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 75
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 

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EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: PERMETHRIN 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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PERMETHRIN FINAL 

ii

TABLE OF CONTENTS 

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

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

I. Introduction.......................................................................................4 

II. Sources of Scientific Data and Technical Information......................................5 

A. EDSP Tier 1 Screening Assays............................................................5 

B. Other Scientifically Relevant Information (OSRI)......................................5 

III. Weight of Evidence (WoE) Evaluation.......................................................5 

A. EDSP Tier 1 Screening Assays.............................................................8 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9 

1. Effects on Estrogen Pathway.....................................................9 

2. Effects on Androgen Pathway..................................................15 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................20 

IV. Committee’s Assessment of Weight of Evidence..........................................22 

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................22 

1. Tier 1 in vivo Assays.............................................................23 

2. OSRI...............................................................................23 

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

C. Androgen Pathway.........................................................................26 

D. Thyroid Pathway...........................................................................26 

E. Conclusions.................................................................................27 

V. EDSP Tier 2 Testing Recommendations....................................................27 

VI. References.......................................................................................28 

LIST OF TABLES: 

 Table 1. Tier 1 Assays for Permethrin.................................................................8 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Permethrin..................................12 

 Table 3. Androgenic/Anti-Androgenic Pathway for Permethrin.................................17 

 Table 4. Thyroid Pathway for Permethrin..........................................................21 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................32 

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

APPENDIX 3. References Not Utilized in the Permethrin WoE Analysis............................65 

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iii

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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iv

Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

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v

Abbreviation Terminology 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

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Abbreviation Terminology 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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Page 1 of 67

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 hypothalamicpituitary thyroid (HPT) axes. In addition to the available Tier 1 assay data, other 

scientifically relevant information (OSRI), including general toxicity data and open 

literature studies of sufficient quality were considered in this weight of evidence (WoE) 

assessment. 

In determining whether permethrin interacts with E, A or T hormone pathways, the 

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

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

effects occur were considered, in particular, whether or not endocrine-related responses 

occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity. 

On July 23, 2014, the EDS{ 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 

permethrin 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).

For the estrogen pathway, a number of OSRI in vitro assays evaluating estrogen receptor 

activation resulted in conflicting results, i.e. some showed activation of estrogen signaling while 

others did not. The OSRI assays included estrogen receptor (ER) binding, cellular proliferation 

of estrogen sensitive MCF-7 cells, expression of estrogen sensitive pS2 mRNA in the cancer 

cells, and interaction with either ERα or ERβ in transfered HeLa cells. Permethrin was negative 

in the Tier I in vitro aromatase assay, whereas in the steroidogenesis assay permethrin caused an 

increase (p<0.05) in estradiol production. In vivo, permethrin was negative in both the 

uterotrophic and female pubertal rat assays, with no effects on uterine weight, mean age at 

vaginal opening, percent cycling, or histopathological changes in the ovaries and uterus. In the 

fish short-term reproduction assay (FSTRA), all effects in females were seen only at a dose that 

caused overt toxicity (i.e, mortality, clinical signs of toxicity). With the exception of one 

reproductive study in mallard duck which had a slight reduction in the number of eggs laid by 

the hen (which has limited ability to inform about potential estrogen-related effects by itself and 

decreases in food consumption also observed at this concentration), no estrogen-related effects 

were noted in the in vivo studies in mammals, fish, or birds that occurred in the absence of overt 

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Page 2 of 67

and/or systemic toxicity. Therefore, although several of the in vitro studies [estrogen receptor 

(ER) binding, ER transactivation assay (ERTA), and steroidogenesis] indicated that permethrin 

had the potential to affect the E pathway at the cellular level, these effects are not supported by 

the available in vivo studies. 

For the androgen pathway, conflicting results were reported for AR interaction across the in vitro

assays. In an in vitro assay, permethrin reduced sperm motility in a concentration-dependent 

manner (1-64 μmol/L). Multiple Hershberger assays were available for permethrin, in which the 

Tier 1 assays as well as an additional open literature study (Kunimatsu, et al., 2002) were 

negative at doses up to 75 or 120 mg/kg/day, respectively. However, in another OSRI 

Hershberger assay (Kim et al., 2005), significant decreases in AST weights were reported at 

doses of ≥10 mg/kg/day. 

In the male rat pubertal assay, no androgen-related effects were observed. While there does not 

appear to be androgen-related effects in rats with an intact HPG axis (i.e. male pubertal and Part 

158 rat data); published results in the literature (Jin et al., 2012, Wang et al., 2012) reported that 

permethrin caused testicular toxicity in mice following oral exposure at a dose of 35 or 100 

mg/kg/day. In these studies, testicular toxicity manifested as changes in testosterone levels, 

decreased testicular and epididymal weights, changes to the epididymal duct, regression of sperm 

quality and histopathological lesions in the testes. In the FSTRA, all effects in males were only 

observed at the high concentration in the presence of overt toxicity. 

Therefore based on the available in vitro and mammalian in vivo data, there appears to be a 

potential interaction with the androgen pathway in mammals. There was no convincing evidence 

of a potential interaction with the androgen pathway in wildlife. 

 

There was no convincing evidence of an interaction of permethrin with the thyroid pathway in 

mammals or wildlife in the absence of overt toxicity. The only thyroid-related effect observed in 

the absence of overt toxicity was a decrease in serum T4 levels in the male and female pubertal 

assays. No thyroid related effects were observed in the mammalian Part 158 studies. No 

treatment-related thyroid effects were seen in the AMA. 

Overall, there is no convincing evidence of potential interaction with the estrogen or thyroid 

pathways in mammals or wildlife. There is evidence for potential interaction with the androgen 

pathway in mammals. There is no convincing evidence for a potential interaction with the 

androgen pathway in wildlife.

For permethrin, a point of departure (POD) of 25 mg/kg/day for human health risk assessment is 

based on neurotoxic clinical signs (abnormal and/or decreased movement, increased body 

temperature, and aggression) observed at 75 mg/kg/day (LOAEL) in an acute neurotoxicity study 

in rats. This POD is used for deriving the chronic Reference Dose (RfD) for chronic dietary risk 

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assessment. A second POD of 11 mg/kg/day based on clinical signs of toxicity (body 

temperature and hypersensitivity to noise) seen at 154 mg/kg/day (LOAEL) in a subchronic 

inhalation toxicity study in rats. This POD is used for calculating the Margins of Exposure 

(MOEs) for non-dietary risk assessment. These PODs are lower than the dose (100 mg/kg/day) 

that caused the testicular effects in mice. Consequently, the current RfD and MOEs are 

protective of the androgen-mediate effects observed in the published literature study. 

Additionally, there was no evidence of interaction with the androgen pathway in the EDSP Tier 1 

assays. Therefore, there is no concerns for the findings reported in the literature study. 

Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is not 

recommended for permethrin since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. 

Given the lack of evidence for a potential interaction with the estrogen and androgen pathway in 

fish and thyroid pathway in amphibians, no additional testing is recommended. 

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

duck are available for permethrin. In the bobwhite quail, no reproductive effects were observed, 

but effects on reproductive parameters were observed in the mallard duck. The type of data 

obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient 

for evaluating potential reproductive effects to birds from permethrin exposure. Relative to the 

EDSP, additional testing is not recommended. 

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I. Introduction 

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

provide the necessary empirical data to evaluate the potential of chemicals to interact 

with the estrogen (E), androgen (A) or thyroid (T) signaling pathways. This interaction 

includes agonism and antagonism at the estrogen and androgen receptors, altered 

steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamicpituitary thyroid (HPT) axes. In addition to the available Tier 1 assay data, other 

scientifically relevant information (OSRI), including general toxicity data and open 

literature studies of sufficient quality were considered in this weight of evidence (WoE) 

assessment. 

In determining whether permethrin interacts with E, A or T hormone pathways, the 

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

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

effects occur were considered, in particular, whether or not endocrine-related responses 

occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity. 

On July 23, the Tire 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 permethrin 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).

Permethrin has a water solubility of <1 mg/L, and a vapor pressure of is 2.15 x 10−8 mm Hg 

(25°C). Permethrin was immobile in several soils tested (Koc >5000). It is also slow to 

hydrolyze and biodegrade (anaerobic aquatic study half-lives of 113-175 days). Based upon its 

Henry’s law constant (1.4x10−6

 atm-m3/mol) and vapor pressure, permethrin is expected to have 

a relatively low potential for volatilization from soil and water surfaces. The potential of 

permethrin for volatilization is also reduced significantly because it adsorbs strongly to soils and 

suspended solids or sediment in the water column. 

The available information considered in determining the potential interaction of permethrin with 

the E, A, or T pathways include submitted EDSP Tier 1 assays and/or other scientifically 

relevant information (OSRI) such as general toxicity studies and other published articles. These 

data are summarized in Sections III.A through III.C. An analysis of the data submitted to the 

agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in 

Section IV. The EDSP Tier 2 study recommendations are presented in Section V. 

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II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

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

agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix 

1. 

B. Other Scientifically Relevant Information (OSRI) 

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

Screening 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 permethrin (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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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 includes a battery of 11 assays 

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

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

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

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

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

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

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

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

endpoints are measured in each assay. 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

the whole organism level. The extent of expectation 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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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 in 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., change 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. 

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

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

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

which supports a conclusion of whether a substance has the potential to interact with the E, A, or 

T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for 

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

A. EDSP T1 Screening Assays 

The Tier 1 assays submitted in response to the agency’s test order for permethrin are shown 

below in Table 1. 

Table 1. Tier 1 Screening Assays for Permethrin 

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay (Rat uterine cytosol) OSCPP 

890.1250 

Requirement Satisfied by OSRI 

(Saito et. al., 2000) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OSCPP 

890.1300 

Requirement Satisfied by OSRI 

(Tyler et. al., 2000) 

AR Binding Assay (Rat prostate cytosol) OSCPP 

890.1150 

Requirement Satisfied by OSRI 

(Xu et. al., 2008; Zhang et. al., 

2008a; Tyler et. al., 2000; Zhang 

et. al., 2008b; Kim et. al., 2005) 

Steroidogenesis Assay (Human cell line 

H295R) 

OSCPP 

890.1550 

Requirement Satisfied (MRID 

No. 48618603) 

Aromatase Assay (human recombinant 

microsomes) 

OSCPP 

890.1200 

Requirement Satisfied 

(MRID No. 48618601) 

Uterotrophic Assay (Rat) OSCPP 

890.1600 

Requirement Satisfied by OSRI 

(Kim et. al., 2005; Kunimatsu et. 

al., 2002) 

Hershberger Assay (Rat) OSCPP 

890.1400 

Requirement Satisfied 

 (MRID No. 48618602) 

Pubertal Female Assay (Rat) OSCPP 

890.1450 

Requirement Satisfied 

(MRID No. 48672901) 

Pubertal Male Assay (Rat) OSCPP 

890.1500 

Requirement Satisfied 

(MRID No. 48687101) 

Fish Short-term Reproduction Assay OSCPP 

890.1350 

Requirement Satisfied 

 (MRID No. 48702301) 

Amphibian Metamorphosis Assay (Frog) OSCPP 

890.1100 

Requirement Satisfied 

(MRID No. 48988601) 

Page 16 of 75
PERMETHRIN FINAL 

Page 9 of 67

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

1. Effects on Estrogen Pathway 

The potential interactions of permethrin with the estrogen pathway are 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. 

The requirement for the Tier 1 ER binding and ERTA assays were satisfied by Saito et al., 2000 

and Tyler et al., 2000, respectively. In the study by Saito et al., permethrin was negative for 

interaction with the ER based on the results of three in vitro assays utilizing human ER (hERα): 

1) a mammalian cell-based luciferase reporter assay, 2) a yeast two-hybrid method, and 3) a 

competitive ligand-binding assay. Conversely, in the study by Tyler et al., permethrin showed 

weak activity in a recombinant yeast reporter assay with a half maximal effective concentration 

(EC50) of 2 × 10−3

 M. In other published studies considered as OSRI, the effects of permethrin in 

ER binding, ER transcriptional activation, cell proliferation, and ER-responsive pS2 gene 

expression showed conflicting results regarding the estrogenic potential of permethrin. The 

findings from the additional in vitro OSRI considered to evaluate potential ER activity are 

summarized below. 

In an ER competitive-binding study using rat uterine cytosol (Chen et al., 2002), permethrin 

exhibited equivocal evidence of specific binding to the ER. The percent [3H]-E2 binding to the 

ER was ≥ 67% at all concentrations up to 10-3 M permethrin. However, in another ER 

competitive binding assay also using rat uterine cytosol, Kim et al. (2004) reported that 

permethrin did not competitively bind to the ER, while in a whole cell competitive binding 

study, Lemaire et al. (2006), reported that permethrin did not bind to ERα or ERβ. 

Permethrin was also evaluated in several ER transactivation test systems. In a study by Du et al. 

(2010), permethrin was tested for potential interaction with the ERα using an ER transactivation 

assay in African green monkey CV-1 cells. Permethrin exhibited weak agonist effects at low 

concentrations (10−8

 to 10−5 M), and exhibited weak antagonist effects at higher concentrations 

(10−6

 to 10−5 M). In a study by Lemaire et al. (2006), permethrin was evaluated for ERα and 

ERβ activity in transfected HeLa-derived (HELN) cells. In this study, the authors reported that 

permethrin showed no agonist or antagonist activity for ERα or ERβ at the single concentration 

(10−5

 M) tested. 

Page 17 of 75
PERMETHRIN FINAL 

Page 10 of 67

Published studies also showed conflicting results regarding the ability of permethrin to induce 

ER-responsive cell proliferation. In a MCF-7 cell proliferation assay by Chen et al. (2002), 

permethrin induced proliferation at and above 10−10 M, with maximal proliferation at 10−8

 M. In 

a study by Go et al. (1999), permethrin induced modest increases in MCF-7 proliferation at 10−4

M and in a study Jin et al. (2010), permethrin induced proliferation of MCF-7 cells at 

concentrations of 10−8

 to 10−6 M. Conversely, in a study by Kim et al. (2004), permethrin did 

not induce proliferation in MCF-7 cells, but it did antagonize the proliferative response of 

estradiol at 10−6

 M. 

Several of the published studies also measured pS2 mRNA levels as a biomarker for estrogenic 

activity in MCF-7 cells. The pS2 gene is strongly expressed in response to estrogen or estrogenlike compounds. In a study by Chen et al. (2002), pS2 mRNA expression increased 1.3-fold 

after treatment with 10−7 M permethrin. Go et al. (1999), reported permethrin weakly induced 

pS2 expression at 10−4 M and Jin et al. (2010) reported that permethrin induced pS2 expression 

2.6-fold and decreased ERα mRNA expression levels at 10−6 M. 

In another in vitro study, Garey and Wolf (1998) evaluated the potential for permethrin to act as 

an estrogen agonist or antagonist using the Ishikawa Var-1 human endometrial adenocarcinoma 

cell line. This cell line produces a dose-dependent increase in alkaline phosphatase in response to 

estrogens. Permethrin did not show estrogen agonist or antagonist activity in this test system. 

Permethrin was negative in the Tier 1 aromatase assay; while in the Tier 1 steroidogenesis assay, 

permethrin produced an increase (p<0.05) in estradiol production in two runs at 1 and 10 μM 

(average 1.16-fold for 1 μM and 1.51-fold for 10 μM). 

The test order requirement for the uterotrophic assay was satisfied by the results of two literature 

studies submitted as OSRI: Kim et al., 2005; and Kunimatsu et al., 2002. In Kim et al., 

permethrin (administered s.c.) increased relative (to body) uterus weights at a dose level of 800 

mg/kg/day. Permethrin also enhanced E2-induced uterine weight increases at a lower dose (200 

mg/kg); permethrin-induced uterine weight increases were inhibited by co-administration of the 

anti-estrogen ICI 182,780, but inhibition was only statistically different at a lower dose (50 

mg/kg). Kim also reported that permethrin treatment had no effects on relative vagina weights. 

In Kunimatsu et al., no estrogenic or anti-estrogenic effects were observed on uterine weights 

with oral administration up to 150 mg/kg/day permethrin which also caused clinical signs of 

toxicity (3-6/6 rats displaying tremor). No estrogen-related effects were observed in the Tier 1 

female pubertal assay. 

In the Tier 1 FSTRA, increased GSI and effects on gonadal histopathology along with a 42% 

decrease in fecundity were observed at the highest concentration tested. However, overt toxicity 

(i.e., mortality, clinical signs of toxicity) was noted at this dose level. 

Page 18 of 75
PERMETHRIN FINAL 

Page 11 of 67

In a Part 158 developmental toxicity study in rabbits, post-implantation loss was increased 

(p<0.05) at the mid (1200 ppm) and high (1800 ppm) dose levels; however, rabbits in all dose 

groups (including 600 ppm) had decreases in body weight gains of 50-91% during GD 0-18. 

There were no estrogen-related effects noted in the other Part 158 studies. 

