UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: June 29, 2015 

SUBJECT: EDSP Weight of Evidence Conclusions on the Tier 1 Screening Assays for the 

List 1 Chemicals 

PC Code: See table, Attachment A 

Decision No.: NA 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

Environmental Fat~~fec~~~n ~ Office of Pesticide Program 

THROUGH: Jess Rowland ~ S ~~~ Deputy Division Director 

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

EPA has completed its Weight of Evidence (WoE) assessment evaluating results of the 

Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. 

The WoE documents for the 52 chemicals are listed in Attachment A along with the chemical 

and report identifiers. 

Page I of2 

Page 1 of 67
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

Page 2 of 67
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAY 

CHEMICAL: BIFENTHRIN 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

Page 3 of 67
BIFENTHRIN FINAL 

ii 

TABLE OF CONTENTS 

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

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

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

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

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

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

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

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

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

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

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

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................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-Estrogenic Pathway for Bifenthrin.....................................10 

 Table 3. Androgenic/Anti-Androgenic 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............................56 

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BIFENTHRIN 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 Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

F2 Second filial generation 

Fcd Fecundity

Page 5 of 67
BIFENTHRIN FINAL 

iv 

Abbreviation Terminology 

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

N Negative 

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BIFENTHRIN FINAL 

v 

Abbreviation Terminology 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

TR Thyroid Receptor

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BIFENTHRIN FINAL 

vi 

Abbreviation Terminology 

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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BIFENTHRIN FINAL 

Page 1 of 59 

Executive Summary 

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

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

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

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

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) perturbations. 

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

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

this weight of evidence (WoE) assessment. 

In determining whether bifenthrin interacts with E, A, or T hormone pathways, the number and 

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

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

in particular, whether or not endocrine-related responses occurred at dose(s) that also resulted in 

general systemic toxicity or overt toxicity. 

On 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 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 member of the synthetic pyrethroid class of insecticide having similar toxicity 

profiles that elicit neurotoxic behaviors in laboratory animals. This assessment includes studies 

conducted with two enantiomers: 1S-cis-bifenthrin and 1R-cis-bifenthrin. 

For the 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-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 uterotrophic assay was negative and, in the female pubertal rat assay, there 

were no treatment-related estrogen-related effects observed in the absence 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 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 studies, the only effect 

observed was an increase in number of cracked eggs, but no differences in mean egg weight or 

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BIFENTHRIN FINAL 

Page 2 of 59 

egg shell thickness or other reproductive parameter. Therefore, there is a lack of convincing 

evidence for potential interaction with the estrogen pathway. 

For the androgen pathway, bifenthrin 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 not reported. No treatment-related effects were 

observed in the male pubertal rat study, in the absence 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 evidence for potential interaction with the androgen 

pathway. 

Based on mammalian Tier 1 assays and the OSRI mammalian studies, bifenthrin does not appear 

to affect the thyroid or the HPT axis. Developmental 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 (decreased snout-vent length and body 

weight). Treatment-related differences in thyroid 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 atrophy 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 commonly affected in the presence of a thyroidrelated 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 histopathology. Serum thyroxine (T4) concentration was decreased only in the 

low-dose groups of both sexes (↓16%), with an increase in thyroid stimulating hormone (TSH) 

concentration occurring only in the high-dose males and which was associated with overt 

toxicity. Furthermore, there were no effects 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 potential interaction with 

the estrogen, androgen or thyroid pathways. 

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BIFENTHRIN FINAL 

Page 3 of 59 

I. Introduction 

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

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

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

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

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) perturbations. 

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

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

this weight of evidence (WoE) assessment. 

In determining whether bifenthrin interacts with E, A or T hormone pathways, the number and 

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

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

in particular, whether or not endocrine-related responses occurred at dose(s) that also resulted in 

general systemic toxicity or overt toxicity. 

On 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 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 pyrethroid (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 commercial products consist of predominantly the 1R-cisbifenthrin 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 coefficient (log Kow) of 6.00. The 

estimated bioconcentration factor for bifenthrin is 6,089. Bifenthrin is expected to be removed 

from the atmosphere by photodegradation and deposition. Volatilization from water is also 

expected to contribute to the dissipation of bifenthrin in the environment. Bifenthrin has soil 

half-life estimates of approximately 100 to >300 days. 

The available information considered in determining the potential interaction 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 studies 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, 

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BIFENTHRIN FINAL 

Page 4 of 59 

using the WoE approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The 

EDSP Tier 2 studies recommendations are presented in Section V. 

II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the 

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

presented in Appendix 1. 

B. Other Scientifically Relevant Information (OSRI) 

In response to the Agency’s Test Orders, data believed to be relevant to one or more of the Tier 1 

assays were submitted as OSRI by the Test Order recipients and/or the public. This included 

studies published in the open literature and/or data submitted to support pesticide registration 

(e.g., Part 158 guideline studies). The Agency’s review of the initial OSRI is provided in the 

Report of the Endocrine Disruptor Review Team on bifenthrin (USEPA, 2010). 

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

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

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

listed in Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

above. 

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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 include published literature studies as well as 

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

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

relevance for informing interactions with the E, A or T pathway. Additionally, the 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 specific endpoint for discerning whether or not the 

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

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

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

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

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

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

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

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

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

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

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

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

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

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

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

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

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

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

biological organization were the responses observed and whether organisms exhibit 

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

conditions did the responses occur including consideration of whether the responses were 

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

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BIFENTHRIN FINAL 

Page 6 of 59 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

forms of toxicity (e.g., changes in organ weights or blood chemistry). The circumstances for 

which a dose/concentration was considered overtly toxic for a particular study are described in 

Section IV.A. 

In summary, EPA considers multiple lines of evidence in including the observed responses in 

the Tier 1 assays and OSRI in the context of a chemical’s physical/chemical properties and its 

known modes of action in its overall characterization of a chemical’s potential to interact with 

the E, A or T pathway. Adequately addressing the three main components described above is 

fundamental to the WoE process and in determining whether additional data are needed. In 

addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their 

potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or 

conflicting data; 4) overall strength of evidence supporting a conclusion; and, 5) what, if any, 

additional data are needed and why. Assessing the need for additional data is based on a caseby-case analysis which took all available toxicity data into account. 

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BIFENTHRIN FINAL 

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

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

considered scientifically and biologically relevant 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 the presence of overt toxicity are discussed in the text for each 

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

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays submitted in response to the agency’s test order for 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) 

ERα 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) 

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

1. Effects on Estrogen Pathway 

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

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

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

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

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

considered further in the WOE assessment. 

Bifenthrin was negative (or not interactive) in the in vitro ER binding, ERTA, aromatase, and 

steroidogenesis assays. Several published, non-guideline in vitro estrogen-responsive assays 

were available for bifenthrin. Brander et al. (2012) reported that bifenthrin was negative for an 

estrogenic response in the ER–based CALUX mammalian cell bioassay, but observed an antiestrogenic response at bifenthrin 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-cisbifenthrin 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, proliferation was blocked by co-administration 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 signs of toxicity noted 24 hours post-dose. While a delay in VO 

was observed at the low dose, in the absence of overt toxicity, it is noted that this 2 day delay is 

at the upper range of normal biological variability, no other estrogen-related effects were 

observed in this study. 

In the FSTRA, there was a significant 60% increase in fecundity at the medium concentration 

(0.10 μg a.i./L) and a 74% reduction at the high concentration (3.0 μg a.i./L). There was also a 

51% increase in female GSI at the high concentration. Female body weight was increased at the 

high-concentration (↑28%, p<0.05), but no other growth endpoints were affected by treatment. 

