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

## ~ S

## ~~~ 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

PATHWAY

CHEMICAL: BIFENTHRIN

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYBIFENTHRIN 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...........................17

IV. Committee’s Assessment of Weight of Evidence..........................................19

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI....................19

1. Tier 1 in vivo Assays.............................................................20

2. OSRI................................................................................20

B. Estrogen Pathway...........................................................................22

C. Androgen Pathway.........................................................................22

D. Thyroid Pathway............................................................................23

E. Conclusions..................................................................................23

V. EDSP Tier 2 Testing Recommendations.....................................................23

VI. References........................................................................................24

LIST OF TABLES: Table 1. Tier 1 Assays for Bifenthrin.................................................................7
Table 2. Estrogenic/Anti-Estroge nic Pathway for Bifenthrin.....................................10
Table 3. Androgenic/Anti-Androge nic Pathway for Bifenthrin..................................14
Table 4. Thyroid Pathway for Bifenthrin.............................................................18

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................28
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................40

APPENDIX 3. References Not Utilized in the Bifenthrin WoE Analysis............................56BIFENTHRIN FINAL

iii 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 eneration
F2 Second filial g eneration
Fcd Fecundit yBIFENTHRIN FINAL

iv Abbreviation Terminology

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 Concentration
MTD Maximum Tolerated Dose
N Ne g ativeBIFENTHRIN FINAL

v Abbreviation Terminology

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 Tie r 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)

TP Testosterone Pro p ionate
T R Th y roid Rece p to rBIFENTHRIN FINAL

vi Abbreviation Terminology

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-EvidenceBIFENTHRIN FINAL

Page 1 of 59 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 recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (H PG) and hypothalamic-pituitary thyroid (HPT) perturbations.
In addition to the available Ti er 1 data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether bifenthrin interacts with E, A, or T hormone path ways, the number and type of effects induced, the magnitude and pattern of responses observed acr oss studies, taxa, and
sexes were considered. Additi onally, the conditions under which effects occu r were considered, in particular, whether or not e ndocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On June 11, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) analysis of the potential inte raction of bifenthrin with the E, A or T hormone pathways. The T1 WoERC 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).

Bifenthrin is a member of the synthetic pyrethr oid class of insecticide having similar toxicity profiles that elicit neurotoxic be haviors in laboratory animals. This assessment includes studies conducted with two enantiomers: 1S-cis -bifenthrin and 1R -cis-bifenthrin.

For the estrogen pathway, bifent hrin was negative in the Tier 1 in vitro assays [estrogen receptor (ER) binding, ER transactivation assay (ERT A), steroidogenesis and aromatase assays].Although, potential estrogenic and/or anti-estrogenic activity were reported in several nonGuideline in vitro assays, these positive in vitro responses were not supported by the in vivo assays. The Tier 1 uterotrophi c assay was negative and, in the female pubertal rat assay, there were no treatment-related estrogen-related effect s observed in the absenc e of overt toxicity. Furthermore, in the fish short-term reproduction assay (FSTRA), all effects in females occurred in the presence of overt toxicity. In the ma mmalian Part 158 studies, th e only estrogen-related
effect in the absence of overt t oxicity was a slight (9%) decrease in absolute ovarian weight in the two-generation rat reproduction study. In the avian reproduction studies, the only effect observed was an increase in number of cracked eg gs, but no differences in mean egg weight orBIFENTHRIN FINAL

Page 2 of 59 egg shell thickness or other reproductive parameter. Therefore, there is a lack of convincing evidence for potential interac tion with the estrogen pathway.

For the androgen pathway, bifenthr in was negative in the Tier 1 in vitro AR binding and steroidogenesis assays. Additionally, the Tier 1 Hershberger assay was negative. In a nonguideline Hershberger assay submitted as OSRI, while decreases in AST weights were reported, chemical purity and numerical AST data were no t reported. No treatment-related effects were observed in the male pubertal rat study, in the ab sence of overt toxicity. No androgen specific effects were observed in the FSTRA, in the absence of overt toxicity, or in the Part 158 wildlife studies. As such, there is no convincing eviden ce for potential interact ion with the androgen pathway.

Based on mammalian Tier 1 assays and the OSRI ma mmalian studies, bifenthr in does not appear to affect the thyroid or the HPT axis. Devel opmental findings in the amphibian metamorphosis assay (AMA) included decreased developmental stage and normalized hind limb length at Day 21 (test termination), concurrent with effects on growth (decr eased snout-vent length and body weight). Treatment-related differences in th yroid gross pathology and histopathology analyzed statistically by the study authors indicated a slight trend (p = 0.11) towards mild gland atrophy in the high treatment group relative to the negative control group with a decrease in maximum follicle size (p=0.024). Although findings of thyroid gland atr ophy are identified in the AMA guideline as diagnostic criteria, the differences between control and treated tadpoles in this study were slight. Moreover, no effects were seen on the incidence of follicular cell hypertrophy or hyperplasia, which are expected to be more co mmonly affected in the presence of a thyroidrelated developmental delay. Overall, the thyroi d and developmental find ings in the AMA with
bifenthrin are inconsistent with a direct, thyroid-related delay and may be more reflective of treatment-related growth inhibition.

In both the female and male pubertal rat assays, no treatment-related effects were noted for thyroid weight or histopa thology. Serum thyroxine (T 4 ) concentration was de creased only in the low-dose groups of both sexes ( ↓ 16%), with an increase in t hyroid stimulating hormone (TSH) concentration occurring only in the high-dose males and which was associated with overt toxicity. Furthermore, there were no e ffects on thyroid weight or histopathology.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not recommended for bifenthrin since there was no convincing evidence of pote ntial interaction with the estrogen, androgen or thyroid pathways

.BIFENTHRIN FINAL

Page 3 of 59 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 (H PG) and hypothalamic-pituitary thyroid (HPT) perturbations.
In addition to the available Ti er 1 data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether bifenthrin interacts with E, A or T hormone path ways, the number and type of effects induced, the magnitude and pattern of responses observed acr oss studies, taxa, and
sexes were considered. Additi onally, the conditions under which effects occu r were considered, in particular, whether or not e ndocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On June 11, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WOE) an alysis of the potenti al interaction of bifenthrin with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Bifenthrin is a synthetic pyrethr oid (Type I) insecticide/ acaricide and is a chiral compound with
two enantiomers: 1S-cis-bifenthrin and 1R-cis-bifenthrin. The 1R- cis -bifenthrin enantiomer is more effective as a pesticide and commercia l products consist of predominantly the 1R- cis

bifenthrin enantiomer. Bifenthrin has a vapor pressure of 1.8 × 10

− 7 mm Hg at 20 to 25°C and it will exist as both a vapor and a particulate if released to the atmosphere. The water solubility of bifenthrin is 0.0140 μg/L and ! has an octanol/water partition co efficient (log Kow) of 6.00. The
estimated bioconcentration factor for bifenthrin is 6,089. Bifenthrin is ex pected to be removed from the atmosphere by photodegradation and depos ition. Volatilization from water is also expected to contribute to the diss ipation of bifenthrin in the e nvironment. Bifenthrin has soil half-life estimates of approxi mately 100 to >300 days.

The available information considered in determin ing the potential interacti on of bifenthrin with the E, A, T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxicity stud ies and/or other published articles. These data are summarized in Sections III.A through III.C. An analysis of the data submitted to the agency,BIFENTHRIN FINAL

Page 4 of 59 using the WoE approach outlined by the Agency (U SEPA, 2011), is presented in Section IV. The
EDSP Tier 2 studies r ecommendations are presen ted 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 for bifenthrin are show n 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 Revi ew Team on bifenthrin (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any additional relevant info rmation. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/ studies considered but not uti lized 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.BIFENTHRIN FINAL

Page 5 of 59 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 concordance or consistency (complementarity) 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 mol ecular initiating event 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 releva nt, 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 biological organization? Finally, under what conditions did the responses occur including co nsideration of whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemicBIFENTHRIN FINAL

Page 6 of 59 toxicity introduces uncertainty in the ability to distinguish eff ects specifically related 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 ., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular 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 addressing the three main components described 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.BIFENTHRIN FINAL

Page 7 of 59 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 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 th e presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not re ported in the table for E, A or T.

A. EDSP Tier 1 Screening Assays

The Tier 1 assays submitted in response to the ag ency’s test order for bifenthrin are shown below
in Table 1.

Table 1. Tier 1 Screening Assays for Bifenthrin.

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID 48615003)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID 48615004)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID 48615001)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID 48615006)

Aromatase Assay (human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID 48615002)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID 48615007)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID 48615005) and by OSRI (Zhang et al ., (2008)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID 48669401)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID 48669401)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID 48675902)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied (MRID 48675901)BIFENTHRIN FINAL

Page 8 of 59 . B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for bifenthrin to interact w ith the estrogen pathway is summarized in Table 2. The various targets of the estrogen pathway across the relevant Tier 1 a ssays 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.

Bifenthrin was negative (o r not interactive) in the in vitro ER binding, ERTA, aromatase, and steroidogenesis assays. Seve ral published, non-guideline in vitro estrogen-res ponsive assays
were available for bifenthrin. Brander et al. (2012) reported that bifent hrin was negative for an estrogenic response in the ER –based CALUX mammalian cell bioa ssay, but observed an antiestrogenic response at bi fenthrin concentrations of 1–100 ng/L. In an in vitro cell proliferation assay with MCF-7 cells (E-Screen), Wang et al. (2007) examined the potential estrogenic activity of the two bifenthrin enantiomers (1R- cis -bifenthrin and 1S- cis -bifenthrin). 1S- cis

bifenthrin and 1R- cis -bifenthrin increased cell proliferation 3.59- and 1.73-fold relative to vehicle control, respectively, which translated to 10% and 1% of the maximal induction by estradiol. Furthermore, prolif eration was blocked by co-adminis tration with the ER-antagonist ICI 182,780, suggesting interaction with the estrogen receptor. In another E-Screen assay (Zhao

et al. , 2010), 1S- cis -bifenthrin exhibited similar estrogenic activity.

Bifenthrin was negative in the Tier 1 in vivo uterotrophic assay. In the female pubertal rat assay, age at attainment of vaginal opening (VO) was delayed by 2.0 to 2.3 days in the low- (2.5 mg/kg/d) and high-dose (10 mg/kg/ d) animals, with no change in body weight. However, at the high dose, 9/16 animals showed tremors at 6 hours post-dose on at least one occasion during the study period, with no clinical si gns of toxicity noted 24 hours pos t-dose. While a delay in VO was observed at the low dose, in th e absence of overt toxicity, it is noted that this 2 day delay is at the upper range of normal biological variab ility, no other estrogen-related effects were observed in this study.

In the FSTRA, there was a significant 60% increa se in fecundity at the medium concentration (0.10 μg a.i./L) and a 74% reducti on at the high concentration (3.0 μg a.i./L). There was also a 51% increase in female GSI at the high concentr ation. Female body weight was increased at the high-concentration ( ↑ 28%, p<0.05), but no other gr owth endpoints were a ffected by treatment. There was an increase in oocyte atresia in 87% of the fish in the high-concentration group along
with an inc rease in post-atretic follicles and gr anulomatous inflammation which was attributed to the increased oocyte atresia. However, at the highest concentration, c linical signs of overt toxicity were observed beginning on Day 5 a nd included erratic swimming, lethargy, loss of equilibrium, and surfacing.BIFENTHRIN FINAL

Page 9 of 59 In the Part 158 subchronic dog study, there we re decreases (signifi cance NR) of 41.1% and 58.8% in mean absolute ovary weights in the medium-high and high-dose females, respectively,
however these effects co-occurred with tremors. Additionally, in the Part 158 two-generation reproductive study in rats, mean absolute ovary we ights were slightly de creased at the medium and high doses (9% and 12%, respectively) in the F 1 parental generation; at the high dose this cooccurred with tremors. Increased (55%) absolute ovary weights were also observed in the highdose group F 1b female offspring which co-occurred with tremors. Non-significant, non-dosedependent increases in ovarian weight of 45 and 30% were obser ved in the low- and mediumdose groups, respectively. However, no histopathol ogical changes were noted in these animals, and there were no treatment-related effects on fertility or fecundity. Bifenthrin did not alter other reproductive organ histopathol ogy, including the pituitary, ovaries, and uterus.

In a Part 158 avian reproduction study with the mallard duck, there were no significant effects on any reproductive parameter up to and including the highest treatment group (75 ppm). In a Part 158 northern bobwhite quail re production study, the number of cracked or broken eggs was significantly higher in th e middle (50 ppm) and high (75 ppm) treatment groups as compared to the control, but no differences in mean egg weig ht or egg shell thickness. There were no other significant effects on any other reproductive parameter.

In Brander et al ., 2012, silverside fish ( Menidia beryllina ) exposed for 14 days to 1 ng/L of bifenthrin exhibited sign ificantly greater chorioge nin (precursor to chori on, which is an estrogen dependent protein that forms the layer between the embryo and the outer layer of the egg) expression than 1 ng/L of the positive control, ethi nylestradiol. It is wo rth noting that this study did not have a negative control but rather comparisons were made to a methanol solvent control. Additionally, the measured test c oncentrations reported appear to reflect results from a single 24 hour renewal cycl e under abiotic ( i.e. without fish) conditions. Since, these results are confounded by several uncertainties including the absence of a negative control and limited exposure measurements, and this study is not reported in the table below.BIFENTHRINFINALPage 10 of 59Table 2. Estrogenic/Anti-Estrogenic Pathway for BifenthrinLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Bifenthrin1Study Type/Literature Citation ER Binding ER Activation/ Gene expression Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48615003)NERTA (MRID48615004)N4Aromatase(MRID 48615002)NSteroidogenesis(MRID 48615006)NUterotrophic(MRID 48615007)NNEN NFemale Pubertal Rat(MRID 48669401)NENNNN N NNX(H)FSTRA(MRID 48675902)NENNNFcd:↑60%(M)NX(H)OSRIERTA (CALUX)(Branderet al., 2012)PCell Proliferation(Wanget al., 2007)PCell Proliferation(Zhaoet al., 2010)PER-related geneexpression (Liuet al.,2011ab)PBIFENTHRINFINALPage 11 of 59Table 2. Estrogenic/Anti-Estrogenic Pathway for BifenthrinLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Bifenthrin1Study Type/Literature Citation ER Binding ER Activation/ Gene expression Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Subchronic toxicity(Rat; MRID00141199)NE N N NENX(MH,H)Subchronic toxicity(Dog; MRID00141200)NENNNENNX(ML,MH, H)Developmentaltoxicity (Rat; MRID00141201)NENNX(H)Developmentaltoxicity (Rat; MRID45352301)NRNNX(H)Developmentaltoxicity (Rabbit;MRID 00145997)NENNX(H)DevelopmentalNeurotoxicity (Rat;MRID 46750501)NENNNX(MH, H)Two-generationreproduction (Rat;MRID 00157225)NR↓9%(M)NNRNNX(H)Chronictoxicity/oncogenicity(Rat; MRID00157226)NENNNENX(H)Carcinogenicity(Mouse; MRID00157227)NRNRNRNRNX(MH, H)BIFENTHRINFINALPage 12 of 59Table 2. Estrogenic/Anti-Estrogenic Pathway for BifenthrinLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Bifenthrin1Study Type/Literature Citation ER Binding ER Activation/ Gene expression Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Chronic toxicity(Dog; MRID00163065/40266601)NENNNNX(MH, H)Avian reproduction(Duck; MRID00163099)NN NAvian reproduction(Quail; MRID00163097)P5N 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 is parameter isnot routinely evaluated or is not applicable in this assay. Changesin 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 Xin this 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. The responsiveness of the cells to the very weak positive control 17α-methyltestosterone was lower than the expected values. Although the conditions ofthis assay were not optimal to detect very weak activity, bifenthrin responses were similar to those of the negative control corticosterone and notcomparable to the responses of 17α-methyltestosterone, which was able toreach a maximum of 8.1-52.8% PC. Bifenthrin was only able to reach amaximum of 3.8-5.0% PC when tested up to the highest concentration possible based on cytotoxicity (10-4M). Because the RPCMax< PC10in both assayruns, bifenthrin was considered negative for estrogen receptor transcriptional activation in this test system.5. The number of cracked or broken eggs was significantly higher (p<0.05) in the middle (50 ppm) and high (75 ppm) treatment groups as compared to thecontrolP Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedBIFENTHRIN FINAL

Page 13 of 59 2. Effects on Androgen Pathway

The potential for bifenthrin to interact w ith 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.

