate 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 attain ment was recorded. Following sacrifice on PND 42 or 43, blood was collected for clinical ch emistry analyses, including total thyroxine (T 4 ) and thyroid stimulating hormone (TSH), which were analyzed using an electrochemiluminescent immunoassay or a radioimmunoassay, respectivel y. Kidney, liver, adrenal gland, thyroid,ESFENVALERATE FINAL

Page 37 of 62 pituitary, uterus, and ovary weights were r ecorded, and microscopic examinations were performed on the thyroid, kidney, ovary, and uterus.

No treatment-related effect was observed on su rvival, body weight, body weight gain, age of attainment of VO, body weight at VO, or gross pathology. No treatment-re lated effect was noted on estrous cycle status at necropsy, mean age at first estrus, mean cycle length, or percent cycling. Serum T 4 and TSH were not affected by treatment. No treatment-related microscopic lesions were observed on the thyr oid, 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, f our 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 observ ed. No effects were observed on body weights or body weight gain in the study.

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

Male Pubertal A ssay; (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 es fenvalerate (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 exam ined for preputial separation (PPS) daily beginning on PND 30 and weight at day of atta inment was recorded. Following sacrifice on PND 53 or 54, blood was collected for clinical chemistry analyses, incl uding total thyroxine (T 4 ), testosterone, thyroid stimulating hor mone (TSH). Testosterone and T 4 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, b ody weight gain, organ weights, or gross pathology. Ag e and body weight at PPS in the tr eated groups were similar to controls. Thyroid hormone levels, colloid area an d 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 microscopi c findings were noted in the epididymis, testis, and kidney.ESFENVALERATE FINAL

Page 38 of 62 At 9.0 mg/kg/day, tremors were obs erved almost exclusively at ≥ 3 hours after dosing for 12 animals on PND 23-31. During the daily examina tions, 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 si gn of clear material around mouth was increased for 9.0 mg/kg/day animals at up to 3 hours afte r dosing starting at PND 35 and continuing sporadically during the study. Red material ar ound the mouth was observed at 3.0 mg/kg/day (6 males) and 9.0 mg/kg/day (5 males) dur ing the daily examinations on PND 26-53.

The clinical sign of tremors observed at 9.0 mg/kg/day indi cated that the high dose was adequate.

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 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 dimet hyl sulfoxide (DMSO) was used as the vehicle for the positive controls, at a final concentra tion of 0.05%. Precipitates were observed after the exposure period

at 10 μ M and 100 μ M. Cytotoxicity great er 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 r un of a test chemical plate to demonstrate that the assay responded properly to pos itive control agents at two concentration levels; positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol a nd testosterone production.

Guideline acceptability recommendations and requirements were generally met, including adequate production of testosterone and estrad iol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with positive controls. The ex ceptions 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 increa se of 1.7-fold rather than the 2-fold increase
Guideline criterion.

In the three assay runs compari ng esfenvalerate treatment to the solvent control (SC), inhibition (p ≤ 0.05) of testosterone producti on was noted at 0.0001, 0.01, and 1 μM , as well as inhibition ofESFENVALERATE FINAL

Page 39 of 62 estradiol at 0.01 μM. However, th ese responses were not concentr ation-dependent; therefore, the apparent effects are not considered treatment-re lated. 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 re producible 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.ESFENVALERATE FINAL

Page 40 of 62 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 ge neral toxicity studies considered relevant for determining the potential interaction of esfenvale rate (or fenvalerate) with the endocrine system are summarized below. Given the breadth of toxicity data av ailable on fenvalerate a nd esfenvalerate, the only Part 158 studies discussed in this appendix are those that fulfill current guideline requirements and are directly relevant to asse ssing 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 (hER α and
hER β ) and one human androgen receptor (hAR) in tr ansactivation assays with Chinese hamster ovary cells (CHO-K1 cells) was inve stigated. For detection of hER α or hER β activity, cells were transfected with pcDNAER α or pcDNAER β , pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were transfected with pZ eoSV2AR, pIND-ARE, an d 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 17 β -estradiol (E 2 ; purity >97%) or 5 α -dihydrotestosterone (DHT; purity 95%) for the ER or AR assays, respectively. For meas urement of antagonistic activity to hER α , hER β and hAR, either 10

− 11 or 10

− 10 M E 2 or 10

− 10 M DHT was added to the cell cultures along with the test compound, respectively. A gonistic activities were eval uated by the relative activity expressed as 20% relative effective concentration (REC 20 ), the concentration of the compound demonstrating 20% of the activity of 10