There was an increase in oocyte atresia in 87% of the fish in the high-concentration group along 

with an increase in post-atretic follicles and granulomatous inflammation which was attributed to 

the increased oocyte atresia. However, at the highest concentration, clinical signs of overt 

toxicity were observed beginning on Day 5 and included erratic swimming, lethargy, loss of 

equilibrium, and surfacing. 

Page 16 of 67
BIFENTHRIN FINAL 

Page 9 of 59 

In the Part 158 subchronic dog study, there were decreases (significance 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 weights were slightly decreased at the medium 

and high doses (9% and 12%, respectively) in the F1 parental generation; at the high dose this cooccurred with tremors. Increased (55%) absolute ovary weights were also observed in the highdose group F1b female offspring which co-occurred with tremors. Non-significant, non-dosedependent increases in ovarian weight of 45 and 30% were observed in the low- and mediumdose groups, respectively. However, no histopathological changes were noted in these animals, 

and there were no treatment-related effects on fertility or fecundity. Bifenthrin did not alter other 

reproductive organ histopathology, 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 reproduction study, the number of cracked or broken eggs was 

significantly higher in the middle (50 ppm) and high (75 ppm) treatment groups as compared to 

the control, but no differences in mean egg weight 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 significantly greater choriogenin (precursor to chorion, 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, ethinylestradiol. It is worth noting that this study 

did not have a negative control but rather comparisons were made to a methanol solvent control. 

Additionally, the measured test concentrations reported appear to reflect results from a single 24 

hour renewal cycle 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.

Page 17 of 67
BIFENTHRIN FINAL 

Page 10 of 59 

Table 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) 

EDSP Tier 1 Assay 

ER Binding (MRID 48615003) N ERTA (MRID 48615004) N4 Aromatase (MRID 48615002) N Steroidogenesis (MRID 48615006) N Uterotrophic (MRID 48615007) N NE Female Pubertal Rat (MRID 48669401) NE N N N N N N FSTRA (MRID 48675902) NE N N N Fcd: ↑60

(M) 

OSRI 

ERTA (CALUX) (Brander et al., 2012) P Cell Proliferation (Wang et al., 2007) P Cell Proliferation (Zhao et al., 2010) P ER-related gene expression (Liu et al., 2011ab) 

P 
BIFENTHRIN FINAL 

Page 11 of 59 

Table 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) 

Subchronic toxicity (Rat; MRID 00141199) 

NE N N NE 

Subchronic toxicity (Dog; MRID 00141200) 

NE N N NE N 

Developmental toxicity (Rat; MRID 00141201) 

NE N 

Developmental toxicity (Rat; MRID 45352301) 

NR N 

Developmental toxicity (Rabbit; MRID 00145997) 

NE N 

Developmental Neurotoxicity (Rat; MRID 46750501) 

NE N N 

Two-generation reproduction (Rat; MRID 00157225) 

 NR ↓9% (M) N NR N 

Chronic toxicity/oncogenicity (Rat; MRID 00157226) 

NE N N NE 

Carcinogenicity (Mouse; MRID 00157227) 

NR NR NR NR 
BIFENTHRIN FINAL 

Page 12 of 59 

Table 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) 

Chronic toxicity (Dog; MRID 00163065/40266601) 

NE N N N 

Avian reproduction (Duck; MRID 00163099) 

 N 

Avian reproduction (Quail; MRID 00163097) 

 P5 

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

or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicabin weight are absolute unless otherwise indicated. 

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

in this column. For details see Section IV. A 

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

details see Section IV. A 

4. The responsiveness of the cells to the very weak positive control 17α-methyltestosterone was lower than the expected values. A

this assay were not optimal to detect very weak activity, bifenthrin responses were similar to those of the negative control cortico

comparable to the responses of 17α-methyltestosterone, which was able to reach a maximum of 8.1-52.8% PC. Bifenthrin was o

maximum of 3.8-5.0% PC when tested up to the highest concentration possible based on cytotoxicity (10-4 M). Because the RPC

runs, 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 gr

control 

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

NE Not examined 

 NR Not reported 
BIFENTHRIN FINAL 

Page 13 of 59 

2. Effects on Androgen Pathway 

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

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

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

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

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

table and not considered further in the WOE assessment. 

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 concentrations were not determined. In a non-guideline Hershberger assay 

submitted as OSRI (Zhang et al. 2008), treatment with bifenthrin (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 the 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 preputial separation (PPS), 

body weight at PPS or serum testosterone concentration. There was a statistically significant 

increasing trend in LABC (adjusted) and dorsolateral prostate (unadjusted and adjusted) weight, 

however pairwise comparison to controls was not statistically significant and there were no 

treatment-related macroscopic observations or histopathological changes observed. Pituitary 

weight increased 11% at the high dose in this study. 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 males in the high-concentration group (0.402 μg/L) 

and one death (male) at the medium concentration (0.075 μg/L). In the surviving males, there 

were no significant effects on male VTG, male GSI, median tubercle scores or treatment-related 

effects in male gonadal histopathology. 

No androgen-related effects on reproductive parameters due to bifenthrin treatment have been 

reported in existing Part 158 studies.

Page 21 of 67
BIFENTHRIN FINAL 

Page 14 of 59 

Table 3. Androgenic/Anti-Androgenic Pathway for Bifenthrin

Lines of Evidence Indicating Potential Interaction with the 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 

Villi(VTG) 

EDSP Tier 1 Assay 

AR Binding (MRID 48615001) N Steroidogenesis (MRID 48615006) N Hershberger (MRID 48615005) NE N Male Pubertal Rat (MRID 48669401) N N N N N N N N FSTRA (MRID 48675902) NE N N N N N 
BIFENTHRIN FINAL 

Page 15 of 59 

Table 3. Androgenic/Anti-Androgenic Pathway for Bifenthrin

Lines of Evidence Indicating Potential Interaction with the 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 

Villi(VTG) 

OSRI 

Subchronic toxicity (Rat; MRID 00141199) 

N N NE N N N 

Subchronic toxicity (Dog; MRID 00141200) 

N N N N N NE 

Developmental Neurotoxicity (Rat; MRID 46750501) 

N 

Two-generation reproduction (Rat; MRID 00157225) 

N N NR NR NR NR N 

Chronic toxicity/ oncogenicity (Rat; MRID 00157226) 

N N NE N NE NE 

Carcinogenicity Mouse; MRID 00157227) 

 NR NR NR NR NR NR 

Chronic toxicity (Dog; MRID 00163065/40266601) 

N N N N N N 

Avian reproduction (Duck; MRID 00163099) 

 N 
BIFENTHRIN FINAL 

Page 16 of 59 

Table 3. Androgenic/Anti-Androgenic Pathway for Bifenthrin

Lines of Evidence Indicating Potential Interaction with the 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 

Villi(VTG) 

Avian reproduction (Quail; MRID 00163097) 

 N 

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

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

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

(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 OS

this column. For details see Section IV. A 

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

details see Section IV. A 

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

NE Not examined NR Not reported 
BIFENTHRIN FINAL 

Page 17 of 59 

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

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

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

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

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

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

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

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

HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of 

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

further in the WOE assessment. 

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) developmental stage by 1.5 stages at 21 days in 

the highest treatment concentration (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 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 concentration, particularly at the highest treatment 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 high 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 significant (p<0.05) at the 

middle and high treatment concentrations (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 (T4) concentration was 

decreased 16% in the low-dose groups only. Thyroid stimulating hormone (TSH) levels (↑51%) 

and pituitary weight (↑11%) were increased in the high-dose males only and co-occurred with 

increased incidence of tremors (7/16 animals). 