In a Part 158 avian reproduction toxicity study with quail, no treatment-related systemic or 

reproductive effects were noted at dietary levels up to 500 ppm. In the mallard duck 

reproduction study, there appeared to be a slight reduction (NS, but considered biologically 

significant) in the number of eggs laid by hens at the high dose (500 ppm) during the last two 

weeks of egg production, which was correlated with an increase in the number of hens with a 

regressing ovary. However, feed consumption at this dose was observed to be slightly decreased 

throughout the study, with a significant difference from controls during Weeks 6, 12, 14, and 19. 

In a Part 158 fish life-cycle toxicity study with fathead minnows, at the mean measured 

concentration 0.41 ppb, percent survival of fry were reduced in the first and second generations 

after 30 days. In a study by Nillos et al. (2010), permethrin stereoisomers were examined for 

estrogenic activity in vivo using VTG protein measurements in male medaka. After exposure of 

medaka for 8 days to the individual permethrin stereoisomers and permethrin mixture at 10 μg/L, 

hepatic VTG production was increased (p<0.05) compared to controls. Additionally, significant 

differences in the relative estrogenic potential of the enantiomers were reported: 1S-cis- and 1Strans-permethrin had responses 2.5 and 1.3 times greater than their respective R enantiomers. 

However, this study was conducted under static conditions and as such, given the physical 

properties of permethrin (low solubility, propensity to bind), there is concern about the actual 

exposure concentrations (study not included in table below). 

In Brander et al., 2012, silverside fish (Menidia beryllina) exposed for 14 days to 0.1 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. 

In Jin et al. 2009, zerbrafish embryo-larvae were exposed to permethrin for 7 days under staticrenewal conditions exhibited upregulation of several estrogen-responsive genes (e.g., vtg1, esrα, 

cyp19b) at 250 ng/L and above. In this study, as in other studies, only a solvent control was used 

and given the static-renewal test design without reported measured test concentrations, there is 

uncertainty in the actual exposure concentrations. As such, this study is not reported in table 

below. 

Page 19 of 75
PERMETHRIN FINAL 

Page 12 of 67

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

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Permethrin1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Vitellogenin 

EDSP Tier 1 Assay 

ER Binding Requirement satisfied by OSRI (Saito et al., 2000); see below. ERTA Requirement satisfied by OSRI (Tyler et al., 2000); see below. Aromatase (MRID 48618601) N Steroidogenesis (MRID 48618603) P Uterotrophic Requirement satisfied by OSRI (Kim et al., 2005; Kunimatsu et al., 2002); see below. Female Pubertal Rat (MRID 48672901) N N N N N N FSTRA (MRID 48702301) NE N N N N N 

OSRI 

ER Binding and Transactivation (Saito et al., 2000) 

N N 

Recombinant Yeast Transactivation Assay (Tyler et al., 2000) 

 P 

ER Binding/ Proliferation/PS2  (Chen et al., 2002) 

E P4

ER Binding/ Proliferation/PS2 (Kim et al., 2004) 

N P4 
PERMETHRIN FINAL 

Page 13 of 67

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

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Permethrin1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Vitellogenin 

ER Binding/ ER Transactivation (Lemaire et al., 2006) 

N N5

ER Transactivation (Du et al., 2010) P6Proliferation/PS2 (Jin et al., 2010) P4 Proliferation/PS2 (Go et al., 1999) P4 Estrogenic Potential (Garey and Wolff, 1998) N7Uterotrophic (Kim et al., 2005) N Uterotrophic (Kunimatsu et al., 2002) N Three-Generation Reproduction (Rat; MRID 00120271) 

NE NE N NE NE N 

Developmental Toxicity 

(Rat; MRID 40943603) N 

Developmental Toxicity (Rabbit; MRID 40943602) 

 P 

(M)

Chronic Toxicity/ Carcinogenicity (Rat; MRID 00069701) 

 NE N N N 

Chronic Toxicity (Dog; MRID 00129600) NE N N N 
PERMETHRIN FINAL 

Page 14 of 67

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

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Permethrin1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Vitellogenin 

28-Day Feeding (Rat; MRID 00120267) NE N N N Avian Reproduction (Quail; MRID 2322901) N Avian Reproduction (Duck; MRID 2322902) P8 Fish Full Life Cycle (Minnow; MRID 00102096) 

 N 

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

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

in weight are absolute unless otherwise indicated. LW= Liver weight 

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

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 

Section IV. A 

4. Reported increased MCF-7 proliferation and induction of PS2 expression. 

5. Negative at the single permethrin concentration tested (10-5 M) 

6. Permethrin exhibited weak agonist effects at low concentrations (10−8 to 10−5 M), and exhibited weak antagonist effects at higher concentr

CV-1 cells. 

7. Negative for estrogen agonist or antagonist using the Ishikawa Var-1 human endometrial adenocarcinoma cell line test system. 

8. Slight reduction (NS, but considered biologically significant) in the number of eggs laid by hens at the high dose (500 ppm) during the last 

production, which was correlated with an increase in the number of hens with a regressing ovary. 

9. Feed consumption at this dose was slightly decreased throughout the study, with a significant difference from controls during Weeks 6, 12

E Equivocal P Positive findings N Negative findings (in vitro)/ No effects (in vivo) NE Not Examined
PERMETHRIN FINAL 

Page 15 of 67

2. Effects on Androgen Pathway 

The potential interactions of permethrin with the androgen pathway are 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. This table also 

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

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

considered further in the WOE assessment. 

The requirement for the AR binding assay was satisfied by the results of four studies submitted 

as OSRI: Xu et al., 2008; Zhang et al., 2008; Tyler et al., 2000; and Kim et al., 2005. In an in 

vitro AR transcriptional activation assay Xu et al., reported that permethrin showed AR 

antagonist activity by attenuating dihydrotestosterone (DHT)-related chloramphenicol 

transferase (reporter gene) expression with an IC50 value of 5.86 × 10−5 M. In Zhang et al., 2008, 

permethrin significantly inhibited DHT-induced hAR transcriptional activation in vitro at a 

concentration of 10−5 M. In Tyler et al., high concentrations of permethrin (~10−3 M) induced 

slight increases in activity above baseline values, thus suggesting very weak agonist activity. 

Permethrin also exhibited weak anti-androgenic activity in this assay with an IC50 of 7.3 × 10−4

M. Additionally, two published studies examining the effects of permethrin on AR binding 

and/or AR transactivation were considered as OSRI. In a study by Bauer et al. (2002), 

permethrin did not displace DHT from immobilized recombinant hAR. Conversely, in a receptor 

transactivation study by Du et al. (2010), permethrin showed AR antagonist activity with a 

calculated 20% relative inhibitory concentration (RIC20) of 1.88 x 10−6

 M. In the Tier 1 in vitro

steroidogenesis assay, permethrin induced a slight, but significant (p<0.05) increase in 

testosterone production at 10 μM in two runs (average 1.1-fold). 

Permethrin was negative in the Tier 1 Hershberger assay and no androgen related effects were 

noted in the Tier 1 male pubertal assay. Additionally, in a published 5-day Hershberger assay by 

Kunimatsu et al. submitted as OSRI, no effects were noted on the accessory sex tissues at dose 

levels that caused tremors (75 mg/kg/day). In another published 10-day Hershberger assay, Kim 

et al. (2005) reported statistically significant reductions in the five sex accessory tissue weights 

at all doses tested (10, 50, and 100 mg/kg/day), although the decreases were not dose dependent. 

In addition, permethrin caused significant decreases in seminal vesicle and ventral prostate 

weights in a modified Hershberger assay conducted by Zhang et al. (2008) using a single dose 

level (50 mg/kg/day) of permethrin; however, no chemical purity information was provided 

which reduces confidence in the study and therefore the results are not included in the table 

below. 

Page 23 of 75
PERMETHRIN FINAL 

Page 16 of 67

In the Tier 1 FSTRA, effects were noted in males at the highest concentration tested (2.6 μg 

a.i./L), including an increase in the GSI and decreased nuptial tubercle score. However, only 

63% of the males survived at the high dose, and clinical sings of toxicity were noted at this dose, 

including erratic swimming, loss of equilibrium, surfacing, and excess feed noted in the test 

chambers. There were no androgen-related effects determined in the Part 158 ecotoxicity studies. 

The only androgen-related effect in the Part 158 mammalian studies was decreased testicular 

weight seen in a rat 28-day feeding toxicity study at doses that also caused neurotoxicity 

(hypersensitivity). 

In addition to the Part 158 toxicity studies, published studies were reviewed that specifically 

examined effects of permethrin on testes and/or sperm parameters in rodents. In a testicular 

toxicity study by Jin et al., (2012), (+)-cis, (−)-cis, and (−)-trans permethrin exposure at 100 

mg/kg significantly reduced serum testosterone levels, and decreased both absolute and relative 

weights of testes in the (+)-cis, (−)-cis and (−)-trans-permethrin treated groups. A decreased 

number of spermatogenic cells and large interstitial spaces in the seminiferous tubules were also 

observed in the 100 mg/kg (+)-cis, (−)-cis, and (−)-trans-permethrin treated groups. In a 

testicular toxicity study by Wang et al. (2012), the percentage of seminiferous tubules in stage 

group VII-VIII was significantly decreased in the cis-permethrin group (oral dosing at ~35 

mg/kg/day), and in the caput epididymis, cytoplasmic vacuolation was observed more frequently 

in the cis-permethrin group, and median vacuolation scores were significantly higher. Caudal 

epididymal sperm count was significantly reduced in the cis-permethrin group (↓60%), and 

sperm motility was significantly decreased. 

Page 24 of 75
PERMETHRIN FINAL 

Page 17 of 67

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

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Permethrin1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histo

pathology

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight 

EDSP Tier 1 Assay 

AR Binding Requirement satisfied by OSRI (Xu et al., 2008; Zhang et al., 2008a; Tyler et al., 2000; Zhang et al., 2008b; Steroidogenesis (MRID 48618603) P Hershberger (MRID 48618602) NE N Male Pubertal Rat (MRID 486687101) N N N N N N N FSTRA (MRID 48702301) NE N N N N 

OSRI 

AR Binding (Bauer et al., 2002) N Recombinant Yeast AR Assay (Tyler et al., 2000) 

 P 

Receptor Transactivation (Du et al., 2010) P AR Transactivation (Xu et al., 2008) P Hershberger (Kim et al., 2005) NE P (L, M, H) Hershberger (Kunimatsu et al., 2002) NE N 
PERMETHRIN FINAL 

Page 18 of 67

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

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Permethrin1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histo

pathology

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight 

Three-Generation Reproduction (Rat; MRID 00120271)

 NE N NE N NE NE N 

Chronic Toxicity / Carcinogenicity (Rat; MRID 00069701) 

 N N NE N N 

Chronic Toxicity (Dog; MRID 00129600) N N NE N N 28-Day Feeding (Rat; MRID 00120267) 

↓10% 

(MH) NE NE NE N 

Testicular Toxicity (Mouse; Jin et al., 2012) 

P 

(H) 

P 

(H) 

P 

(H) 

Testicular Toxicity (Mouse; Wang et al., 2012) 

 N N P N P 

Avian Reproduction (Quail; MRID 42322901) N Avian Reproduction (Duck; MRID 42322902) N Fish Full Life Cycle (Minnow; MRID 00102096)

 N 
PERMETHRIN FINAL 

Page 19 of 67

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

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

absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). 

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

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 thi

Section IV. A 

4. Feed consumption at this dose was slightly decreased (NS) throughout the study, with a significant difference from controls during Week

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

Page 20 of 67

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

The potential interactions of permethrin 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. 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. 

In the Tier 1 male pubertal assay, serum T4 levels were decreased by 28 and 42% at the low (75 

mg/kg/day) and high (120 mg/kg/day) doses, respectively, and serum TSH levels were increased 

by 77% at the high dose. However, the thyroid hormone changes at the high dose occurred in the 

presence of overt toxicity. No changes were seen in thyroid weight or histopathology. Similarly, 

in the female pubertal assay, serum T4 levels were decreased by 26 and 42% at the low (75 

mg/kg/day) and high (150 mg/kg/day) doses, respectively, and serum TSH levels were increased 

by 40% at the high dose. In addition, thyroid weights in females were also decreased by 22% at 

the high dose, but without a change in thyroid histopathology. It was determined that the high 

dose in the female pubertal was overtly toxic. 

In the AMA, Day-21 normalized hind-limb length (HLL) was increased by 21-23% compared to 

negative control, however, it was not dose-dependent. Additionally, when Day-21 normalized 

HLL results were compared to the solvent control, no significant differences were observed. 

There was no significant acceleration or delay of median Nieuwkoop-Faber (NF) developmental 

stage at Day 7 or 21 for any treatment level. Further, no asynchronous development was 

observed. A statistically significant increase in Day 21 wet weight and snout-to-vent length 

(SVL) was observed at the lowest concentration tested (0.054 μg a.i./L). Finally, effects on 

thyroid gland histopathology were observed in both controls and all treatment groups. 

Histopathological effects included thyroid gland atrophy and hypertrophy, follicular cell height 

increases and decreases, and follicular cell asymmetry. These effects were of low frequency and 

did not exhibit a concentration-dependent response; therefore, they are not considered to be 

treatment-related. 

In a receptor transactivation study by Du et al. (2010), permethrin was observed to have 

antagonist effects at the thyroid receptor in a transfected African green monkey kidney CV-1 cell 

line. 

Page 28 of 75
PERMETHRIN FINAL 

Page 21 of 67

In a Part 158 chronic toxicity study in dogs, thyroid weights were increased in a non-dose 

dependent manner in the low (5 mg/kg/day; ↑27%), mid (100 mg/kg/day; ↑28%) and high (1000 

mg/kg/day; ↑17%) dose males, and in the mid (↑27%) and high (↑33%) dose females; 

additionally, no corroborating histopathological changes were observed. 

Table 4. Thyroid Pathway for Permethrin.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Permethrin1

Study Type/ 

Literature Citation 

TR Binding Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, SVL) Systemic Toxicity2 

Overt Toxicity Observed3

EDSP Tier 1 Assay 

Male Pubertal Rat 

(MRID 48687101) N N ↓28% 

(L) N N ↑LW (L, H)

X 

(H)

Female Pubertal 

Rat (MRID 

48672901) 

 N N ↓26% 

(L) N N ↑LW (H) 

X 

(H)

AMA 

(MRID 48988601) N N4 

Day 21: 

↑BW, 

SVL (L) 

 N 

OSRI

Receptor 

Transactivation (Du 

et al., 2010) 

P 

Chronic Toxicity/ 

Carcinogenicity 

(Rat; MRID 

00069701) 

 N N ↑LW; LE 

( H) 

X 

(H) 

28-Day Feeding 

(Rat; MRID 

00120267) 

 N N X 

(MH, H)

X 

(H) 

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

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

parameter is not routinely evaluated or is not applicable in this assay. LW= Liver weight; LE= Liver enzyme 

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

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

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

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

4. Day-21 normalized hind-limb length (HLL) was increased by 21-23%, however, it was not dose-dependent. 

Additionally, when Day-21 normalized HLL results were compared to the solvent control, no significant differences 

were observed 

P Positive findings 

N Negative findings 

NE Not examined 

Page 29 of 75
PERMETHRIN FINAL 

Page 22 of 67

IV. Committee’s Assessment of Weight of Evidence 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

whether or not the responses are 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 Tier 1 assays stated that “In summary, 

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

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

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

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

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

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

OSRI studies are described below. Generally, one parameter (i.e., death or >10% decrease in 

mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. 

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

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

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

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1. Tier 1 in vivo Assays 

Overt and/or systemic toxicity was not observed in the AMA at concentrations up to 0.68 μg 

a.i./L or in the Hershberger assay at doses up to 120 mg/kg/day. In the FSTRA, overt toxicity 

was noted at the highest tested concentration (2.6 μg a.i./L) as survival was reduced to 63% for 

males and 50% for females. Clinical signs of toxicity were also noted in the surviving fish at the 

high concentration, including erratic swimming, loss of equilibrium and surfacing. Excess feed 

was also noted in the test chambers on Day 10 and Days 17-19. 

In the female pubertal rat assay, systemic toxicity was evident at the high dose (150 mg/kg/day) 

based the 10% increase (p<0.05) in absolute liver weights (both adjusted and unadjusted). 

Additionally, overt toxicity was noted at the high dose in the female and male pubertal assays. 

For females, tremors were observed in 4 animals at the low dose (75 mg/kg/d) and in all animals 

at the high dose. In the male pubertal, three high dose (150 mg/kg/day) rats were found dead 

after 2 days of dosing; therefore, the high dose was reduced to 120 mg/kg/day. At this reduced 

dose, one rat was considered uncoordinated on one day. Tremors were observed in 3 animals at 

the low dose (75 mg/kg/day) and mild to severe tremors in all animals at the high (120 

mg/kg/day) at 6 h post-dosing. Additionally, absolute and relative (to body) liver weights were 

increased (p<0.05) by 6 and 11% at the low and high dose, respectively. 