Bifenthrin was classified as negative in the Tier 1 in vitro AR binding and steroidogenesis assays. The Tier 1 in vivo Hershberger assay was negative [ up to 10 mg/kg/day (highest dose tested)]; testosterone co ncentrations were not determined. In a non-guideline Hershberger assay submitted as OSRI (Zhang et al. 2008), treatment with bifenthr in (13.5 mg/kg/day) decreased weights of all the accessory sex tissues (AST). However, the extent of the decreases in AST weights is unknown as numerical data were not reported, also th e purity of bifenthrin was not reported. Therefore, these findings are not reported in the table below. In the Tier 1 male pubertal rat assay, no treatment-related effects were noted on age at prep utial separation (PPS), body weight at PPS or serum testosterone concen tration. There was a statistically significant increasing trend in LABC (adjusted) and dorsolate ral prostate (unadjusted and adjusted) weight, however pairwise comparison to controls was not statistically signifi cant and there were no treatment-related macroscopic observations or histopathological changes observed. Pituitary weight increased 11% at the high dose in this st udy. In the high-dose group (10 mg/kg/day), 7/16 animals displayed tremors on at least one day.

In the FSTRA, there was 100% mortality of male s in the high-concentration group (0.402 μg/L) and one death (male) at the medium concentratio n (0.075 μg/L). In the surviving males, there were no significant effects on male VTG, male GSI, median tuberc le scores or treatment-related effects in male gona dal histopathology.

No androgen-related effects on reproductive paramete rs due to bifenthrin treatment have been reported in existing Part 158 studies.BIFENTHRINFINALPage 14 of 59Table 3. Androgenic/Anti-Androgenic Pathway for BifenthrinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Bifenthrin1Study Type/Literature Citation AR Binding 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 Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48615001)NSteroidogenesis(MRID 48615006)NHershberger(MRID 48615005)NENN NMale Pubertal Rat(MRID 48669401)NNNNNNNNX(H)X(H)FSTRA(MRID 48675902)NENNNNNX(H)BIFENTHRINFINALPage 15 of 59Table 3. Androgenic/Anti-Androgenic Pathway for BifenthrinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Bifenthrin1Study Type/Literature Citation AR Binding 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 Observed 2 Overt Toxicity Observed 3OSRISubchronic toxicity(Rat; MRID00141199)NNNENNNNX(MH, H)Subchronic toxicity(Dog; MRID00141200)NNNNNNENX(ML,MH, H)DevelopmentalNeurotoxicity (Rat;MRID 46750501)NNX(H)Two-generationreproduction (Rat;MRID 00157225)NNNRNRNRNR NN NChronic toxicity/oncogenicity (Rat;MRID 00157226)N N NENNE NENX(H)CarcinogenicityMouse; MRID00157227)NR NR NR NR NR NRNX(L, MH,H)Chronic toxicity(Dog; MRID00163065/40266601)NNNNNNNX(MH, H)Avian reproduction(Duck; MRID00163099)NN NBIFENTHRINFINALPage 16 of 59Table 3. Androgenic/Anti-Androgenic Pathway for BifenthrinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Bifenthrin1Study Type/Literature Citation AR Binding 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 Observed 2 Overt Toxicity Observed 3Avian reproduction(Quail; MRID00163097)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. A shaded cell indicatesthat is parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventralprostate (VP), Dorsal prostate(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. Fordetails see Section IV. AN Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedBIFENTHRIN FINAL

Page 17 of 59 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 bifenthrin to interact with thyroid regulation are summarized in Table 4. The various targets of the th yroid 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.

In the AMA, there were significant increases in growth [snout-vent length (SVL) and/or body weight] between the negative and solvent control. As result, the effects discussed reflect the results of the treatment concentrations compared to solvent control. Bifenthrin significantly delayed (p<0.05) median Nieuwkoop-Faber (NF) deve lopmental stage by 1.5 stages at 21 days in the highest treatment concentra tion (0.235 μg a.i./L). In addition, day 21 normalized HLL was significantly decreased (p<0.05) by 18.7% in the highest concentration. Asynchronous development (stages 54 and 55) was observed in one tadpole at test termination in the middle treatment (0.0506 μg a.i./L). Mild to moderate thyroid gland at rophy and follicle size decrease were observed in the negative and solvent controls and all treatment groups with a slight increase in incidence with increasing con centration, particul arly at the highest tr eatment concentration.
Treatment-related differences in thyroid gross pathology and histopathology were analyzed statistically by the study authors and included a significant decrease in maximum follicle size (p=0.024) relative to the negative control. A non-significant (p=0.11) increase in the incidence of mild thyroid atrophy was also observed (compared to negative control). Day 7 SVL was significantly decreased at the middle and hi gh treatment concentration (4.4 and 13%, respectively) while at Day 21 a significant decreased was observed at all treatment concentrations (4-7%). Conversely, Day 7 body weight was significantly decreased at all treatment concentrations (13-28%) while at Day 21, this decrease was sign ificant (p<0.05) at the middle and high treatment concentrati ons (6 and 11%, respectively).

In both the female and male pubertal rat assays, no treatment-related effects were noted for thyroid weight or histopathology. In both sexes, serum thyroxine (T 4 ) concentration was decreased 16% in the low-dose groups only. T hyroid stimulating hormone (TSH) levels ( ↑ 51%) and pituitary weight ( ↑ 11%) were increased in the high-dos e males only and co-occurred with increased incidence of tremors (7/16 animals).BIFENTHRIN FINAL

Page 18 of 59 Table 4. Thyroid Pathway for Bifenthrin

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

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

EDSP Tier 1 Assays

Female Pubertal Rat (MRID 48669401) N N ↓ 16% (L) N N N X (H)
Male Pubertal Rat (MRID 48669401 ) N N ↓ 16% (L) N N N X (H)

AMA (Frog; MRID 48675901) P 4 (H) Day 21: Stage: ↓ 1.5 (H);
HLL: ↓ 19%

(H) SVL: Day 7 - ↓ 4.4, 13% (M, H), Day 21 - ↓ 4, 5, 7% (L, M, H); BW: Day 7 - ↓ 17, 13, 28% (L, M, H), Day
21 - ↓ 6, 11% (M, H) N

Subchronic toxicity (Rat; MRID 00141199) N X (H)

Subchronic toxicity (Dog; MRID 00141200) N N X (MH, H)

Two-generation reproduction (Rat; MRID 00157225) NE N X (females; H)

Chronic toxicity /oncogenicity (Rat; MRID 00157226) N N NE N X (L, M, H)

Carcinogenicity (Mouse; MRID 00157227) NR NR NR N X (ML, MH, H) Chronic toxicity (Dog; MRID 00163065/40266601) N N N N X (MH, H)

1. Key to responses: L=Low treatment, ML=Medium- low treatment, M=Medium treatment, MH=Medium high treatment, H=High treatment. Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated.
2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A
3. The overt toxicity in the Tier 1 assa ys 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. ABIFENTHRIN FINAL

Page 19 of 59 4. Mild to moderate thyroid gland atrophy and follicle size decrease were observed in the negative and solvent controls and all treatment groups with a slight increase in incidence with increasing conc entration, particularly at the highest treatment concentration. P Positive findings N Negative findings NE Not examined NR Not reported

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 bifenthrin to interact with endocri ne 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 co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substance may contribute to the WoE 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 a sequelae of system ic effects. Endocrine modes of action may evoke 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 ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence eval uation of the results of the Tire 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l 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. In addition to the endocrine -related effects described above
for the in vivo Tier 1 assays and OSRI, other eff ects observed which may be considered overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10%BIFENTHRIN FINAL

Page 20 of 59 decrease in mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. However, in other instances, mo re than one parameter was needed to determine overt toxicity. Effects which were considered to be signs of system ic toxicity were generally less severe forms of toxicity (e.g., changes in organ weights or blood chemistry).

1. Tier 1 in vivo Assays

In the female and male pubertal rat studies, all animals survived until scheduled sacrifice. No clinical signs of toxicity we re observed at the 24-hour post- dose observation period. During
clinical observations 6 hours post dose, 7/16 males and 9/16 females in the 10 mg/kg/day group
were found to have tremors on at least one da y. These animals appeared normal on all other days of the study. No systemic or overt toxicity was noted in the Hershbe rger assay at dose up to 10 mg/kg/day or in the ut erotrophic assay at doses up to 15 mg/kg/day.

In the FSTRA study, there was 100% mortality of males in the high-concentration group (0.402 μg/L), and one death (male) at the medium con centration (0.075 μg/L). This was observed with several females in the high treatment group exhib iting clinical signs of toxicity that included erratic swimming, lethargy, and lo ss of equilibrium. Additiona lly, there was a significant (p<0.05) increase in female body we ight at the highest treatment concentration. No signs of overt toxicity were observed in the AMA.

2. OSRI

The most common sign of systemic toxicity in the general toxicity studies was an increased incidence of tremors and other signs of neur otoxicity. Mortality di d not occur during the subchronic studies in rats and dogs. In the subc hronic rat study, tremors were noted in animals (3/sex) during the first two weeks of the study at the medium-high dose (7.5/8.5 mg/kg/day in males and females, respectively) and in all an imals (15/sex) throughout the study at the high dose (15/17.2 mg/kg/day). In the subchronic dog stud y, tremors were noted in 3 dogs/sex at the medium-low dose (4.42 mg/kg/day) and in 4 dogs/sex in the me dium-high and high-dose groups (8.84 and 17.7 mg/kg/day, respectivel y), with an indication of a cumulative effect in the two higher dose groups. Ataxia was noted in 4 dogs/s ex at the medium-high and high-doses and in one female at the medium-low dose, and langui dness occurred in medium-dose and high-dose animals. Other dose-related clinical signs included blinking, mydriasis, nystagmus, lacrimationand polypnea in th e two highest dose groups.

There was no mortality observed du ring the two rat developmental studies. In the first, maternal toxicity was characterized as tremors in 18/ 25 dams in the high-dose group (1.77 mg/kg/day) beginning at GD 10 until GD 19 (four days after the last dose was administered). In the second rat developmental study, clinical signs indicative of neurotoxici ty, were observed at the highBIFENTHRIN FINAL

Page 21 of 59 dose (15.5 mg/kg/day). These included tremors (22/25) from GD 9-20; hype rsensitivity to sound (5/25) from GD 18-20; splayed hindlimbs (1/25) from GD 15-20; a nd piloerecti on (1/25) observed at GD 19. In a rabbit developmental study, treatment-related clinical signs of toxicity consisted of head and forelimb twitching (medium and high doses; 4.0 and 8.0 mg/kg/day, respectively) and tremors (high dose only); 19/2 0 animals in the high-dose group displayed at
least one of these clinical signs.

In a developmental neurotoxicity study in rats , no dams died during the study. Tremors were observed during the daily examinations in 8/25 fe males at the medium dose (7.2/16.2 mg/kg/day during gestation and lactation, respectively) beginning on lact ation day (LD) 14 and in 23/25 females in the high-dose group (9.0/20.7 mg/kg/da y) beginning on LD 4. In the medium-dose group, the tremors were graded as slight, resolv ing in half (4/8) of the females after one
occurrence and in the remaini ng females after 3-7 times. In the high-dose group, the tremors were graded slight to moderate and continue d for 2-18 consecutive days during lactation.
Piloerection also was observed on ce or twice in 6/25 females at the high dose, primarily during LD 14-17. During the Functional Operational Batte ry (FOB) evaluations, slight piloerection was observed in 4/25 high-dose females on GD 15 a nd in 2/25 and 1/25 females on LD 10 and 21, respectively. Clonic convulsi ons (limb tremors) and tremor s were noted in 2/25 and 7/25 females, respectively, in the high-dose group on LD 10. On LD 21, the number of females with these findings was 10/25 and 13/25, respectively. Clonic convulsions (limb tremors) and tremors
were noted in 2/23 and 3/23 female s, respectively, in the medium-dose group on LD 21. In two of 20 female pups in the high-dose group, tr emors were observed during the physical examinations on PND 28. During the FOB, an in crease in the incidence of tremors and clonic convulsions was observed in male pups at the high dose on PND 21. There was no treatmentrelated mortality in either the P or F 1 generation in the multi-ge neration reproduction study in rats. Tremors were noted only in females of both generations at the high dose (5.0 mg/kg/day) with one P generation rat observed to have clon ic convulsions. The tremors occurred during Day 9-35 after delivery in the P generation and during Day 3- 35 after delivery in the F 1 generation.

In the chronic rat study, there were no treatment -related differences in survival. Tremors were noted in all high-dose males (9.7 mg/kg/day) during Days 4-28 and in all high-dose females (12.7 mg/kg/day) during Days 4-30 and 36-38. Indi vidual animals in the low- and/or mediumlow dose groups (0.6/2.3 mg/kg/day in males and 0.7/3.0 mg/kg/day in females) exhibited tremors late in the study; one female in the medium-high dose group (6.1 mg/kg/day) exhibited tremors during Days 3-5 similar to the high-dose animals. Other clinical signs in the high-dose
males and females included increased incidences of abrasion, alopecia and tail laceration. Mean body weights of high-dose females were generally decreased starting at Week 13 and were
decreased ≥ 10% by study termination, in addition to a decreased total body weight gain ( ↓ 17%).

In the mouse carcinogenicity study, there were no treatment-related effect s on survival; however, two females and two males in the high-dose group (97.2 and 81.3 mg/kg/day, respectively) andBIFENTHRIN FINAL

Page 22 of 59 one female in the medium-dose group (82.2 mg/kg/ day) died with compound-related symptoms. Tremors were noted in all medium-high (65.4 mg/kg/day; male) and high-dose males and females during the first three months of the study . Tremors were also noted in two males and two females at the medium-low dose (25.6 and 32.7 mg/kg/day, respectively) and in one male at the low dose (6.7 mg/kg/day) late in the study. Tremors in the medium-low males were observed on Days 37-39, a period when tremors were quite frequent in the higher-dose animals. Highdose males and females also displayed cloni c convulsions, jerks and twitching.

In the chronic dog study, there were no mortalities. Tremors were noted in all males and females (4/4 per sex) in the high-dose group (4.4 mg/kg/day) during Weeks 15-29, and in 1/4 males and 2/4 females at the medium-high dose (2.7 mg/kg/ day) during Weeks 16-23. The lack of tremors after 29 weeks suggests that dogs may have develope d a tolerance to treatment. In seven of the dogs (two from the medium-high dose group and five from the high-dose group), tremors were noted prior to the daily dose, indicating a persiste nt effect from the previous day’s dose.

There were no treatment-related mortalities or cl inical signs of toxic ity noted in the avian reproduction studies with mallard ducks and bobwhite quail.

B. Estrogen Pathway

Bifenthrin was negative in the Tier 1 in vitro assays [estrogen receptor (ER) binding, ER transactivation assay (ERTA), steroidogenesis and aromatase assays]. Although, potential estrogenic and/or anti-estroge nic activity were reported in several non-Guideline in vitro assays, these positive in vitro responses were not supported by the in vivo assays. The Tier 1 uterotrophic assay was negative and, in the fema le pubertal rat assay, there were no treatmentrelated estrogen-related effects obs erved in the absence of overt t oxicity. Furthermore, in the fish short-term reproduction assay (FSTRA), all e ffects in females occurred in the presence of overt toxicity. In the mammalian Part 158 studies, the only estrogen- related effect in the absence of overt toxicity was a slight (9%) decrease in absolute ovarian weight in the two-generation rat reproduction study. In the avian reproduction studie s, the only effect observed was an increase in number of cracked eggs, but no differences in m ean egg weight or egg sh ell thickness or other reproductive parameter. Therefore, there is a lack of convincing evidence for potential interaction with the estrogen pathway.

C. Androgen Pathway

Bifenthrin was negative in the Tier 1 in vitro AR binding and steroidogenesis assays. Additionally, the Tier 1 Hershberger assay was ne gative. In a non-Guide line Hershberger assay submitted as OSRI, while decreases in AST we ights were reported, chemical purity and numerical AST data were not reported. No treat ment-related effects were observed in the male pubertal rat study, in the absence of overt toxicity. No androgen specific e ffects were observedBIFENTHRIN FINAL

Page 23 of 59 in the FSTRA in the absence of overt toxicity, or in the Part 158 wildlife studies. As such, there is no convincing evidence for potential inte raction with the androgen pathway.