Page 25 of 67
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Page 18 of 59 

Table 4. Thyroid Pathway for Bifenthrin

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Bifenthrin1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

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

Overt Toxicity Observed 3 

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) 

P4 

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

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

Page 26 of 67
BIFENTHRIN 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 concentration, 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 weight of evidence (WoE) determination on the 

potential of bifenthrin to interact with endocrine related processes (i.e., E, A, or T hormonal 

pathways) as well as recommendations regarding Tier 2 testing. The results of the Tier 1 assays 

are considered, along with other scientifically relevant information (e.g., 40 CFR Part 158 test 

guidelines and published or publicly available peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence (i.e., specific Tier 1 assays and 

OSRI), then performing an integrated analysis of those lines of evidence. 

The WoE evaluation includes considerations of biological plausibility of the findings from the 

different lines of evidence by examining the consistency, coherence, and interrelationships 

among the measured endpoints within and across studies. The available findings from standard 

toxicology studies on the substance may contribute to the WoE evaluation in helping elucidate if 

effects seen in the Tier 1 assay are related to perturbations of the endocrine system per se or 

alternatively a sequelae of systemic effects. Endocrine modes of action may evoke a number of 

phenotypic consequences other than those evaluated in the Tier 1 assays. 

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to 

whether or not the responses are 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 evaluation of the results of the Tire 1 assays stated that “In summary, 

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

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

terms of primary interactions with the endocrine system vs. secondary effects related to toxicity 

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

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and 

OSRI studies are described below. In addition to the endocrine-related effects described above 

for the in vivo Tier 1 assays and OSRI, other effects observed which may be considered 

overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% 

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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, more than one parameter was needed to determine 

overt toxicity. Effects which were considered to be signs of systemic toxicity were generally less 

severe forms of toxicity (e.g., changes in organ weights or blood chemistry). 

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 were 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 day. These animals appeared normal on all other 

days of the study. No systemic or overt toxicity was noted in the Hershberger assay at dose up to 

10 mg/kg/day or in the uterotrophic 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 concentration (0.075 μg/L). This was observed with 

several females in the high treatment group exhibiting clinical signs of toxicity that included 

erratic swimming, lethargy, and loss of equilibrium. Additionally, there was a significant 

(p<0.05) increase in female body weight 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 neurotoxicity. Mortality did not occur during the 

subchronic studies in rats and dogs. In the subchronic 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 animals (15/sex) throughout the study at the high dose 

(15/17.2 mg/kg/day). In the subchronic dog study, tremors were noted in 3 dogs/sex at the 

medium-low dose (4.42 mg/kg/day) and in 4 dogs/sex in the medium-high and high-dose groups 

(8.84 and 17.7 mg/kg/day, respectively), with an indication of a cumulative effect in the two 

higher dose groups. Ataxia was noted in 4 dogs/sex at the medium-high and high-doses and in 

one female at the medium-low dose, and languidness occurred in medium-dose and high-dose 

animals. Other dose-related clinical signs included blinking, mydriasis, nystagmus, 

lacrimationand polypnea in the two highest dose groups. 

 

There was no mortality observed during 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 neurotoxicity, were observed at the high 

Page 28 of 67
BIFENTHRIN FINAL 

Page 21 of 59 

dose (15.5 mg/kg/day). These included tremors (22/25) from GD 9-20; hypersensitivity to sound 

(5/25) from GD 18-20; splayed hindlimbs (1/25) from GD 15-20; and piloerection (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); 19/20 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 females at the medium dose (7.2/16.2 mg/kg/day 

during gestation and lactation, respectively) beginning on lactation day (LD) 14 and in 23/25 

females in the high-dose group (9.0/20.7 mg/kg/day) beginning on LD 4. In the medium-dose 

group, the tremors were graded as slight, resolving in half (4/8) of the females after one 

occurrence and in the remaining females after 3-7 times. In the high-dose group, the tremors 

were graded slight to moderate and continued for 2-18 consecutive days during lactation. 

Piloerection also was observed once or twice in 6/25 females at the high dose, primarily during 

LD 14-17. During the Functional Operational Battery (FOB) evaluations, slight piloerection was 

observed in 4/25 high-dose females on GD 15 and in 2/25 and 1/25 females on LD 10 and 21, 

respectively. Clonic convulsions (limb tremors) and tremors 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 females, respectively, in the medium-dose group on LD 21. In two 

of 20 female pups in the high-dose group, tremors were observed during the physical 

examinations on PND 28. During the FOB, an increase 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 F1 generation in the multi-generation 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 clonic convulsions. The tremors occurred during Day 

9-35 after delivery in the P generation and during Day 3-35 after delivery in the F1 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. Individual 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 effects on survival; however, 

two females and two males in the high-dose group (97.2 and 81.3 mg/kg/day, respectively) and 

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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 clonic 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 developed 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 persistent effect from the previous day’s dose. 

There were no treatment-related mortalities or clinical signs of toxicity 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-estrogenic 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 female pubertal rat assay, there were no treatmentrelated estrogen-related effects observed in the absence 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 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 studies, the only effect observed was an increase 

in number of cracked eggs, but no differences in mean egg weight or egg shell 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 negative. In a non-Guideline Hershberger assay 

submitted as OSRI, while decreases in AST weights were reported, chemical purity and 

numerical AST data were not reported. No treatment-related effects were observed in the male 

pubertal rat study, in the absence of overt toxicity. No androgen specific effects were observed 

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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 interaction with the androgen pathway. 

D. Thyroid Pathway 

Based on mammalian Tier 1 assays and the OSRI studies, bifenthrin does not appear to affect the 

thyroid or the HPT axis. Developmental findings in the AMA included decreased developmental 

stage and normalized hind limb length at Day 21 (test termination), concurrent with effects on 

growth (decreased snout-vent length and body weight). Treatment-related differences in thyroid 

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 atrophy 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 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 histopathology. Serum thyroxine (T4) concentration was decreased only in the 

low-dose of both sexes (↓16%), with an increase in thyroid stimulating hormone (TSH) 

concentration occurring only in the high-dose males and which was associated with overt 

toxicity. Furthermore, there were no effects 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 potential interaction with 

the estrogen, androgen or thyroid pathways. 

 

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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 activity 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. Unpublished study performed by FMC 

Toxicology Laboratory, Somerville NJ. Study No. A83-977. January 31, 1986. (MRID 

00157225) 

Freeman, C., DeProspo, J.R., Nadasky, N., McConnell, R.F., Fletcher, M.J., Malloy, A.V., and 

Bullock, W.L. (1984) Teratology study in rabbits with FMC 54800 technical. Unpublished study 

performed by FMC Toxicology Laboratory, Somerville, N.J. A83-l092, Report dated February 

24, 1984. (MRID 00145997) 

Freeman, C., DeProspo, J.R., Nadasky, N., Fletcher, 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., and Liu, W. (2011b) Enantioselective endocrinedisrupting effects of bifenthrin on hormone 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 neurotoxicity study of bifenthrin technical in rats. 

Unpublished study performed by WIL Research Laboratories, LLC, Ashland, 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 Technical. Unpublished study performed by FMC 

Toxicology Laboratory, Somerville, N.J. FMC Study No. A83-818; January 31, 1984. (MRID 

00141199) 

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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 dietary inclusion of FMC 54800 on reproduction in the 

Bobwhite quail (Revised). Unpublished study performed by FMC Toxicology Laboratory, 

Somerville, N.J. FMC Study No. A84-1259. (MRID 00163097) 

Schneider, S.Z., T. Z. Kendall, and H.O. Krueger. (2012) Bifenthrin: Amphibian Metamorphosis 

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

International, Ltd., Easton, Maryland 21601. Laboratory project number 701A-106. Study 

sponsored by Consumer Specialty Products Association for the Bifenthrin Task Force Steering 

Committee/Joint Venture. Study completed 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 International, Ltd., Easton, Maryland, Report No.: 701A-105. Submitted 

by Consumer Specialty Products Association 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 America, 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 America, Inc. Study No. A83-821. June 17, 1985. MRID 

(00163065) 

USEPA (2010) Bifenthrin - Report of the Endocrine 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. Environmental Protection Agency, Washington, D.C. 