2. OSRI 

In the three-generation reproduction toxicity study in rats, treatment-related clinical signs in 

high-dose parental animals were limited to whole body tremors, occurring in all parental 

generations (exception: tremors were not observed in the P males) during the first few days of 

the premating period. In the 2500 ppm groups, the incidence rates for the tremors were 20/24 P 

females, 11/12 and 24/24 F1 males and females, respectively, and 12/12 and 24/24 F2 males and 

females, respectively. Tremors were also observed in pregnant and lactating females exposed to 

2500 ppm permethrin. Microscopic examination of F3b weanlings revealed slight to moderate 

centrilobular hypertrophy, ranging from 0 to 80% for the males and from 10 to 100% for the 

females. 

In a developmental toxicity study in rats, clinical signs of toxicity seen between GD 8-19 

included tremors in 21/24 rats and head flicking in 6/24 rats in the high-dose group (150 

mg/kg/day). Body weight gains and food consumption by the high-dose dams were less 

(p<0.05) than that of the controls throughout the dosing interval. For GD 7-10, 10-13, and 13-

16, body weight gains were decreased by 88%, 32%, and 18%, respectively. Mean fetal body 

weight of the high-dose group was 3.2% (p ≤ 0.05) less than that of the controls. 

In a developmental toxicity study in rabbits, a total of 0, 5, 5, or 4 does died or were sacrificed 

moribund in the control, low-, mid- or high-dose groups, respectively. Due to the lack of a doseresponse, the deaths could not be definitively attributed to test article administration. Clinical signs 

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of toxicity included body tremors observed in 5 of the high-dose (1800 ppm) animals. Body 

weight gains by the low- (600 ppm), mid- (1200 ppm) and high-dose groups were decreased by 

79%, 50% and 91%, respectively, during GD 0-18 with significance (p<0.05) attained for the 

low- and high-dose groups. During the post-dosing interval, recovery of body weights was noted 

for the low- and mid-dose groups, but not for the high-dose group. Post-implantation loss was 

increased (p<0.05) in the mid- and high-dose groups to 155% and 248% of the control level. 

Correspondingly, the number of early and late resorptions was higher in these groups as 

compared to the control group values. Mean fetal body weights in the high-dose group were 

slightly (↓9%; NS) less than that of the controls and attributed to maternal body weight 

decreases. 

In a chronic toxicity/carcinogenicity study in rats, treatment-related tremors and hypersensitivity 

were observed in the high-dose (2500 ppm) males and females during the first two weeks of the 

study. Liver changes suggestive of adaptive hypertrophy included increased aminopyrine-Ndemethylase activity in all male treatment groups in the mid- (1000 ppm) and high-dose female 

at 52 weeks, and in the high-dose male and female groups at 104 weeks. This was coupled with 

modestly increased absolute and relative liver weights in the high-dose males and high and lowdose females at 52 weeks and in all male treatment groups and mid-dose females at 104 weeks. 

Further evidence for adaptive changes included hypertrophy of centrilobular hepatocytes with 

increased cytoplasmic eosinophilia in the mid- and high-dose male and females at 104 weeks’ 

exposure and increased smooth endoplasmic reticulum proliferation in all treatment groups 

except low-dose males at 52 weeks and high-dose groups at 104 weeks. Liver changes also 

included fatty vacuoles confirmed by electron microscopy in the mid- and high-dose males at 

both 52 and 104 weeks and in the high-dose females at 104 weeks. 

In a chronic toxicity study in dogs, neurological clinical signs (tremors, uncoordinated gait, 

nervousness and convulsions, also excessive salivation and vomiting) were observed in the highdose group (1000 mg/kg/day). Other findings of systemic toxicity at the high-dose included: 

decreased body weight gains (↓37% males; ↓33% females), decreased food consumption 

(increased food left uneaten), increased liver weight (↑30% males; ↑36% females) and alkaline 

phosphatase levels (↑377% males; ↑220% females). At mid-dose (100 mg/kg/day), increased 

liver weight (↑25% both sexes) and alkaline phosphatase levels (↑134% males; ↑99% females) 

were observed. Microscopic evaluation of the adrenals showed focal degeneration and necrosis 

in the cortex with variable inflammatory cell infiltration along with swelling and vacuolization of 

the cells in the inner cortex at high-dose males and females (5/5 males and 4/6 females) and at 

mid-dose males (1/5). The liver also showed hepatic cellular swelling in the mid- and high-dose 

males and females. 

In a 28-day feeding toxicity study in rats, one female and four males died by Day 18 at the high 

dose (5000 ppm). Surviving rats receiving 2500 ppm and above became hypersensitive during 

the first week of treatment and remained so throughout the study. Body weight of the 5000 ppm 

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males was 16-22% below that of controls throughout the treatment period, and overall body 

weight gain of that group was decreased by 31%. Liver weight was increased 28-33% in the 

2500 and 5000 ppm females. 

In an avian reproduction toxicity study in quail, systemic toxicity was not observed at the highest 

dose tested (500 ppm). However, in an avian reproduction toxicity study in ducks, feed 

consumption was slightly decreased throughout the study, with a significant difference from 

controls during Weeks 6, 12, 14 and 19 at the high dose (500 ppm). 

In a fish full life-cycle toxicity study on fathead minnows, mean measured concentrations of 0.41 

ppb significantly reduced the percent survival of fry during the initial 30 days of exposure in the 

first and second generations. However, minnows which survived the initial exposure period 

demonstrated normal ranges of measured parameters of survival, growth, reproduction and egg 

hatchability. 

In the published uterotrophic and Hershberger studies in rats by Kim et al. (2005), systemic 

toxicity was not mentioned despite dosing the females at up to 800 mg/kg/day via s.c. injection 

and the males at up to 100 mg/kg/day via oral gavage. However in a similar study by Kunimatsu 

et al. (2002), tremors were observed in 3-6/6 female rats at the highest dose tested (150 

mg/kg/day) and in 2-6/6 male rats at the highest dose tested (75 mg/kg/day). Systemic toxicity 

was also not observed in the published Hershberger study on rats by Zhang et al. (2008) at a 

single dose level of 50 mg/kg/day (oral dosing). In two other published testicular toxicity studies 

(Y. Jin et al., 2012, and Wang et al., 2012), systemic toxicity was not noted in mice dose orally 

at up to 100 and 35 mg/kg/day, respectively. In a published study by Nillos et al. (2010) in 

medaka, a slight decrease in survival was observed at a concentration of 10 μg/L. 

B. Estrogen Pathway

A number of OSRI in vitro assays evaluating estrogen receptor activation resulted in conflicting 

results, i.e. some showed activation of estrogen signaling while others did not. The OSRI assays 

included estrogen receptor (ER) binding, cellular proliferation of estrogen sensitive MCF-7 cells, 

expression of estrogen sensitive pS2 mRNA in the cancer cells, and interaction with either ERα

or ERβ in transfered HeLa cells. Permethrin was negative in the Tier I in vitro aromatase assay, 

whereas in the steroidogenesis assay permethrin caused an increase (p<0.05) in estradiol 

production. In vivo, permethrin was negative in both the uterotrophic and female pubertal rat 

assays, with no effects on uterine weight, mean age at vaginal opening, percent cycling, or 

histopathological changes in the ovaries and uterus. In the fish short-term reproduction assay 

(FSTRA), all effects in females were seen only at a dose that caused overt toxicity (i.e, mortality, 

clinical signs of toxicity). With the exception of one reproductive study in mallard duck which 

had a slight reduction in the number of eggs laid by the hen (which has limited ability to inform 

about potential estrogen-related effects by itself and decreases in food consumption also 

observed at this concentration), no estrogen-related effects were noted in the in vivo studies in 

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mammals, fish, or birds that occurred in the absence of overt and/or systemic toxicity. 

Therefore, although several of the in vitro studies [estrogen receptor (ER) binding, ER 

transactivation assay (ERTA), and steroidogenesis] indicated that permethrin had the potential to 

affect the E pathway at the cellular level, these effects are not supported by the available in vivo

studies. 

 C. Androgen Pathway

For AR binding, conflicting results were reported across the in vitro assays with different cell 

lines. In an in vitro assay, permethrin reduced sperm motility in a concentration-dependent 

manner (1-64 μmol/L). Multiple Hershberger assays were available for permethrin, in which the 

Tier 1 assays as well as an additional open literature study (Kunimatsu, et al., 2002) were 

negative at doses up to 75 or 120 mg/kg/day. However, in another OSRI Hershberger assay 

(Kim et al., 2005), significant decreases in AST weights were reported at doses of ≥10 

mg/kg/day. 

In the male rat pubertal assay, no androgen-related effects were observed. While there does not 

appear to be androgen-related effects in rats with an intact HPG axis (i.e. male pubertal and Part 

158 rat data); published results in the literature (Jin et al., 2012, Wang et al., 2012) reported that 

permethrin caused testicular toxicity in mice following oral exposure at a dose of 35 or 100 

mg/kg/day. In these studies, testicular toxicity manifested as changes in testosterone levels, 

decreased testicular and epididymal weights, changes to the epididymal duct, regression of sperm 

quality and histopathological lesions in the testes. In the FSTRA, all effects in males were only 

observed at the high concentration in the presence of overt toxicity. 

Therefore based on the available in vitro and mammalian in vivo data, there appears to be a 

potential interaction with the androgen pathway in mammals. There was no convincing evidence 

of a potential interaction with the androgen pathway in wildlife. 

 

D. Thyroid Pathway 

There was no convincing evidence of an interaction of permethrin with the thyroid pathway in 

mammals or wildlife in the absence of overt toxicity. The only thyroid-related effect observed in 

the absence of overt toxicity was a decrease in serum T4 levels in the male and female pubertal 

assays. No thyroid related effects were observed in the mammalian Part 158 studies. No 

treatment-related thyroid effects were seen in the AMA. 

 

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E. Conclusions 

There is no convincing evidence of potential interaction with the estrogen or thyroid pathways in 

mammals or wildlife. There is evidence for potential interaction with the androgen pathway in 

mammals. There is no convincing evidence for a potential interaction with the androgen 

pathway in wildlife.

V. EDSP Tier 2 Testing Recommendations 

For permethrin, a point of departure (POD) of 25 mg/kg/day for human health risk assessment is 

based on neurotoxic clinical signs (abnormal and/or decreased movement, increased body 

temperature, and aggression) observed at 75 mg/kg/day (LOAEL) in an acute neurotoxicity study 

in rats. This POD is used for deriving the chronic Reference Dose (RfD) for chronic dietary risk 

assessment. A second POD of 11 mg/kg/day based on clinical signs of toxicity (body 

temperature and hypersensitivity to noise) seen at 154 mg/kg/day (LOAEL) in a subchronic 

inhalation toxicity study in rats. This POD is used for calculating the Margins of Exposure 

(MOEs) for non-dietary risk assessment. These PODs are lower than the dose (100 mg/kg/day) 

that caused the testicular effects in mice. Consequently, the current RfD and MOEs are 

protective of the androgen-mediate effects observed in the published literature study. 

Additionally, there was no evidence of interaction with the androgen pathway in the EDSP Tier 1 

assays. Therefore, there is no concerns for the findings reported in the literature study. 

Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is not 

recommended for permethrin since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. 

Given the lack of evidence for a potential interaction with the estrogen and androgen pathway in 

fish and thyroid pathway in amphibians, no additional testing is recommended. 

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

duck are available for permethrin. In the bobwhite quail, no reproductive effects were observed, 

but effects on reproductive parameters were observed in the mallard duck. The type of data 

obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient 

for evaluating potential reproductive effects to birds from permethrin exposure. Relative to the 

EDSP, additional testing is not recommended. 

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VI. References 

 (Authors not provided) (1977a) Chronic toxicity of FMC 33297 to the fathead minnow 

(Pimephales promelas). E. G. and G. Bionomics, Aquatic Toxicology Laboratory, Wareham, 

MA. Sponsored by FMC Corporaton, Princeton, NJ. FMC Study No. ACT092.72, October 

1977. MRID 00102096. Unpublished. 

Bauer, E, Bitsch, N, Brunn, H, et al. (2002) Development of an immuno-immobilized androgen 

receptor assay (IRA) and its application for the characterization of receptor binding affinity of 

different pesticides. MRID 48176001. Published: Chemosphere 46: 1107+1115. 

Beavers, J, Foster, J, Lynn, S, et al. (1992) Permethrin: A one generation reproduction study 

with the northern bobwhite (Colinus virginianus). Wildlife lnternational Ltd., Easton, MD. 

Sponsored by FMC Corporaton, Princeton, NJ. Laboratory Project No. 104166, May 20, 1992. 

MRID 42322901. Unpublished. 

Beavers, J, Foster, J, Lynn, S, et al. (1992). Permethrin: A one generation reproduction study 

with the Mallard (Anas platyrhynchos) Wildlife lnternational Ltd., Easton, MD. Sponsored by 

FMC Corporaton, Princeton, NJ. Laboratory Project No. 104166, May 20, 1992. MRID 

42322902. Unpublished. 

Brander, S, He, G, Smalling, K, et al. (2012) The in vivo estrogenic and in vitro anti-estrogenic 

activity of permethrin and bifenthrin. Environ. Toxicol. Chem. 31 (12): 2848-2855. 

Chen, H, Xiao, J, Hu, G, et al. (2002) Estrogenicity of organophosphorous and pyrethroid 

pesticides. MRID 48160705. Published: J. Toxicol. Environ. Health, Part A. 65: 1419-1435. 

Clapp, M, Banham, P, Chart, I, et al. (1977) PP557: 28-Day feeding study in rats. Imperial 

Chemical Industries, Ltd., Central Toxicology Laboratory (location not given). Laboratory 

Report No. CTL/P/355, December 20, 1977. MRID 00120267. Unpublished. 

Du, G, Shen, O, Sun, H, et al. (2010) Assessing hormone receptor activities of pyrethroid 

insecticides and their metabolites in reporter gene assays. MRID 48196301. Published: 

Toxicol. Sci. 116 (1): 58-66. 

Garey, J, and Wolff, M (1998) Estrogenic and antiprogestagenic activities of pyrethroid 

insecticides. MRID 48176005. Published: Biochem. and Biophys. Res. Comm. 251: 855-859. 

Go, V, Garey, J, Wolff, M, et al. (1999) Estrogenic potential of certain pyrethroid compounds in 

the MCF-7 human breast carcinoma cell line. MRID 48176004. Published: Environ. Health 

Perspect. 107 (3): 173-177. 

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Hodge, M (1988) Permethrin: teratogenicity study in the rat. ICI Central Toxicology 

Laboratory, Alderley Park Macclesfield, Cheshire, UK. Laboratory Study No. CTL/P/2269, 

September 20, 1988. MRID 40943603. Unpublished. 

Hodge, M, Banham, P, Glaister, J, et al. (1977) Permethrin (PP557): 3-generation reproduction 

study in rats. ICI Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, UK. 

CTL Study Number: RR0015, December 1977. MRID 00120271. Unpublished. 

Jin, M, Li, L, Xu, C, et al. (2010) Estrogenic activities of two synthetic pyrethroids and their 

metabolites. J. Environ. Sci. (China) 22 (2): 290-296. 

Jin, Y, Chen, R, Sun, L, et al. (2009) Enantioselective induction of estrogen-responsive gene 

expression by permethrin enantiomers in embryo-larval zebrafish. Chemosphere 74 (9): 1238-

1244. 

Jin, Y, Liu, J, Wang, L, et al. (2012) Permethrin exposure during puberty has the potential to 

enantioselectively induce reproductive toxicity in mice. Environ. Int. 42: 144-151. 

Kalinowski, A.E, et al. (1982) Permethrin: one year oral dosing study in dogs. ICI Central 

Toxicology Laboratory Alderley Park, Macclesfield, Cheshire, UK. Laboratory Study No. 

CTL/P/647, February 24, 1982. MRID 00129600. Unpublished. 

Kim, S, Lee, R, Lim, K, et al. (2005) Potential estrogenic and androgenic effects of permethrin 

in rats. MRID 48176002. Published: J. Reprod. Dev. 51 (2): 201-210. 

Kim, Y, Shin, J, Kim, H, et al. (2004) Assessing estrogenic activity of pyrethroid insecticides 

using in vitro combination assays. MRID 48176007. Published: J. Reprod. Dev. 50 (2): 245-

255. 

Kunimatsu, T, Yamada, T, Ose, K, et al. (2002) Lack of (anti-) androgenic or estrogenic effects 

of the three pyrethroids (esfenvalerate, fenvalerate, and permethrin) in the Hershberger and 

uterotrophic assays. MRID 48176008. Published: Regul. Toxicol. Pharm. 35: 227-237. 

Lemaire, G, Minf, W, Mauvais, P, et al. (2006) Activation of α- and β-estrogen receptors by 

persistent pesticides in reporter cell lines. MRID 48074114. Published Life Sciences 79: 1160-

1169. 

Nillos, M, Chajkowski, S, Rimoldi, J, et al. (2010) Stereoselective biotransformation of 

permethrin to estrogenic metabolites in fish. Chem. Res. Toxicol. 23 (10): 1568-1575. 