D. Thyroid Pathway

Based on mammalian Tier 1 assays and the OSRI studi es, bifenthrin does not appear to affect the thyroid or the HPT axis. Developmental finding s in the AMA included decreased developmental stage and normalized hind limb length at Day 21 (test termination), conc urrent with effects on growth (decreased snout-vent length and body weight). Treatment -related differences in thyroid gross pathology and histopathology analyzed statis tically by the study author s indicated a slight trend (p = 0.11) towards mild gland atrophy in the high treatment group relative to the negative control group with a decrease in maximum follicle size (p=0.024). Although findings of thyroid gland atrophy are identified in th e AMA guideline as diagnostic cr iteria, the differences between control and treated tadpoles in this study were slight. Moreov er, no effects were seen on the incidence of follicular cell hypertrophy or hyperpla sia, which are expected to be more commonly affected in the presence of a thyroid-related developmental delay. Overall, the thyroid and developmental findings in the AMA with bifenthrin are inconsistent with a direct, thyroid-related delay and may be more reflective of treatment-related growth inhibition.

In both the female and male pubertal rat assays, no treatment-related effects were noted for thyroid weight or histopa thology. Serum thyroxine (T 4 ) concentration was de creased only in the low-dose of both sexes ( ↓ 16%), with an increase in t hyroid stimulating hormone (TSH) concentration occurring only in the high-dose males and which was associated with overt toxicity. Furthermore, there were no e ffects on thyroid weight or histopathology.

E. Conclusions

There was no convincing evidence for a potential interaction with the estrogen, androgen or thyroid pathways.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not recommended for bifenthrin since there was no convincing evidence of pote ntial interaction with the estrogen, androgen or thyroid pathways.BIFENTHRIN FINAL

Page 24 of 59 VI. References

Brander, S.M., He, G., Smalling, K.L., Denison, M.S., and Cherr, G.N. (2012) The in vivo estrogenic and in vitro anti-estrogenic act ivity of permethrin and bifenthrin. Environ. Toxicol.
Chem. 31(12):2848-2855.

DeProspo, J.R., Barbera, J., Ballester, E.J ., and Geiger, L.E. (1986) Multi-Generation Reproduction Study with FMC 54800 technical in rats. Unpublis hed study performed by FMC Toxicology Laboratory, Somerville NJ. Study No. A83-977. January 31, 1986. (MRID 00157225)

Freeman, C., DeProspo, J.R., Nadasky, N., McConne ll, R.F., Fletcher, M.J., Malloy, A.V., and Bullock, W.L. (1984) Teratology study in ra bbits with FMC 54800 technical. Unpublished study performed by FMC Toxicology Laboratory, Some rville, N.J. A83-l092, Report dated February 24, 1984. (MRID 00145997)

Freeman, C., DeProspo, J.R., Nadasky, N., Fletch er, M.J., Barta, W.D., and Bullock, W.L. (1984) Teratology Study in rats with FMC 54800 technical. Unpublished study performed by FMC Toxicology Laboratory, Somerville, N.J. A83-1091. February 24, 1984. (MRID 00141201)

Geiger, L.E., Barbera, J., and Ballester, E.J. (1986) Oncogenicity study of FMC54800: lifetime feeding study in albino mice. Unpublished study performed by FMC Toxicology Laboratory, Somerville NJ. Study No. A83- 974. February 3, 1986. (MRID 00157227)

Liu, J., Yang, Y., Zhuang, S., Yang, Y., Li, F., a nd Liu, W. (2011b) Enantioselective endocrinedisrupting effects of bifenthrin on ho rmone synthesis in rat ovarian cells. Toxicology 290(1):42-

49.

McCarty, J.D., Barbera, J., and Ballester, E.J ., and Geiger, L.E. (1986) Oncogenicity study of FMC 54800: 2 year (734 day) feeding study in albino rats. Unpublished study performed by FMC Toxicology Laboratory, Somerville NJ. Study No. A83-952. January 31, 1986. (MRID 00157226)

Nemec, M. (2006). A dietary developmental neurotoxi city study of bifenthr in technical in rats. Unpublished study performed by WIL Research La boratories, LLC, Ashla nd, OH. Study number WIL-105021, January 13, 2006. (MRID 46750501)

Rand, G.M., Seaman, L.R., Ballester, E.J., Kikta, E.J., Norvell, M.J., Fletcher M.J. (1984) Ninety Day Feeding Study in Rats with FMC 54800 Tec hnical. Unpublished study performed by FMC Toxicology Laboratory, Somerville, N.J. FM C Study No. A83-818; January 31, 1984. (MRID 00141199)BIFENTHRIN FINAL

Page 25 of 59 Roberts, N.L., Phillips, C.N.K., Anderson, A., MacDonald, I., Dawe, I.S., and Chanter, D.O. (1986a) The effects of dietary inclusion of FMC 54800 on reproduction in the mallard duck. Unpublished study performed by FMC Toxicology Laboratory, Somerville, N.J. FMC Study No. A84-1260. (MRID 00163099)

Roberts, N.L., Phillips, C.N.K., Anderson, A., MacDonald, I., Dawe, I.S., Chanter, D.O., and Haynes, S.M. (1986b). The effects of dietar y inclusion of FMC 54800 on reproduction in the Bobwhite quail (Revised). Unpublished st udy performed by FMC Toxicology Laboratory, Somerville, N.J. FMC St udy No. A84-1259. (MRID 00163097)

Schneider, S.Z., T. Z. Kendall, and H.O. Kruege r. (2012) Bifenthrin: Amphibian Metamorphosis Assay for the Detection of Thyroid Active Subs tances. Unpublished stu dy performed by Wildlife International, Ltd., Easton, Maryland 21601. Laboratory project number 701A-106. Study sponsored by Consumer Specialty Products Associ ation for the Bifenthrin Task Force Steering Committee/Joint Venture. Study co mpleted August 14, 2012. (MRID 48675902)

Schneider, S.Z., K.H. Martin, T.Z. Kendall, and H.O. Krueger. (2012) Bifenthrin: Fish Shortterm Reproduction Assay with the Fathead Minnow ( Pimephales promelas ). Unpublished study performed by Wildlife Intern ational, Ltd., Easton, Maryla nd, Report No.: 701A-105. Submitted by Consumer Specialty Products Associati on for Bifenthrin Task Force Steering Committee/Joint Venture, Washington, DC. Completion date August 14, 2012. (MRID 48675901)

Serota, D.G. (1984) 13-Week Subchronic Oral Toxicity Study in Dogs. Unpublished study performed by Hazleton Laboratories Ameri ca, Inc. Project No. 104-217. February 7, 1984. MRID (00141200)

Serota, D.G. (1985) 52-Week Chronic Oral Toxicity Study in Dogs . Unpublished study performed by Hazleton Laboratories Ameri ca, Inc. Study No. A83-821. June 17, 1985. MRID (00163065)

USEPA (2010) Bifenthrin - Report of the Endoc rine Disruptor Review Team; Test Order# EDSP-128825-66, 67, 68, 70, 71, and 73. September 23, 2010. Office of Chemical Safety and
Pollution Prevention. U.S. Environmenta l Protection 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 1BIFENTHRIN FINAL

Page 26 of 59 Screening. Office of Chemical Safety and Pollution Pr evention. U.S. Environmental Protection Agency, Washington, DC.

Wagner, H. (2011) Bifenthrin: H295R Steroi dogenesis Assay. Unpublished study performed by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9146V-100382STER, November 17, 2011. (MRID 48615006)

Watt, B. and Freeman, C. (2001) Bifenthrin Tech nical Prenatal Developmental Toxicity Study in Rats. Unpublished study performed by FMC Corpor ation Toxicology Laboratory, Princeton, NJ. Laboratory Study No.: A2000-5263, February 22, 2001. (MRID 45352301)

Weight of Evidence Evaluation for EDSP Tier 1 Screening of Bifenthrin Data Requirement
EPA - EDSP Test Order for Bifenthrin (Order/DC#: EDSP-128825-66, 67, 68, 70, 71 and 73) Prepared for the Bifenthrin Task Force Steer ing Committee/Joint Venture Operating under the auspices of Consumer Specialty Products Association Prepared by Science Strategies, LLC Quill Law Group, LLC Date Completed April 11, 2013 (MRID: 48698901)

Wilga, P. (2011) Bifenthrin: Human Recombinant Aromatase Assay. Unpublished study performed by CeeTox, Inc., Kalamazoo, MI . Laboratory Study No.: 9141V-100382AROM; November 17, 2011. (MRID 48615002)

Willoughby, J.A. (2011) Bifenthrin: Androgen Receptor Binding (Rat Prostate Cytosol). Unpublished study performed by CeeTox, Inc., Ka lamazoo, MI. Laboratory Study No.: 9146V100382ARB, November 17, 2011. (MRID 48615001)

Willoughby, J.A. (2011) Bifenthr in: Estrogen Receptor Bindi ng (Rat Uterine Cytosol). Unpublished study performed by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9146V100382ERB, November 17, 2011. (MRID 48615003)

Willoughby, J.A. (2012) Supplemental Information - Laboratory Proficiency Data for ERTA assays and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. Unpublished study performed by CeeTox, In c., Kalamazoo, MI. July19, 2012. (MRID 48843501)

Willoughby, J.A. (2011) Bifenthr in: Estrogen Receptor Transc riptional Activation Assay (Human Cell Line (HeLa-9903)). Unpublished st udy performed by CeeTox, Inc. Kalamazoo, MI. Laboratory Report No.: 9146V-100382ERT A, November 17, 2011. (MRID 48615004)

Willoughby, J.A. (2012) Estrogen Receptor Transcri ptional Activa tion (Human Cell Line (HeLa9903)) Proficiency Data Report. Unpublished st udy performed by CeeTox, Inc. Kalamazoo, MI. May 15, 2012.BIFENTHRIN FINAL

Page 27 of 59 Zhang, J., Zhu, W., Zheng, Y., Yand, J., and Z hu, X. (2008) The antiandrogenic activity of pyrethroid pesticides cyfluthrin and β -cyfluthrin. Reproductive Toxicology 25: 491-496.

Zhao, M., Chen, F., Wang, C., Zhang, Q., Gan, J., and Liu, W. (2010) Inte grative assessment of enantioselectivity in endocrine disruption a nd immunotoxicity of s ynthetic pyrethroids. Environ.
Pollut. 158(5):1968-1973.

Zorrilla, L. (2011) The Hershberger Bioassa y for Bifenthrin (OPPTS Guideline 890.1400). Unpublished study performed by Integrated Labora tory Systems, Inc., Durham, NC. Project Study No. C196-200, November 18, 2011. (MRID 48615005)

Zorrilla, L. (2011) The Uterotrophic Assay fo r Bifenthrin. Unpublished study performed by Integrated Laboratory Systems, Inc., Durh am, NC. Laboratory Proj ect Study No.: C196-100, November 14, 2011. (MRID 48615007)

Zorrilla, L. (2012) Pubertal Development a nd Thyroid function with Bifenthrin in Juvenile/Peripubertal CRL: CD(SD)IGS Female and Male Rats. Unpublished study performed by Integrated Laboratory Systems, Inc., Durham, NC. ILS Study No. C196-300, January 19, 2012. (MRID 48669401)BIFENTHRIN FINAL

Page 28 of 59 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100)

The 21-day assay (MRID 48675901) of bifenthrin on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under fl ow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) Stage 51 (80/treatment group, 20/replicate) were e xposed to bifenthrin (93.6% purity) at nominal concentrations of 0 (negative and solvent controls), 0.000030, 0.00010, and 0.00030 mg a.i./L. The solvent control consisted of dimethylformamide at a final
concentration of 0.02 mL/L. Time -weighted average (TWA) concen trations, calculated by the reviewer, were <0.0000150 (<LOQ; control), 0.0000133, 0.0000506, and 0.000235 mg a.i./L. The test system was ma intained at 21.9 to 22.7 o C and a pH of 7.9 to 8.1.

According to the study author, “O n Day 2 there was an unusual mort ality event in replicate A of the negative control. By Day 3, there were 7 mort alities in the replicate. However there were no mortalities in the remaining replicates of the ne gative control or the solvent control.” One of the AMA validity criterion states: “For any given treatment (includi ng controls), mortality should not exceed 10%. For any given replicate, mort ality should not exceed three tadpoles, otherwise the replicate may be considered compromised.” A ccordingly, replicate A of the negative control was considered to be compromised and was excl uded from descriptive statistics as well as statistical analyses.

There were no treatment-related effects on survival of tadpoles during the 21-day test. After 7 days of exposure, survival was 100% in the nega tive control (excluding re plicate A; 91.1% with replicate A), 100% in the solvent control, and 100, 96.25, and 100% in the low, mid, and high treatment groups, respectively. From Day 8 to test termination, survival was 95.6% in the negative control (excluding repli cate A; 92.3% with replicate A), 100% in the solv ent control, and 100, 96.5, and 100% in the low, middle, and high treatment groups, respectively. In the negative and solvent control groups, one and two ta dpoles, respectively, were incidentally killed during siphoning and were not consid ered in survival calculations.

At test termination, there was no treatment-rela ted effect on the incidence of tail curvature, which was present in 63, 59, 78, 65 and 63% of tadpoles in the negati ve control (excluding replicate A), solvent control, and low, mid, and high treatment groups, respectively.

There were significant differences (p<0.05) in Day 7 and 21 growth parameters ( i.e. , snout-vent length [SVL] and body weight) between the negative and solvent controls. Compared to the negative control, Day 7 SVL and body weight were significantly in creased by 10.5 and 26.2%, respectively, in the solvent control, and Day 21 SVL and body weight were significantly increased by 8.1 and 22.2%, respectively, in the solv ent control. Therefore, the treatment groupsBIFENTHRIN FINAL

Page 29 of 59 were compared separately to the negative and solvent controls for all growth and developmental
parameters.

Day 21 body weights were significantly increa sed (p<0.05) by 24.4, 21.0, and 14.2% in the low, mid, and high treatment groups, respectively, relativ e to the negative control. However, at the mid and high treatment concentrations, significan t decreases (p<0.05) were observed in Day 7 SVL (4.4 and 12.5%), Day 7 body weight (12.5 and 27.7%), Day 21 SVL (4.8 and 6.6%), and Day 21 body weight (5.9 and 11.2%), relative to the solvent control. Furthermore, Day 7 body weight and Day 21 SVL were significantly decr eased (p<0.05) by 16.8 and 4.4%, respectively, at
the low treatment concentration rela tive to the solvent control.

There were no significant differenc es in developmental endpoints ( i.e. , NF developmental stage, hind limb length [HLL], normalized HLL) in any treatment group when compared to the negative control. However, bifenthrin significan tly delayed (p<0.05) median NF developmental stage at 21 days in the high treatment group (m edian NF=57) relative to the solvent control (median NF=58.5). In addition, Day 21 HLL and normalized HLL were significantly decreased (p<0.05) by 22.1 and 18.7%, respectively, in the hi gh treatment group relative to the solvent control. Asynchronous development (stages 54 a nd 55) was observed in one tadpole at test termination in the mid treatment group.

Mild to moderate thyroid gland atrophy was noted in the high treatment group (9/20) relative to the negative control (4/15) and solvent contro l (4/20). Thyroid gland hypertrophy and follicle size increase were observed in the negative and solv ent controls as well as in almost all of the treatment groups. Increases or decreases in t hyroid gland and follicle cell asymmetry were observed sporadically in a ll control and treated groups.

There were significant differences between the negative and solvent cont rol groups in growth parameters, which showed a consistent pattern of promotion in the solvent control relative to the negative control. Additionally, there was unaccept ably high mortality in one replicate of the negative control, compromising sample size and statistical power for analyzing Day 21 endpoints. However, the confounding effects of the solvent on growth parameters were addressed by comparison of the treated groups with the solvent control; th erefore, the excessive mortality in the one negative control replicate had less of an impact on the results.

Androgen Receptor (AR) Bindi ng Assay; (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48615001), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment, which measured the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (10

− 10 to 10

− 3 M) of bifenthrin (93.6% a.i., Batch #: PL09-0251).BIFENTHRIN FINAL

Page 30 of 59 Dimethyl sulfoxide (DMSO) was used as the solvent at a final a ssay concentration of approximately 3.2%. A total of three independe nt runs were performed, and each run included dexamethasone as a weak positive control, a nd R1881 as the ligand reference standard.