− 10 M E 2 , 10

− 9 M E 2 , or 10

− 9 M DHT for ER α , ER β , and AR, respectively. Antagonistic activities were evaluated similarly by the relative activity expressed as 20% relative i nhibitory concentration (RIC 20 ), the concentrat ion of the compound showing 20% inhibition of the activity of E 2 or DHT against ER α , ER β , and AR, respectively. In these assays, fenvalerate was negative for ER β activity, but induced a positive ER α agonist response with a REC 20 of 3.7 x 10 -6 M and a positive AR antagonism response with an RIC 20 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. Constr uction of a hAR expression plasmid fo r 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 repor ter 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 tran scriptional control of a TK promoter was also prepared (pGL3-TK). HeLa cells were maintained in cell culture in an atmosphere of 5% CO 2 :95% air at saturated humidity.

HeLa cells were plated at a density of 2×l0 6 cells per 10-cm plate with the assay medium that contained 10% (v/v) twice-dextran:charcoal-treated PCS, and incubated overnight. Cells wereESFENVALERATE FINAL

Page 41 of 62 transfected with 3.75 ng of recep tor expression plasmid (pRc/RSV-hAR Kozak) and 3.25 μg of reporter plasmid (pGL3-MMTV) per dish. For cont rol assays to evaluate plasmid transfection efficiency, cell toxicity of a chemical, non-sp ecific 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 2×10 4 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 a nd added to the transfecte d cells for the agonist assay. The solubility of each chemical was vi sually confirmed under the light microscope to assess precipitation. The selected doses of fenvale rate were determined based on solubility and cell toxicity results from a control assa y. For the antagonist assay, DHT (100 pM; 10

− 10 M) was incubated with fenvalerate (10

− 5 M) or the anti-androge n, hydroxyflutamide (10

− 6 M). Cells were cultured for an additional 40-hour peri od, followed by removal of medium, washing with phosphate-buffered saline, and preparation for ev aluation by treatment with cell lysis buffer. After incubation for 30 minutes at room temperat ure, 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 saturate d at 1 nM (approximately 34-fold induction). Based on these results, a DHT concentration of 100 pM was select ed for evaluation of antagonistic activity. The relative fold-induction of luciferase by 10 μM fe nvalerate 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 hydroxyf lutamide (1 μM) decreased DHT luciferase activity by 80%.
There was no difference between vehicle contro l and 10 μM of fenvalerate, indicating that fenvalerate was also negative for anta gonistic 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 th e pCAT3-Basic vector was c onstructed 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 cy totoxicity of the test substances) and the hAR expression plasmid AR/pcDNA3.1 containing the full open readi ng frame of hAR cDNA were also prepared. The CV-1 cell lin e (African monkey kidney cell) wa s maintained at 37°C in an atmosphere of 5% CO 2 :95% air at saturated humidity, and transfected by incubation for 24 hours with the three plasmids.ESFENVALERATE FINAL

Page 42 of 62 The reported gene assay was conducted with vari ous 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 c oncentrations in culture medium did not exceed 0.1% (v/v). In order to dete rmine AR antagonistic activity, 0.5 nM DHT was added along with fenvalerate. Cells were ha rvested after incubati on for 24 hours, lysed, centrifuged, and the cell lysates we re 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 (EC 50 ) 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 inhi bition of CAT activity induced by 0.5 nM DHT.
Fenvalerate did not affect DHT-induced CAT ac tivity at a fenvalerate concentration of 10

− 7 M, but CAT activity was decreased (p<0.01) at 10

− 5 M, with an IC 50 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 y east two-hybrid assay was conducted with cDNA containing the LBD of hAR (amino acids N o. 623-919). The plasmid (pGBT9-hARLBD) expresses the fusion protein of the LBD of hA R and the DNA binding domain of GAL4. Yeast cells (Y190) were transformed with two pl asmids (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 agonis t 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 fo r evaluation of antagonistic activity.

The relative fold-induction of β -galactosidase by 10 μM fenva lerate was 1.0±0.1. Therefore, fenvalerate was negative in the y east two-hybrid assay for evaluati on of agonistic activity. The antagonistic activity of hydroxyf lutamide (1 μM) decreased β -galactosidase ac tivity by 70%.ESFENVALERATE FINAL

Page 43 of 62 There was no difference between vehicle contro l and 10 μM of fenvalerate, indicating that fenvalerate was also negative for anta gonistic activity in this assay.