Richards, D, Banham, P, Chart, I, et al. (1977) PP557: Two year feeding study in rats. Imperial 

Chemical Industries Limited, Central Toxicology Laboratory. Sponsored by FMC Corporaton, 

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Saito, K, Tomigahara, Y, Ohe, N, et al. (2000) Lack of significant estrogenic or anti-estrogenic 

activity of pyrethroid insecticides in three in vitro assays based on classic estrogen receptor αrelated mechanisms. MRID 48176003. Published: Toxicol. Sci. 57: 54-60. 

Schneider, S, Kendall, T, and Kruger, H (2012) Permethrin: Amphibian Metamorphosis Assay 

for the Detection of Thyroid Active Substances. Wildlife International, Ltd., Easton, MD. 

Sponsored by Consumer Specialty Products Association, For the Permethrin Data Group II Task 

Force Steering Committee/Joint Venture, Washington, DC. Laboratory Report No. 701A-109A, 

November 6, 2012. MRID 48988601. Unpublished. 

Schneider, S, Martin, K, Kendall, T et al. (2012) Permethrin: fish short-term reproduction assay 

with the fathead minnow (Pimephales promelas). Wildlife International, Ltd., Easton, MD. 

Sponsored by Consumer Specialty Products Association for the Permethrin Data Group II Task 

Force Steering Committee/Joint Venture, Washington, DC. Laboratory report No. 701A-108, 

September 11, 2012. MRID 48702301 Unpublished 

Tyler, C, Beresford, N, Van der Woning, M, et al. (2000) Metabolism and environmental 

degradation of pyrethroid insecticides produce compounds with endocrine activities. MRID 

48213601. Published: Environ. Toxicol. Chem. 19 (4): 801–809. 

USEPA (2010) Permethrin: Report of the Endocrine Disruptor Review Team; Test Order # 

EDSP- 109701-83, 84, 85, 87, 89, 90 and 91, December 6, 2010. Office of Chemical Safety and 

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

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 Science Coordination and Policy. U.S. Environmental 

Protection Agency, Washington, DC. 

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

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

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

Agency, Washington, DC. 

Wagner, H (2011) Permethrin: H295R steroidogenesis assay. CeeTox, Kalamazoo, MI. 

Sponsored by Consumer Specialty Products Association for the Permethrin Data Group II Task 

Force Steering Committee/Joint Venture, Washington, DC. Laboratory Report No.: 9146V100383STER, November 17, 2011. MRID 48618603. Unpublished. 

Wang, D, Kamijima, M, Okamura, A, et al. (2012) Evidence for diazinon-related inhibition of 

cis-permethrin metabolism and its effects on reproductive toxicity in adult male mice. Reprod. 

Toxicol. 34 (4): 489-497. 

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Wilga, P (2011) Permethrin: human recombinant aromatase assay. CeeTox, Inc., Kalamazoo, 

MI. Laboratory Report No. 9146V-100383AROM, November 17, 2011. MRID 48618601. 

Unpublished. 

Xu L, Liu L, Ren X, et al. (2008) Evaluation of androgen receptor transcriptional activities of 

some pesticides in vitro. MRID 48176006. Published: Toxicology 243 (l-2): 59-65. 

Zhang, J, Zhu, W, Zheng, Y, et al. (2008) The anti-androgenic activity of pyrethroid pesticides 

cyfluthrin and β-cyfluthrin. MRID 48196302. Published: Reprod. Toxicol. 25: 491-496. 

Zorrilla, L (2011) The Hershberger bioassay for permethrin. Integrated Laboratory Systems, 

Inc., Durham, NC. Sponsored by Consumer Specialty Products Association for the Permethrin 

Data Group II Task Force Steering Committee/Joint Venture, Washington, DC. Laboratory 

Project Study ID: C195-200, November 15, 2011. MRID 48618602. Unpublished. 

Zorilla, L (2012) Pubertal development and thyroid function in intact juvenile/peripubertal 

female rats; permethrin. Integrated Laboratory Systems, Inc., Durham NC. Sponsored by 

Consumer Specialty Products Association for the Permethrin Data Group II Task Force Steering 

Committee/Joint Venture, Washington, DC. Laboratory Report No.: C195-300, April 13, 2012. 

MRID 48672901. Unpublished. 

Zorrilla, L (2012) Pubertal development and thyroid function in intact juvenile/peripubertal male 

rats with permethrin. Integrated Laboratory Systems, Inc., Durham, NC. Sponsored by 

Consumer Specialty Products Association for the Permethrin Data Group II Task Force Steering 

Committee/Joint Venture, Washington, DC. Laboratory Study No.: C195-800, September 5, 

2012. MRID 48687101. Unpublished. 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay; (AMA, OCSPP 890.1100) 

The 21-day assay (MRID 48988601) of permethrin (97.7% purity) on amphibian metamorphosis 

of African clawed frogs (Xenopus laevis) was conducted under flow-through conditions. 

Amphibian larvae at developmental Nieuwkoop-Faber Stage (NF) 51 (80; 20/replicate) were 

exposed to control (water only; negative), a solvent control (0.020 mL/L dimethylformamide; 

DMF), and permethrin at nominal concentrations of 0.00011, 0.00033, and 0.0010 mg a.i./L; 

mean-measured concentrations were <0.0000600 (<LOQ; controls), 0.000052, 0.00022, and 

0.00063 mg a.i./L. The reviewer-calculated time-weighted average concentrations were 

<0.0000600 (<LOQ; controls), 0.000054, 0.00022, and 0.00068 mg a.i./L. The test system was 

maintained at 21.5 to 23.0°C and a pH of 7.9 to 8.2. 

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

water) control. 

Two non-incidental mortalities were observed in the test, one each in the low and high treatment 

groups. Clinical signs included tail curvature, which was observed in both controls and all 

treatment groups (12-28% of individuals), with no indication of a dose-dependent pattern. 

Compared to the negative control, permethrin significantly promoted Day 21 normalized hindlimb length (HLL) at the low and mid treatment groups (by 21% and 23% respectively). There 

were no effects on normalized HLL on Day 7. However, there was an apparent solvent effect 

(21% stimulation of normalized HLL, p=0.01) relative to the negative control at Day 21. When 

Day 21 normalized HLL results were compared to the solvent control, no significant differences 

were observed. There was no significant acceleration or delay of median NF developmental stage 

at Day 7 or 21 for any treatment level. Further, no asynchronous development was observed. 

Compared to the negative control, there was a statistically significant increase in Day 21 wet 

weight and snout-to-vent length (SVL) at the low treatment group. No solvent effect was seen for 

SVL. 

Thyroid gland histopathology observations included thyroid gland atrophy and hypertrophy, 

follicular cell height increases and decreases, and/or follicular cell asymmetry in both controls 

and all treatment groups. These effects were of low frequency and did not exhibit a 

concentration-dependent response; therefore, they are not considered to be treatment-related. 

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

Requirement satisfied by OSRI (Xu et al., 2008; Zhang et al., 2008a; Tyler et al., 2000; Zhang 

et al., 2008b; Kim et al., 2005); see Appendix 2. 

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Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48618601), permethrin (97.7% a.i., Lot # 

PMPS000014) was incubated with human recombinant aromatase and tritiated androstenedione 

(1-β [3

H(N)]-androst-4-ene-3,17-dione; [3H]ASDN) in dimethyl sulfoxide (DMSO) at log 

concentrations of 10−10 to 10−3 M for 15 minutes to assess the potential of permethrin 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 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 permethrin series 

(10−10 to 10−3 M) with three repetitions per concentration. 

Aromatase activity in the full activity controls was 0.588 ± 0.079 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, and the activity in the background controls ranged from 0.21 to 0.31% and averaged 

0.28% of the full activity. 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 −7.3 M and the Hill slope was −1.0. 

Precipitation was noted at the two highest concentrations of permethrin tested (10−4 and 10−3 M); 

therefore, the maximum concentration used for data analysis was 10−4.5 M. The lowest portion of 

the response curve was greater than 75% activity at all soluble concentrations of permethrin. 

Aromatase activity averaged 101.3% and 101.9% activity at concentrations of 10-10 and 10−4.5 M 

permethrin, respectively, indicating that permethrin did not inhibit aromatase activity in this 

assay. 

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

aromatase activity in this assay. 

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

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

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

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

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

The 21-day short-term reproduction assay (MRID 48702301) of permethrin with fathead 

minnow (Pimephales promelas) was conducted under flow-through conditions. Adult fish (6 

months old, 2 males and 4 females per replicate) were exposed to permethrin (97.7% purity) at 

nominal concentrations of 0 (negative and solvent controls), 0.00033, 0.00099, and 0.0030 mg 

a.i./L. Time-weighted average (TWA) concentrations were <0.000150 (<LOQ; controls), 

0.00022, 0.00075, and 0.0026 mg a.i./L. The solvent control consisted of dimethylformamide 

(DMF) at a final concentration of 0.02 mL/L. The test system was maintained at 24.6 to 25.8°C 

and a pH of 7.9 to 8.1. 

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

water) control. Survival in the negative control and the low and mid treatment groups was 100%, 

but there was 63% survival for males (5 of 8) and 50% survival for females (8 of 16) at the high 

treatment concentration. Additionally, clinical signs of toxicity occurring in the high treatment 

group included erratic swimming (5 fish), loss of equilibrium (2 fish), and surfacing (2 fish). 

There were also observations of excess feed noted in the test chambers of the high treatment 

group on Day 10 and Days 17-19. These observations indicate that overt toxicity was present 

during the course of this study at the high treatment concentration. 

Fecundity in the negative control averaged 19.8 eggs/female/reproductive day with all replicates 

having at least 15 eggs/female/reproductive day; spawning occurred at least every 4 days. 

Fertilization success in the negative control averaged 95.4%. Permethrin had no significant effect 

on fecundity or fertility in any treatment group relative to the negative control (p>0.05). While 

not statistically significant (p>0.05), there was a 42% decrease on fecundity at the high treatment 

concentration. 

Plasma vitellogenin (VTG) concentrations were comparable between the negative control and 

treatment groups for both sexes. While not statistically significant (p>0.05), there was a 37.8% 

reduction in VTG as compared to the negative control at the high treatment concentration. The 

data from this treatment group were based on three replicates and not four (as the rest of the 

treatment groups) as 3 of 4 female fish from Replicate A died during the course of the study. 

Effects on gonadal histopathology were observed in female fish at the high treatment level, 

including increased post-atretic follicles, decreased post-ovulatory follicles, and granulomatous 

inflammation. Microsporidia were observed in one fish each of the control and treatment 

concentrations that showed a minimal to mild severity of granulomatous inflammation. There 

were no effects of treatment on gonadal histopathology in male fish. 

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A significant increase (p<0.05) in gonado-somatic index (GSI) was detected at the high treatment 

level for both sexes (↑48.9% for males and 28.4% for females). Nuptial tubercle score was 

significantly decreased in males at the high concentration (<0.05). Permethrin exposure was not 

associated with any significant changes (p>0.05) in body weight or length for either sex. 

Other clinical signs (i.e., behavioral and other sublethal effects) including bloody lower jaw and 

weakness were observed in 1 to 3 of the 8 surviving females at the high treatment level. 

Hershberger Assay; (OCSPP 890.1400) 

In a Hershberger assay (MRID 48618602) screening for androgenic activity, permethrin (97.7% 

a.i., Lot# PMPS000014) in corn oil was administered daily via oral gavage to groups of 59 to 60-

day old, castrated male Sprague Dawley rats (8/group) at dose levels of 0 (vehicle), 37.5, or 

120 mg/kg/day. An androgenic positive control group consisting of eight castrated rats exposed 

to 0.4 mg/kg/day testosterone propionate (TP) by subcutaneous (s.c.) injection was also included 

as a positive control. 

To screen for potential anti-androgenic activity, permethrin in corn oil was also administered 

daily via oral gavage to 59 to 60-day old, castrated male Sprague Dawley rats (8/group) at dose 

levels of 12, 37.5, 120 mg/kg/day in conjunction with a daily dose of reference androgen TP at 

0.4 mg/kg/day by s.c. injection. The control group consisted of eight castrated male rats dosed 

daily with TP (0.4 mg/kg/day) by s.c. injection in corn oil, and the anti-androgenic positive 

control group consisted of eight castrated male rats dosed orally with flutamide (FT) in corn oil 

at 3 mg/kg/day in conjunction with a daily s.c. dose of TP (0.4 mg/kg/day). 

For both components of the assay, the animals were dosed for 10 consecutive days and 

necropsied approximately 24 hours after the final dose administration to determine weights of 

the five androgen-dependent tissues. 

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

the permethrin dose groups of the androgen agonist or antagonist assays. Body weights and 

overall body weight gains in the treated groups were similar to controls in both the androgen 

agonist and anti-androgen assays. 

In the androgen agonist assay, animals dosed with TP had increased (p<0.05) body weights 

(↑12%) on Day 11, and increased (p<0.05) overall body weight gains (↑110%) compared to 

controls. No significant increases in the organ weights were observed in any of the five 

accessory sex tissues of animals dosed with permethrin. Animals in the TP group had increases 

(p≤0.05) in accessory sex organ weights as follows: 1002% in seminal vesicles; 898% in ventral 

prostate; 187% in levator ani-bulbocavernosus (LABC); 486% in Cowper’s gland; and 51% in 

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glans penis. For the vehicle control, the maximum guideline-specified %CV was exceeded for 

the LABC (45% vs. 30%). All other %CV values were less than the maximum permissible 

values. 

In the anti-androgen assay positive control group (TP + FT), overall body weight gains were 

decreased (p≤0.05) by 35%. No significant decreases in the organ weights were observed in any 

of the five accessory sex tissues of animals dosed with permethrin+TP. Animals dosed with TP 

+ FT (positive control) had decreased (p≤0.05) accessory sex organ weights as follows: 84% in 

seminal vesicles; 77% in ventral prostate; 60% in LABC; 62% in Cowper’s gland; and 20% in 

glans penis. The maximum guideline-specified %CV was exceeded for the Cowper’s gland in 

the 120 mg/kg/day permethrin group (22% vs. 20%). All other %CV values were less than the 

maximum permissible values. 

The dosing for this study (high-dose of 120 mg/kg/day) was considered adequate based on data 

from two earlier studies showing either mortality at doses ≥200 mg/kg/day or clinical signs of 

neurotoxicity at 150 mg/kg/day. 

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

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

Hershberger assay. 

Female Pubertal Assay; (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID48672901), 16 Sprague-Dawley (Crl:CD® [SD] IGS) female 

rats/dose group were treated daily via oral gavage with permethrin (Lot # PMPS00014, 97.7% 

a.i.) at doses of 0 (corn oil) 75, or 150 mg/kg/day from post-natal day (PND) 22 to 42/43. 

Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and 

weight at attainment of VO were recorded. Following sacrifice on PND 42/43, blood was 

collected for clinical chemistry analyses, including analysis of total thyroxine (T4) and thyroid 

stimulating hormone (TSH) using a radioimmunoassay (RIA). Adrenal, liver, thyroid, pituitary 

and urogenital organ weights were recorded, and microscopic examinations were performed on 

the ovaries, uterus, thyroid, and kidneys. 

All rats administered permethrin survived until schedule termination. From PND 34 onward, 

tremors were observed in 4 animals administered 75 mg/kg/day and all animals administered 150 

mg/kg/day at 4-6 hours post-dosing on at least one day. These findings did not persist to the 

daily examinations, which were conducted just prior to dosing. 

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No treatment-related effects were observed on body weights, body weight gains, age of 

attainment of VO, or weight at day of attainment at any dose. All surviving animals attained 

complete VO. 

Absolute liver weights (adjusted and unadjusted) were increased (p<0.05) by 10% at 150 

mg/kg/day. Relative (to body weight) liver weights were increased (p<0.05) by 4 and 8% at 75 

and 150 mg/kg/day. Both unadjusted and adjusted thyroid weights were decreased (p<0.05) by 

22% at 150 mg/kg/day. 

Administration of permethrin did not change the day of first estrus compared to the vehicle 

control group. Animals exhibited similar cycle lengths and regularity. The majority of females 

in the 150 mg/kg/day group were in diestrus at necropsy. 

Serum T4 levels were decreased (p<0.05) by 26 and 42% at 75 and 150 mg/kg/day, respectively, 

and, serum TSH levels were increased (p<0.05) by 40% at 150 mg/kg/day. Additionally, at 150 

mg/kg/day, increases (p<0.05) were observed in sodium (↑3%), chloride (↑4%), and sorbitol 

dehydrogenase (SDH; ↑20%), alkaline phosphatase (ALP) was decreased (↓16%). 

There were no effects of treatment in the thyroid on either colloid area or follicular cell height in 

any treated group. No effects of treatment were observed microscopically in the ovaries, uterus 

or kidney. 

The dose levels tested were determined to be adequate since they were based on the results of the 

range finding study where mortality was seen at doses greater than 200 mg/kg/day and tremors at 

150 mg/kg/day. 