A saturation binding experiment was conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and wa s functioning with appropriate affinity for the radio-labeled reference androgen (R1881) prior to routinely conducting AR competitive binding experiments. Saturation binding data were not provided in the original study report; however, summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency. As separate cytosolic preparations were used for each of the three competitive binding runs, the data from all three saturation binding experiments were evaluated. For the cytosolic prepara tion made on March 30, 2011, the mean dissociation constant (K d ) for [ 3 H]-R1881 was 0.614±0.065 nM, and the estimated B max was 1.080±0.109 fmol/100 μg protein. For the cytosolic preparation made on July 23, 2011, mean K d for [ 3 H]-R1881 was 1.080 nM (±0.690), and the estimated B max was 1.071 fmol/100 μg protein (±0.167). For the cytosolic preparation made on September 24, 2011, the mean K d for [ 3 H]- R1881 was 0.613 nM (±0.041), and the estimated B max was 0.817 fmol/100 μg protein (±0.049).
The mean and individual K d values were generally below the range reported in the EPA validation program (0.685 to 1.57 nM), although two individual values were within the range and one value was above the range. Confidence in these numbers is generally high based on the goodness of fit (R 2 = 0.880-0.992) and the sm all variation in R 2 and K d estimates among runs (although greater variability was observed in the July 23, 2011 satu ration binding data).

In the competitive binding experiment, precipitati on of bifenthrin was visually observed in Run 1

at 10

− 4 and 10

− 3 M; therefore the concentrations used in Runs 2 and 3 were 10

− 4 M to 10 -11 M. The suitable top concentrations used for evaluation were 10

− 5 M in Run 1 and 10 -4 M in Runs 2 and 3. In Run 1, specific binding of [ 3 H]-R1881 was >75% at bifent hrin concentrations of ≤ 10

− 5 M, classifying bifenthrin as a “non-bi nder”. In Run 2, specific binding of [ 3 H]-R1881 was
74.9% at a concentration of 10

− 4 M, classifying bife nthrin as “equivocal” for binding. In Run 3, specific binding of [ 3 H]-R1881 was >75% at bifenthrin concentrations of ≤ 10

− 4 M, classifying
bifenthrin as a “non-binder ”. An estimated log IC 50 and relative binding affinity (RBA) could
not be calculated.

The estimated mean log IC 50 s for R1881 and the weak positive control (dexamethasone) were

− 9.3 and

− 4.4 M, respectively, and the mean RBA for the dexamethasone was 0.0012%. The performance criteria were generally met.

Based on the results from the three runs, bifenthrin is classified as a Non-Binder in the Androgen Receptor Binding Assay.BIFENTHRIN FINAL

Page 31 of 59 Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP19) assay (MRID 48615002), bifenthrin (93.6% a.i. Lot # PL09- 0251) was incubated with human recombinant aromatase and tritiated androstenedione (1- β [ 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) in ≤ 1% dimethyl sulfoxide (DMSO) at final log
concentrations of 10

− 10 to 10

− 3 M for 15 minutes to assess the potenti al of bifenthrin to inhibit aromatase activity.

Aromatase activity was determined by measuring the amount of tritiated water produced at the
end of a 15-minute incubation for each concentra tion of test chemical. Tritiated water was quantified using liquid scintillation counting (LSC ). Three runs were conducted and each run included a full activity c ontrol, a background activity control, a positive control series (10

− 10 to
10

− 5 M) using a known inhibito r (4-hydroxyandrostenedione; 4- OH ASDN), and the test chemical series (10

− 10 to 10

− 3 M) with three repetitions per concentration. In itially, the two highest concentrations (10 -3 and 10 -4 M) of bifenthrin tested in Run 1 showed precipitation during the 15-minute incubation period and we re not used by the st udy author in data interpretation. Subsequently, th e highest test concentration used for study interpretation by the study author was 10 -4.5 M, as this concentration di d not result in precipitation.

Aromatase activity in the fu ll activity controls for the three runs averaged 0.578 ± 0.099 nmol·mg-protein

− 1 ·min

− 1 . The response of each full activity control within a run was between 90 to 110% of the average full activ ity. Activity in the background controls averaged 0.28 ± 0.20% of the full activity controls.

For the positive control substance (4-OH ASDN), aromatase activity results were within the recommended ranges for the performance criteria. The estimated log IC 50 for 4-OH ASDN averaged -7.27 M and the Hill slope was -1.00.

For bifenthrin, aromatase activity averaged 0.526 ± 0.053 nmol·mg-protein

− 1 ·min

− 1 at the lowest
tested concentration 10

− 10 M and 0.568 ± 0.027 nmol·mg-protein

− 1 ·min

− 1 at the highest successfully tested concentration, 10

− 4.5 M. As a percent of control, aromatase for bifenthrin was

≥ 100.09 % at concentrations from 10

− 10 to 10

− 4.5 M.

Based on data from the average response curve, bi fenthrin is classified as a Non-inhibitor of aromatase activity in this assay.

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

In an estrogen receptor (ER) binding assa y (MRID 48615003), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiments. A competitiveBIFENTHRIN FINAL

Page 32 of 59 binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-17 β

estradiol (1 nM) in the presence of increasing concentrations (10

− 11 to 10

− 3 M) of bifenthrin (93.6% purity, Batch #PL09-0251). DMSO was us ed as the solvent vehicle at a final concentration of approximately 4%. A total of three runs were performed, and each run included 19-norethindrone as a weak positive control, oc tyltriethoxysilane as a negative control, and 17 β

estradiol as the natural ligand reference material.

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. The protein concentrations used in the saturation binding runs varied between each run, and were approxima tely 3- to 6-fold greater than recommended (160 to 320 μg versus 50±10 μg). The K d for [ 3 H]-17 β -estradiol was 0.331±0.061 nM and the
estimated B max was 74.55±3.03 fmol/100 μg protein for the prepared rat uterine cytosol. The K d for each run was within the expected Guideline range of 0.03 to 1.5 nM.

In the competitive binding experiment, precip itation of bifenthrin was detected at 10 -4 and 10 -3 M
in Run 1 and at 10 -4 M in Runs 2 and 3. Therefore, the highest concentra tion evaluated for binding was 10 -5 M. The estimated mean log IC 50 for 17 β -estradiol and 19-norethindrone was

− 9.03 M and

− 5.50 M, respectively. The mean RBA was 0.030% for 19-norethindrone, compared to the natural ligand. All perfor mance criteria were generally met for 17 β -estradiol, and the negative control octyltriethoxysilane.

Bifenthrin was tested over a concentration range that fully defined the top of the curve. The percent binding at this top plateau (87.3-106.5%) was within 25 percentage points of the lowest
concentration of the estradiol st andard (95.1-107.4%). The lowest average percent radiolabeled estradiol binding in the presence of bifent hrin was >75% at concentrations up to 10

− 5 M in all

runs.

Based on the results from the three runs, bifenthrin is classified as Not Interactive in the Estrogen Receptor Binding Assay.

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

In an estrogen receptor transcript ional activation assay (MRID 48615004), hER α -HeLa-9903
cells cultured in vitro were exposed to bifent hrin (93.6% a.i., Lot #PL 09-0251) at logarithmically increasing concentrations from 10

− 11 to 10

− 4 M in 0.1% DMSO for approximately 24 hours in two independent runs. The experiments were pe rformed using 96-well plates and each bifenthrin concentration was tested in 6 wells/plate in each run. Cells were exposed to the test agent for 24±2 hours at 37±1°C to induce repo rter (luciferase) gene produc ts. Luciferase expression in response to activation of the estrogen receptor by bifenthrin was measured using a proprietaryBIFENTHRIN FINAL

Page 33 of 59 luciferase assay. Bifenthrin was tested up to th e limit of cytotoxicity, which was observed at 10 -4 M in a range finding test and in the first run.

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, bifenthrin responses were 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 8.1-52.8% PC. Bifenthrin wa s only able to reach a maximum of 3.8-5.0% PC when tested up to the highest concen tration possible based on cytotoxicity (10 -4 M). Because
the RPC Max < PC 10 in both assay runs, bifenthrin was considered negative for estrogen receptor transcriptional activation in this test system.

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

The 21-day short-term reproduction assay (MRI D 48675902) of bifenthrin with fathead minnows ( Pimephales promelas) was conducted under continuous flow -through conditions. Six-month old fish in 20 spawning groups (2 males and 4 females in each group) were exposed to bifenthrin (93.6% purity) at nominal concentrations of 0 (control), 0 (solvent, dimethylformamide [DMF], <20 μL/L), 0.00003, 0.00010, and 0.00030 mg a.i./L. Time-weighted average (TWA) concentrations, calculated by the reviewer, were <LOQ (<0.000015 mg a.i./L negative and solvent control), 0.000017, 0.000075, and 0.000402 mg a.i./L . The test system was maintained at

24.5 to 25.5 o C and a pH of 8.0 to 8.4.

In the negative control, solvent control, and low- and mid-concentration treatment groups, there
were no notable observations with regards to changes in behavior or appearance, specifically color/banding, ovipositor appearance, or size of dorsal nape pad. Clin ical signs of toxicity in the high-concentration group were noted begi nning on Day 5 and included erratic swimming, lethargy, loss of equilibrium, surfacing, weak be havior and changes in coloration. In the highconcentration group, there was 100% mortality in males. Survival was also decreased (p<0.05) in the medium-concentration males to 88%, compared to 100% in the negative control. Female survival was unaffected by treatment with survival in the high-concentration females identical to the negative control (94 %). Data for male specific endpoints were subsequently dropped from analysis in the highest treatment concentration.

Spawning occurred at least two times in 7 days during the pre-exposure period. In the negative control, fecundity was 16.1 eggs/female/day/repl icate and fertilization success averaged 98.3%. There were statistically significan t effects (p<0.05) on fecundity in females, with a 60% increase
at the mid concentration and a 74% reduction at th e high concentration, compared to the negative
control. These effects were observed in con centrations with 88 and 0% male survival,BIFENTHRIN FINAL

Page 34 of 59 respectively. There were two cont rol replicates (Replicates A and D) that were observed to have a period of at least 5 days in which no eggs we re produced. These two replicates had an average fecundity of 11.7 eggs/female/reproductive day, falling just below the guideline recommended criteria of 15 eggs/female/day, although as stat ed previously, the mean control fecundity was 16.1 eggs/female/day. There were no statisticall y significant reductions (p>0.05) in fertilization

success.

There was a significant (p<0.05) increase of 51% in female gonado-somatic index (GSI) at the high concentration, compared to the negative control. No statis tically significant differences were noted for the other developmental endpoints including male GSI and male tubercle score (p>0.05). Additionally, there were no signifi cant effects (p>0.05) on male and female vitellogenin (VTG) levels. Female body weight was significantly increa sed by 28% (p<0.05) at the high concentration relative to the negative control. No other growth endpoints were affected by treatment. As stated previously, data for ma le-specific endpoints at the highest treatment
concentration were not available due to total mortality at that concentration.

In males, there were no statistically signifi cant differences (p>0.05) between histopathology severity scores or gonadal stages in treated groups compared to the negative control. There was a significant increase in oocyte atresia (p<0.05; Jonc kheere-Terpstra trend test) in 13/15 female in the high treatment group (compared to 4/15 fish in the negative control) . There was also an increase in post-atretic follicl es and granulomatous inflammation, almost all of which was due to the increased oocyte atresia. Th ere were no other statistically significant differences between severity scores or gonadal stages in treated groups compared to th e negative or solvent controls
for females. Microsporidia spores were identified in one fish in each of the control groups, as
well as two fish in the low treatment con centration, one fish in the middle treatment concentration, and one fish in the high treatment concentration. All of these cases coincided with some level of granulomatous inflammation.

Hershberger Assay; (OCSPP 890.1400)

In a Hershberger Assay (MRID 48615005) screeni ng for androgenic activit y, bifenthrin (93.6% purity; Lot # PL09-0251) in corn oil was administer ed daily via oral gavage to groups of eight 59- or 60-day old, castrated male Sprague Dawley (S D) rats at dose levels of 0 (vehicle), 3.2, or 10 mg/kg/day. An androgenic positi ve control group consisted of ei ght castrated rats exposed to the vehicle by oral gavage plus 0.4 mg/kg/day of testosterone propionate (TP) by subcutaneous (s.c.) injection.

To screen for potential anti-androge nic activity, bifenthrin in corn oil was administered daily via oral gavage to groups of eight 59- or 60-day old, castrated male SD rats at dose levels of 0 (vehicle), 1, 3.2, or 10 mg/kg/day in conjunction with a daily dose of reference androgen TP atBIFENTHRIN FINAL

Page 35 of 59 0.4 mg/kg/day by s.c. injection. The positive control group for androgenic activity served as the negative control group for the anti-androgenic activity. An anti-andr ogenic positive control group consisted of eight castrated rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg/day in conjunction with a s. c. dose of TP (0.4 mg/kg/day).

For both components of the assay, body weights were determined da ily. Animals were dosed for 10 consecutive days and terminated approximately 24 hours after the final dose administration to determine weights of the five androgen-dependent tissues. All animals survived until scheduled termination and no clinical signs of toxicity were observed in a ny of the groups. Measurements of food consumption, serum hormone concentrati ons, and liver, kidney, and adrenal weights are optional according to the Guideline and were not performed in this study.

In both the androgenic and anti -androgenic assays, no gross e ffect was reported on the sexrelated tissues. No treatment-related effect wa s observed on mortality, cl inical signs, or sex organ weights (seminal vesicles, ventral prosta te, LABC, Cowper’s gla nds, and glans penis). The Guideline criteria for %CV in organ weights were met in all cases for the vehicle/negative control and high dose groups.

In the androgen agonist assay, overall (Days 1- 11) body weight gain was increased (p<0.05) by 29% at 10 mg/kg/day and increased (not statisti cally significant [NS]) by 16% at 3.2 mg/kg/day. Only minor increases were noted in terminal body weights by 3.2 or 10 mg/kg/day bifenthrin (<4%). No treatment-related effects were obser ved in any of the accessory sex organs of bifenthrin-dosed animals. The positive control group had increases (p<0.05) in the weights of the seminal vesicles ( ↑ 1347%), ventral prostate ( ↑ 918%), LABC ( ↑ 241%), Cowper’s glands ( ↑ 661%), and glans penis ( ↑ 75%), indicating that the test system was sensitive to an androgenic

response.

In the anti-androgen assay, there was no effect on terminal body weights or overall (Days 1-11) body weight gain in the bifenthrin with TP gr oups compared to the ne gative control. No
treatment-related effects were obs erved in any of the accessory se x organs of animals dosed with bifenthrin + TP. The FT positive control group ha d decreases (p<0.05) in weights of the seminal vesicles ( ↓ 85%), ventral prostate ( ↓ 79%), LABC ( ↓ 53%), Cowper’s glands ( ↓ 68%), and glans penis ( ↓ 24%), indicating that the test system was sensitive to an anti-androgenic response.

Although no systemic toxicity was seen in this study, the dose leve ls were selected based on the results of a range-finding study in which SD rats were administered bife nthrin at doses ranging from 2.5 to 20 mg/kg/day for 14 days (ILS Study No. C196-500). Animals in the 15 mg/kg/day and 20 mg/kg/day exhibited tremors 24 hours post-dose. In addition, tremors were seen in rats at 10 mg/kg/day in the male (7 of 16 animals) and fe m ale (9 of 16 rats) rats in the pubertal assays. Therefore, the high dose tested is determ ined to be adequate for this study.BIFENTHRIN FINAL

Page 36 of 59 Statistically significant changes were not seen in two of more of the five androgen sensitive tissues. Bifenthrin was negative for androgenici ty and anti-androgenicity in the Hershberger

assay.

Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48669401), 16 Sprague Dawley Crl:CD(SD) IGS rats/dose group were treated daily by oral gavage (5 mL/k g) with bifenthrin ( 93.6% purity, Lot No. PL09- 0251) in corn oil at doses of 0 (vehicle), 2.5 or 10 mg/kg/day from post-natal day (PND) 22 to 42/43. Animals were weighed daily and examined for clinical signs of toxicity. Beginning on PND 22, animals were also examined daily for va ginal opening (VO), and age and weight at day
of attainment was recorded. Af ter completion of VO, the status of each animal’s estrous cycle was determined daily. Animals were sacrifi ced on PND 42 or 43 and blood was collected for analysis of clinical chemistry. Total serum thyroxine (T4) and thyr oid stimulating hormone (TSH) concentrations were also determined by radioimmunoassay. Liver, kidneys, thyroid, pituitary, adrenals, and urogenital organs we re collected and weighed, and microscopic
examinations were conducted on the ovaries, uterus, t hyroid, and kidneys.

All animals survived until scheduled termination. No clinical signs of toxicity were observed at the 24-hour post-dose observation period. Howeve r, during clinical obs ervations at 6 hours post-dose, 9/16 animals in the 10 mg/kg/day grou p were found to have tremors on at least one day. These animals appeared normal on all other days of the study.

There was a significant trend (p <0.05) in delay of VO with attainment occurring on PND 35.1, 37.1 ( ↑ 2.0 days) and 37.4 ( ↑ 2.3 days) in the 0, 2.5 and 10 mg /kg/day groups, respectively; pairwise comparisons were not significantly different. No changes in body weights were
observed in treated animals relative to control.

There were no effects of treatmen t on any organ weights in this study. There were no significant differences in age at first estrus in either treatment group relati ve to controls. There were no effects of treatment on cy cle length or regularity.

At 2.5 mg/kg/day, serum T4 concentrations were decreased (p<0.05) by 16%; however, no effect was observed at 10 mg/kg/day. No significant effects of treatment were noted for TSH concentrations. Sorbitol dehydrogenase was incr eased (p<0.05) at 2.5 mg/kg/day by 55%, but not at 10 mg/kg/day. At 10 mg/kg/ day, increases in serum calcium ( ↑ 5%), creatinine ( ↑ 41%), total bilirubin ( ↑ 200%) and total protein ( ↑ 8%) were observed. Sodium and chloride were increased (p<0.05) by 3-5% at 2.5 and 10 mg/kg/day. There were no other si gnificant changes inBIFENTHRIN FINAL

Page 37 of 59 clinical chemistry parameters. No treatment re lated histopathological ch anges were observed in the thyroid, uterus, ovaries or kidneys.

The high dose of 10 mg/kg/day is considered an adequate based on the results of a female rangefinding study in which doses of 15 and 20 mg/kg/ day resulted in advers e clinical signs and mortality, and clinical signs including tremors in this assay.

Male Pubertal A ssay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48669401), 16 Sprague Dawley Crl:CD(SD) IGS rats/dose group were treated daily via oral gavage with bifenthrin (93.6% purity, Lot No. PL09-0251) in
corn oil at doses of 0 (vehicle), 2.5 or 10 mg/kg/ day from post-natal day (PND) 23 to 53/54. Animals were weighed daily and examined for preputial separation (PPS) daily beginning on PND 30, and age and weight at day of attainme nt was recorded. Follo wing sacrifice on PND 53 or 54, blood was collected for analysis of clinic al chemistry, and serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were analyzed using radiometric assays. Liver, kidneys, thyroid, pituit ary, adrenals, and urogenital orga ns were collected and weighed, and microscopic examinations were conducted on the testes, epididymides, thyroid, and kidneys.

All animals survived unt il study termination. No c linical signs of toxicity were observed at the 24-hour post-dose observation period. During cl inical observations 6 hours post dose, 7/16 animals in the 10 mg/kg/day group were found to have tremors on at least one day. These animals appeared normal on al l other days of the study.

No significant changes were obs erved in body weights at PPS or final body weights in the 2.5 or 10 mg/kg/day groups. The mean age at PPS wa s 46.5 and 45.9 days at 2.5 and 10 mg/kg/day, respectively; the mean age at PPS for the control group was 46.7 days. There was no effect of treatment on age or weight at PPS.

At 10 mg/kg/day, increases (p<0.05) were noted in absolute a nd adjusted pituitary weights ( ↑ 11% each); relative weights were not significantly different. The increase in pituitary weight can be explained by the slight in crease in terminal body weights ( ↑ 4%) as reflected in the lack of significance in relative pituitary weights. Although androgen-sensi tive tissue weights in treated groups were not statistically different from contro ls in pairwise comparisons, significant trends (p<0.05) were noted for increased weights of the dorsolateral prostate (unadjusted and adjusted) and LABC (adjusted). Adjusted liver weights also showed an increasing trend (p<0.05).

No changes were observed in seru m testosterone levels. Serum T 4 was significantly decreased (p<0.05) by 16% at 2.5 mg/kg/day, but was not different from c ontrol at 10 mg/kg/day. Serum TSH was significantly increased (p<0.05) by 51% at 10 mg/kg/day.BIFENTHRIN FINAL

Page 38 of 59 At 10 mg/kg/day, increases (p<0.05) in sodium concentration ( ↑ 2%) and sorbitol dehydrogenase ( ↑ 38%) were observed. No other changes in c linical chemistry parameters were observed.

Bifenthrin appeared to be associ ated with a slight increase in follicular cell height although this was not statistically significant. No findings were observed in the testes, epididymides or kidneys at either dose level.

Based on the results of male and female range finding studies in which doses of 15 and 20 mg/kg/day resulted in adverse c linical signs (both se xes) and mortality (females), the high dose of 10 mg/kg/day was adequate.

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48615006), H295R cells cultured in vitro in 24-well plates
were incubated with bifenthrin (93.6% purity, Batch # PL09-0251) at concentrations of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 μM for 48 hours in triplicat e for four independent experiments. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.05%. Precipitates were observed under the micros cope after the exposure period at 10 μ M in Run 2
and at 100 μ M in all runs. Cytotoxicity greater th an 20% was not observed. Run 1 was not analyzed because one of the forskolin concentrat ions on the quality control plate was incorrect (3.33 μ M was used instead of 1 μ M).

Testosterone and estradiol levels were measured using HPLC-MS/MS. A Quality Control (QC) plate was run concurrently with ea ch independent run of a test chem ical plate to demonstrate that the assay responded properly to positive contro l agents at two concentration levels; positive
controls included the known inhi bitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production.

Guideline acceptability recommendations and requirements were met, including adequate production of testosterone and estradiol, acceptabl e reproducibility (low %CV), and appropriate
induction and inhibition with positive controls.

Statistically significant induction of testosterone was noted at 0.1 μM bifenthrin in Run 4 (1.13- fold) and at 1 μM bifenthrin in Runs 2 and 4 (1.45- and 1.32-fold). Statistically significant induction of estradiol was noted at 1 μM bi fenthrin in Run 3 (1.39-fold). Hormone concentrations at 10 μM bifenthrin were sim ilar to control in Runs 3 and 4, but were not evaluated in Run 2 due to preci pitation. These differences were not considered related to
treatment because the effect was not concentrat ion-dependent and/or not reproducible between

runs.BIFENTHRIN FINAL

Page 39 of 59 Based on hormone responses in three independent runs, bifenthrin treatment did not result in statistically significant and repr oducible alterations in testoste rone or estradiol production.

Uterotrophic Assay; (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48615007), bi fenthrin (93.6% a.i., Lot/Batch # PL09-0251) in corn oil was administered daily via oral gavage (5 mL/kg) to groups of 8 ovariectomized female Sprague-Dawley rats at dose levels of 0 (vehicle), 2.5, or 15 mg/ kg/day on post-natal days (PND) 56-58. A positive control group was tr eated with a daily dose of 17 α -ethynyl estradiol (EE) at 0.075 mg/kg/day in corn oil by oral gavage. Body weights were determined daily. All animals were terminated on PND 59 approximately 24 hours after the final dos e administration to
determine wet and blo tted uterine weights.

No treatment-related effects were observed on mortality, clinical signs , body weights, or body weight gains.

No abnormal gross observations were seen in th e uterus. Uterine wei ghts in the bifenthrin treated groups were comparable to the vehicl e controls. Absolute wet and blotted uterine weights for the positive control (EE) group were increased (p<0.05) by 154% and 108%, respectively.

The high dose of bifenthrin selected for this stud y (15 mg/kg/day) is adequate based on data from a dose range-finding study s howing mortality at doses ≥ 20 mg/kg/day.

No statistically significant changes were seen in uterine weight in this assay. Bifenthrin is negative in the uterotrophic assay.BIFENTHRIN FINAL

Page 40 of 59 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 ge neral toxicity studies considered relevant for determining the potential interaction of bifenthr in with the endocrine system are summarized below. Given the breadth of toxicity data ava ilable on bifenthrin, th e only Part 158 studies discussed in this appendix are those that fulfill current guideline requirements and are directly relevant to assessing potential endocrine effects ( i.e. , multiple-generation rodent reproductive studies, development studies, chronic toxicity studies, etc.).

ERTA Assays

In a non-Guideline in vitro ER binding assay ( Brander et al. , 2012 ), the ER–based CALUX mammalian cell bioassay [a human ovarian carcinoma (BG-1) cell li ne, stably-transfected with an ER luciferase reporter plasmid] was used to assess the estrogenic a nd anti-estrogenic activity of bifenthrin (99% purity; 50/ 50 mixture of isomers). The CALUX bioassay expresses mRNA
for both ER α - and β -isoforms. The CALUX bioassay was us ed to determine the concentrationdependent agonist (test material) an d antagonist (test material in th e presence of estradiol) effects
of bifenthrin. Bifenthrin was tested at final concentr ations of 1, 10, 100, 1000, and 10,000 ng/L (0.0024, 0.024, 0.24, 2.4 and 24 nM); the concentration of 17 β -estradiol was 1 nM. Plated cells were incubated with carrier solvent (EtOH; 1% final solvent concentration), bifenthrin, or bifenthrin in the presence of estradiol for 24 hours at 37°C.

The CALUX assay did not detect ER agonism for any of bifenthrin concentra tions tested. In the ER antagonism assay, an initial bifenthrin concen tration-dependent decrea se in the ability of estradiol to induce ER-depe ndent reporter gene activity was observed at bifenthrin concentrations of 1–100 ng/L (0.0024-0.24 nM). At bifenthrin concentrations >100 ng/L, there was a recovery of estrogenic activity. These re sults demonstrate that bifenthrin acts as an estrogen antagonist in this in vitro assay. The Good Laboratory Pract ices (GLP) status of this
research is unknown.

In a non-Guideline in vitro cell proliferation assay [estroge n-dependent, human breast carcinoma MCF-7 cells (E-Screen)] conducted by Wang et al. , 2007 , the potential for differential, enantioselective estrogenic activity of the two bifenthrin enantiomers (1R- cis -bifenthrin and 1Scis -bifenthrin) was examined. An analytical stan dard of racemic bifenthrin (99.5% purity) was separated by HPLC with a variable wavelength circular dichro ism detector; purity of the isolated enantiomers was assessed by GC-ECD. MCF-7 cell pr oliferation was determined for the isolated bifenthrin enantiomers against 17 β -estradiol (positive control), and in the presence or absence of ICI 182,780 (ER antagonist) to assess ER binding.

The dose-response relationships of cell proliferation induced by th e bifenthrin enantiom ers (10

− 13

to 10

− 5 mol/L) and the combined effects with 10 -6 mol/L of ICI 182,780 were determined. Proliferative effect (PE) values of 1S- cis -bifenthrin and 1R- cis -bifenthrin were 3.59 and 1.73,BIFENTHRIN FINAL

Page 41 of 59 respectively (2.1-fold difference). In comparison with the PE of 17 β -estradiol synchronously (4.49), the relative prolifer ative effect (RPE) was dete rmined to be 74.2% for 1S- cis -bifenthrin
and 20.9% for 1R -cis -bifenthrin. In addition, by comparing with the concentration needed for maximal cell proliferation of 17 β -estradiol (1×10

− 10 mol/L), relative proliferative potency (RPP)
was calculated to be 10% for 1S- cis -bifenthrin and 1% for 1R -cis -bifenthrin. Therefore, the estrogenic potential of 1S- cis -bifenthrin was greate r than that of 1R- cis- bifenthrin. The proliferation induced by bifenthrin en antiomers was completely blocked by 10 -6 mol/L of ICI 182,780, supporting a direct binding eff ect of bifenthrin enantiome rs with the ER. The GLP
status of this research is unknown.

A similar response with bifenthrin enantiomers was demonstrated by Zhao et al. (2010) in
another non-Guideline in vitro MCF-7 cell proliferation assay (E-Screen). An analytical standard of racemic bifenthrin (99.5% purity) was separated by HPLC with a method similar to Wang et al. (2007). The dose-response relationships of cell proliferation induced by the bifenthrin enantiomers (10

− 9 to 10

− 5 mol/L) were determined. This study included a positive
control of 10

− 9 mol/L 17 β -estradiol and a vehicle control of 0.1% v/v ethanol. In addition,
estrogen response gene expression (pS2 and ER α ) were determined with qRT-PCR.

The proliferative response to the cis -bifenthrin enantiomers was consistently greater for 1S- cis

bifenthrin relative to 1R- cis -bifenthrin at every tested concen tration. At a concentration of

10

− 8 mol/L, 1S- cis -bifenthrin exhibited estrog enic activity of approximately 2.2-fold greater than

1R- cis -bifenthrin (and the 1S-enant iomer response was approximately equivalent to the 17 β

estradiol response). For cis -bifenthrin (10

− 7 mol/L), pS2 gene was up-regulated by 1S- and 1Rcis -bifenthrin by 2.65- and 1.3-fold, resp ectively. Down-regulation of ER α was about 3.05-fold stronger for 1S- cis -bifenthrin compared to 1R- cis -bifenthrin. The combined results from the MCF-7 cell proliferation and qRT-P CR assays showed that in the cis -bifenthrin enantiomer pair, the 1S-enantiomer consistently s howed a higher estrogenic activity than its counterpart (1R).
The GLP status of this research is unknown.

Hershberger Assay

In a study examining the potential anti-androgeni c effects of several pyrethroid pesticides ( Zhang et al. , 2008 ), bifenthrin (purity not reported) was assessed in an in vivo Hershberger assay. The study evaluated the androgenic (b ifenthrin not included) and anti-androgenic activities of cyfluthrin, β -cyfluthrin, and four structurally-related pyr ethroids (permethrin, cypermethrin, β -cypermethrin, and bifenthrin). All test substances were administered in peanut
oil.

Three-week old male SD rats were maintain ed under standard la boratory conditions and provided with standard la boratory diet and water ad libitum . After an acclimation period of seven days, the rats were castr ated on PND 28 and allowed to recover for two weeks prior to initiation of dosing on PND 42. After the reco very period, rats were weighed and randomlyBIFENTHRIN FINAL

Page 42 of 59 assigned to dose groups (n=6). The androgenic portion of the Hershberger assay was conducted with a sham-operated control gr oup, but this assessment did not in clude bifenthrin. The antiandrogenic portion of the Hershberg er assay included eight treatme nt groups: a vehicle control group (peanut oil only); a positiv e control group dosed orally w ith flutamide (50 mg/kg/day); five groups dosed orally with other pyrethroids each at 50 mg /kg/day; and one group dose with bifenthrin at 13.5 mg/kg/day. Each group was dosed orally for seven days in addition to a daily s.c. injection of TP at 0.5 mg/kg/day. After the fi nal dose, rats were anaesthetized and sacrificed by exsanguination. Blood from each rat was coll ected for serum analysis of testosterone concentration. The seminal vesicles, ventral pr ostate, dorsolateral pr ostate, LABC, Cowper’s glands, glans penis, liver, paired adrenal glan ds, spleen, and paired kidneys of each rat were dissected and weighed. Serum testosterone c oncentrations were analyzed by time-resolved fluorescence with commercially available FIA kits.

No mortality or treatment-related clinical si gns were noted in any of the groups in the Hershberger assays. The text states there were no significant differences between initial or final body weights, or weight gains of the pyrethroid-treated groups co mpared to the control groups,
but the actual data for the bi fenthrin treated group were not presented. Serum testosterone
concentrations were not shown for the bifenthr in-treated rats, but it was reported that no
treatment-related effects were observed. Treatme nt with bifenthrin at 13.5 mg/kg/day decreased (p<0.05) absolute weights of all the androgen-se nsitive accessory sex tissues compared to the castrated control + TP. However, the exact percentage decreases, relative to cont rol, could not
be ascertained as the data we re only reported graphically. Th e weights of all accessory sex
tissues for the castrated control + TP were simila r to the uncastrated males, indicating that the dosage of TP (0.5 mg/kg/day) was appropriate, and the positive control (castrated + TP + flutamide) displayed a greater decrease in tissu e weights compared to bi fenthrin. The authors
concluded that bifenthr in displayed an anti-androgenic effect in the in vivo assay.