MDA-MB-453 cells (used for the hAR Competitive Bi nding assay) were maintained in culture at 37°C in an atmosphere of 5% CO 2 :95% air at saturated humidity. Cells were grown to 70-80%
confluence in tissue culture dishes. Co mpetitive binding was determined by testing concentrations of fenvalerate (10

− 9 to 10

− 5 M) and unlabeled DHT against [ 3 H]-DHT (50 nM). Binding was determined by LSC after incubation fo r 1 hour at 4°C. DHT binding inhibition was assessed in the assay by the a ddition 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 [ 3 H]-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 pyrethroi d (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 de termined by competitive displacement of [ 3 H]R1881 from receptor sites on cultured human fibroblasts with a dispersed, intact cell system. Binding
studies were conducted at 22°C to minimize metabo lism 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 experi ments, fixed amounts of competitor or vehicle (ethanol) were added to incr easing 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 inhib itor to assess binding to high affinity sites (specific binding). The amount of R1 881 required for 50% displacement of [ 3 H]R1881 was
determined to be 0.50±0.08 nM. The RBA of fe nvalerate was calculated as the ratio of the
concentration of unlabeled R1881 to the concen tration of fenvalerate required for 50% displacement of high a ffinity 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 2×10 9 M − 1

.
The SHBG assay was conducted with pooled citr ated plasma from which endogenous steroids had been removed by charcoal adsorption. Bind ing to SHBG was determined at 37°C with [ 3 H]testosterone and a solid-phase method with C on-A-Sepharose. Cortisol (2 μg) was included in order to minimize the possible cont ribution of transc ortin to total [ 3 H]testosterone binding ofESFENVALERATE 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 bi nding with a similar slope to unlabeled R1881, but with an approximate 10

− 5 lower potency. Fenvalerate had an intermediate potency with a K i 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 ER α pathway using three diffe rent assay systems: a mammalian ERTA assay with HeLa cells and a lu ciferase reporter, a yeast two-hybrid system (Nishikawa procedure), and hER competitive liga nd-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 areporter gene assay with the same sequence as human prostate cDNA was completed (pRc/RSVhER α 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 expr essing a reporter gene under the tr anscriptional c ontrol of a TK promoter was also prepared (pGL3-TK). HeLa cells were maintained in cell culture in an atmosphere of 5% CO 2 :95% air at saturated humidity. Cons truction of a plasmid for hER in a yeast two-hybrid assay was conducted w ith cDNA containing the LBD of hER α (amino acids No. 249-596). The plasmid (pGBT9-hER α LBD) 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-hER α LBD and pGAD424-TlF2RID), and yeast tran sformants were grown overnight at

30°C.

Positive controls were run for agonism (17 β -estradiol and 4-nonyl phenol) and antagonism (4-hydroxytamoxifen). 17 β -estradiol displayed the expected re sponses in all three assays, with dose-dependent induction in the ERTA and th e yeast two-hybrid assay, and dose-dependent inhibition (displacement of fl uorescence standard) in the co mpetitive 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 th e yeast two-hybrid assay; tested dose(s) were determined on the basis of solubility. The result s were displayed graphica lly; 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 estroge nic effects of several pyrethroid pesticides ( Kunimatsu et al. , 2002 ), permethrin, fenvalerate (97.3% purity), and esfenvalerate (94.2%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 stan dard laboratory diet and water ad libitum . After an acclimation period of seven days, the rats were ovariectom ized on PND 42 and allowed to recover for two weeks prior to initiati on 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). Te st and reference substances were suspended or dissolved daily in vehicles (corn oil includi ng 10% ethanol for ethynyl estrad iol; corn oil only for other compounds). Clinical signs, body weight, food c onsumption, and weights of liver and kidneys were assessed as indices of systemic toxicit y. Clinical signs includi ng 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, et hynyl estradiol, or methoxychl or 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 st udy. Ethynyl estradiol (0.03 mg/kg/day) and methoxychlor (125 mg/kg/day) were administered as reference chemicals for estrogenicity; doses
of fenvalerate and esfenvaler ate were 20, 40, and 80 or 5, 10, a nd 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 uter us, 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 clin ical 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 estradio l and methoxychlor increased (p<0.01)
both wet and blotted uterin e weights as expected ( ↑ 74 and 73%, and ↑ 185 and 140, respectively).