 

Male Pubertal Assay; (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48687101), 16 Sprague-Dawley [Crl:CD(SD) IGS] rats/dose 

group were treated daily via oral gavage with permethrin (97.7% a.i., Lot # PMPS00014) in corn 

oil at doses of 0, 75, or 150 mg/kg/day from postnatal day (PND) 23 to 53/54. However, three 

150 mg/kg/day rats were found dead after two days of dosing. Therefore, the high dose was 

reduced to 120 mg/kg/day on PND 25. Animals were examined for preputial separation (PPS) 

daily beginning on PND 30, and age and weight on day of attainment were recorded. Following 

sacrifice on PND 53/54, blood was collected for clinical chemistry and hormone analyses. Total 

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

determined using radioimmunoassays. Adrenal, liver, pituitary, thyroid, and urogenital organs 

were weighed and examined macroscopically. The left testes, left epididymides, left kidney, and 

thyroid were examined microscopically. 

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No treatment-related related effects were observed on body weights, body weight gains, age or 

weight at attainment of PPS, organ weights (except liver), clinical chemistry, and gross or 

histopathology parameters. 

After reduction of the high dose to 120 mg/kg/day, all remaining animals survived to study 

termination. At 6 hours post-dose, tremors were observed at 75 and 120 mg/kg/day in a dosedependent manner, with incidence, frequency, and severity increasing with dose. At 75 

mg/kg/day, three animals exhibited mild tremors on one day and one animal exhibited mild 

tremors on two days. At 120 mg/kg/day, very mild to severe tremors were observed in all 

surviving animals on several days. Additionally, one 120 mg/kg/day rat was considered 

uncoordinated on one day. 

Serum testosterone levels in the treated groups were similar to controls. Mean serum T4 levels 

were decreased (p<0.001) by 28 and 42% at 75 and 120 mg/kg/day, respectively, and serum TSH 

level was increased (p<0.05) by 77% at 120 mg/kg/day. The T4 and TSH values were all within 

or just below their respective reference ranges. Additionally, there were no concomitant effects 

observed on thyroid weights or thyroid follicular cell height or colloid area. Absolute and 

relative liver weight were increased (p<0.05) by 6-11% at 75 and 120 mg/kg/day. There were no 

changes in clinical chemistry parameters that suggested hepatotoxicity due to permethrin 

administration. The increased liver weights were considered to be the result of hypertrophy and 

associated microsomal enzyme induction, a common finding with permethrin. 

The 150 mg/kg/day dose was an excessive dose level as three rats were found dead on Day 2. 

Therefore, the dose was lowered to 120 mg/kg/day which was an adequate high dose based on 

the clinical signs (tremor and in coordination) seen in these animals. Tremors were also noted at 

75 mg/kg/day. 

Steroidogenesis Assay; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48618603) H295R cells cultured in vitro in 24-well plates 

were incubated with permethrin (97.7% a.i.; Batch PMPS000014) at concentrations of 0.0001, 

0.001, 0.01, 0.1, 1, 10 and 100 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was 

used as the vehicle, at a final concentration of 0.05%. 

Testosterone and estradiol levels were measured using HPLC/MS-MS. Three independent 

experiments were performed (Run #s 2-4). 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. 

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Guideline acceptability recommendations and requirements were met, including lack of 

cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low 

%CV) and appropriate induction and inhibition with positive controls. 

No cytotoxicity was observed over the range of tested permethrin concentrations. However, 

precipitation of the permethrin was observed at the 100 μM concentration in each run and at 

10 μM in Run #2. Accordingly, the data from these concentrations were not included in the 

analysis of the respective runs. For permethrin, there was a statistically significant (p<0.05) 

increase in testosterone production at 10 μM in Runs #3 and #4 (average 1.14-fold), and a 

statistically significant increase in estradiol production at 1 and 10 μM in Runs #3 and #4 

(average 1.16-fold for 1 μM and 1.51-fold for 10 μM). 

Based on hormone responses in two of the three independent runs, permethrin treatment resulted 

in statistically significant and reproducible increases in testosterone and estradiol production. 

Uterotrophic Assay; (OCSPP 890.1600) 

Requirement satisfied by OSRI (Kim et al., 2005; Kunimatsu et al., 2002); see Appendix 2.

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

ER Transactivation and Binding Assays 

In a published study by Saito et al. (MRID 48176003), the estrogenic and anti-estrogenic 

activity of permethrin (>93% purity) was evaluated using three in vitro assays based on hERαrelated mechanisms: 1) a mammalian cell-based luciferase reporter assay, 2) a yeast two-hybrid 

method, and 3) a competitive ligand-binding assay. For the luciferase reporter assay, HeLa cells 

were transiently transfected with an expression vector, a reporter plasmid for hERα, and a 

luciferase control plasmid. Permethrin was added to the medium at concentrations of 100 nM to 

10 μM. For anti-estrogenic activity measurements, 100 pM of E2 was incubated along with 

permethrin. For the yeast two-hybrid assay, yeast transformants were grown overnight and then 

treated with permethrin at 10 μM (maximum solubility). For antagonist assays, 100 pM of E2

was incubated with permethrin. After incubation, induced β-galactosidase was measured by 

chemiluminescent detection. Competitive binding to hERα was assayed by a fluorescence 

polarization method. Test chemical-dependent displacement of a labeling ligand, fluoromone 

ES1, from ERα was measured by changes in fluorescence anisotropy. Dose-dependent 

displacement of fluormone ES1 from hERα was detected with the positive controls DES, pnonylphenol, and HTM. 

In the luciferase reporter assay, permethrin was tested at a maximum dose of 30 μM and no 

statistically significant estrogenic or anti-estrogenic activity was detected. No statistically 

significant estrogenic or antiestrogenic activity for permethrin was detected in the yeast twohybrid assay or the receptor binding assay. The lack of statistically significant effects at 

concentrations of 100 nM-10 μM indicates that permethrin is not estrogenic or anti-estrogenic by 

classic ER-related pathways in vitro. 

Recombinant Yeast Assay 

In a published study by Tyler et al. (MRID 48213601), permethrin was tested for estrogen and 

androgen receptor binding activity (both agonist and antagonist) using a recombinant yeast 

assay. Two yeast strains were stably transfected to express either the human ER (hER) or the 

AR (hAR) genes along with the lacZ reporter gene. In addition to permethrin, three major 

derivatives of permethrin resulting from metabolism and/or environmental degradation were 

similarly tested: cyclopropane permethrin acid, 3-PBOH, and 3-phenoxybenzoic acid (3-PBA). 

Permethrin from four separate chemical suppliers was tested (purities 97.3-99.4%). In screens 

for agonistic activity, E2 and 5α-dihydrotestosterone (DHT) were used as positive controls. The 

positive controls in the anti-estrogen and anti-androgen screens were hydroxytamoxifen and 

flutamide, respectively. EC50s and IC50s were calculated relative to the reference standards, and 

for all chemicals inducing a response, LOEC (agonist) and/or LOIC (antagonists) were 

determined relative to the reference standards. 

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Permethrin was a very weak estrogen agonist with potencies between seven and eight orders of 

magnitude less than E2. In the anti-estrogen screen, increasing concentrations of permethrin 

produced a progressive increase in the E2-stimulated background absorbance, indicating an 

additive estrogenic response. In the hAR assay, very high concentrations of permethrin (~10−3

M) induced slight increases in activity above baseline values, thus indicating very weak agonist 

activity. Permethrin also exhibited weak anti-androgenic activity with a potency approximately 

500-fold less than flutamide. The following activities were presented for permethrin: Estrogenic 

LOEC = 3.1 ± 0.4 × 10−4

 M; estrogenic EC50 = 2 × 10−3 M; anti-androgenic LOIC = 3.0 ± 0.57 × 

10−5

 M; anti-androgenic IC50 = 7.3 ± 1.4 × 10−4 M. 

All three of the permethrin metabolites had endocrine activity. 3-Phenoxybenzyl alcohol had 

both estrogenic and anti-androgenic activity and was approximately 10−5-fold less potent than E2, 

but only 4-fold less potent than flutamide. 3-Phenoxybenzoic acid and permethrin cyclopropane 

acid both had anti-estrogenic activity and were approximately 100-fold and 1,000-fold less 

potent than 4-OH-tamoxifen, respectively. In the anti-estrogen screen, an increasing 

concentration of 3-PBOH produced progressive enhancement of the E2-stimulated absorbance, 

showing an additive estrogenic response. At concentrations greater than 8 × 10−3

 M, 3-PBOH 

was toxic to the yeast and caused lysis of the cells. 

Uterotrophic Assay (Rat) 

In a multi-component study, Kim et al. (MRID 48176002, 2005) examined the estrogenic 

effects of permethrin (96.6% purity) using Northern blot analysis of uterine Calbindin-D9k gene 

expression (CaBP-9k) and an uterotrophic assay using immature (18-day-old) female SD rats. 

CaBP-9k is an intracellular calcium binding protein that is estrogen-responsive in the rat uterus. 

Uterine tissues obtained during the uterotrophic assay were used for the Northern blot analysis. 

Permethrin (5, 10, 50, 100, 200, or 800 mg/kg) alone, plus E2 (3 μg/kg), or plus ICI 182,780 (3 

μg/kg) was administered to immature female rats by s.c. injection. For the uterotrophic assay, 

animals were sacrificed 24 h after the 3-day treatment, and the uterus and vagina were removed 

and weighed. For the CaBP-9k assay, total RNA from uterine tissues was extracted for Northern 

blot analysis of CaBP-9k mRNA. A description of the probe used was not provided. 

High dose (800 mg/kg) permethrin increased (p<0.05) relative (to body) uterine weights (data 

presented graphically). Permethrin also enhanced E2-induced uterine weight increases (data 

presented graphically); however, this was maximal at 200 mg/kg (p<0.05). It was stated that the 

permethrin-induced increases in relative uterine weights were inhibited by co-treatment with ICI 

182,780; however, the inhibitory effect was statistically different only at 50 mg/kg (p<0.01), and 

control weights were not provided. Relative vaginal weight was unaffected by treatment with 

permethrin. Northern blot analysis showed an up-regulation in CaBP-9k mRNA following 

permethrin exposure that was maximal at the lowest dose (5 mg/kg). Permethrin also enhanced 

the E2-induced increase in CaBP-9k mRNA levels in a non-dose-dependent fashion. 

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Uterotrophic (Rat) 

In a multi-component study, Kunimatsu et al. (MRID 48176008, 2002) performed a 

uterotrophic assay with permethrin (97.4% purity) using 5-week-old ovariectomized female 

Sprague-Dawley [Crj:CD(SD)IGS] rats. Groups of six females were dosed with permethrin in 

corn oil via oral gavage at dose levels of 0, 37.5, 75, or 150 mg/kg/day for 3 days. Two 

additional groups received ethynyl estradiol (30 μg/kg/day) or methoxychlor (125 mg/kg/day) as 

reference chemicals. One day after the final dose, rats were euthanized and uterus, liver, and 

kidneys were dissected and weighed. For the uterus, both wet (with luminal contents) and 

blotted (without luminal contents) weights were determined. 

Tremor was observed in 3-6/6 rats in the high-dose (150 mg/kg/day) group. No increases in 

uterus weight (wet or blotted) were observed. Reference controls ethynyl estradiol and 

methoxychlor both showed the expected effects. The results of the study provide no evidence 

that permethrin exhibits any potential to cause adverse (anti-) estrogenic effects at dose levels 

below those causing excessive systemic toxicity. 

Receptor Transactivation Assays 

In a published study by Du et al. 2010 (MRID 48196301), permethrin and its metabolite 3-PBA 

were tested for ER, AR, and thyroid hormone receptor (TR) binding activity. Agonist and 

antagonist interactions with the ER and TR were examined using transfected African green 

monkey kidney CV-1 cells and AR activation was determined using transfected MDA-kb2 cells. 

The agonist assays were conducted using permethrin (94.0% purity) at concentrations ranging 

from 10−9 M to 10−5 M; to test antagonist activity, the ER, AR, and TR tests were conducted at 

the same concentrations of permethrin in the presence of E2, DHT, and T3, respectively. The 

REC20 (relative effective concentration, agonist) and RIC20 (relative inhibitory concentration, 

antagonist) were calculated. 

Permethrin exhibited weak estrogen agonistic effects at concentrations from 10−8

 to 10−5 M 

(REC20 = 8.10×10−7

 M) and also exhibited weak antagonist effects at 10−6 and 10−5 M, in the 

presence of 1×10−9

 M E2 (RIC20 >10−5 M). The metabolite 3-PBA showed antagonist effects at 

10−6

 and 10−5 M, in the presence of 1×10−9 M E2 (RIC20 = 8.84×10−8 M). No AR agonist activity 

was found for permethrin or 3-PBA; however, permethrin and 3-PBA showed AR antagonist 

activity with a calculated RIC20 of 1.88 x 10−6

 M for permethrin and >10−5 M for 3-PBA. 

Similarly, permethrin and 3-PBA did not exhibit TR agonist activity but did show TR antagonist 

activity with a calculated RIC20 of 1.50×10−9 M for permethrin and 4.76×10−6 M for 3-PBA. 

ER Transactivation and Binding Assays 

In a published study by Chen et al. (MRID 48160705), the effect of permethrin (purity ≥90%) 

on cell proliferation in human breast cancer estrogen-sensitive (MCF-7) cells in the absence of 

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estrogens (negative control) and in the presence of 17β-estradiol (E2, positive control) was 

investigated (E-Screen assay). A binding assay using rat uterine cytosol ER was also studied and 

estrogen-responsive pS2 mRNA expression in MCF-7 cells treated with permethrin was 

measured by RT-PCR. 

For the cell proliferation assay, MCF-7 cells were plated and permethrin was tested in log 

increments at concentrations from 10−11 to 10−6 M. The bioassay was terminated on Day 6 and 

proliferation was determined by absorbance measurements after addition of thiazolyl blue 

solution. The proliferative effect (PE) is measured as the ratio between the highest cell yield 

obtained with the test chemical and the hormone free control and is expressed as -fold 

proliferation. Estrogenic activity was assessed by relative proliferative potency (RPP) which is 

the ratio of maximum PE for the chemical to PE for 17β-estradiol (E2). 

For the ER competitive-binding assay, 10−9

 M [3H]-estradiol was incubated with increasing 

concentrations of permethrin, uterine cytosol preparation, and buffer. The bound ligand was 

separated from the free ligand and radioactivity was measured. Data for permethrin and the E2

standard curve were plotted as percent [3H]-E2 bound vs. molar concentration, and the IC50 was 

determined. Permethrin was tested in log increments at concentrations from 10−12 to 10−3

 M. 

For the pS2 mRNA assay, MCF-7 cells in medium were treated with permethrin or E2 at various 

concentrations. All cells were harvested at the same time, total RNA was extracted, and mRNA 

for pS2 and β-actin were amplified by RT-PCR. 

In the E-Screen assay, an inverse U-shaped dose response curve was obtained that was 

significant at 10−10 to 10−7 M, with a maximum proliferation response at 10−8 M. The PE value 

for permethrin was 2.69. In comparison with the PE of E2 (3.46), permethrin showed a partial 

agonistic response. In the ER competitive-binding assay, permethrin inhibited the binding of 

[

3

H]-E2 at concentrations greater than 10−7 M, at a maximum of <67%. The pS2 mRNA 

expression increased 1.28-fold after 10−7 M permethrin treatment. 

ER Transactivation and Binding Assays 

In a published study by Kim et al. (MRID 48176007), the estrogenic activity of permethrin was 

evaluated using the E-screen assay (with the MCF-7 cell line) and a competitive ER binding 

assay. Cells were incubated for six days in the presence of permethrin (98% purity; 

concentrations not reported) prior to being assayed for cell proliferation. The competitive ER 

binding assay was conducted using uterine cytosols isolated from Sprague-Dawley rats. 

Aliquots of rat uterus cytosol were incubated with selected concentrations of permethrin and 

[

3

H]-estradiol. 

In the E-screen assay, the positive control, E2, induced cell proliferation in a dose dependent 

manner. Permethrin did not induce MCF-7B BUS cell proliferation at any of the concentrations 

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tested. To evaluate potential anti-estrogenic activity, cells were treated for six days at a 

concentration of 10−6

 M permethrin in the presence of either 10−10 or 10−11 M E2. 10−6 M 

permethrin significantly inhibited E2-induced cell proliferation. In the competitive ER binding 

assay, permethrin did not compete with [3

H]-estradiol at any of the concentrations tested. 

ER Transactivation Assays 

In a published study by M. Jin et al. (2010), permethrin (99% purity) and its metabolites 

PBCOH (98% purity), PBCHO (≥97% purity), and 3-PBA (98% purity) were tested for 

estrogenic activities in the MCF-7 cell line. A cell proliferation assay was conducted using 

MCF-7 cells incubated with medium containing 10−9

 M E2 (positive control), the test chemicals 

at 0.001-10 μM, or ethanol (0.1%, v/v; negative control). After 5 days, cell proliferation was 

determined by measuring the absorbance with a micro plate reader. The proliferative effect (PE), 

the ratio of the maximal cell yield of the test compound to the cell yield of the negative control, 

and the relative proliferative effect (RPE), the ratio of the PE for the test chemical to the PE of 

E2, were calculated for assessment of the estrogenicity of the chemicals. 