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

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

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

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

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

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Page 26 of 59 

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

Agency, Washington, DC. 

Wagner, H. (2011) Bifenthrin: H295R Steroidogenesis 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 Technical Prenatal Developmental Toxicity Study in 

Rats. Unpublished study performed by FMC Corporation 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 Steering 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., Kalamazoo, MI. Laboratory Study No.: 9146V100382ARB, November 17, 2011. (MRID 48615001) 

Willoughby, J.A. (2011) Bifenthrin: Estrogen Receptor Binding (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, Inc., Kalamazoo, MI. July19, 2012. (MRID 

48843501) 

Willoughby, J.A. (2011) Bifenthrin: Estrogen Receptor Transcriptional Activation Assay 

(Human Cell Line (HeLa-9903)). Unpublished study performed by CeeTox, Inc. Kalamazoo, MI. 

Laboratory Report No.: 9146V-100382ERTA, November 17, 2011. (MRID 48615004) 

Willoughby, J.A. (2012) Estrogen Receptor Transcriptional Activation (Human Cell Line (HeLa9903)) Proficiency Data Report. Unpublished study performed by CeeTox, Inc. Kalamazoo, MI. 

May 15, 2012. 

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Page 27 of 59 

Zhang, J., Zhu, W., Zheng, Y., Yand, J., and Zhu, 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) Integrative assessment of 

enantioselectivity in endocrine disruption and immunotoxicity of synthetic pyrethroids. Environ. 

Pollut. 158(5):1968-1973. 

Zorrilla, L. (2011) The Hershberger Bioassay for Bifenthrin (OPPTS Guideline 890.1400). 

Unpublished study performed by Integrated Laboratory Systems, Inc., Durham, NC. Project 

Study No. C196-200, November 18, 2011. (MRID 48615005) 

Zorrilla, L. (2011) The Uterotrophic Assay for Bifenthrin. Unpublished study performed by 

Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study No.: C196-100, 

November 14, 2011. (MRID 48615007) 

Zorrilla, L. (2012) Pubertal Development and 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) 

 

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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 flow-through conditions. Amphibian larvae at 

Nieuwkoop-Faber (NF) Stage 51 (80/treatment group, 20/replicate) were exposed 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) concentrations, calculated by the 

reviewer, were <0.0000150 (<LOQ; control), 0.0000133, 0.0000506, and 0.000235 mg a.i./L. 

The test system was maintained at 21.9 to 22.7oC and a pH of 7.9 to 8.1. 

According to the study author, “On Day 2 there was an unusual mortality event in replicate A of 

the negative control. By Day 3, there were 7 mortalities in the replicate. However there were no 

mortalities in the remaining replicates of the negative control or the solvent control.” One of the 

AMA validity criterion states: “For any given treatment (including controls), mortality should 

not exceed 10%. For any given replicate, mortality should not exceed three tadpoles, otherwise 

the replicate may be considered compromised.” Accordingly, replicate A of the negative control 

was considered to be compromised and was excluded 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 negative control (excluding replicate 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 replicate A; 92.3% with replicate A), 100% in the solvent 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 tadpoles, respectively, were incidentally killed 

during siphoning and were not considered in survival calculations. 

At test termination, there was no treatment-related effect on the incidence of tail curvature, 

which was present in 63, 59, 78, 65 and 63% of tadpoles in the negative 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 increased 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 solvent control. Therefore, the treatment groups 

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were compared separately to the negative and solvent controls for all growth and developmental 

parameters. 

Day 21 body weights were significantly increased (p<0.05) by 24.4, 21.0, and 14.2% in the low, 

mid, and high treatment groups, respectively, relative to the negative control. However, at the 

mid and high treatment concentrations, significant 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 decreased (p<0.05) by 16.8 and 4.4%, respectively, at 

the low treatment concentration relative to the solvent control. 

There were no significant differences 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 significantly delayed (p<0.05) median NF developmental 

stage at 21 days in the high treatment group (median 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 high treatment group relative to the solvent 

control. Asynchronous development (stages 54 and 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 control (4/20). Thyroid gland hypertrophy and follicle 

size increase were observed in the negative and solvent controls as well as in almost all of the 

treatment groups. Increases or decreases in thyroid gland and follicle cell asymmetry were 

observed sporadically in all control and treated groups. 

There were significant differences between the negative and solvent control groups in growth 

parameters, which showed a consistent pattern of promotion in the solvent control relative to the 

negative control. Additionally, there was unacceptably 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; therefore, the excessive 

mortality in the one negative control replicate had less of an impact on the results. 

Androgen Receptor (AR) Binding 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). 

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Dimethyl sulfoxide (DMSO) was used as the solvent at a final assay concentration of 

approximately 3.2%. A total of three independent runs were performed, and each run included 

dexamethasone as a weak positive control, and R1881 as the ligand reference standard. 

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

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

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 preparation made on March 30, 2011, the mean dissociation 

constant (Kd) for [3H]-R1881 was 0.614±0.065 nM, and the estimated Bmax was 1.080±0.109 

fmol/100 μg protein. For the cytosolic preparation made on July 23, 2011, mean Kd for 

[

3

H]-R1881 was 1.080 nM (±0.690), and the estimated Bmax was 1.071 fmol/100 μg protein 

(±0.167). For the cytosolic preparation made on September 24, 2011, the mean Kd for [3

H]-

R1881 was 0.613 nM (±0.041), and the estimated Bmax was 0.817 fmol/100 μg protein (±0.049). 

The mean and individual Kd 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 (R2 = 0.880-0.992) and the small variation in R2 and Kd estimates among runs 

(although greater variability was observed in the July 23, 2011 saturation binding data). 

In the competitive binding experiment, precipitation 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 bifenthrin concentrations of ≤10−5

M, classifying bifenthrin as a “non-binder”. In Run 2, specific binding of [3H]-R1881 was 

74.9% at a concentration of 10−4

 M, classifying bifenthrin 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 IC50 and relative binding affinity (RBA) could 

not be calculated. 

The estimated mean log IC50s 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. 

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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; [3H]ASDN) in ≤1% dimethyl sulfoxide (DMSO) at final log 

concentrations of 10−10 to 10−3 M for 15 minutes to assess the potential 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 concentration of test chemical. Tritiated water was 

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

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

10−5

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

chemical series (10−10 to 10−3

 M) with three repetitions per concentration. Initially, 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 were not used by the study author in data 

interpretation. Subsequently, the highest test concentration used for study interpretation by the 

study author was 10-4.5 M, as this concentration did not result in precipitation. 

Aromatase activity in the full 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 activity. 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 IC50 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, bifenthrin is classified as a Non-inhibitor of 

aromatase activity in this assay. 

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

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

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binding experiment was conducted to measure the binding of a single concentration of [3H]-17βestradiol (1 nM) in the presence of increasing concentrations (10−11 to 10−3

 M) of bifenthrin 

(93.6% purity, Batch #PL09-0251). DMSO was used 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, octyltriethoxysilane as a negative control, and 17βestradiol as the natural ligand reference material. 

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

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

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

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

(160 to 320 μg versus 50±10 μg). The Kd for [3H]-17β-estradiol was 0.331±0.061 nM and the 

estimated Bmax was 74.55±3.03 fmol/100 μg protein for the prepared rat uterine cytosol. The Kd

for each run was within the expected Guideline range of 0.03 to 1.5 nM. 