The highest doses of fenvalerate and esfenvalerate each exhibite d 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 a nd 2-4/6 rats in the 20 mg/ kg/day esfenvalerate group. However, no changes were observed in final body weight, food consum ption, or liver, kidney, and uterine weights in all treat ed groups. One minor exception was a slight, non-dose-dependent decrease (p<0.05) in food consumption at th e medium dose of fenvalerate. Under theseESFENVALERATE FINAL

Page 46 of 62 experimental conditions, fenvalera te and esfenvalerate had no e ffects 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% A α ) was administered for seven weeks (interim sacrifice; 10/sex/dose) or thirteen weeks (15/sex/dose) in the diet to SD rats at nominal concentra tions 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, a ll rats were weighed, and sacrificed for gross and histopathological evalua tion. Organs that were weighed in cluded the liver, kidneys, testes, ovaries, and uterus; gross and mi croscopic pathological examina tions were conducted on these organs as well.

There was no mortality during the study. Neur ological signs were noted after Week 10; hyperactivity was observed in females only (n=5) between Weeks 10 a nd 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; decr eases were only significant (significance not reported, with only selected data reported) for ma les at Weeks 1 and 2 and for females at Weeks 3, 4, 5, 7, and 12. Slight decreases in food cons umption were consider ed negligible. No treatment-related effects were noted for hematology, clinical chemistry, or urinalysis parameters. Changes in relative liver weight s in males were not considered to be related to treatment. Treatment-related increases (significance not repo rted) in absolute kidney weights were seen in high-dose females ( ↑ 8 and 9%), and relative kidney weight s were increased in high-dose males and females. Gross pathology was negative, a nd microscopic histopathol ogy was not conducted.

The LOAEL is 300 ppm (7.5 mg/kg/day for male s; 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;

A α =24.2%) or esfenvalerate (94.5% purity; A α =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 esfenva lerate were 10.5/12.6, 30.5/36.8, and 106/113 mg/kg/day; achieved doses of fenvale rate 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.ESFENVALERATE FINAL

Page 47 of 62 No deaths occurred during the study. Clin ical signs were observed in the high-dose esfenvalerate group and the fenvalerate gr oup: fibrillation, tremors, convulsions, hypersensitivity, abnormal gait, and salivation (W eek 1); scratching and lic king (after Week 3); and alopecia, scabs, and sores (after Week 4). Hypersensiti vity 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 dur ing 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 ma les and high-dose esfenvalerate females by Days 63-90. No treatment-related differences in food consumption were obs erved. Decreases in
water intake were noted for the firs t few weeks, but increased by Week 5.

Absolute liver weights were decreased in th e 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 we ights were also increased in the fenvalerate-treated animals.
Some increases were also observed in absolute a nd relative spleen weights, and absolute/relative salivary gland weights in the high-dose esfenvalerate and fenvalerate males and females. Gross pathology findings were primarily as sociated with the long-term e ffects 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 neoplas tic 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 (equ ivalent to 106/113 mg/kg/day, males/females), based on clinical signs of toxi city in both sexes, increased water intake, skin lesions, and stomach lesions. The NOAEL is 150 ppm for es fenvalerate (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; % A α 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 (1 05 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 a ssessed at pretest and just prior to sacrifice. Organs that were weighed include d the liver, kidneys, testes, ep ididymides, 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. A bnormal 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, medi um-high, and high-dose groups, respectively.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 wa s not noted in the FOB assessmen t 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 sing le incident in the cont rol male group was noted on Day 10 after most of the males stopped s howing 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 incide nt at 25 mg/kg/day is consider ed related to treatment. Other clinical signs noted included hyperactivity/hype rreactivity and vocalizati on in the medium-high and high-dose females. Motor activity data we re confounded by the pretest baseline data being elevated in the higher dose groups, but even afte r accounting for the higher baseline there was an
increased mean number of movements and durati on of movements in the female medium-high and high-dose groups. Howeve r, it could not be determin ed whether the property of esfenvalerate to produce a dermal sensory stimula tion 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 we re the result of system ic 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. Howeve r, because the effect was no cumu lative, 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 irrita tion is >1000 mg/kg/day. However, no NOAEL and LOAEL is being assigned for sensory stimulatio n 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 pe riod. Organs that we re weighed included the liver, kidneys, adrenal, and testes. Gross and microscopic pathologic al examinations were conducted on these tissues as well as the pituitary, thyroi d, 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 er ythema was present in all animals except the control and low-dose males, with no dose response. Mild edema, superfic ial necrosis, and scar tissue were noted in medium-high and high-dose fe males. No deleterious, treatment-related effects on body weight, body weight gain, food c onsumption, organ weights, or microscopic pathology were noted.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 irr itation 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; % A α 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/da y on GDs 6-15, inclusive. The rats were sacrificed at GD 21 of gestati on and subjected to gross necrop sy. All fetuses were weighed, sexed, crown-rump measurement taken, and examined for gross external alte rations. One-half of
the fetuses in each litter were examined for visceral soft tissue alterations by the Wilson technique. The remaining fetuses in each litt er were examined for skeletal alterations.