The Good Laboratory Practices status of this research is unknown. The study was conducted prior to the establishment of the OPPTS 890 Test Guideline for the Hershberger (890.1400) assay; however, the in vivo portion of the study generally conforms to the Guideline.
Differences from the Guideline were that the ra ts were castrated at PND 28 instead of PND 42, as recommended by the Guideline, and dosing was conducted for seven days instead of the recommended ten days. In addition, this study in vestigated only the an ti-androgenic activity of bifenthrin (no androgenic portion) , and the castrated rats were dosed with TP at 0.5 mg/kg instead of 0.2-0.4 mg/kg/day. However, because the control group (castr ated + TP) exhibited similar growth of accessory sex tissues to the sham-operated group (uncas trated), the 0.5 mg/kg dose of TP was acceptable. The study is consider ed acceptable for use in a WoE evaluation. We note that the dose utilized in the published study (13.5 mg/kg/day) wa s slightly above the
maximum dose utilized for the Guide line Hershberger assa y (10 mg/kg/day).BIFENTHRIN FINAL

Page 43 of 59 Subchronic Oral Toxicity in Rodents (Rat; OCSPP 870.3050)

In an oral toxicity study in rats ( MRID 00141199 ), bifenthrin (91.4% pur ity) was administered for 90 days in the diet to SD rats (15/sex/ dose) at nominal doses of 0, 12, 50, 100, and 200 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). An additional 10 rats/sex were admi nistered the diet containing bifenthrin at 0 or 200 ppm for 90 days, and then fed a recovery (con trol) diet for an additional 28 days. After 90 days, all rats were weighed, and sacrificed for gross and histopathologi cal evaluation. Organs that were weighed included the liver, kidne ys, testes, ovaries, and adrenals. Gross and microscopic pathological examinations were conducted on these as well as the pituitary, thyroid,
and uterus.

There was no mortality during the study. Tremors were noted in some males and females (3/sex) during the first two weeks of th e study at the medium-high dose, and in all animals (15/sex) throughout the study at the high dose. Tremors were noted to be reversib le within three days after discontinuation of dosing in the high-dose recovery group. Males and females in the highdose group had decreased mean bod y weights compared to control; decreases were only significant (p ≤ 0.05; data NR) for males at Weeks 1 and 2 and for females at Week 1. Overall mean body weight gains were slightly decr eased (NS) at the high dose for females ( ↓ 6.0%) relative to control. High-dose females had de creased food consumption after Week 4, but this was only significant during Week 9; no differe nces in food consumption were observed for males. There were no treatment-related organ weight or histopathol ogical effects.

The LOAEL for this 90-day study is 100 ppm ( 7.5 mg/kg/day for males; 8.5 mg/kg/day for females) based on the increased incidence of tremors in both sexes. The NOAEL is 50 ppm (3.8 mg/kg/day for males; 4.3 mg/kg/day for females).

Subchronic Oral Toxicity in Non-Rodents (Dog; OCSPP 870.3150)

In an oral toxicity study in dogs ( MRID 00141200 ), bifenthrin (88.35% pur ity) was administered by gelatin capsule to Beagle dogs (4/sex/dos e) at doses of 0, 2.5, 5, 10, or 20 mg/kg/day (equivalent to 2.21, 4.42, 8.84, and 17.7 mg/kg/day base d on purity) for 13 weeks. Organs that were weighed included the liver, ki dneys, testes/epididymides, ovari es, and thyroid/ parathyroids. Gross and microscopic pathological examinati ons were conducted on these as well as the pituitary, adrenals, prostate, mammary gland, and uterus.

There was no mortality during the study. Tremors were noted in 3 dogs/sex at the medium-low dose and in 4 dogs/sex in the medium-high a nd high-dose groups. The incidence of tremors increased in the medium-high and high-dose groups as the study continued, indicating a cumulative effect. Ataxia was noted in 4 dogs/sex at the medium-high and high-doses and in one female at the medium-low dose. Languidness occu rred primarily at the hi gh dose in both sexes, but also occasionally at the medium-high dose. A ll of these symptoms occurred more frequentlyBIFENTHRIN FINAL

Page 44 of 59 during the last three weeks of the study. Other dose-related c linical signs included blinking, mydriasis, nystagmus, lacrimation, and polypne a in the two highest dose groups. Possible treatment-related decreases (NS) in mean body weight gain were noted in males and females relative to control ( ↓ 20 and 54%, respectively). None of the females at the medium-high or high dose showed cyclic activity or signs of estrus, but cyclic activity and/or estrus was observed in 1-2 females in the control, low, and medium-l ow dose groups. There were no treatment-related changes noted in food consumption, and gross or microscopic parameters. Organ weights were generally unchanged except for decreases (significance NR) of 41.1 and 58.8% in mean absolute ovary weights in the medium-high and high-dose females, respectively, presumably because estrus did not occur, as well as decrease d (NS) mean absolute thyroid weights ( ↓ 11.1 and 23.1%, respectively). A slight increase in relative liver weights of was reported, but may be due to slightly lower body weights.

The LOAEL for this 13-week study is 4.42 mg/k g/day based on the increased incidence of tremors in both sexes, and the NOAEL is 2.21 mg//kg/day.

Prenatal Development in Rodents (Rat; OSCPP 870.3700)

In a developmental toxicity study in rats ( MRID 00141201 ), bifenthrin (88.35% purity) in corn oil was administered by gavage to pregnant female SD rats (25/dose) at doses of 0 (corn oil), 0.5, 1.0, or 2.0 mg/kg/day (calculated equivalency to 0, 0.44, 0.88, and 1.77 mg/kg/day adjusted for purity), or 250 mg/kg/day aspirin (positive cont rol) in 2% carboxymethylcellulose, during GD 6 through 15, inclusive. On GD 20, the dams were euthanized and subjected to gross necropsy. All fetuses were weighed, sexed and examined for gross external alteration s. One-half of the
fetuses in each litter were examined for visceral soft tissue alterations using a variation of Wilson's sectioning technique. The remaining one-half of the fetuses in each litter were
examined for skeletal alterations.

There was no mortality during the study. Maternal toxicity was characte rized as tremors in 18/25 dams in the high-dose group during GD 10-19 (four days after the last dose was administered). No significant differences between groups or dose-related trends with respect to mean maternal body weight gains or food consum ption were noted. The maternal LOAEL is 1.77 mg/kg/day (HDT) based on the incidence of tremors, and the maternal NOAEL is 0.88 mg/kg/day. Slight fetotoxicity wa s noted at the high dose and was characterized as an increased fetal and litter incidence (NS) of hydroureter with out hydronephrosis (3.55% vs 0% control). In addition, this finding involved five fetuses (from five different litters) in high-dose dams. There
were no other treatment-related malformations or variations noted, and no group differences or dose-related trends with respect to pregnancy rates, numbers of corpora lutea, implantation sites and resorptions, litter sizes, sex ratios, fetal body weights, or viab ility. The positive control gave the appropriate responses of incr eased early resorptions, depresse d fetal body weights, external, visceral, and skeletal malformations and variations.BIFENTHRIN FINAL

Page 45 of 59 The developmental LOAEL is 1.77 mg/kg/day ( HDT) based on the increased fetal and litter incidence of hydroureter without hydronephrosis. The developmental NOAEL is 0.88 mg/kg/day.

In a second developmental toxicity study ( MRID 45352301 ), bifenthrin (95.3% purity) was
administered in the diet to female SD CD rats (25/dose group) at nominal doses of 0, 30, 60, 90, or 200 ppm (0, 2.4, 4.8, 7.1 or 15.5 mg/kg/day adjusted for purity) on GD 6 through 20,
inclusive. All dams were sacrificed on GD 20 and their fetuses were removed by cesarean and examined as described previously.

No mortality was observed during the study. Clinic al signs, indicative of neurotoxicity, were observed at the high dose. These included tremors (22/25), observed from GD 9-20; hypersensitivity to sound (5/25), observed from GD 18-20; splayed hindlimbs (1/25), observed from GD 15-20; and piloerec tion (1/25), observed at GD 19. None of these findings was observed in any control an imal. A negative trend (p ≤ 0.05) in body weight gain was observed during GD 6-9, 15-18, and 18-20 with a decrease of 44, 17, and 14%, respectively, in the highdose group relative to control. In addition, a negative trend (p ≤ 0.001) was observed in adjusted (for gravid uterine weight) body weight gain with a 22% decrease at the high dose compared to control. Food consumption was decreased at th e high dose at the beginning (GD 6-9) and end (GD 18-20) of treatment ( ↓ 11-12%, relative to control). In addition, a negative trend (p ≤ 0.05) was observed during GD 6-9, 9-12, 18-20, and for the overall treatment inte rval (GD 6-20). No treatment-related changes were observed in gross pathology, th e number of corpora lutea, number of implantations, number of live and dead fetuses, number of resorptions, fetal weights, sex ratios, or post-implantation losses relative to control. Pregnancy rate was decreased in the low-dose group compared to control (68 vs. 92% control), but was 92-10 0% in all higher dose

groups.

Dose-dependent (p ≤ 0.05 for negative trend) decreases in food consumption were observed; however, the decreases noted at doses <200 ppm did not result in decreased body weight gains
and were considered not to be toxicologically relevant.

The maternal LOAEL is 200 ppm (15.5 mg/kg/day) based on clinical si gns and decreased food consumption, body weight gain, and adjusted (for gravid uterine weight) body weight gain. The maternal NOAEL is 90 ppm (7.1 mg /kg/day). No treatment-relate d developmental findings were noted at any dose tested. The developmenta l toxicity LOAEL was not observed, and the developmental toxicity NOAEL is 200 ppm.

Prenatal Development in No n-Rodents (Rabbit; OCSPP 870.3700)

In a developmental toxicity study ( MRID 00145997 ), pregnant New Zealand White rabbits (20/dose group) were administered bifenthrin (88.35% purity) by ga vage at doses of 0 (corn oil), 2.67, 4.0, or 8.0 mg/kg/day (adjustment for purity not reported), or 3.0 mg/kg/dayBIFENTHRIN FINAL

Page 46 of 59 6-aminonicotinamide (positive control) in 2% car boxymethylcellulose, on GD 7 to 19, inclusive. Mating was conducted with male New Zealand white rabbits, and c onfirmation of mating defined GD 0. On GD 29, all surv iving does were sacrificed, and all fetuses were weighed and
examined for external malformation/variations. Each fetus was examined viscerally by fresh
dissection and the sex determined. All carcasses were eviscerated and processed for skeletal
examination.

Ten does died during the study (3 control; 2 low- dose; 1 medium-dose; and 4 each high-dose and positive control); one high-dose death was due to an unknown cause, and the remaining deaths
were attributed to Pasteurella multocida infection. All other animals survived to terminal sacrifice, but some animals at necr opsy had lung damage suggestive of P. multocida infection. Treatment-related clinical signs of toxicity consisted of h ead and forelimb twitching (medium and high dose) and tremors (high dose); 19/20 an imals in the high-dose group displayed at least one of these clinical sign s. It was reported that body weight means were different (p<0.05) over
the GD 21-28 interval and for the overall GD0-29 in terval, but the only appa rent decrease is in the low-dose group (BWG; ↓ 31%). No differences in pregnancy rate were noted. The maternal toxicity LOAEL was 4.0 mg/kg/da y based clinical signs (head a nd forelimb twitching), and the maternal toxicity NOAEL is 2.67 mg/kg/day.

No treatment-related differences were observed between the treated and control groups for total implantations, number of resorbed fetuses, total live fetuses, fetal body weights, or fetal sex
ratios. No treatment-related increases in external , visceral, or skeletal malformations/variations were observed in the fetuses; treatment groups di splayed a lower inciden ce of minor variations compared to control. Theref ore, the developmental toxic ity NOAEL was 8.0 mg/kg/day and a developmental toxicity is not determined.

Developmental Neurotoxicity in Rodents (Rat; OCSPP 870.6300)

In a developmental neurotoxicity study ( MRID 46750501 ), bifenthrin (94.8% purity) was administered in the diet to female Crl:CD®(S D) rats (25/dose group) at doses of 0, 50, 100 and 125 ppm (mean-measured concentrations of 0, 3.6, 7.2 and 9.0 mg/kg/day, respectively, during gestation and 0, 8.3, 16.2 and 20.7 mg/kg/day, respect ively, during lactation) from GD 6 through LD 21. A FOB was conducted on all dams on GD 10 and 15, and on LD 10 and 21. On PND 4, litters were culled to yield four males and four females (as closel y as possible). Offspring were
allocated for detailed clinical observations (FOB) a nd assessment of moto r activity, auditory startle reflex habituation, learning and memo ry (water maze testing), and neuropathology at termination (PND 72). On PND 21, the whole br ain was collected from 10 pups/sex/group for micropathologic examination and morphometric analysis. Pup physical development was evaluated by body weight, and th e age of sexual maturation (VO in females and PPS in males)
was assessed.BIFENTHRIN FINAL

Page 47 of 59 No dams died during the study. Tremors were observed during the daily examinations in 8/25 females at the medium dose beginning on LD 14 and in 23/25 females in the high-dose group beginning on LD 4. In the medium-dose group, the tremors were graded as slight and resolved in half (4/8) of the females after one occurrence; slight tremors were obs erved in the remaining females 3-7 times. In the high-dose group, the tr emors were graded slight to moderate and continued on multiple occasions (2-18 consecutive days) during lactation. Piloerection also was
observed once or twice in 6/25 females at the hi gh dose, primarily during LD 14-17. During the FOB, the mean number of grooming counts was in creased (p<0.01) in females at the medium and high doses during gestation and lactation. In addition, slight piloer ection was observed in 4/25 high-dose females on GD 15 and in 2/25 a nd 1/25 females on LD 10 and 21, respectively. Clonic convulsions (limb tremors) and tremors were noted in 2/25 and 7/25 (p<0.01) females, respectively, in the high-dose group on LD 10. On LD 21, the number of females with these findings (p<0.01) was 10/25 and 13/25, respectivel y. Clonic convulsions (limb tremors) and tremors were noted (NS) in 2/23 and 3/23 fema les in the medium-dose group, respectively, on LD 21. Maternal body weight, body weight gain, food consumption, and reproductive performance were unaffected by treatment.

The maternal LOAEL for bifenthrin in rats was 100 ppm (7.2 mg/kg/da y during gestation and 16.2 mg/kg/day during lactation) based on clinical signs of ne urotoxicity (tremors, clonic convulsions, and increased grooming counts). The maternal NOAEL is 50 ppm (3.6 mg/kg/day during gestation and 8.3 mg/ kg/day during lactation).

The mean number of delivered pups per dam, per centage of live-born and stillborn pups, and sex ratio were not affected by treatment. Ther e was no treatment-related effect on offspring body weight or body weight ga in; decreased body weights ( ↓ 6-9%) and body weight gains ( ↓ 16-17%) in the medium-dose group males and females wa s minimal and not dose dependent. The mean day for reaching sexual maturation (VO in fema les and PPS in males) was not affected by
treatment. Two of 20 fema les in the high-dose group had tremors during the physical examinations on PND 28. During the FOB, an in crease in the incidence of tremors and clonic convulsions was observed in males at the high dose on PND 21. A significant increase in mean grooming counts was noted in females at the me dium and high doses on PND 21. No treatmentrelated effects on motor activity, acoustic startle response, or learning an d memory testing were observed. Brain weight, length, and width and macroscopic fi ndings were not affected by

treatment.

The offspring LOAEL for bifent hrin in rats is 100 ppm (7.2 mg/kg/day during gestation and 16.2 mg/kg/day during lactation; maternal dose) based on clini cal signs of neurotoxicity (increased grooming counts). The offspring NOAEL is 50 ppm (3.6 mg/kg/day during gestation and 8.3 mg/kg/day during lact ation; maternal dose).BIFENTHRIN FINAL

Page 48 of 59 Multi-Generation Reproduction in Rodents (Rat; OCSPP 870.3800)

In a two-generation study ( MRID 00157225 ), bifenthrin (88.35% purity) mixed with acetone
was administered in the diet to TAC(SD)fBR rats (25/sex/dose) at nominal doses of 0, 30, 60, or 100 ppm as FMC 54800 technical (approximately equivalent to 0, 1.5, 3.0 and 5.0 mg/kg/day;
the amount of test material added to the diet wa s adjusted to account for purity). For the P generation, dosing began 8 weeks prior to mating and for the F 1 generation, 11 weeks prior to mating. P generation animals were bred twice, and F 1 parental animals were selected from the

F 1 b pups.