In the competitive binding experiment, precipitation 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 concentration evaluated for 

binding was 10-5 M. The estimated mean log IC50 for 17β-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 performance criteria were generally met for 17β-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 standard (95.1-107.4%). The lowest average percent radiolabeled 

estradiol binding in the presence of bifenthrin 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. 

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

In an estrogen receptor transcriptional activation assay (MRID 48615004), hERα-HeLa-9903 

cells cultured in vitro were exposed to bifenthrin (93.6% a.i., Lot #PL09-0251) 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 performed 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 reporter (luciferase) gene products. Luciferase expression in 

response to activation of the estrogen receptor by bifenthrin was measured using a proprietary 

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luciferase assay. Bifenthrin was tested up to the 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 the cells to the very weak positive control 17αmethyltestosterone was lower than the expected values, indicating a decreased sensitivity of the 

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

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

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

reach a maximum of 8.1-52.8% PC. Bifenthrin was only able to reach a maximum of 3.8-5.0% 

PC when tested up to the highest concentration possible based on cytotoxicity (10-4 M). Because 

the RPCMax < PC10 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 (MRID 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 25.5oC 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. Clinical signs of toxicity in the 

high-concentration group were noted beginning on Day 5 and included erratic swimming, 

lethargy, loss of equilibrium, surfacing, weak behavior 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/replicate and fertilization success averaged 98.3%. 

There were statistically significant effects (p<0.05) on fecundity in females, with a 60% increase 

at the mid concentration and a 74% reduction at the high concentration, compared to the negative 

control. These effects were observed in concentrations with 88 and 0% male survival, 

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respectively. There were two control replicates (Replicates A and D) that were observed to have 

a period of at least 5 days in which no eggs were 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 stated previously, the mean control fecundity was 

16.1 eggs/female/day. There were no statistically 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 statistically significant differences 

were noted for the other developmental endpoints including male GSI and male tubercle score 

(p>0.05). Additionally, there were no significant effects (p>0.05) on male and female 

vitellogenin (VTG) levels. Female body weight was significantly increased 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 male-specific endpoints at the highest treatment 

concentration were not available due to total mortality at that concentration. 

In males, there were no statistically significant 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; Jonckheere-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 follicles and granulomatous inflammation, almost all of which was due to 

the increased oocyte atresia. There were no other statistically significant differences between 

severity scores or gonadal stages in treated groups compared to the 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 concentration, 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) screening for androgenic activity, bifenthrin (93.6% 

purity; Lot # PL09-0251) in corn oil was administered daily via oral gavage to groups of eight 

59- or 60-day old, castrated male Sprague Dawley (SD) rats at dose levels of 0 (vehicle), 3.2, or 

10 mg/kg/day. An androgenic positive control group consisted of eight 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-androgenic 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 at 

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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-androgenic 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 daily. 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 any of the groups. Measurements 

of food consumption, serum hormone concentrations, and liver, kidney, and adrenal weights are 

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

In both the androgenic and anti-androgenic assays, no gross effect was reported on the sexrelated tissues. No treatment-related effect was observed on mortality, clinical signs, or sex 

organ weights (seminal vesicles, ventral prostate, LABC, Cowper’s glands, 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 statistically 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 observed 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 groups compared to the negative control. No 

treatment-related effects were observed in any of the accessory sex organs of animals dosed with 

bifenthrin + TP. The FT positive control group had 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 levels were selected based on the 

results of a range-finding study in which SD rats were administered bifenthrin 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 female (9 of 16 rats) rats in the pubertal assays. 

Therefore, the high dose tested is determined to be adequate for this study. 

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

tissues. Bifenthrin was negative for androgenicity 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/kg) 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 vaginal opening (VO), and age and weight at day 

of attainment was recorded. After completion of VO, the status of each animal’s estrous cycle 

was determined daily. Animals were sacrificed on PND 42 or 43 and blood was collected for 

analysis of clinical chemistry. Total serum thyroxine (T4) and thyroid stimulating hormone 

(TSH) concentrations were also determined by radioimmunoassay. Liver, kidneys, thyroid, 

pituitary, adrenals, and urogenital organs were collected and weighed, and microscopic 

examinations were conducted on the ovaries, uterus, thyroid, and kidneys. 

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

the 24-hour post-dose observation period. However, during clinical observations at 6 hours 

post-dose, 9/16 animals in the 10 mg/kg/day group 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 treatment on any organ weights in this study. There were no significant 

differences in age at first estrus in either treatment group relative to controls. There were no 

effects of treatment on cycle 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 increased (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 significant changes in 

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clinical chemistry parameters. No treatment related histopathological changes 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 adverse clinical signs and 

mortality, and clinical signs including tremors in this assay. 

Male Pubertal Assay; (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 attainment was recorded. Following sacrifice on PND 53 

or 54, blood was collected for analysis of clinical chemistry, and serum testosterone, thyroxine 

(T4), and thyroid stimulating hormone (TSH) levels were analyzed using radiometric assays. 

Liver, kidneys, thyroid, pituitary, adrenals, and urogenital organs were collected and weighed, 

and microscopic examinations were conducted on the testes, epididymides, thyroid, and kidneys. 

All animals survived until study termination. No clinical signs of toxicity were observed at the 

24-hour post-dose observation period. During clinical 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 all other days of the study. 

No significant changes were observed in body weights at PPS or final body weights in the 2.5 or 

10 mg/kg/day groups. The mean age at PPS was 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 and adjusted pituitary weights 

(↑11% each); relative weights were not significantly different. The increase in pituitary weight 

can be explained by the slight increase in terminal body weights (↑4%) as reflected in the lack of 

significance in relative pituitary weights. Although androgen-sensitive tissue weights in treated 

groups were not statistically different from controls 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 serum testosterone levels. Serum T4 was significantly decreased 

(p<0.05) by 16% at 2.5 mg/kg/day, but was not different from control at 10 mg/kg/day. Serum 

TSH was significantly increased (p<0.05) by 51% at 10 mg/kg/day. 

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At 10 mg/kg/day, increases (p<0.05) in sodium concentration (↑2%) and sorbitol dehydrogenase 

(↑38%) were observed. No other changes in clinical chemistry parameters were observed. 

Bifenthrin appeared to be associated 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 clinical signs (both sexes) and mortality (females), the high dose 

of 10 mg/kg/day was adequate. 

Steroidogenesis Assay; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 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 triplicate for four independent experiments. 

Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.05%. 

Precipitates were observed under the microscope after the exposure period at 10 μM in Run 2 

and at 100 μM in all runs. Cytotoxicity greater than 20% was not observed. Run 1 was not 

analyzed because one of the forskolin concentrations on the quality control plate was incorrect 

(3.33 μM was used instead of 1 μM). 

Testosterone and estradiol levels were measured using HPLC-MS/MS. A Quality Control (QC) 

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

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

controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol and 

testosterone production. 

Guideline acceptability recommendations and requirements were met, including adequate 

production of testosterone and estradiol, acceptable 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 bifenthrin in Run 3 (1.39-fold). Hormone 

concentrations at 10 μM bifenthrin were similar to control in Runs 3 and 4, but were not 

evaluated in Run 2 due to precipitation. These differences were not considered related to 

treatment because the effect was not concentration-dependent and/or not reproducible between 

runs. 

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

statistically significant and reproducible alterations in testosterone or estradiol production. 