Estrogenic activity was also evaluated using a gene expression assay. MCF-7 cells were 

incubated for two days with the test chemicals at 0.001-10 μM, E2 at 10−9

 M (positive control), 

or ethanol at 0.1% (v/v; negative control). Total RNA was extracted and reverse transcribed for 

quantitative PCR analyses for the genes pS2 (an estrogen-responsive protein in breast tumors) 

and ERα. Based on the results of preliminary experiments, quantitative PCR measurements were 

conducted with the test chemicals at 10−6

 M. 

Permethrin induced proliferation (p<0.05) of MCF-7 cells at 10−8 to 10−6 M compared to the 

solvent control; RPE values at 10−7 M were determined to be 56.3% and 55.4%, compared to 

10−9 M E2 (100%). The three metabolites induced cell proliferation to various degrees with RPE 

values for PBCOH, PBCHO, and 3-PBA determined as 62.5%, 15.2%, and 28.6%, respectively. 

Permethrin and the metabolites PBCOH and 3-PBA induced proliferation of MCF-7 cells in a 

concentration-related manner; it was noted that the nonlinear response seemed to follow an 

inverted U-shaped dose-response pattern with the most significant effects observed at a 

concentration of 10−7

 M. 

All test chemicals were found to up-regulate pS2 transcription. Permethrin and the metabolite 

PBCOH induced pS2 expression 2.59- and 3.45-fold, respectively, compared to the control. The 

effects of the metabolites PBCHO and 3-PBA on the pS2 expression were much lower (1.37- and 

1.85-fold, respectively) than the other test chemicals. Permethrin and PBCOH at 10−6

 M 

decreased the ERα mRNA expression to levels similar to E2, which was found to down-regulate 

ERα by 50% compared to the control. Metabolites PBCHO and 3-PBA down-regulated the ERα

expression levels at lower levels (3.7% and 12.0%, respectively). The relative inductive 

efficiency of the test chemicals on the up-regulation of pS2 expression compared to that of E2

and the relative inhibitory efficiency of the test chemicals on the down-regulation of ERα

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regulation compared to that of E2 were calculated. In the pS2 expression assay, PBCOH showed 

a fully estrogenic response (82%), and permethrin and 3-PBA showed a partially estrogenic 

response (29-53%); PBCHO showed only a weakly estrogenic response (13%). Similarly, in the 

ERα expression assay, PBCOH a showed fully estrogenic response (89%), and permethrin 

showed a partially estrogenic response (69%); 3-PBA showed only a weakly estrogenic response 

(24%), and the PBCHO response was <10% (negative response). 

Estrogenic Potential

In a published study by Garey and Wolff (MRID 48176005, 1998), permethrin (95.7% purity) 

was evaluated for estrogen and progesterone agonist/antagonist activities using two in vitro

systems based on human cells (Ishikawa Variant-I human endometrial cancer cell line for 

estrogen effects and T47D human breast cancer cell line for progesterone effects). Both cell 

lines produce alkaline phosphatase (ALP) as an indicator of hormonal activity. Test compounds 

and standards were dissolved in ethanol and diluted in medium, added to cells in plates, and 

incubated. After a 48-h exposure period, cells were rinsed and a p-nitrophenyl phosphate 

solution was added to each well. The resulting formation of p-nitrophenol was detected using a 

plate reader. 

In Ishikawa Var-I cells, permethrin did not demonstrate estrogen agonist or antagonist activities; 

in T47D cells, permethrin did not exhibit progesterone agonist or antagonist activities. 

Estrogenic Potential

In a published study by Go et al. (MRID 48176004), permethrin (95.7% purity) was evaluated 

for estrogenic potential in the MCF-7 estrogen-sensitive cell line, as measured by expression of 

pS2 and cell proliferation. For the pS2 expression assay, MCF-7 cells were incubated for 3 days 

and then exposed to permethrin for 48 hours. Total RNA was extracted and pS2 RNA was 

detected by Northern blot and autoradiography with quantification by densitometry. Estradiol 

(E2) was used as the positive control. Results were expressed as the ratio of permethrin to the 

vehicle control. In the MCF-7 cell proliferation assay, cells were exposed to permethrin at 

concentrations of 0.001-100 μM. Cell proliferation was determined on Day 6 by counting with a 

Coulter counter. 

Permethrin did not induce pS2 above the negative control or inhibit E2 induction of pS2 in initial 

experiments at 5 μM, but at higher concentrations (100 μM), it weakly induced pS2 expression. 

In combination with 0.1 nM E2, permethrin had little effect on pS2 expression levels stimulated 

by E2. At concentrations below 10 μM, permethrin did not induce MCF-7 cell growth, but 

induced modest increases in proliferation at 100 μM. 

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ER Transcriptional Activation Assay

In a published study by Lemaire et al. (MRID 48074114), permethrin was evaluated for ERα

and ERβ activation or inhibition in stable reporter cell lines. Stable transfection of the ERα and 

ERβ constructs together with an estrogen reporter luciferase vector into HeLa cells resulted in 

two E2-sensitive cell lines. The cells were incubated with permethrin (≥95% purity) for 16 h. At 

the end of incubation, a commercial luciferase assay system was added and luciferase activity 

was measured with a luminometer. For the ERα and ERβ competitive-binding assay, ERα and 

ERβ cells were treated with 0.1 nM [3

H]-E2 in the presence or absence of increasing 

concentrations of unlabeled E2 (as control) or permethrin. Total cell lysate was counted by liquid 

scintillation. 

With the transactivation activity of E2 set at 100%, the estrogenic activity percentages of 10 μM 

permethrin were 13.0 ± 0.4% for ERα and 14.2 ± 2.4% for ERβ; these were not statistically 

different from the control values of 9.3 ± 1.3% and 10.8 ± 1.6%, respectively. Permethrin 

showed no anti-estrogenic activity in either ERα or ERβ. 

AR Binding Assay 

In a published study by Bauer et al. (MRID 48176001, 2002), permethrin (97.5% purity) was 

evaluated for its AR binding capability using an immuno-immobilized androgen receptor assay 

(IRA) with recombinant human androgen receptor (rhAR). The receptor preparation was diluted 

and incubated with 3H-DHT in the presence or absence of increasing concentrations of 

permethrin or DHT. After washing unbound radioactivity, wells were separated and transferred 

into scintillation vials and radioactivity was counted. Specific binding was calculated as the 

difference of total binding and non-specific binding in the presence of a 200-fold surplus of 

unlabelled DHT. The relative binding affinity of permethrin was compared to DHT using the 

inhibition constants (Ki) of each compound to calculate the relative binding affinity, where RBA 

= (Ki(DHT) × 100) ÷ Ki(permethrin). 

The Ki (permethrin) could not be determined because there was no detectable displacement of 

DHT up to the highest concentration tested (unreported). Therefore the permethrin RBA was 

reported as <0.01%. 

Androgen Receptor Transactivation Assays 

In a published study by Xu et al. (MRID 48176006), the potential androgenic and antiandrogenic activities of permethrin (>98% purity) were evaluated using a transiently-transfected 

human AR reporter gene assay in an African monkey kidney cell line (CV-1). After a 24 h 

incubation period, 0.1% ethanol (vehicle control) or 1 nM DHT (positive control), and various 

concentrations of permethrin (10−7

 to 10−5 M) were added for measurement of agonistic or 

antagonistic activity. The cells were harvested 24 h after dosing, lysed, and the lysate analyzed 

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for chloramphenicol transferase (CAT) and β-galactosidase (β-Gal) activities using commercial 

kits. The amount of CAT for each lysate was normalized to the β-Gal activity. 

There were no indications of cytotoxicity at the dose levels tested; there was no significant 

difference in the amounts of β-Gal between the treated groups and vehicle control groups. 

Permethrin did not exhibit androgenic activity at the tested concentrations. Permethrin did 

attenuate an increase in the CAT amount induced by DHT; the inhibitory effect was detected at 

1 × 10−5

 M. The IC50 value was (5.86 ± 2.20) × 10−5 M. 

Androgen Receptor Transactivation Assays 

As part of a multi-component published study by Zhang et al. (MRID 48196302, 2008), the 

potential anti-androgenic effects of permethrin (purity not reported) were examined using an 

androgen-responsive cell line (MDA-kb2). Various concentrations of permethrin with and 

without DHT (1 nM) were tested. 

Permethrin was found to significantly inhibit DHT-induced transcriptional activation at a 

concentration of 10−5

 M (data presented graphically). 

Hershberger Assay (Rat) 

In a multi-component study, Kim et al. (MRID 48176002) conducted a Hershberger assay with 

permethrin (96.6% purity) using 5-week-old male SD rats. Male SD rats were castrated at 5 

weeks of age. Permethrin was administered by oral gavage at 10, 50 or 100 mg/kg/day for 10 

days (n=6–8). As a reference androgen, 0.4 mg/kg TP was injected subcutaneously. To examine 

anti-androgenic activity in a second group of rats, TP-treated rats (n=6–8) were administered 

permethrin at 10, 50 or 100 mg/kg/day for 10 days. Flutamide (25 mg/kg/day) and p,p ́-DDE 

(100 mg/kg/day) were used as reference anti-androgens. Animals were evaluated daily for 

mortality, injury and general appearance, and individual body weights were recorded every three 

days. Twenty-four hours after the final dose the five androgen-dependent tissues were excised 

and weighed. 

No statistically significant changes in body weights were noted in any of the treatment groups. 

The positive control, TP, induced statistically significant increases in the absolute and relative 

weights of all of the androgen-dependent sex accessory tissues. Permethrin did not show an 

agonist effect on androgen-dependent tissue weights at any dose tested. Flutamide and p,p ́-

DDE reduced sex accessory tissue weights in TP-treated castrated males verses the TP-treated 

group. Administration of permethrin to TP-treated castrated rats led to statistically significant 

reductions (antagonism) in the five sex accessory tissue weights compared to TP at all doses 

tested, though not in a dose-dependent manner, nor to the extent of flutamide or p,p ́-DDE. 

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Hershberger Assay (Rat) 

In a multi-component study, Kunimatsu et al. (MRID 48176008) conducted a 5-day 

Hershberger assay with permethrin (97.4% purity) using 9-week-old castrated male SpragueDawley [Crj:CD(SD)IGS] rats. Groups of six males were dosed with permethrin in corn oil via 

oral gavage at dose levels of 0, 25, 50, or 75 mg/kg/day for 5 days with or without 0.25 

mg/kg/day TP administered by s.c. injection as a reference androgen (eight groups). For the 4 

groups treated with TP, an additional group received 100 mg/kg/day p,p ́-DDE (gavage) as a 

reference control for assessing weak anti-androgenic activity. For the groups receiving no TP, an 

additional group received 100 mg/kg/day methyltestosterone via gavage as a reference control 

for androgenicity. One day after the final administration of permethrin, rats were euthanized, the 

sex accessory glands/tissues (ventral prostate, dorsolateral prostate, seminal vesicles with 

coagulating glands and LABC), liver and kidneys were dissected and weighed. 

Tremor was noted in 2-6/6 rats at 75 mg/kg/day. No toxicologically significant changes were 

observed in body weight, food consumption, or organ weights. No androgenic or antiandrogenic effects resulted from treatment with permethrin. Reference controls p,p ́-DDE and 

methyltestosterone produced the expected effects. The results of the study provide no evidence 

that permethrin exhibits any potential to cause adverse (anti-) androgenic effects at dose levels 

below those causing excessive systemic toxicity. 

Hershberger Assay (Rat) 

As part of a multi-component published study by Zhang et al. (MRID 48196302, 2008), a 

Hershberger assay was conducted with permethrin (purity not reported) using 3-week-old male 

Sprague-Dawley rats. After 7 days acclimation, the rats were castrated and allowed to recover 

for 2 weeks. Following recovery, rats were weighed and randomly assigned to experimental 

groups (n=6). The groups consisted of controls (0.5 mg/kg/day TP s.c.), TP + flutamide (50 

mg/kg/day p.o.), and TP + permethrin (50 mg/kg/day orally). The treatment period was 7 days 

for all groups. Following the last dose, rats were euthanized by exsanguination. The seminal 

vesicles, ventral prostate, dorsolateral prostate, LABC muscle, Cowper’s glands, glans penis, 

liver, paired adrenal glands, spleen and paired kidneys were dissected and weighed. Serum 

testosterone was determined using commercially available FIA kits. 

No mortality or treatment-related clinical signs were noted in any of the groups. It was reported 

that there were no significant differences between initial or final body weights, or weight gains 

of the groups compared to the control group. Permethrin caused significant decreases in seminal 

vesicle and ventral prostate weight (data presented graphically). 

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Three-Generation Reproduction Toxicity (Rat, OCSPP 870.3800) 

In a two-generation reproduction toxicity study (MRID 00120271), permethrin (94.0-98.8% 

purity) was administered to groups of 12 male and 24 female Wistar rats in the diet at 

concentrations of 0, 500, 1000, or 2500 ppm (0, 25, 50, and 125 mg/kg/day, respectively, using a 

standard conversion factor of 0.05). Two litters were produced by each generation. Parental 

animals in each generation received test or control diet for 12 weeks post-weaning and were then 

paired for mating to produce the A litters. After various rest periods, the parental animals were 

mated again to produce the B litters. Test diets were administered during mating, gestation and 

lactation for three successive generations throughout the study. The F2 parents were mated for a 

third time, using the same breeding pairs as for the B litters, producing the C litters for a 

developmental toxicity evaluation. Ten males of the F1 generation were maintained on 

experimental diets until they were 54-55 weeks old and were submitted for microscopic 

examination of selected neurological tissues. 

There were no treatment-related parental deaths. A few parental animals were euthanized for 

humane reasons. There were no treatment-related effects on body weights, body weight gains, 

food consumption, or food efficiency. 

Treatment-related clinical signs in high-dose parental animals were limited to whole body 

tremors, occurring in all parental generations (exception: tremors were not observed in the P 

males) during the first few days of the premating period. In the 2500 ppm groups, the incidence 

rates for the tremors were 20/24 (P females), 11/12 and 24/24 (F1 males and females, 

respectively), and 12/12 and 24/24 (F2 males and females, respectively). Tremors were also 

observed in pregnant and lactating females exposed to 2500 ppm permethrin. The tremors were 

intermittent and transient. Neuropathy was not observed in a special microscopic examination of 

selected neurological tissues from F1 males continued on test for one year. 

Gross examination at necropsy did not reveal any dose- or treatment-related findings, nor did 

microscopic examination of grossly abnormal tissues from all parents surviving to scheduled 

termination and of reproductive tissues from animals suspected of infertility. 

The LOAEL for systemic toxicity is 2500 ppm (125 mg/kg/day) based on tremors observed in 

the P females, and the F1 and F2 males and females. The systemic toxicity NOAEL is 1000 ppm 

(50 mg/kg/day). 

There were no treatment-related effects in the reproductive performances (male or female 

fertility indices, gestation index, gestation length, live birth index, viability index, lactation 

index, parturition, or fetal sex ratio) of the F1, F2 and F3 generations. In the F3c litters, there were 

no developmental effects associated with the administration of permethrin over three 

generations. The percentages of male fetuses of the 1000 and 2500 ppm groups (39.0 and 

44.7%, respectively) were lower than the control value (53.2%), but the effect was not associated 

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with increased resorptions and was not dose-related. Also, no consistent effect on sex ratios was 

observed in other litters or generations of the study. 

Microscopic examination of F3b weanlings revealed dose-related increases in centrilobular 

hypertrophy of the liver. The incidences of slight and moderate centrilobular hypertrophy were 

dose-related, ranging from 0 to 80% for the males and from 10 to 100% for the females. The 

hypertrophy of the liver was an adaptive and reversible effect and was not considered adverse. 

The reproductive toxicity and offspring toxicity NOAEL is ≥2500 ppm (125 mg/kg/day) and the 

reproductive toxicity and offspring toxicity LOAEL is not identified. 

Developmental Toxicity (Rat, OCSPP 870.3700) 

In a developmental toxicity study (MRID 40943603), 24 presumed pregnant Wistar rats per 

group were administered 0, 15, 50, or 150 mg/kg/day of permethrin (93.9% purity; 38 cis:62 

trans isomers) in corn oil by oral gavage on gestation days (GD) 7-16, inclusive. On GD 22, all 

surviving dams were sacrificed and all fetuses were weighed, sexed, and examined for external, 

visceral, and skeletal malformations/variations. 

All dams survived to scheduled termination and no treatment-related abnormalities were noted at 

necropsy. There were no effects of treatment on clinical signs, body weight gains, or food 

consumption in the low- or mid-dose groups. In the high-dose group, clinical signs of toxicity 

seen between GD 8-19 included tremors in 21/24 rats and head flicking in 6/24 rats. Body 

weight gains and food consumption by the high-dose dams were less (p<0.05) than that of the 

controls throughout the dosing interval. For GD 7-10, 10-13, and 13-16, body weight gains were 

decreased by 88%, 32%, and 18%, respectively. 

The maternal LOAEL is 150 mg/kg/day based on clinical signs of toxicity and decreased body 

weight gain and food consumption. The maternal NOAEL is 50 mg/kg/day. 