There was no treatment-related mortality in either the P or F 1 generation. Tremors were noted only in females of both generations at the high do se with one P generation rat observed to have
clonic convulsions. The tremors occurred during Days 9-35 after delivery in the P generation and during Days 3-35 after delivery in the F 1 generation. Premating body weights were comparable to control for both sexes of both generations in all dose groups. P generation females at the high dose had lower mean body weights ( ↓ 4%, p<0.05) at Week 17 (after gestation and lactatio n), lower body weights ( ↓ 5-6%, p<0.05 or 0.01) duri ng the first lactation period (Day 7 and 14); and lower body weights du ring the second gestation and lactation periods (p<0.01 on LD 14, ↓ 5%). There was no correlation between lower body weight and frequency of tremors. Lower body weights in females at th e medium dose (NS) frequently paralleled body weight depression at the high dose. There were no clinical signs or effects on body weight at the low dose. There were no significant differen ces in mean body weights between treated F 1 animals and control. There were generally no treatment-related differences in food consumption in any parental group. In the high-dose P genera tion females, there was an increase (p<0.01) in absolute brain weight ( ↑ 5%). Mean absolute ovary wei ghts were slightly decreased ( ↓ 9%, p<0.05 or ↓ 12%, p<0.01) at the medium and high doses, respectively, in the F 1 parental generation. In the high-dose group F 1 b female progeny, absolute adre nal and heart weights were increased (p<0.05; ↑ 31 and 15%, respectively) compared to control, with non-dose-dependent
increases in the lower dose groups. Increased (p<0.01; ↑ 55%) absolute ovary weights were also observed in these animals, with non-dose-depend ent increases of 45 and 30% in the low- and medium-dose groups, respectively. There were no treatment-related gross or microscopic findings in either adults or progeny. There were no treatment -related effects on reproductive parameters (mating, male fertility, female fer tility, and gestation indices), and there were no treatment-related gross or microscopic findi ngs in either sex in either the P or F 1 generations.

The systemic LOAEL is 100 ppm (approximately 5.0 mg/kg/day) based on the incidence of tremors and marginally lower body weights in P and F 1 generation females during gestation and lactation. The systemic NOAEL is 60 ppm (appr oximately 3.0 mg/kg/day). The reproductive NOAEL is 100 ppm (HDT); a reproduc tive LOAEL is not observed.BIFENTHRIN FINAL

Page 49 of 59 Chronic Oral Toxicity/Carcinogenici ty in Rodents (Rat; OCSPP 870.4300)

In a chronic toxicity/carci nogenicity study in rats ( MRID 00157226 ), bifenthrin (88.35% purity) was administered for 734 days in the diet to SD rats (50/sex/dose group) at doses (adjusted for purity) of 0, 12, 50, 100, or 200 ppm (mean-measur ed concentrations of 0, 0.6, 2.3, 4.7, and 9.7 mg/kg/day for males and 0, 0.7, 3.0, 6.1, and 12.7 mg/kg/day for females). At study termination, all surviving rats in all groups were sacrificed for gross and histopathological evaluation. Select organs from 10 animals/ sex/dose group were weighed at termination (including liver, kidneys, testes, ovaries, and brain). Gross and microscopic pathological examination included the liver, kidneys, pituita ry, thyroid/parathyroids, adrenals, mammary gland, testes, epididymides, prostate, semi nal vesicles, uterus, vagina, and ovaries.

There were no treatment-related differences in survival. Tremors were noted in all high-dose males during Days 4-28 and in all high-dose fe males during Days 4-30 and 36-38. Individual animals in the low and/or medium-low dose groups exhibited tremors late in the study; one female in the medium-high dose group exhibite d tremors during Days 3-5 similar to the highdose animals. Other clinical signs in the high-dose males and fema les included increased incidences of abrasion, alopecia, and tail lace ration. Mean body weights of females at 200 ppm were generally decreased (p<0.05 or 0.01, ↓ 4-11%) from Week 13 to study termination. The 200 ppm females also exhibited a decrease d total body weight gain (significance NR, ↓ 17%). Mean body weights of the 200 ppm males were slightly decreased (NS; ↓ 2-4%) relative to
control from Weeks 1-92. There was no treatmen t-related effect on food consumption during the study. Males at 200 ppm had increased (NS) mean liver ( ↑ 11%) and kidney ( ↑ 28%) weights at study termination, but similar incr eases in these organ weights we re also observed in the lowdose males. There were no differences in brai n or gonadal weights, and no treatment-related findings in gross pathology or non-neoplastic change s. The incidences of pancreatic islet cell adenoma and fibrosarcoma in high-dose males we re increased (NS; 3/ 50 vs. 1/47 control and 3/50 vs 0/50 control, respectively) , and historical contro l data indicated that these are not rare
tumors in this strain.

The chronic LOAEL is 100 ppm (4.7 mg/kg/day in males; 6.1 mg /kg/day in females) based on
the increased incidence of tremors in females. The chronic NOAEL is 50 ppm (2.3 mg/kg/day in males; 3.0 mg/kg/day in females). Under the c onditions of this study, there was no conclusive evidence of carcinogenic potential.

Carcinogenicity in Rodents (Mouse; OCSPP 870.4200)

In a carcinogenicity study in mice ( MRID 00157227 ), bifenthrin (88.35% purity) was administered in the diet to Swiss-Webster, Ta c(SW)fBR mice (50/sex/do se) at doses (adjusted for purity) of 0, 50, 200, 500, or 600 ppm (mean-m easure concentrations of 0, 6.7, 25.6, 65.4, and 81.3 mg/kg/day for males and 0, 8.8, 32.7, 82.2, and 97.2 mg/kg/day for females) for 87 weeks (males) or 92 weeks (females).BIFENTHRIN FINAL

Page 50 of 59 There were no treatment-related effects on surviv al; however, two females and two males in the high-dose group and one female in the medium-dos e group were reported to have died due to compound-related symptoms. Tremors were noted in all males and females in the medium-high and high-dose groups during the first three months of the study. Tremors we re also noted in two
males and two females at the medium-low dose a nd in one male at the low dose. However,
tremors in the low-dose male occurred late in th e study and this animal died soon after with fluid detected in the lungs. Tremors in the medium -low males were observed on Days 37-39, a period when tremors were quite frequent in the higher-dose animals. High-dose males and females also displayed clonic convulsions, jerks and twitching . Mean body weight in high-dose males was decreased (significance NR) from Weeks 2 to 20, and was also decreased (significance NR) in the medium-dose males during Weeks 5, 8, 9, 12, and 13. Mean body weight of the females was decreased (significance NR) in the medium-high and high-dose groups during Weeks 1 to 6.
Treated male mice exhibited an increased tre nd (p=0.054) for hemangiopericytomas (originally reported as leiomyosarcomas) of the urinar y bladder (15, 12, 17, 16, and 31%, respectively).
Combined bronchioalveolar adenomas and adenocar cinomas were increased in treated females, with increases (p<0.05) at the low, medium-h igh, and high doses. Males had a dose-related increasing trend (p=0.022) in combined hepatoce llular adenomas and adenocarcinomas. Males also had an increased, but not dose-related, incidence (p ≤ 0.05) of bilateral germinal epithelial degeneration of the testes of 8, 25, 31, 21 and 24%, respectively.

The chronic LOAEL is 200 ppm (25.6 mg/kg/day in males; 32.7 mg /kg/day in females) based on
the incidence of tremors in both sexes. The ch ronic NOAEL is 50 ppm (6.7 mg/kg/day in males; 8.8 mg/kg/day in females). Carcinogenic potenti al was evidenced by a dose-related increase in the incidence of hemangiopericytomas in the urinary bladder, a significant dose-related trend for combined hepatocellular adenomas and carcinomas in males, and a significantly higher incidence of combined lung adenomas and carcinomas in females.

Chronic Oral Toxicity Non- Rodents (Dog; OCSPP 870.4100)

In a chronic toxicity study in dogs ( MRID 00163065 ), bifenthrin (88.35% purity) was administered for 52 weeks by capsule to Beagle dogs (4/sex/dose) at doses of 0, 0.75, 1.5, 3, or 5 mg/kg/day (equivalent to 0.66, 1.3, 2.7, or 4.4 mg/ kg/day after adjustment for purity). Organ weights determined included adrenals, kidneys, liver/gall bladder, ovaries, testes/epididymides, pituitary, and thyroid/parathyr oid; histopathological examinations were conducted on these tissues as well as the prostate, uterus, and mammary gland.

No mortality occurred during the study and there were no tr eatment-related effects on food consumption, organ weights, and gross or micr oscopic pathology. Tremors were noted in all males and females (4/4 per sex) in the high- dose group during Weeks 15-29 and in 1/4 males and 2/4 females at the medium-high dose during Weeks 16-23. The lack of tremors after 29 weeks suggests that dogs may have developed a tolerance to treatment. In seven of the dogs (two fromBIFENTHRIN FINAL

Page 51 of 59 the medium-high dose group and five from the high- dose group), tremors were noted prior to the daily dose, indicating a persistent effect from the previous day’s dose. Mean body weight gain in the high-dose males was decreased 42% compared to control during Weeks 0-24 and decreased 56% during Weeks 0-52 (significance NR); no treat ment-related effects were noted on female body weights. The LOAEL for this 52-week study is 2.7 mg/kg/day based on the increased
incidence of tremors in both se xes; the NOAEL is 1.3 mg/kg/day.

Mechanistic Studies: Ovarian Gene Expre ssion and Hormone Synthesis (Rat)

In a published non-Guideline study ( Liu et al. , 2011 ), the concentration-response of ovulatory gene expression and prostaglandi n synthesis in rat ovarian gra nulosa cells after exposure to bifenthrin (99.5% pur ity) was evaluated in vivo and in vitro . For the in vivo study, female SD
rats were administered bifent hrin in corn oil by i.p. on PND 21-26, inclusive, at doses of 0 (vehicle), 0.5, and 5.0 mg/kg/day. Animals we re injected with pr egnant mare serum gonadotropin (PMSG; 10 IU s.c.) on PND 24, and received human chorionic gonadotropin (hCG; 10 IU s.c.) 48 hours after PMSG administ ration. PMSG and hCG are used to mimic the effects of FSH and LH, respectively, in this mode l. Control animals were sacrificed 48 hours after PMSG administration for isolation of pr e-ovulatory granulosa cells, and treated animals were sacrificed at 6 hours post-hCG administra tion for isolation of peri-ovulatory granulosa cells. The pre-ovulatory granulosa cells (isolated from vehicle c ontrol animals) were prepared for cell culture. Cultured cells were pretreated wi th bifenthrin (in EtOH at a final concentration of 0.1% v/v) at concentrations of 1, 10, 100, and 1000 nM for 30 minutes, followed by incubation with hCG (1 IU/mL) or forskolin ( 10 μM) for 6 hours. Conc entrations of mRNA were quantitated in ovarian granulosa cells after in vitro or in vivo exposure to bifenthrin with a qRT-PCR method. The genes examined have roles in the regulation of progesterone, growth factors, transcriptional factors, cell-cycle, and estrogen and pros taglandin. Concentrations of prostaglandin E2 (PGE2) in cell culture media (after in vitro incubation) were determined with
an ELISA method.

The expressions of hCG-induced genes in the cu ltured granulosa cells were decreased in the presence of bifenthrin in eith er dose-dependent (p< 0.05) or non-dose dependent (NS) responses. Responses of housekeeping genes were not affected by bifenthrin exposure. In vivo
administration of bifenthrin for six days generally yielded minima l decreases (NS) of mRNA in the low-dose group (0.5 mg/kg/day) and significant decreases (p <0.05) or a clear trend to decrease in the high-dose group (5.0 mg /kg/day). Bifenthrin treatment in vitro also inhibited (p<0.05) PGE2 production in the cultured rat ovu latory granulosa cells in a non-dose-dependent

manner.

In summary, low-dose exposure to bifenthr in at 1 nM inhibited the hCG-induced in vitro expression of m ost of the ovulator y genes that were examined, and a moderate concentration of 100 nM attenuated hCG-stimulated expression of all tested genes. Furthermore, in vivo studiesBIFENTHRIN FINAL

Page 52 of 59 demonstrated a trend of inhibitory action due to bifenthrin on LH/hCG -stimulated rat ovulatory gene expression. These observati ons support the disrupti ng effects of bifenthrin on the network of ovulatory gene expression patter ns, with potential targets at pr otein kinase A-related signaling, gene transcription, and/or ovulat ory gene expression. The GLP st atus of this research is
unknown.

In a related, published non-Guideline study ( Liu et al. , 2011b ), the effects of exposure to bifenthrin enantiomers on rat ovarian granulosa cell hormone synthesis (progesterone and PGE2) were evaluated. An analytical standard of r acemic bifenthrin (99.5% purity) was separated by HPLC; quantification and purity of the isolated enantiomers was assessed by GC-ECD. Immature female SD rats (PND 22 or 23) were administered PMSG (10 IU s.c.), and were
sacrificed 48 hours after PMSG administration. The pre-ovulatory ovarian granulosa cells were isolated prepared for cell culture. Cultured cel ls were pretreated with individual bifenthrin enantiomers (in EtOH at a final concentrati on of 0.1% v/v) at a concentration of 10

− 7 M for 30 minutes, followed by incubation with hCG (1 IU /mL) for 6 hours. C oncentrations of mRNA were quantitated in ovarian granulosa cells after in vitro exposure to bifenthrin enantiomers with a qRT-PCR method. Concentrations of progester one and PGE2 in cell culture media (after in

vitro incubation) were determ ined with EIA methods.

The baseline concentration of progesterone in control cells was 8.0±2.9 ng/mL, and treatment with hCG increased the progesterone concentr ation approximately 8-fold (65.1±4.2ng/mL). Exposure to 1S- cis -bifenthrin decreased (p<0.05) hCG-i nduced production of progesterone by 55% to 35.5±4.2 ng/mL; however, exposure to1R- cis -bifenthrin had no effect compared to hCG alone. Effects on gene expression were determined by examination of the mRNA levels of genes that encode CYP450 side chain cleavage en zyme (P450scc), steroidogenic acute regulatory protein (StAR), peripheral benzodiazepine receptor (PBR), and diazepam binding inhibitor (DBI). As expected, hCG induced the expression of these progesterone-related genes. Exposure

to 1S- cis -bifenthrin resulted in approximately 50-60% decreases (p<0.05) in mRNA expression levels compared to levels in cells trea ted with hCG alone. Treatment with 1R- cis -bifenthrin had no effect on mRNA expression levels of these gene s. Similarly, the baseline concentration of PGE2 in control cells was 92.7±4.2 pg/mL, and PGE2 accumulation in the culture media of hCG-incubated cells was increased (p<0.05) approximately 30% (121.1±10.9 pg/mL). Exposure

to 1S- cis -bifenthrin decreased (p<0.05) hCG-s timulated PGE2 production to 93.3±7.6 pg/mL,
but 1R- cis -BF did not affect PGE2 production. Cycl ooxygenase-2 (COX-2), the presumed ratelimiting step in the synthesis of prostaglandins, was also examined. Th e expression levels of COX-2 mRNA were down-regulated (p<0.05) in the cells incubated with 1S- cis -bifenthrin (relative to hCG induction), but no difference was observed in COX-2 mRNA gene expression
due to 1R- cis -bifenthrin-exposure.BIFENTHRIN FINAL

Page 53 of 59 Avian Reproduction (OCSPP 850.2300)

In an avian reproduction study ( MRID 00163099 ), 26-week old Mallard ducks ( Anas platyrhynchos ) were administered bifenthrin (88 .35% purity) in the feed at nominal concentrations of 0 (corn oil), 25, 50, or 75 ppm (adjusted for purity was not reported) for 22 weeks (ten weeks prior to the start of egg production and during twelve weeks of egg production). Each treatment group was comprised of two replicates, with two drakes and five hens per pen. A progressive photo period was em ployed in this study: 7 hours/day until Week 8 and 16 hours/day until the end of the study (Day 154). Temperatur e was maintained at 18±3°C throughout the study and humidity was maintained at 73±6%. Individual adult body weights were recorded on Days -7, 0, 14, 28, 42, 56, 70, and 154; food consumption in each replicate was measured weekly throughout the pre-treatment and treatment pe riods of the adult phase. Individual chick body weights we re recorded within 24 hours of hatching and on Day 14 after hatching; egg shell thickness was measured at Weeks 11, 13, 15, 17, 19, and 21 of dosing (during egg production). Adult birds and chicks were obser ved daily for mortality and clinical signs; all adult birds were subjected to post-mortem examination.