Maternal toxicity was observed at all doses. Behavioral/CNS in cidences in the low-dose group included erratic jerking and exte nsion of forelimbs (22/25), rapi d 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 th e 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 atax ia, ataxia, and convulsions. Incidence and frequency of these behaviors increased with incr easing dose. Dams in the pilot study displayed
similar behaviors at esfenva lerate concentrations of ≥ 4 mg/kg/day, but not ≤ 3 mg/kg/day. Decreased (p<0.05) body weight gains of 8% we re observed in the medium-high and high-dose dams during GDs 6-15. Overall body weight gain s were not affected, and no treatment-related changes in food consumption were reported. Th ere were no treatment-related findings at

necropsy.

Reproductive (cesarean section) pa rameters were not affected by treatment with esfenvalerate; there were no abortions or early deliveries. No treatment-related e ffects 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 matern al 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 No n-Rodents (Rabbit; OCSPP 870.3700)

In a developmental toxicity study in rabbits ( MRID 43211503 ), esfenvalerate (97.1% purity; %

A α not reported) was administered to artifici ally-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 sa crificed, and all fetuses were weighed, sexed, crown-rump measurements were taken, and extern al examinations were conducted. Heads fromESFENVALERATE FINAL

Page 50 of 62 one-half of the fetuses in each litter were exam ined for visceral soft tissue alterations by the Wilson technique. All carcasses were eviscera ted and processed for skeletal examination.

One high-dose rabbit died on GD 17, but the death di d not appear to be related to treatment. Maternal toxicity was observed at all doses. Behavioral/CNS in cidences in the low-dose group included erratic jerking and exte nsion of forelimbs (11/20), rapi d 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 hypersens itivity 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 increas ing 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 decrease d 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 in creases during GDs 19-29 (overall gains ranged from ↓ 14 to ↑ 28%). Decreases (p<0.01 or 0.05) in f ood 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 fi ndings at necropsy.

Reproductive (cesarean section) pa rameters 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 treatmen t was not established. No treatment-related effects were noted during external, visceral, or sk eletal examinations. The ma ternal LOAEL is 3.0 mg/kg/day, based on behavioral/CNS clinical signs, and th e maternal NOAEL is 2.0 mg/kg/day (developed from the pilot study results). The developmen tal 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;
% A α not reported) was administered to Crl:C D (SD) BR rats (30/sex/dose) at nominal concentrations of 0, 75, 100, or 350/150 ppm (dieta ry concentration reduc ed to 150 ppm after approximately four months of dosing). M ean 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, F 1 generation). Two successive generations were produced, each consisting of one breeding (two sets of litters).

Seventy-three days after in itiation of the study, each P 1 female was mated with a randomly selected male in the same dose group until a c opulatory plug was observed or until three weeks had passed. F 1 rats were mated in a similar manner afte r they had been administered their diets for 105 days. Siblings were not mated.ESFENVALERATE FINAL

Page 51 of 62 P 1 males in the high-dose group exhibited abnormal gait/mobility, alopecia, scabs and sores. Two males in the high-dose group died; one deat h was due to genitourinary tract inflammation,but the second death was believed to be due to treatment-related ulcerative dermatitis. Highdose P 1 females exhibited abnormal gait. One P 1 female in the medium-dose group was sacrificed in extremis (attributed to dystocia). F 1 males and females in the high-dose group exhibited various clinical signs of neurotoxicity (abnormal gait/m obility, ataxia, barrel rolls, general spasms, hyperactivity, hypersensitivity, p opcorn 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 we re sacrificed in extremis by Da y 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 P 1 and F 1 animals were available. Minimal effects
occurred in the low- and medium-dose animal s throughout the study. Body weight gains in the high-dose (350 ppm) P 1 animals during the premating peri od were generally decreased during the initial weeks, but overall body weight gains in high-dose P 1 males and females were only decreased (p<0.05) by 19 and 32%, respectivel y. Decreased body weights in the high-dose females during gestation were attributed to pr e-existing conditions. Body weight gain was decreased (p<0.05) in the high-dose P 1 females during lactation. Body weight gains in the highdose (150 ppm) F 1 animals during the premating period were slightly decreased during the initial weeks, but increased mortality occurred in high-dose F 1 animals by approximately Week 5. Food consumption was generally decr eased in the high-dose animals.