Uterotrophic Assay; (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48615007), bifenthrin (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 treated with a daily dose of 17α-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 dose administration to 

determine wet and blotted 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 the uterus. Uterine weights in the bifenthrin 

treated groups were comparable to the vehicle 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 study (15 mg/kg/day) is adequate based on data from 

a dose range-finding study showing 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. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

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

potential interaction of bifenthrin with the endocrine system are summarized below. Given the 

breadth of toxicity data available on bifenthrin, the only Part 158 studies discussed in this 

appendix are those that fulfill current guideline requirements and are directly relevant to 

assessing potential endocrine effects (i.e., multiple-generation rodent reproductive studies, 

development studies, chronic toxicity studies, etc.). 

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 line, stably-transfected with 

an ER luciferase reporter plasmid] was used to assess the estrogenic and 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 used to determine the concentrationdependent agonist (test material) and antagonist (test material in the presence of estradiol) effects 

of bifenthrin. Bifenthrin was tested at final concentrations of 1, 10, 100, 1000, and 10,000 ng/L 

(0.0024, 0.024, 0.24, 2.4 and 24 nM); the concentration of 17β-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 concentrations tested. In the 

ER antagonism assay, an initial bifenthrin concentration-dependent decrease in the ability of 

estradiol to induce ER-dependent 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 results demonstrate that bifenthrin acts as an 

estrogen antagonist in this in vitro assay. The Good Laboratory Practices (GLP) status of this 

research is unknown. 

In a non-Guideline in vitro cell proliferation assay [estrogen-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 standard of racemic bifenthrin (99.5% purity) was 

separated by HPLC with a variable wavelength circular dichroism detector; purity of the isolated 

enantiomers was assessed by GC-ECD. MCF-7 cell proliferation was determined for the isolated 

bifenthrin enantiomers against 17β-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 the bifenthrin enantiomers (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, 

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respectively (2.1-fold difference). In comparison with the PE of 17β-estradiol synchronously 

(4.49), the relative proliferative effect (RPE) was determined 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 17β-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 greater than that of 1R-cis-bifenthrin. The 

proliferation induced by bifenthrin enantiomers was completely blocked by 10-6 mol/L of ICI 

182,780, supporting a direct binding effect of bifenthrin enantiomers 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 17β-estradiol and a vehicle control of 0.1% v/v ethanol. In addition, 

estrogen response gene expression (pS2 and ERα) were determined with qRT-PCR. 

The proliferative response to the cis-bifenthrin enantiomers was consistently greater for 1S-cisbifenthrin relative to 1R-cis-bifenthrin at every tested concentration. At a concentration of 

10−8

 mol/L, 1S-cis-bifenthrin exhibited estrogenic activity of approximately 2.2-fold greater than 

1R-cis-bifenthrin (and the 1S-enantiomer response was approximately equivalent to the 17βestradiol 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, respectively. Down-regulation of ERα 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-PCR assays showed that in the cis-bifenthrin enantiomer pair, 

the 1S-enantiomer consistently showed 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-androgenic 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 (bifenthrin not included) and anti-androgenic 

activities of cyfluthrin, β-cyfluthrin, and four structurally-related pyrethroids (permethrin, 

cypermethrin, β-cypermethrin, and bifenthrin). All test substances were administered in peanut 

oil. 

Three-week old male SD rats were maintained under standard laboratory conditions and 

provided with standard laboratory diet and water ad libitum. After an acclimation period of 

seven days, the rats were castrated on PND 28 and allowed to recover for two weeks prior to 

initiation of dosing on PND 42. After the recovery period, rats were weighed and randomly 

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assigned to dose groups (n=6). The androgenic portion of the Hershberger assay was conducted 

with a sham-operated control group, but this assessment did not include bifenthrin. The antiandrogenic portion of the Hershberger assay included eight treatment groups: a vehicle control 

group (peanut oil only); a positive control group dosed orally with 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 final dose, rats were anaesthetized and sacrificed 

by exsanguination. Blood from each rat was collected for serum analysis of testosterone 

concentration. The seminal vesicles, ventral prostate, dorsolateral prostate, LABC, Cowper’s 

glands, glans penis, liver, paired adrenal glands, spleen, and paired kidneys of each rat were 

dissected and weighed. Serum testosterone concentrations were analyzed by time-resolved 

fluorescence with commercially available FIA kits. 

No mortality or treatment-related clinical signs 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 compared to the control groups, 

but the actual data for the bifenthrin treated group were not presented. Serum testosterone 

concentrations were not shown for the bifenthrin-treated rats, but it was reported that no 

treatment-related effects were observed. Treatment with bifenthrin at 13.5 mg/kg/day decreased 

(p<0.05) absolute weights of all the androgen-sensitive accessory sex tissues compared to the 

castrated control + TP. However, the exact percentage decreases, relative to control, could not 

be ascertained as the data were only reported graphically. The weights of all accessory sex 

tissues for the castrated control + TP were similar 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 tissue weights compared to bifenthrin. The authors 

concluded that bifenthrin 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 rats 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 investigated only the anti-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 (castrated + TP) exhibited 

similar growth of accessory sex tissues to the sham-operated group (uncastrated), the 0.5 mg/kg 

dose of TP was acceptable. The study is considered acceptable for use in a WoE evaluation. We 

note that the dose utilized in the published study (13.5 mg/kg/day) was slightly above the 

maximum dose utilized for the Guideline Hershberger assay (10 mg/kg/day). 

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Subchronic Oral Toxicity in Rodents (Rat; OCSPP 870.3050) 

In an oral toxicity study in rats (MRID 00141199), bifenthrin (91.4% purity) 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 administered the diet containing bifenthrin at 0 or 

200 ppm for 90 days, and then fed a recovery (control) diet for an additional 28 days. After 90 

days, all rats were weighed, and sacrificed for gross and histopathological evaluation. Organs 

that were weighed included the liver, kidneys, 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 the study at the medium-high dose, and in all animals (15/sex) 

throughout the study at the high dose. Tremors were noted to be reversible within three days 

after discontinuation of dosing in the high-dose recovery group. Males and females in the highdose group had decreased mean body 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 decreased (NS) at the high dose for females (↓6.0%) 

relative to control. High-dose females had decreased food consumption after Week 4, but this 

was only significant during Week 9; no differences in food consumption were observed for 

males. There were no treatment-related organ weight or histopathological 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% purity) was administered 

by gelatin capsule to Beagle dogs (4/sex/dose) 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 based on purity) for 13 weeks. Organs that 

were weighed included the liver, kidneys, testes/epididymides, ovaries, and thyroid/parathyroids. 

Gross and microscopic pathological examinations 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 and 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 occurred primarily at the high dose in both sexes, 

but also occasionally at the medium-high dose. All of these symptoms occurred more frequently 

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during the last three weeks of the study. Other dose-related clinical signs included blinking, 

mydriasis, nystagmus, lacrimation, and polypnea 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-low 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 decreased (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/kg/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 control) 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 alterations. 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 characterized 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 consumption 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 was noted at the high dose and was characterized as an increased 

fetal and litter incidence (NS) of hydroureter without 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 viability. The positive control gave 

the appropriate responses of increased early resorptions, depressed fetal body weights, external, 

visceral, and skeletal malformations and variations. 

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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. Clinical 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 piloerection (1/25), observed at GD 19. None of these findings was 

observed in any control animal. 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 the 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 interval (GD 6-20). No 

treatment-related changes were observed in gross pathology, the 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 92-100% 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 signs 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-related developmental findings were 

noted at any dose tested. The developmental toxicity LOAEL was not observed, and the 

developmental toxicity NOAEL is 200 ppm. 

Prenatal Development in Non-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 gavage 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/day 

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6-aminonicotinamide (positive control) in 2% carboxymethylcellulose, on GD 7 to 19, inclusive. 

Mating was conducted with male New Zealand white rabbits, and confirmation of mating 

defined GD 0. On GD 29, all surviving 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 necropsy had lung damage suggestive of P. multocida infection. 