No dose- or treatment-related effects were observed on gravid uterine weights, fetal sex ratios, 

pre- or post-implantation losses, or numbers of corpora lutea/dam or live fetuses/dam. Mean 

fetal body weight of the high-dose group was 3.2% (p ≤ 0.05) less than that of the controls. 

However, mean litter weight of the high-dose group was 3% (NS) greater than that of the 

controls. Therefore, the reduced fetal body weights were considered a questionable toxic 

response. 

No treatment-related external or visceral fetal malformations/variations were noted. The fetal and 

litter incidence rates of short length extra ribs were increased (p<0.05) in the high-dose group as 

compared with the controls. Short length extra ribs were observed in 31% of the high-dose 

fetuses vs. 11% of the control fetuses and in 87% of high-dose litters vs. 57% of control litters. 

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The developmental LOAEL is 150 mg/kg/day based on decrease in fetal body weights and an 

increase in the incidence rate of short length extra ribs. The developmental NOAEL is 50 

mg/kg/day. 

Developmental Toxicity (Rabbit, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 40943602), presumed pregnant Dutch rabbits 

were administered 0, 600, 1200, or 1800 mg/kg/day of permethrin (92.5% purity; 32.3 cis:60.2 

trans isomers) in 0.5% aqueous Tween 80 by oral gavage on GD 6-18, inclusive. The number of 

does mated for each group was 19, 21, 20, and 23, respectively. On GD 29, all surviving does 

were sacrificed and all fetuses were weighed and examined for external 

malformations/variations. Approximately one-half of the fetuses were processed for skeletal 

examination and the remaining one-half was fixed and examined for visceral anomalies. 

A total of 0, 5, 5, or 4 does died or were sacrificed moribund in the control, low-, mid-, or highdose groups, respectively. Due to the lack of a dose-response, the deaths could not be 

definitively attributed to test article administration. Clinical signs of toxicity included body 

tremors observed in 5 of the high-dose animals only. Little or no feces or urine was noted on at 

least one occasion for 2/19 (11%), 4/21 (19%), 6/20 (30%), and 8/23 (35%) animals in the 

control, low-, mid-, and high-dose groups, respectively. 

Absolute body weights were similar between the treated and control groups throughout the study. 

However, after examining the replotted body weight data, there was a sharp drop in weight for 

the low, mid, and high dose groups after Day 6 and only a slight drop for the control that was 

noticeable after Day 12. Body weight gains by the low-, mid-, and high-dose groups were 

decreased by 79%, 50%, and 91%, respectively, during GD 0-18 with significance (p<0.05) 

attained for the low- and high-dose groups. During the post-dosing interval, recovery of body 

weights was noted for the low- and mid-dose groups, but not for the high-dose group. 

The maternal LOAEL is estimated to be <600 mg/kg/day based on decreased body weight gain. 

The maternal NOAEL is not identified. 

The number of live fetuses and mean litter size was decreased for all dose groups compared to 

the control group (110(15), 80(13), 69(14), and 72(13) for control, low-, mid-, and high-dose 

groups, respectively). However, no dose-response was evident or statistical significance noted. 

Post-implantation loss was increased (p<0.05) in the mid- and high-dose groups to 155% and 

248% of the control level. Correspondingly, the number of early and late resorptions was higher 

in these groups as compared to the control group values. Mean fetal body weights in the highdose group were slightly (↓9%; NS) less than that of the controls and attributed to maternal body 

weight decreases. No dose-related or statistical differences were observed between the treated 

and control groups for number of fetuses/litter or mean gravid uterine weights. 

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No treatment-related external or visceral fetal malformations/variations were noted. In the midand high-dose groups, reduced ossification of the fore- and hindlimbs was indicated by slightly 

(NS) greater ossification scores as compared with the controls. Mean scores for the control, 

low-, mid-, and high-dose groups were 1.92, 1.99, 2.00, and 2.25, respectively, for the forelimb 

and 1.65, 1.56, 1.89, and 1.90, respectively, for the hindlimb. 

Therefore, the developmental LOAEL is 1200 mg/kg/day based on increased post-implantation 

loss, greater numbers of early and late resorptions and an equivocal decrease in ossification of 

the fore- and hindlimbs. The developmental NOAEL is 600 mg/kg/day. 

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300) 

In a combined chronic toxicity/carcinogenicity study (MRID 00069701), permethrin was 

administered to 60 rats/sex/group in the feed at doses of 0, 500, 1000, or 2500 ppm (mean 

estimated intake 0/0, 19.4/19.1, 36.9/40.2, and 91.5/104 mg/kg/day in males/females, 

respectively). Twelve rats/sex/group were sacrificed at 52 weeks and the surviving rats were 

sacrificed at 104 weeks exposure. 

There was no treatment-related effect on mortality or tumor induction. During the first two 

weeks of the study, treatment-related tremors and hypersensitivity were observed in the highdose males and females. There were no effects on body weight, body weight gain, food 

consumption or food efficiency, ophthalmologic endpoints, hematologic endpoints, clinical 

chemistry, or urinalysis parameters. 

Liver changes suggestive of adaptive hypertrophy included increased aminopyrine-Ndemethylase activity in all male treatment groups, in the mid- and high-dose female at 52 weeks, 

and in the high-dose male and female groups at 104 weeks. This was coupled with modestly 

increased absolute and relative liver weights in the high-dose males and high and low-dose 

females at 52 weeks and in all male treatment groups and mid-dose females at 104 weeks. 

Further evidence for adaptive changes included hypertrophy of centrilobular hepatocytes with 

increased cytoplasmic eosinophilia in the mid- and high-dose male and females at 104 weeks’ 

exposure and increased smooth endoplasmic reticulum proliferation in all treatment groups 

except low-dose males at 52 weeks and high-dose groups at 104 weeks. Liver changes also 

included fatty vacuoles confirmed by electron microscopy in the mid- and high-dose males at 

both 52 and 104 weeks and in the high-dose females at 104 weeks. It was concluded that the 

increased liver weight and hypertrophy observed in the liver were adaptive and reversible and 

were not adverse. 

Under the conditions of this study, the LOAEL is 2500 ppm (91.5/104 mg/kg/day in 

males/females, respectively) based on tremors and hypersensitivity. The NOAEL is 1000 ppm 

(36.9/40.2 mg/kg/day in males/females, respectively). 

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At the doses tested, permethrin did not affect the incidence of tumor-bearing animals or the 

incidence of any specific tumor type in either sex. Permethrin was not carcinogenic to the rat. 

Dosing was considered adequate based on liver effects and on tremors and hypersensitivity in 

male and female rats. 

No treatment-related changes were seen in absolute or relative weights of the testes, ovaries, 

pituitary, or adrenal glands at any dose level. No treatment-related histopathological lesions 

were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, uterus, cervix, 

mammary, thyroid, adrenal or pituitary glands at any dose level. 

Chronic Toxicity (Dog, OCSPP 870.4100) 

In a chronic toxicity study (MRID 00129600), permethrin (92.5%, purity, cis/trans 32.3/60.2) 

was administered to beagle dogs (6/sex/group) in corn oil by gelatin capsule at dose levels of 0, 

5, 100, or 1000 mg/kg/day for one year. The high dose was lowered from 2000 mg/kg/day after 

2 days due to overt toxic reaction to the test material. 

There were no mortalities. Neurological clinical signs (tremors, uncoordinated gait, nervousness 

and convulsions, also excessive salivation and vomiting) were observed in the high-dose group. 

At the high-dose, decreased body weight gain (37% for males and 33% for females less than 

control, respectively), decreased food consumption (increased food left uneaten), increased liver 

weight (+30% and +36% for males and females, respectively) and alkaline phosphatase level 

(+377% and +220% for males and females, respectively) were reported. At mid-dose, increased 

liver weight (+25% both sexes) and alkaline phosphatase levels (+134% for males and +99% for 

females) were observed. 

The thyroid showed apparent increases in weight for the males (+27%, +28% and +17%) and the 

females (-5%, +27% and +33%) for the low, mid and high dose test groups. No treatmentrelated changes were seen in absolute or relative weights of the testes, ovaries, adrenal or 

pituitary glands at any dose level. No treatment-related histopathological lesions were seen in 

the testes, epididymides, prostate, ovaries, uterus, cervix, mammary, thyroid, or pituitary glands 

at any dose level. Microscopic evaluation of the adrenals showed focal degeneration and 

necrosis in the cortex with variable inflammatory cell infiltration along with swelling and 

vacuolization of the cells in the inner cortex at high-dose males and females (5/5 males and 4/6 

females) and at mid-dose males (1/5). The liver also showed hepatic cellular swelling at midand high-dose males and females. 

The observations of increased liver weight, alkaline phosphatase levels, and hepatic cellular 

swelling were considered adaptive and reversible and were not adverse. Therefore, the systemic 

toxicity LOAEL is 1000 mg/kg/day based on clinical neurotoxic signs and decreased body 

weight gain and food consumption. The NOAEL is 100 mg/kg/day. 

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28-Day Oral (Feeding) Toxicity (Rat) 

In a 28-day oral toxicity range-finding study (MRID 00120267), permethrin (90.5% purity) was 

administered to Wistar rats (8/sex/dose) in the diet at dose levels of 0, 200, 500, 1000, 2500, 

5000, or 10,000 ppm (equivalent to 0/0, 20/20, 51/51, 106/76, 267/207, and 319/428 mg/kg/day 

in males/females, respectively). The mg/kg equivalent of the 10,000 ppm dose could not be 

calculated, since all rats receiving that dose died within three days. 

At 5000 ppm, one female and four males died by Day 18. No other deaths were observed. 

Surviving rats receiving 2500 ppm and above became hypersensitive during the first week of 

treatment and remained so throughout the study. Body weight of the 5000 ppm males was 16-

22% below that of controls throughout the treatment period, and overall body weight gain of that 

group was decreased by 31%. Liver weight was increased 28-33% in the 2500 and 5000 ppm 

females. The relative (to body) liver weight was increased in the 2500 and 5000 ppm males, and 

in all treated females except those receiving 500 ppm. The liver changes were judged to be an 

adaptive response. Testis weight was decreased 10-13% in 2500- and 5000-ppm males, and 

relative adrenals weight was decreased 29% in 5000-ppm females; neither of these organ weight 

changes showed a dose-response relationship. There were no treatment-related changes in food 

consumption, hematology, clinical chemistry, or urinalysis of any group of rats. No treatmentrelated changes were seen in absolute or relative weights of the ovaries or pituitary glands at any 

dose level. No treatment-related histopathological lesions were seen in the testes, epididymides, 

seminal vesicle, prostate, ovaries, uterus, mammary, thyroid, adrenal or pituitary glands at any 

dose level. 

Under the conditions of this study, the LOAEL for permethrin in rats is 2500 ppm (equivalent to 

267/207 mg/kg/day for males/females, respectively) based on hypersensitivity. The NOAEL is 

1000 ppm (equivalent to 106/76 mg/kg/day for males/females, respectively). 

Testicular Toxicity (Mouse) 

In a published study by Y. Jin et al. (2012), 5 male ICR mice/dose group were administered one 

of the four permethrin enantiomers, (+)-cis-, (−)-cis-, (+)-trans-, and (−)-trans-permethrin 

(separated from racemic permethrin, >95% purity), in DMSO orally at dose levels of 0 (DMSO), 

25, 50, or 100 mg/kg/day from PND 21 to PND 42. Mice were sacrificed on the last day of 

treatment. Blood was collected, and serum testosterone levels were measured using a 

radioimmunoassay. The testes were weighed, and the left testis in each group was fixed, 

processed, sectioned, and stained with hematoxylin and eosin for histological analysis. Total 

RNA was isolated from the testes of mice and then reverse transcribed for quantitative PCR to 

determine the expression of 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase), 3-

hydroxy-3-methylglutaryl-CoA reductase (HMC-CoA reductase), low-density lipoprotein 

receptor (LDL-R), scavenger receptor class B type 1 (SR-B1, high-density lipoprotein receptor), 

peripheral benzodiazepine receptor (PBR), steroidogenic acute regulatory protein (StAR), 

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cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid 

dehydrogenase (3β-HSD), cytochrome P450 17α-hydroxysteroid dehydrogenase (P450 17α), and 

17β-hydroxysteroid dehydrogenase (17β-HSD). 

No clinical signs of toxicity were observed and there were no effects of treatment on body 

weight. At 100 mg/kg, both absolute and relative weights of testes in the (+)-cis, (−)-cis and 

(−)-trans-permethrin treated groups were decreased significantly (p<0.05) compared to the 

control; no significant differences were found in the (+)-trans-permethrin treated group. No 

histological changes in the testes were observed in mice exposed to 25 or 50 mg/kg of any of the 

permethrin enantiomers. However, the number of spermatogenic cells was decreased in the 

(+)-cis, (−)-cis, and (−)-trans-permethrin treated groups at 100 mg/kg. Large interstitial spaces 

in the seminiferous tubules were also observed in the 100 mg/kg (+)-cis, (−)-cis, and (−)-transpermethrin treated groups. At 100 mg/kg (+)-cis, (−)-cis, and (−)-trans permethrin exposure, 

serum testosterone levels were significantly reduced compared to the control. However, when 

the groups treated with the different enantiomer types were compared to each other, no 

significant differences in testosterone concentration were observed at any dose. 

The effects of permethrin enantiomer exposure on the mRNA levels of the main genes related to 

cholesterol synthesis in the testes were evaluated, as cholesterol is the main precursor for steroid 

hormones such as testosterone. The mRNA levels of HMG-CoA synthase decreased 

significantly in the testes of the mice after treatment with 25, 50, and 100 mg/kg (+)-cis, (−)-cis, 

(+)-trans, and (−)-trans enantiomers compared to the control; however, no significant differences 

were observed between the 4 enantiomers. For HMG-CoA reductase, significant differences in 

mRNA levels were not observed between the 25 mg/kg enantiomer-treated mice and control or 

between the different enantiomer groups. At 50 and 100 mg/kg, enantiomer-specific responses 

were observed. The mRNA levels of HMG-CoA reductase were only significantly decreased 

compared to the control in the 50 and 100 mg/kg (+)-cis enantiomer treated groups (↓40% and 

48%, respectively). Significant differences were also found between the (+)-cis and (−)-cis or 

(−)-trans groups at 50 mg/kg dosage. The mRNA levels of testicular SR-B1 in all permethrin 

enantiomer treated groups were slightly inhibited, but did not attain statistical significance. No 

significant differences in LDL-R mRNA levels were observed in any permethrin enantiomertreated group compared to the control 

PBR and StAR play key regulatory roles in cholesterol transport from the outer to the inner 

mitochondrial membrane. PBR mRNA levels were decreased by permethrin treatment in dosedependent and enantiomer-specific manners. At 25 and 50 mg/kg exposure levels, the testicular 

mRNA level of PBR was decreased significantly only in the (+)-cis enantiomer treatment group, 

while at 100 mg/kg it was significantly decreased in all enantiomer groups compared with the 

control group (↓39%, 47%, 47%, and 33%, respectively). An enantiomer-specific effect on PBR 

expression was observed at both 25 and 50 mg/kg concentrations; a significant difference in PBR 

mRNA expression was found between the (+)-cis and (−)-trans groups. The testicular StAR 

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mRNA levels were down-regulated significantly in the 25, 50, and 100 mg/kg (+)-cis and 

(−)-trans enantiomer-treated groups compared to control. Additionally, StAR mRNA levels 

were down-regulated in the (−)-cis and (+)-trans enantiomer-treated groups at 50 mg/kg and the 

(−)-cis enantiomer group at 100 mg/kg. Moreover, significant differences were also found 

between the (+)-trans and (−)-trans groups at 100 mg/kg. 

Key genes in the testosterone synthetic pathway, including P450scc, 3β-SHD, P450 17α and 

17β-HSD, in the testes of mice were also examined. A significant decrease in P450scc mRNA 

levels appeared in the 25 mg/kg (+)-cis and (−)-trans enantiomer groups compared to controls. 

At 50 or 100 mg/kg, the testicular P450scc mRNA levels were significantly inhibited by all four 

enantiomers. Treatment with the (+)-cis enantiomer induced a dose-dependent decrease in 3βHSD mRNA levels, with significance reached at 50 and 100 mg/kg (+)-cis enantiomer (↓30% 

and 46%, respectively), and treatment with the (-)-cis enantiomer resulted in a significant 

decrease at 100 mg/kg. The mRNA levels of P450 17α were not influenced significantly 

regardless of the permethrin enantiomers or administration doses. Significant differences in the 

mRNA levels of testicular P450scc, 3β-HSD and P450 17α were not observed between the 

permethrin enantiomers for any dose level. However, the effects on 17β-HSD appeared in a 

different manner. First, a significant decrease was observed in the (+)-cis enantiomer in all 

groups (25, 50, and 100 mg/kg) compared to the control. Second, at 100 mg/kg, gene expression 

was decreased significantly for all the permethrin enantiomers. Third, there was a significant 

difference between the (+)-cis and (+)-trans groups and between the (-)-cis and (+)-trans groups 

at 25 and 50 mg/kg. 