There were no differences in the mating indices, fertility indices, or ge station length of the P 1 or
F 1 groups. The mean number of F 1 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. F 1 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). F 1 pup weights were also decreased in the medium-dose group on PNDs 14 and 21 by approximately 6%. The mean number of F 2 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 avai lable due to mortality). F 2 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 observati ons were noted in the high-do se 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 P 1 animals and a dose-dependent res ponse in a greater number of F 1 parents.ESFENVALERATE FINAL

Page 52 of 62 No differences were noted in gross necropsy fi ndings 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 F 1 females and an increased incidenc e 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 F 1 pup weights on PND 21 of lact ation, decreases in litter
size and F 2 pup weights, and an increas ed 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 diet s continuously until termination (approximately 25 weeks). The parental females were fed the test diets contin uously during pre-mating (10 weeks for the F 1 a litters, 7 weeks for the F 1 b litters), mating, gestation, and lactation. On PND 21, pups (24/sex/dose group) were selected from the F 1 a and F 1 b litters to continue as

F 1 adults. The F 1 a adults were fed the test diets for 203-205 days; the F 1 b adults were fed the test diets for 105 days. This supplemental study was conducted because in the two-generation reproduction toxicity stu dy with esfenvalerate ( MRID 43489001 ), body weights were decreased
in the F 1 generation at 75 ppm (the lowest dose te sted), and a NOAEL was not determined. The effect at the low dose was thought to have been due to reduce d food consumption due to skin paresthesia caused by esfenvalerate in pulve rized 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 F 1 b pups, and
another low-dose P female was euthan ized on GD 23 of production of the F 1 b pups due to the duration of delivery (>24 hours). None of these d eaths were considered to be treatment-related. Additionally, there were no trea tment-related effects on clinic al signs of toxicity, food consumption, body weight, or macroscopic pathol ogy. 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 F 1 a litters, but the overall body weight gain fo r 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 se xes. The NOAEL is 40 ppm (equivalent to 1.89/2.48 mg/kg/day in males/females).

There were generally no treatment-related e ffects on reproductive performance. In the production of the F 1 b litters, the fertility indices were lower (non-dose-depende nt) in the control and all treated groups (27-72%; p<0.01 at the medium dose, ↓ 63%). The fertility index for the

F 1 a litters was also low (76-87%). This effect wa s considered to be due to the advanced age ofESFENVALERATE FINAL

Page 53 of 62 the parental animals at the time of mating for the production of the F 1 b litters. The LOAEL for reproductive toxicity was not observed. The NO AEL for reproductive toxicity is 100 ppm (equivalent to 4.74/6.34 mg/kg/ day in males/females).

F 1 a offspring body weights were decreased in ma les and females on PNDs 0 and 4 in the highdose group, but weights recove red through PND 21. After w eaning, body weights (PNDs 21, 112, and 244) and body weight gain (PNDs 21-244) in females selected as F 1 a adults were decreased (p<0.05 or NS) in th e 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 F 1 b adults, but decreases were <10%. The LOAEL for offspri ng toxicity is 100 ppm (equivalent to 4.74/6.34 mg/kg/day in males/females) based on decrease d body weight and body wei ght 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/Carcinogenici ty in Rodents (Rat; OCSPP 870.4300)

In a chronic toxicity/carci nogenicity study in rats ( MRIDs 00082244 and 00111888 ), fenvalerate (98% purity) was admini stered 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 sacr ificed after three (interim) and six months of dosing. At the interim sacrif ices or at study termination, all selected or surviving rats were examined for gross and histop athological evaluation. In terim 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 wei ghed 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 surv ival. Transient clinical signs were attributed to aging or injury. Body weight determinati ons 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 ha d a 10% decrease after three months. Food consumption was considered unremarkable. No treatment-related effects on urinalysis, hematology, or clinical chemistry parameters we re observed, and organ we ights were generally
unremarkable.

No treatment-related effects on gross and microscopic pathology were observed throughout the study. Pituitary tumors were noted in all te st 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 concu rrent 26-week study at 500 ppm fenvalerate.ESFENVALERATE FINAL

Page 54 of 62 In a complementary chronic toxici ty/carcinogenicity study in rats ( MRID 00079877 ), fenvalerate (98% purity) was ad ministered 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 approxi mately two years. Interim sacr ifices were not reported; at study termination, all selected or surviving rats were examined for gross and histopathological evaluation. Select organs weighe d 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 surviv al; 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 resolv ing within a week) and non-treatment-related alopecia. Body weights in males were decrea sed from Weeks 16-104; weights were decreased generally ≥ 10% by Week 72. Body weights in fema les were decreased from Weeks 44-104, with weights generally decreased ≥ 10% by Week 60. Food consumption was considered unremarkable. In general, no treatment-related effect s on urinalysis, hematology, or clinical chemistry parameters were observed, and or gan weights were generally unremarkable.