Treatment-related clinical signs of toxicity consisted of head and forelimb twitching (medium 

and high dose) and tremors (high dose); 19/20 animals in the high-dose group displayed at least 

one of these clinical signs. It was reported that body weight means were different (p<0.05) over 

the GD 21-28 interval and for the overall GD0-29 interval, but the only apparent 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/day based clinical signs (head and 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 displayed a lower incidence of minor variations 

compared to control. Therefore, the developmental toxicity 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®(SD) 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, respectively, 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 closely as possible). Offspring were 

allocated for detailed clinical observations (FOB) and assessment of motor activity, auditory 

startle reflex habituation, learning and memory (water maze testing), and neuropathology at 

termination (PND 72). On PND 21, the whole brain was collected from 10 pups/sex/group for 

micropathologic examination and morphometric analysis. Pup physical development was 

evaluated by body weight, and the age of sexual maturation (VO in females and PPS in males) 

was assessed. 

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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 observed in the remaining 

females 3-7 times. In the high-dose group, the tremors 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 high dose, primarily during LD 14-17. During the 

FOB, the mean number of grooming counts was increased (p<0.01) in females at the medium 

and high doses during gestation and lactation. In addition, slight piloerection was observed in 

4/25 high-dose females on GD 15 and in 2/25 and 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, respectively. Clonic convulsions (limb tremors) and 

tremors were noted (NS) in 2/23 and 3/23 females 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/day during gestation and 

16.2 mg/kg/day during lactation) based on clinical signs of neurotoxicity (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, percentage of live-born and stillborn pups, and sex 

ratio were not affected by treatment. There was no treatment-related effect on offspring body 

weight or body weight gain; decreased body weights (↓6-9%) and body weight gains (↓16-17%) 

in the medium-dose group males and females was minimal and not dose dependent. The mean 

day for reaching sexual maturation (VO in females and PPS in males) was not affected by 

treatment. Two of 20 females in the high-dose group had tremors during the physical 

examinations on PND 28. During the FOB, an increase 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 medium and high doses on PND 21. No treatmentrelated effects on motor activity, acoustic startle response, or learning and memory testing were 

observed. Brain weight, length, and width and macroscopic findings were not affected by 

treatment. 

The offspring LOAEL for bifenthrin in rats is 100 ppm (7.2 mg/kg/day during gestation and 

16.2 mg/kg/day during lactation; maternal dose) based on clinical 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 lactation; maternal dose). 

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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 was adjusted to account for purity). For the P 

generation, dosing began 8 weeks prior to mating and for the F1 generation, 11 weeks prior to 

mating. P generation animals were bred twice, and F1 parental animals were selected from the 

F1b pups. 

There was no treatment-related mortality in either the P or F1 generation. Tremors were noted 

only in females of both generations at the high dose 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 F1 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 lactation), lower body weights (↓5-6%, p<0.05 or 0.01) during the first lactation 

period (Day 7 and 14); and lower body weights during 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 the 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 differences in mean body weights between treated F1

animals and control. There were generally no treatment-related differences in food consumption 

in any parental group. In the high-dose P generation females, there was an increase (p<0.01) in 

absolute brain weight (↑5%). Mean absolute ovary weights were slightly decreased (↓9%, 

p<0.05 or ↓12%, p<0.01) at the medium and high doses, respectively, in the F1 parental 

generation. In the high-dose group F1b female progeny, absolute adrenal 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-dependent 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 fertility, and gestation indices), and there were no 

treatment-related gross or microscopic findings in either sex in either the P or F1 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 F1 generation females during gestation and 

lactation. The systemic NOAEL is 60 ppm (approximately 3.0 mg/kg/day). The reproductive 

NOAEL is 100 ppm (HDT); a reproductive LOAEL is not observed. 

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Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat; OCSPP 870.4300) 

In a chronic toxicity/carcinogenicity 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-measured 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, pituitary, thyroid/parathyroids, adrenals, mammary 

gland, testes, epididymides, prostate, seminal 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 females 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 exhibited tremors during Days 3-5 similar to the highdose animals. Other clinical signs in the high-dose males and females included increased 

incidences of abrasion, alopecia, and tail laceration. 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 decreased 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 treatment-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 increases in these organ weights were also observed in the lowdose males. There were no differences in brain or gonadal weights, and no treatment-related 

findings in gross pathology or non-neoplastic changes. The incidences of pancreatic islet cell 

adenoma and fibrosarcoma in high-dose males were increased (NS; 3/50 vs. 1/47 control and 

3/50 vs 0/50 control, respectively), and historical control 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 conditions 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, Tac(SW)fBR mice (50/sex/dose) at doses (adjusted 

for purity) of 0, 50, 200, 500, or 600 ppm (mean-measure 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). 

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There were no treatment-related effects on survival; however, two females and two males in the 

high-dose group and one female in the medium-dose 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 were also noted in two 

males and two females at the medium-low dose and in one male at the low dose. However, 

tremors in the low-dose male occurred late in the 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 trend (p=0.054) for hemangiopericytomas (originally 

reported as leiomyosarcomas) of the urinary bladder (15, 12, 17, 16, and 31%, respectively). 

Combined bronchioalveolar adenomas and adenocarcinomas were increased in treated females, 

with increases (p<0.05) at the low, medium-high, and high doses. Males had a dose-related 

increasing trend (p=0.022) in combined hepatocellular 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 chronic NOAEL is 50 ppm (6.7 mg/kg/day in males; 

8.8 mg/kg/day in females). Carcinogenic potential 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/parathyroid; 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 treatment-related effects on food 

consumption, organ weights, and gross or microscopic 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 from 

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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 treatment-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 sexes; the NOAEL is 1.3 mg/kg/day. 

Mechanistic Studies: Ovarian Gene Expression and Hormone Synthesis (Rat) 

In a published non-Guideline study (Liu et al., 2011), the concentration-response of ovulatory 

gene expression and prostaglandin synthesis in rat ovarian granulosa cells after exposure to 

bifenthrin (99.5% purity) was evaluated in vivo and in vitro. For the in vivo study, female SD 

rats were administered bifenthrin 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 were injected with pregnant 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 administration. PMSG and hCG are used to mimic the 

effects of FSH and LH, respectively, in this model. Control animals were sacrificed 48 hours 

after PMSG administration for isolation of pre-ovulatory granulosa cells, and treated animals 

were sacrificed at 6 hours post-hCG administration for isolation of peri-ovulatory granulosa 

cells. The pre-ovulatory granulosa cells (isolated from vehicle control animals) were prepared 

for cell culture. Cultured cells were pretreated with 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. Concentrations 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 prostaglandin. 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 cultured granulosa cells were decreased in the 

presence of bifenthrin in either 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 minimal 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 ovulatory granulosa cells in a non-dose-dependent 

manner. 

In summary, low-dose exposure to bifenthrin at 1 nM inhibited the hCG-induced in vitro 

expression of most of the ovulatory genes that were examined, and a moderate concentration of 

100 nM attenuated hCG-stimulated expression of all tested genes. Furthermore, in vivo studies 

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demonstrated a trend of inhibitory action due to bifenthrin on LH/hCG-stimulated rat ovulatory 

gene expression. These observations support the disrupting effects of bifenthrin on the network 

of ovulatory gene expression patterns, with potential targets at protein kinase A-related signaling, 

gene transcription, and/or ovulatory gene expression. The GLP status 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 racemic 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 cells were pretreated with individual bifenthrin 

enantiomers (in EtOH at a final concentration 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. Concentrations of mRNA 

were quantitated in ovarian granulosa cells after in vitro exposure to bifenthrin enantiomers with 

a qRT-PCR method. Concentrations of progesterone and PGE2 in cell culture media (after in 

vitro incubation) were determined 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 concentration approximately 8-fold (65.1±4.2ng/mL). 