There were no treatment-related effects on adult mortality, food c onsumption, or body weight. There were no effects of treatment on the pr oportion of eggs cracked or broken, mean egg weight, or egg shell thickness. Percentages of infertile eggs/eggs set, early embryonic deaths/fertile eggs, and late embryonic deaths/f ertile eggs did not differ between treatment and
control. There were also no differences between the percentages of hatche d eggs/fertile eggs and the percentage of dead in shell/fertile eggs. Finally, mean body weights at time of hatching and at Day 14 showed no treatment-related difference, as well as no differences in survival of chicks to 14 days. No abnormalities were no ted in chicks that died on study.

The NOAEC for mallard ducks exposed to bifent hrin in the diet for 22 weeks was 75 ppm (75 mg/kg diet; HDT).

In an another avian reproduction study ( MRID 00163097 ), 6-month old Bobwhite quail ( Colinus virginianus ) were administered bifenthr in (88.35% purity) in the feed at nominal concentrations of 0 (corn oil), 25, 50, or 75 ppm (adjusted for purity was not reported) for 24 weeks (twelve weeks prior to the start of egg production and during twelve weeks of egg production). Each treatment group was comprised of twenty mating pa irs, with one pair per pen. A progressive photo period was employed in this study: 7 hours/day until Week 7 and 16 hours/day until the end of the study (Day 168). Temperature was maintained at 24±3°C throughout the study and humidity was maintained at 60± 8%. Individual adult body weight s were recorded on Days -7, 0, 14, 28, 42, 56, 70, 84, and 168; food consumption in each replicate was measured weekly throughout the pre-treatment and treatment peri ods of the adult phase. Individual chick body weights were recorded within 24 hours of ha tching and on Day 14 after hatching; egg shell thickness was measured at Weeks 14, 16, 18, 20, 22, and 24 of dosing (during egg production).BIFENTHRIN FINAL

Page 54 of 59 Adult birds and chicks were observed daily for mort ality and clinical signs; all adult birds were subjected to post-mortem examination.

Individual birds or a complete replicate were replaced during the pre-egg production period due to unscheduled deaths; all were replaced with sp are birds. One replicate replacement was made during the egg production process (Day 91) because of a male that could not stand. There was no reported treatment-related effect on adult mortal ity; there were 3, 3, 4, and 1 deaths during the study in the control, low-, medium-, and hi gh-dose groups, respectively. Adult body weight changes and food consumption during the treatment period were similar in all groups, and there
was no evidence of a treatment-related effect. There was an increase (p<0.05) in the proportion of eggs cracked or broken in the medium- and hi gh-dose groups, but no differences in mean egg weight or egg shell thickness. Percentage s of infertile eggs/eggs set, early embryonic deaths/fertile eggs, and late embryonic deaths/f ertile eggs did not differ between treatment and
control. There were also no differences between the percentages of hatche d eggs/fertile eggs and the percentage of dead in shell/fertile eggs. Finally, mean body weights at time of hatching and at Day 14 showed no treatment-related difference, as well as no differences in survival of chicks to 14 days. No abnormalities were no ted in chicks that died on study.

The reproductive NOAEC for bobwhite quail exposed to bifenthrin in the diet for 24 weeks was 25 ppm (25 mg/kg diet). The LOAEC is 50 ppm based on an increased number of cracked and broken eggs.

Mechanistic Studies: Estrogenic Activity in Fish (Freshwater/Estuarine)

In a published non-Guideline in vivo study ( Brander et al ., 2012 ), evaluation of the concentration response to bifent hrin (99% purity; 50/50 mixture of isomers) with juvenile Menidia beryllina (65- to 70-day old) was examined in a 14-day static aqueous exposure bioassay. The biomarker of estrogenic endocri ne disruption selected was expression of choriogenin (Chg), an estrogen-de pendent egg coat protein, which was assessed with an indirect enzyme-linked immunosorbent assay (ELISA) relativ e to a positive control (ethinylestradiol). Fish were exposed to bifenthrin (1, 10, or 100 ng/L) or ethinylestr adiol (1, 10, 50 ng/L) by adding stock solutions prepared in methanol (a control of 0.01% methanol was included). At study termination, fish were anesthetized (on i ce), frozen, and homogenated in buffer. The resultant supernatant after centrifuga tion was analyzed by an ELISA method.

Mean survival in all treatments after 14 days was ≥ 80%. Juvenile M

. beryllina exposed to all
three concentrations of bifenthrin and ethinylestradiol had higher (p<0.05) relative concentrations of Chg expressed in whole-body homogenate compared to the methanol control.
None of the bifenthrin treatments were differe nt from one another when compared with ANOVA (p >0

. 05); however, a trend toward an inverse co rrelation between response and increasing
concentration with bifenthrin is apparent. Bifenthrin treatments were not significa ntly different from positive control concentrations of 1 and 10 ng/L

, with the exception of 1 ng/L bifenthrin

,BIFENTHRIN FINAL

Page 55 of 59 which had a higher (p=0

. 022) relative Chg concentration compared to 1 ng/L ethinylestradiol

. However, all bifenthrin concentrations trende d toward a higher rela tive expression of Chg compared to the 1 ng/L ethinylestradiol treatment and a lower re lative expression than the 10 ng /L ethinylestradiol treatment. The ethinylestradiol response wa s best fit by a sigmoidal model, and the bifenthrin concentration-response curve was best fit with a unimodal (biphasic)
model. All study concentrations of bifenthrin induced the expr ession of Chg in juvenile M. beryllina relative to control, and 1 ng/L bifenthrin induction yielded greater Chg expression compared to 1 ng/L ethinylestradiol

.BIFENTHRIN FINAL

Page 56 of 59 APPENDIX 3: References Not Utilized in the Bifenthrin 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 Bifenthrin but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the com pound of interest was not us ed in the study; 3) the test material was not adequately described; 4) a formulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6)the experimental conditions were not adequately described; 7) only an abstract of the study was available; 8) the reference is a review article or book 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 da ta on invertebrates.

Abbott, D.H., Padmanabhan, V., and Dumesic, D.A. (2006) Contributi ons of androgen and estrogen to fetal programming of ovarian dysfunction. Reprod Biol Endocrinol 4:17.

Adams, E.R., Gibson, W.R., Koenig, G.R., et al. (1973) A Multi-Gener ation Rat Reproduction Study with Benefin. (Unpublished study receiv ed Aug 5, 1976 under 1471-92; submitted by Elanco Products Co., Div. of Eli Lilly and Co., Indianapolis, Ind.; CDL: 224873-D). (MRID 00037676)

Akhtar, N., Kayani, S.A., Ahmad, M.M., and Shaha b, M. (1996) Insecticide-induced changes in secretory activity of the thyroid gland in rats. J. Appl. Toxicol. 16(5):397-400.BIFENTHRIN FINAL

Page 57 of 59 Bowman, C.J. (2005) Validation of the Pubert al Male Assay in Rats (WA 4-15). WIL-431005. Final report. 13 December 2005.

Burgess, D. (1989) Chronic Toxicity of 54800 to Daphnia magna Under Flow-Through Test Conditions. Prepared by Analytical Bio-Chemistr y Laboratories, Inc., Columbia, Missouri. ABC Report No. 36980. Submitted by FMC Corporation Chemical Research and Development, Princeton, New Jersey. (MRID 41156501)

Carney, E.W., Zablotny, C.L., Marty, M.S., Cri ssman, J.W., Anderson, P., Woolhiser, M., and Holsapple, M. (2004) The Effects of Feed Restriction during in Utero and Postnatal Development in Rats. Toxicol. Sci ., Nov 2004; 82: 237-249.

Deanovic, L.A., Markiewicz, D., Stillway, M., Fong, S., and Werner, I. (2013). Comparing the
Effectiveness of Chronic Water Co lumn Tests with the Crustaceans Hyalella azteca (Order: Amphipoda) and Ceriodaphnia dubia (Order: Cladocera) in Detect ing Toxicity of Current-Use
Insecticides. Environmental Toxicology and Chemistry . 32(3):707-712.

Della Seta, D., Farabollini, F., Dessi-Fulgheri, F., and Fusani, L. (2008) Environmental-like exposure to low levels of estrogen affects se xual behavior and physiolo gy of female rats. Endocrinology 149(11):5592-5598.

Gibson, W.R., Small, R.M., Todd, G.C., et al. ( 1973) A Study of the Effects on Rats from the Ingestion of Benefin for Two Years. (U npublished study received Aug 5, 1976 under 1471-92; submitted by Elanco Products Co., Div. of Eli Lilly and Co., Indianapolis, Ind.; CDL: 224873- C). (MRID 00037675)

Gray, Jr, L.E., Ostby, J., Furr, J., Wolf, C.J., Lambright, C., Parks, L., Veeramachaneni, D.N., Wilson, V., Price, M., Hotchkiss, A., Orlando, E., and Guillette, L. (2001) Effects of environmental antiandrogens on reproductive development in experimental animals. Hum Reprod Update 7(3):248-264.

Hill, R.N., Crisp, T.M., Hurley, P.M., Rosentha l, S.L., and Singh, D.V. (1998) Risk assessment of thyroid follicular cell tumors. Environ. Health Perspect . 106, 447-457.

Hill, R.N., Erdreich, L.S., Paynter, O.E., Roberts, P.A., Rosenthal, S.L., and Wilkinson, C.F. (1989) Thyroid follicular cell carcinogenesis. Fundam. Appl. Toxicol . 12,629-697.

Horsley, E.M.T. (2005) Assessment of Pubertal Development and Thyroid Function in Juvenile Male Rats. Study No. 04-4276. Final report. 22 December 2005. Dow AgroSciences LLC Report ID: AKH020510. Page 25.

Jin, M., Zhang, X., Wang, L., Huang, C., Zha ng, Y., and Zhao, M. (2009) Developmental toxicity of bifenthrin in embr yo-larval stages of zebrafish. Aquat. Toxicol. 13;95(4):347-54.BIFENTHRIN FINAL

Page 58 of 59 Liu, J., Yang, Y., Yang, Y., Zhang, Y., and Liu, W. (2011a) Disrupting eff ects of bifenthrin on ovulatory gene expression and prostaglandin synthesis in rat ovarian granulosa cells. Toxicology 282(1-2):47-55.

Lund, T.D., Munson, O.J., Haldy, M.E., Setchell, K.D.R., Lephart, E.D., and Handa, R.J. (2004) Equol is a novel anti-androgen that inhib its prostate growth and hormone feedback. Biol Reprod 70:1188-1195.

McAllister, W.A. (1988). Full life cycle to xicity of 14C-FMC-54800 to Fathead minnow (Pimephales promelas) in a flow-through system. FMC Study No. A86-21 00. (MRID 160015) ?

Murray, A.G., Seacat, J.L., and Grothe, D.W. (1991) The Toxicity of Benefin to Mallards in a One-generation Reproduction Study. Laborat ory Project No. A01090. Prepared by the Toxicology Research Laboratories, Lilly Resear ch Laboratories, Green field, Indiana. (MRID 42145501)

Murray, A.G., Seacat, J.L., and Grothe, D.W. (1991) The Toxicity of Benefin to Bobwhite in a One-generation Reproduction Study. Laborat ory Project No. A00690. Prepared by the Toxicology Research Laboratories, Lilly Resear ch Laboratories, Green field, Indiana. (MRID 42145502)

O'Connor, J.C., Frame, S.R., and Ladics, G.S. (2002) Evaluation of a 15-day screening assay using intact male rats for identifying antiandrogens. Toxicol Sci 69:92-108.

Orton, F., Rosivatz, E., Scholze, M., and Kortenka mp, A. (2011). Widely used pesticides with previously unknown endocrine activity revealed as in vitro antiandrogens. Environ Health Perspect . 119(6):794-800

Rider, C.V., Furr, J., Wilson, V.S., and Gray, Jr ., L.E. (2008) A mixture of seven antiandrogens induces reproductive malformations in rats. Int J Androl 31:249-262.

Saghir, S.A., Charles, G.D., Battels, M.J., Kan, L.H.L., Dryzga, M.D., Brzak, K.A., Clark, A.J. (2008) Mechanism of trifluralin-i nduced thyroid tumors in rats. Toxicology Letters (30 Jul 2008) Vol. 180, No. 1, pp. 38-45.

Sawaki, M., Noda, S., Muroi, T., Mitoma, H., Takakura, S., Sakamoto, S., and Yamasaki, K. (2003) In utero through lactational exposure to ethi nyl estradiol induces cl eft phallus and delayed ovarian dysfunction in the offspring. Toxicol Sci 75:402-411.

Schlenk, D., Lavado, R., Loyo-Rosales, J.E., Jone s, W., Maryoung, L., Riar, N., Werner, I., and Sedlak, D. (2012) Reconstitution studies of pestic ides and surfactants exploring the cause of estrogenic activity observed in surface wa ters of the San Francisco Bay Delta. Environ. Sci.
Technol. 46(16):9106-9111.

Shin, J.H., Kim, H.S., Moon, H.J, Kang, I.H., Kim, T.S., Seok, J.H., Kim, I.Y., Park, K.L., Han, S.Y., and Nam, S.Y. (2002) Effects of flutamid e on puberty in male rats: An evaluation of theBIFENTHRIN FINAL

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Stoker, T., Parks, L.G., Gray, L.E., Cooper, R.L. (2000) Endocrine-Disrupting Chemicals: Prepubertal Exposures and Effect s on Sexual Maturation and Thyroi d Function in the Male Rat. A Focus on the EDSTAC Recommendations. Critical Reviews in Toxicology , Jan 2000, Vol. 30, No.2, Pages 197-252.

Taylor, J.S., Thomson, B.M., Lang, C.N., Sin , F.Y.T., and Podivinsky, E. (2010) Estrogenic Pyrethroid Pesticides Regulate Expression of Estrogen Receptor Transcripts in Mouse Sertoli Cells Differently From 17 β -Estradiol. Journal of Toxicology & Environmental Health 73(16) p.1075-1089.

Terry, K.K., Cappon, G.D., Hurtt, M.E., Tassinari , M.S., and Gupta, U. (2004) Reproductive toxicity assessment of lasofoxife ne, a selective estrogen receptor modulator (SERM), in female

rats. Birth Defects Res B Dev Reprod Toxicol 71(3):150-60.

Van der Schoot, P. (1992) Disturbe d testicular descent in the rat after prenatal exposure to the antiandrogen flutamide. J Reprod Fert 96:483-496.

Vanage, G.R., Dao, B., Li, X-J., Bardin, C.W., and Koide, S.S. (1997) Effects of Anordiol, an Antiestrogen, on the Reproductive Organs of the Male Rat. Systems Biology in Reproductive Medicine Vol. 38, No. 1, Pages 13-21.

Wang, L., Liu, W., Yang, C., Pan, Z., Gan, J., Xu, C., Zhao, M., and Schlenk, D. (2007) Enantioselectivity in estrogenic pot ential and uptake of bifenthrin. Environ. Sci. Technol. Sep 1; 41(17):6124-8.

Worth, H.M., Pierce, E.C., Harris, P.N., et al. (1966) Subacute Toxicity of Benefin to Rats. (Unpublished study received Mar 15, 1966 unde r unknown admin. no.; submitted by Elanco Products Co., Div. of Eli Lilly and Co., I ndianapolis, Ind.; CDL: 107494-A). (MRID 00024651)

York, R.G. (2006) Interlaboratory Validation of the Pubertal Male Assay. Final Report. 3 January 2006.

Zhao, M., Chen, F., Wang, C., Zhang, Q., Gan, J., and Liu, W. (2010). Inte grative assessment of enantioselectivity in endocrine disruption a nd immunotoxicity of s ynthetic pyrethroids. Environ
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