No treatment-related effects on gross and microscopic pathology were observed throughout the study related to endocrine endpoints. Anterior pitu itary 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 in takes of 0, 0, 1.5, 7.5, 38, or 187.5 mg/kg/day)
for 104 weeks. Interim sacrifices were not reported. At study te rmination, all surv iving mice in all groups were sacrificed for gross and histopath ological evaluation. Sel ect organs weighed at termination included the liver, kidneys, adrenal, and brain. Gross and mi croscopic pathological
examinations were conducted on these tissues as well as the pituit ary, 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 insurvival 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 we ights 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 ofESFENVALERATE FINAL

Page 55 of 62 10% only observed in males and females at W eeks 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 an imals throughout the study, w ith 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 ma les 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 previousl y. Carcinogenicity was not indicated in this study; the only non-neoplastic pathology obser ved 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/da y) and 250 ppm for fe males (38 mg/kg/day) based on the incidences of granulomata (with de creased body weight and increased mortality at the high dose); the NOAELs are 10 and 50 ppm fo r 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 mont hs. 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 terminati on included the liver, kidneys , testes, adrenal, and brain. Gross and microscopic pa thological examinations were c onducted on these tissues as well as the pituitary, thyroid, prosta te, ovaries, epididymides, semina l vesicles, uterus, vagina, and mammary gland.

Clinical signs and lesions observe d in treated mice (medium- and hi gh-dose) were attributable to self-mutilation or secondary effects of self-mutilation. Self-mutilati on occurred due to the induction by esfenvalerate of derm al sensations that stimulate scratching by the mice (a wellknown toxicological effect of pyrethroids). All treated mice exhibite d 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 hi gh- 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 fr om 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 male s and females) by Study Day 56. This excessive mortality wasESFENVALERATE 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 contro l 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 Guid eline requirement (not less than 25%) for this interval. Body weights for selected study days in the low- a nd 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 increase d spleen weights are belie ved 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. Gr oss pathological examination of the medium-dose male and female mice exhibited enlarged spl eens, increased skin ulceration/erosion, and alopecia. The medium-dose females also ex hibited enlarged mandibular lymph nodes. Microscopically, the medium-dose group had incr eased skin erosions/u lcers and eye lesions (erosions/ulcers/keratopathy). Th ese lesions are considered sec ondary physiologic responses to
the chronic inflammation due to self-mutila tion. 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 carcinogeni city 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 fe males); a NOAEL is not establis hed. 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/Carcinogenici ty in Non-Rodents (Dog; OCSPP 870.4100)

In a chronic toxicity/car cinogenicity study in dogs ( MRID 00163855 ), fenvalerate (98.7% purity) was administered to Beagle dogs (6/sex/dose group) in the di et 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 thyr oid; histopathological examinations were conducted on these tissues as well as the pituitary, epidi dymides, prostate, and uterus.ESFENVALERATE FINAL

Page 57 of 62 No mortality occurred during th e study. Control and treated an imals displayed clinical signs including soft stools, mucoid stools, and food-like vomitus; no tr eatment-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 treatmen t-related effects on organ weight s, or on gross or microscopic pathology, and no evidence for esfenvalerate be ing oncogenic in the dog. A LOAEL was not observed, and the NOAEL is greater th an 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 a nd 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 (T 3 ) and

T 4 concentrations were determined by radioimm unoassay. After Day 45, animals were sacrificed
and the brains were removed for determin ation of total and protein- bound calcium concentrations (not discussed further).

Rats administered fenvalerate at 100 and 200 mg/kg/day exhibited hypere xcitability 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 fi rst 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 st udy (Day 45), with the degree of paralytic severity dependent on dose. At Day 0, the concentrations of T 3 and T 4 were similar in all study animals (T 3 = 0.35±0.027 ng/mL, T 4 = 33.0±0.009 μg/dL). Control animals administered corn oil alone for 15, 30 or 45 days did not exhib it any changes in the concentrations of T 3 or T 4