Exposure to 1S-cis-bifenthrin decreased (p<0.05) hCG-induced 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 enzyme (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 treated with hCG alone. Treatment with 1R-cis-bifenthrin had 

no effect on mRNA expression levels of these genes. 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-stimulated PGE2 production to 93.3±7.6 pg/mL, 

but 1R-cis-BF did not affect PGE2 production. Cyclooxygenase-2 (COX-2), the presumed ratelimiting step in the synthesis of prostaglandins, was also examined. The 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. 

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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 employed in this study: 7 hours/day until Week 8 

and 16 hours/day until the end of the study (Day 154). Temperature 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 periods of the adult phase. 

Individual chick body weights were 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 observed 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 consumption, or body weight. 

There were no effects of treatment on the proportion 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/fertile eggs did not differ between treatment and 

control. There were also no differences between the percentages of hatched 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 noted in chicks that died on study. 

The NOAEC for mallard ducks exposed to bifenthrin 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 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 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 pairs, 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 weights 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 periods of the adult phase. Individual chick body 

weights were recorded within 24 hours of hatching 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). 

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Adult birds and chicks were observed daily for mortality 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 spare 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 mortality; there were 3, 3, 4, and 1 deaths during the 

study in the control, low-, medium-, and high-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 high-dose groups, but no differences in mean egg 

weight or egg shell thickness. Percentages of infertile eggs/eggs set, early embryonic 

deaths/fertile eggs, and late embryonic deaths/fertile eggs did not differ between treatment and 

control. There were also no differences between the percentages of hatched 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 noted 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 bifenthrin (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 endocrine disruption selected was expression of 

choriogenin (Chg), an estrogen-dependent egg coat protein, which was assessed with an indirect 

enzyme-linked immunosorbent assay (ELISA) relative to a positive control (ethinylestradiol). 

Fish were exposed to bifenthrin (1, 10, or 100 ng/L) or ethinylestradiol (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 ice), frozen, and homogenated in buffer. The 

resultant supernatant after centrifugation 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 different from one another when compared with ANOVA 

(p>0.05); however, a trend toward an inverse correlation between response and increasing 

concentration with bifenthrin is apparent. Bifenthrin treatments were not significantly different 

from positive control concentrations of 1 and 10 ng/L, with the exception of 1 ng/L bifenthrin, 

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which had a higher (p=0.022) relative Chg concentration compared to 1 ng/L ethinylestradiol. 

However, all bifenthrin concentrations trended toward a higher relative expression of Chg 

compared to the 1 ng/L ethinylestradiol treatment and a lower relative expression than the 

10 ng/L ethinylestradiol treatment. The ethinylestradiol response was 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 expression 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. 

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APPENDIX 3: References Not Utilized in the Bifenthrin WoE Analysis 

In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing 

Test Orders for the EDSP Tier 1 screening battery was made available in a Federal Register 

Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted 

as “other scientifically relevant information (OSRI) was provided in the Report of the Endocrine 

Disruptor Review Team (USEPA, 2010). 

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies 

previously submitted to the agency for registration/reregistration, published literature articles 

and/or Tier 1 assays. The agency also conducted a more recent search (2009 to 2014) of 

available scientific literature for any additional relevant information for their weight of evidence 

(WoE) evaluations. These articles were evaluated in accordance with the agencies Evaluation 

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

(http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species

_reregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance 

for considering and Using Open Literature Toxicity Studies to Support Human Health Risk 

Assessment (http://www.epa.gov/pesticides/science/lit-studies.pdf). 

The following published and unpublished references were considered for use in the WoE 

analysis for Bifenthrin but were not utilized due to one or more of the following reasons: 1) the 

article was not available in English; 2) the compound of interest was not used in the study; 3) the 

test material was not adequately described; 4) a formulated end-use product or mixture of 

chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 

6)the experimental conditions were not adequately described; 7) only an abstract of the study 

was available; 8) the reference is a review article or book chapter and does not contain primary 

study data; 9) insufficient information was available to adequately assess the validity of the study 

results; 10) the 40 CFR Part 158 guideline study was classified as unacceptable/inadequate; 11) 

the study dealt only with non-EDSP assay development; 12) no specific endocrine-related 

endpoints were assessed in the study; and 13) the study contained only data on invertebrates. 

Abbott, D.H., Padmanabhan, V., and Dumesic, D.A. (2006) Contributions 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-Generation Rat Reproduction 

Study with Benefin. (Unpublished study received 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 Shahab, M. (1996) Insecticide-induced changes in 

secretory activity of the thyroid gland in rats. J. Appl. Toxicol. 16(5):397-400. 

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Bowman, C.J. (2005) Validation of the Pubertal 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-Chemistry 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., Crissman, 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 Column Tests with the Crustaceans Hyalella azteca (Order: 

Amphipoda) and Ceriodaphnia dubia (Order: Cladocera) in Detecting 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 sexual behavior and physiology 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. (Unpublished 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., Rosenthal, 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., Zhang, Y., and Zhao, M. (2009) Developmental 

toxicity of bifenthrin in embryo-larval stages of zebrafish. Aquat. Toxicol. 13;95(4):347-54. 

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Liu, J., Yang, Y., Yang, Y., Zhang, Y., and Liu, W. (2011a) Disrupting effects 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 inhibits prostate growth and hormone feedback. Biol Reprod

70:1188-1195. 

McAllister, W.A. (1988). Full life cycle toxicity 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. Laboratory Project No. A01090. Prepared by the 

Toxicology Research Laboratories, Lilly Research Laboratories, Greenfield, 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. Laboratory Project No. A00690. Prepared by the 

Toxicology Research Laboratories, Lilly Research Laboratories, Greenfield, 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 Kortenkamp, 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-induced 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 ethinyl estradiol induces cleft phallus and delayed 

ovarian dysfunction in the offspring. Toxicol Sci 75:402-411. 

Schlenk, D., Lavado, R., Loyo-Rosales, J.E., Jones, W., Maryoung, L., Riar, N., Werner, I., and 

Sedlak, D. (2012) Reconstitution studies of pesticides and surfactants exploring the cause of 

estrogenic activity observed in surface waters 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 flutamide on puberty in male rats: An evaluation of the 

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protocol for the assessment of pubertal development and thyroid function. Toxicol Environ 

Health, PtA 65(5-6):433-445. 

Stoker, T., Parks, L.G., Gray, L.E., Cooper, R.L. (2000) Endocrine-Disrupting Chemicals: 

Prepubertal Exposures and Effects on Sexual Maturation and Thyroid Function in the Male Rat. 

A Focus on the EDSTAC Recommendations. Critical Reviews in Toxicology, Jan 2000, Vol. 30, 

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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 17β-Estradiol. Journal of Toxicology & Environmental Health 73(16) 

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Terry, K.K., Cappon, G.D., Hurtt, M.E., Tassinari, M.S., and Gupta, U. (2004) Reproductive 

toxicity assessment of lasofoxifene, 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) Disturbed 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 

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Wang, L., Liu, W., Yang, C., Pan, Z., Gan, J., Xu, C., Zhao, M., and Schlenk, D. (2007) 

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Worth, H.M., Pierce, E.C., Harris, P.N., et al. (1966) Subacute Toxicity of Benefin to Rats. 

(Unpublished study received Mar 15, 1966 under unknown admin. no.; submitted by Elanco 

Products Co., Div. of Eli Lilly and Co., Indianapolis, Ind.; CDL: 107494-A). (MRID 00024651) 

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Zhao, M., Chen, F., Wang, C., Zhang, Q., Gan, J., and Liu, W. (2010). Integrative assessment of 

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