. Administration of fenvalerate incr eased concentrations of both T 3 and T 4 , with a dose-dependent relation in both dose and time (duration of dosing). Over the 45-day treatment period, T 3 concentrations increased by 54, 119, and 138% in the low-dose group and increased by 144, 210, and 278% in the high-dose gr oup, respectively. Similarly, T 4 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 T 3 and T 4 could be due to a
direct effect of fenvalerate on the mechanis m of thyroid activity or due to some other perturbation of the HPT axis.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 th yroid 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% pur ity, 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 (s even weeks total). Thyroid weight, and concentrations of plasma TSH and total T 4 were not different compared to control at study termination. Total T 3 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 T 3 and T 4 at
an identical dose afte r 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 nom inal concentrations of 0 (acetone, 0.35% v/w), 250, 500, or 1000 mg/kg diet for 25 weeks (fifteen w eeks prior to the start of egg production andten 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. Temper ature was maintained at 16-27°C throughout the study and humidity was maintained at 42-70%. I ndividual adult body weight s were recorded at Week 0, 2, 4, 42, 6, 8, 10, and 25, and body weight ch anges were determin ed for each interim period. Food consumption in each replicate was measured weekly throughout the pre-treatment and treatment periods of the adult phase. Indi vidual chick body weight s were recorded after hatching and on Day 14 after hatching; egg sh ell thickness was measured every two weeks during egg production. Adult birds and chicks were observed routin ely 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, resp ectively. Several of the deaths were attributed to pen-mate aggressi on (cages in the presen t study provided only 13% of the recommended floor space, and overcrowd ing was cited as poten tial confounder). Adult body weight changes and food consumption during the treatment period were generally similar in all groups, and there was no ev idence of a treatment-related e ffect. 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- a nd 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 statis tically significant. Also, food consumption was decreased (p=0.057) by 7% in the low-dose group.ESFENVALERATE FINAL

Page 59 of 62 A decrease (p=0.0483) in the proportion of 14-day ol d survivors of hatchlings in the low-dose group compared to control (89 vs. 95% control) was observed. It was re ported that the reduction was the result of poor hatchli ng 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 contro l 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-da y offspring body weights. No
other treatment-related eff ects were noted on any study parameters. The reproductive NOAEC for bobwhite quail exposed to es fenvalerate in the diet for 25 weeks was 19.1 mg/kg/day (257 ppm). The LOAEC is 38.7 mg/kg/day (504 ppm) ba sed on decreased hatchling weights. All other parameters had a NOAEC of 78.5 mg/kg/day (1000 ppm).

The one-generation reproductive toxi city of Esfenvalerate to 16 pa irs per level of 25-week old mallards ( Anas platyrhynchos ) was assessed over 20 weeks ( MRID 49008802, 2012 ).
Esfenvalerate was administered to the birds in th e diet at nominal concen trations of 0 (negative control), 250, 500, and 1000 mg ai/kg diet. Mean-m easured 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 concentrat ion. 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 di et (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. Thoug h not fully acceptable due to the guideline deviation, it satisfies the guideli ne 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 repr oduction studies were conducted using fenvalerate with both bobwhite quail and mallard duck. In one set of studies, bobwhite quail an d 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-re lated 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 c oncentrations and was re ported in the top two concentrations in the mallard duck study. The review er noted that there were some questions regarding the top concentration (125 mg/kg-diet) as measured sa mples reported concentrations of 60-85 mg/kgdiet. In two other reproduction studies with bobwhite quail and mallard ( MRID 00109832 andESFENVALERATE 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 consumpti on 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 review er indicated that mean eggshell thickness was slightly below the normal range and percent of cracked eggs wa s 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.ESFENVALERATE FINAL

Page 61 of 62 APPENDIX 3: References Not Utilized in the Esfenvalerate WoE Analysis

In 2009, after public review and comment, a fina l list of 67 chemicals and schedule for issuing
Test Orders for the EDSP Tier 1 screening battery was made av ailable in a Federal Register Notice issued October 21, 2009 (74 FR 54422). The ag ency’s review of the initial data submitted as “other scientifically releva nt 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 registra tion/reregistration, published literature articles and/or Tier 1 assays. The agency also c onducted a more recent search (2009 to 2014) of
available scientific literature fo r any additional relevant informa tion for their weight of evidence (WoE) evaluations. These articles were evalua ted in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/s cience/efed/policy_guidance/team _authors/endangered_species _reregistration_workgroup/PDF_ro t/esa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature To xicity Studies to Suppor t 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 wa s not used in the study; 3) the test material was not adequately describe d; 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 adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s 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) th e 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 me thods for endocrine disrupting chemicals in fish and daphnids. Pesticide Chemistr y: Crop Protection, Public H ealth, Environmental Safety (2007), eds. Okawa, H.,Miyagawa, H. and Lee, P., 415-424.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.