Testicular Toxicity (Mouse) 

In a published study by Wang et al. (2012), nine-week-old male Sv/129 mice were dosed by 

daily oral gavage with 10 μmol/kg diazinon (equivalent to 3 mg/kg), 90 μmol/kg cis-permethrin 

(99.5%; equivalent to 35 mg/kg), or diazinon+cis-permethrin at 100 μmol/kg for 6 weeks; a 

control group was dosed with corn oil alone. After the first treatment and just before the last 

treatment, mice were placed in individual cages to collect urine for 24 hours; levels of cispermethrin and metabolite 3-PBA were determined in urine by GC/MS. Mice were terminated 

16 hours after the final treatment. Blood was collected and plasma cis-permethrin (GC/MS) and 

testosterone (enzyme immunoassay) levels were measured. Liver, epididymis, testis, seminal 

vesicle, and prostate were collected and weighed. The levels of cis-permethrin in testis were 

measured by GC/MS. The mRNA levels of StAR (steroidogenic acute regulatory protein), 

TSPO (translocator protein), and P450scc (cytochrome P450 side-chain cleavage) genes in testes 

were measured by real-time quantitative PCR. Epididymal sperm suspension was used to 

microscopically assess sperm count, sperm motility, and abnormalities in sperm morphology. 

Western blot analyses were conducted on testis homogenate with primary antibodies against 

StAR, TSPO, and P450scc. Histopathological evaluation of the right testes and caput 

epididymides was conducted after the tissues were fixed in modified Davidson’s solution, 

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processed, and stained with periodic acid Schiff’s reagent then hematoxylin. Seminiferous 

tubules were classified into three stage groups (I-VI, VII-VIII, and IX-XII) and examined for 

changes of tubule atrophy, cytoplasm vacuolation, and germ cell degeneration. 

No signs of systemic toxicity were observed during dosing, and there were no effects of 

treatment on body weights, body weight gains, or reproductive organ weights. Urinary excretion 

of 3-PBA was decreased (p<0.05) in mice in the mixture group compared to the cis-permethrin 

group (↓30-50%). Levels of cis-permethrin in plasma and testes were 1.5- and 1.8-fold higher, 

respectively, in mice in the mixture group compared to the cis-permethrin group. These results 

indicate that metabolism of cis-permethrin is inhibited by diazinon. Plasma testosterone levels in 

the diazinon and cis-permethrin treatment groups were similar to controls; however, plasma 

testosterone levels were decreased (p<0.01) in the mixture group compared to controls (↓45%) 

and compared to the cis-permethrin group (↓32%). Testicular mRNA and protein expression of 

StAR were not significantly different in any of the treatment groups compared to the controls. 

TSPO mRNA levels and protein expressions were significantly decreased in the cis-permethrin 

(↓46% and 47%, respectively) and mixture (↓53% and 55%, respectively) groups compared to 

controls. There were no significant differences between the cis-permethrin and mixture groups 

for TSPO mRNA and protein expressions. For P450scc, mRNA levels were significantly 

decreased (↓70%) in the mixture group compared to controls; P450scc mRNA levels in the cispermethrin groups were similar to controls. Protein expression of P450scc was significantly 

decreased in all treatment groups compared to controls (↓24% and 43% for the cis-permethrin 

and mixture groups, respectively). Two-way ANOVA did not show any significant interaction 

between exposure and testosterone levels or mRNA or protein expressions of StAR, TSPO, or 

P450scc. 

Histopathological examination of the testes showed no significant increase in abnormalities in 

the cis-permethrin groups compared to controls; however, the numbers of degenerate germ cells 

in seminiferous tubules was significantly increased in the mixture group compared to controls. 

The percentage of seminiferous tubules in stage group VII-VIII was significantly decreased in 

the cis-permethrin and mixture groups. In the caput epididymis, cytoplasmic vacuolation was 

observed more frequently in all treatment groups than in the controls; median vacuolation scores 

were significantly higher in the treatment groups. Two-way ANOVA did not show any 

significant interaction between exposure to both chemicals and testicular or epididymal changes. 

Caudal epididymal sperm count was significantly reduced in the cis-permethrin (↓60%) and 

mixture (↓51%) groups compared to controls. Sperm motility was also significantly decreased in 

the cis-permethrin and mixture groups. The percentage of sperm with normal morphology was 

significantly decreased in the mixture (↓17%) group compared to controls; the decrease in 

normal sperm morphology in the mixture group was also significant when compared to the cispermethrin group. Two-way ANOVA did not show any significant interaction between exposure 

to both chemicals and sperm count, percent motility, or percent abnormal morphology. 

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Avian Reproduction Toxicity (Quail, OCSPP 850.2300) 

In an avian reproduction toxicity study (MRID 42322901, 1992), groups of 32 bobwhite quail 

(Colinus virginianus) were fed diets containing permethrin at 0, 25, 125, or 500 ppm. The birds 

were fed the diets beginning at 20 weeks of age and continuing through egg laying, final 

incubation, hatching, and a 14-day offspring rearing period; the total approximate duration of 

treatment was 27 weeks. The birds were housed with one male and one female per pen, with 16 

pens per dose group. 

Adult quail showed no overt signs of toxicity and no treatment-related clinical signs. There were 

no treatment-related mortalities. There were three deaths in the 25 ppm group which were 

considered to be incidental to treatment as no treatment-related findings were seen at necropsy. 

There were no significant differences in body weights, feed consumption, reproductive 

parameters, egg shell thickness, or offspring body weight between the control and any treatment 

group. 

Avian Reproduction Toxicity (Duck, OCSPP 850.2300) 

In an avian reproduction toxicity study (MRID 42322902), groups of 32 Mallard ducks (Anas 

platyrhyncos) were fed diets containing permethrin at 0, 25, 125, or 500 ppm. The birds were 

fed the diets beginning at 23 weeks of age and continuing through egg laying, final incubation, 

hatching, and a 14-day offspring rearing period; the total approximate duration of treatment was 

24 weeks. The birds were housed with one male and one female per pen, with 16 pens per dose 

group. 

There were two mortalities in the control group and one each in the 25 and 125 ppm groups; 

these mortalities which were considered to be incidental to treatment as no treatment-related 

findings were seen at necropsy. No overt signs of toxicity were observed in any group. One 

female duck in the 125 ppm group had a prolapsed cloaca during week 13; after the prolapse was 

reduced, the duck appeared normal for the remainder of the study. 

There were no significant differences in body weights, egg shell thickness, or offspring body 

weight between the control and any treatment group. At 500 ppm, feed consumption was 

slightly decreased (NS) throughout the study, with a significant difference from controls during 

weeks 6, 12, 14, and 19. There were no significant differences or apparent treatment-related 

effects in reproductive parameters at 25 and 125 ppm. However, while not significant, there 

appeared to be a slight reduction in the number of eggs laid by hens at 500 ppm during the last 

two weeks of egg production. During terminal necropsy, this reduction in egg production was 

correlated with an increase in the number of hens in the 500 ppm group with a regressing ovary. 

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Fish Full Life Cycle Toxicity (Fathead minnow, OCSPP 850.1500) 

In a fish full life cycle toxicity study (MRID 00102096, 1977), groups of 60 fathead minnows 

(Pimephales promelas) embryos (60 embryos/aquarium; 2 aquaria/dose level) were exposed to 

permethrin (95.7% purity) in a flow-through system at nominal concentrations of 0 (control), 0, 

solvent control (7.2 mg/L DMSO), 0.063, 0.13, 0.25, 0.50, and 1.0 μg/L. During the initial 35 

days of the chronic test, mean measured concentrations of permethrin were well below nominal 

concentrations and this trend increased as fish increased in size. To reverse the trend, nominal 

permethrin concentrations were increased to 0.094, 0.19, 0.38, 0.75, and 1.5 μg/L. The 

measured concentration upon which survival and percent hatch were compared to controls were 

0-35 days: <0.023, <0.032, 0.092, 0.14, and 0.41 ppb; and 0-63 days: <0.042, 0.083, 0.17, 0.23, 

and 0.55 ppb. Percent hatch and survival was significantly different at the 0.41 ppb level. After 

156 days exposure, surviving females were returned to spawning chambers in the ratio of 3 

males to 7 females. The levels tested (measured concentrations) were <0.073, 0.16, 0.22, 0.32, 

and 0.87 ppb. At the 0.87 ppb level, there were no female survivors. At ≤0.32 ppb, survival, 

weight, length, and eggs/female did not differ significantly from controls. The second generation 

eggs were then exposed to mean measured concentrations of <0.11, 0.17, 0.30, 0.41 and 0.91 

ppb. The % hatch for these levels did not differ significantly from controls but the survival of 

second generation fry at ≥0.41 ppb was significantly different than controls. To observe if there 

were residual effects of the chemical, fry were transferred from control groups to the 0.41 ppb 

test level and vice-versa; survival of 0.41 ppb fry transferred to control was significantly greater 

than those transferred to the 0.41 ppb test level. 

Mean measured concentrations of 0.91 and 0.41 ppb significantly reduced the percent survival of 

fry during 30 days exposure. Minnows which survived the initial exposure period demonstrated 

normal ranges of measured parameters of survival, growth, reproduction, and egg hatchability. 

Based on the data, the researcher concluded the maximum acceptable concentration of 

permethrin for fathead minnows is estimated to be between 0.30 and 0.41 ppb. Residue analysis 

of fish showed bioaccumulation in female minnows as high as 4600x the water concentration. 

The majority of these residues were eliminated after 14 days in uncontaminated water. 

Estrogenic Effects (Trout and Medaka) 

In a published study by Nillos et al. (2010), permethrin stereoisomers were examined for 

estrogenic activity in vitro using transcriptional measurements of the egg-yolk precursor VTG in 

rainbow trout primary hepatocyte cultures, and in vivo using VTG protein measurements in male 

medaka. Additionally, the estrogenic activity of three permethrin metabolites [3-PBOH), 3-(4’-

hydroxyphenoxy)benzyl alcohol (3,4’-PBOH), and 4-hydroxy permethrin (4-OH permethrin)] 

was investigated in the trout in vitro study. 

For the in vivo VTG expression assay with medaka, adult male medaka were held at static 

conditions for 8 days with individual permethrin enantiomers, 1S-(+)-cis-, 1R-(−)-cis-, 

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1S-(+)-trans-, and 1R-(−)-trans-permethrin (>99% purity), at 10 μg/L, 17-β estradiol (positive 

control; concentration not specified), or 1% acetone (solvent control). Based on the reported 

results, the experiment included “control” and “mixture” test groups which were not defined; the 

study reviewers assumed that the control group included no chemical or solvent and that the 

mixture group was a mixture of all four permethrin enantiomers. On the last day of exposure, 

fish were anesthetized and the livers were excised and pooled for each individual replicate. The 

VTG protein level in the liver was determined using a commercial ELISA kit; the VTG 

concentration was normalized to the total protein in the liver homogenate. VTG production 

between the permethrin enantiomers was compared to determine enantio-selectivity. 

For the in vitro VTG expression assay, trout hepatocytes were isolated from juvenile trout 

(confirmed to be without endogenous expression of VTG) and the hepatocytes were seeded in 

48-well plates. Cells were incubated (18 °C for 24 h) with racemic permethrin at 50 μM, the 

individual permethrin enantiomers at 50 μM each, permethrin metabolites at 5, 10, 25, or 50 μM 

each, 17-β estradiol (positive control) at 0.036 μM, or 1% acetone (solvent control); the 

experiment included a control group and treatments were conducted in triplicate. The VTG 

mRNA expression in fish hepatocytes was measured using quantitative PCR after isolation of 

total mRNA from the cells using a commercial RNA extraction kit. 

Microsomal biotransformation was investigated in livers pooled from five trout. The livers were 

homogenized and the microsomal fractions were resuspended. A reaction mixture of 

microsomal protein, substrate (racemate and enantiomers), NADPH, and Tris-HCl buffer was 

incubated. The supernatant was isolated by centrifugation for reverse-phase HPLC/UV analysis 

for metabolic products. Negative controls omitted the NADPH or included boiled microsomal 

protein. Cytochrome P450 inhibition tests were conducted by co-incubation with ketoconazole, 

which was added after addition of NADPH, and the mixture was incubated for 5 minutes prior to 

addition of the individual permethrin enantiomers. 

After exposure of medaka in vivo for 8 days to the individual permethrin stereoisomers and 

permethrin mixture at 10 μg/L, hepatic VTG production was increased (p<0.05) compared to 

controls. Additionally, significant differences in the relative estrogenic potential of the 

enantiomers were reported: 1S-cis- and 1S-trans-permethrin had responses 2.5 and 1.3 times 

greater than their respective R enantiomers. The expression of VTG mRNA was significantly 

increased in trout primary hepatocytes exposed in vitro to the individual permethrin 

stereoisomers and permethrin mixture at 50 μM compared to controls. Additionally, a 

significant difference in the relative estrogenic potential of 1S-cis-permethrin and 1R-cispermethrin was reported: 1S-cis-permethrin had ~2-fold higher VTG mRNA expression levels 

than 1R-cis-permethrin. A similar (but not significant) trend was observed for the transpermethrin enantiomers. 

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The estrogenic activity of the permethrin metabolites (4-OH permethrin, 3-PBOH, and 3,4’-

PBOH) was also investigated in the in vitro trout hepatocyte experiment. A concentrationrelated increase in the VTG mRNA expression level was observed in trout hepatocytes exposed 

to 4-OH permethrin and 3,4’-PBOH at 5, 10, 25, and 50 μM (the study authors did not state 

whether increases were statistically significant). For 3-PBOH, a significant increase in the VTG 

mRNA expression was only reported at 10 μM; the increase was not dose-dependent. 

Stereoselective formation of three metabolites (4-OH permethrin, 3-PBOH, and 3,4’-PBOH) was 

observed following the incubation of permethrin in rainbow trout liver microsomes. Transpermethrin underwent ester cleavage (resulting in formation of 3-PBOH and 3,4’-PBOH) more 

extensively than cis-permethrin. However, the predominant metabolite produced was the 

hydroxylated derivative of intact permethrin (4-OH permethrin). Furthermore, the cispermethrin diastereomers appeared more susceptible to hydroxylation, yielding >2 times the 

amount of the hydroxylated product than the trans-permethrin diastereomers. 

1S-cis-Permethrin had the highest NADPH-dependent metabolic conversion of the four 

permethrin stereoisomers, with the hydroxylated metabolite (4-OH permethrin) comprising 99% 

of the metabolite profile. Formation of 4-OH permethrin from 1S-cis-permethrin was ~25 times 

greater than the formation from 1R-cis-permethrin, demonstrating a significant stereoselectivity 

to 1S-cis-permethrin for the hydroxylation of permethrin. However, the NADPH-catalyzed 

cleavage of the cis-permethrin enantiomers was enantioselective for 1R-cis-permethrin. 

Of all permethrin stereoisomers, 1S-trans-permethrin was found to be the most susceptible to 

ester cleavage. Although the trans-permethrin stereoisomers appeared more susceptible to ester 

cleavage than the cis-permethrin stereoisomers, hydroxylation in the fish liver microsomes was 

still higher than ester cleavage; 1S-trans-permethrin was more extensively hydroxylated than 1Rtrans-permethrin. 

Generally, ketoconazole significantly inhibited metabolite formation and enantiomer conversion 

when co-incubated with the individual enantiomers. The highest inhibition (98% reduction in 

conversion) was observed for 1S-cis-permethrin, likely due to diminished 4-OH permethrin 

formation. 

The study authors concluded that the results of the in vivo and in vitro studies indicated 

stereoselective estrogenic activity of permethrin resulting from biotransformation of the parent 

compound to more estrogenic metabolites. 1S-cis-Permethrin was observed to have significantly 

higher activity than the 1R-cis enantiomer in both studies. All permethrin enantiomers were 

oxidized to the 4-OH permethrin metabolite and underwent esterase cleavage to 3-PBOH and 

3,4’-PBOH. Racemic 4-OH permethrin as well as 3-PBOH and 3,4’-PBOH possessed 

significant estrogenicity. 

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Estrogen Activity in Fish 

In a published in vivo study by Brander et al. (2012), evaluation of the concentration response to 

permethrin (99% purity; 50/50 mixture of isomers) with juvenile (65- to 70-day old) Menidia 

beryllina was examined in a 14-day static aqueous exposure bioassay. The biomarker selected 

was 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 permethrin (0.1, 1, and 10 μg/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 homogenized in 

buffer, and the resultant supernatant after centrifugation was analyzed by ELISA. An in vitro

chemical-activated luciferase gene expression (CALUX) bioassay was also conducted, using the 

human ovarian carcinoma (BG-1) cell line stably transfected with an estrogen-responsive 

luciferase reporter plasmid. The CALUX bioassay was used to determine the concentrationdependent agonist (pesticide alone) and antagonist (pesticide in presence of estradiol) effects of 

permethrin. Plated cells were incubated with carrier solvent (ethanol) and 17β estradiol (E2; 1 

nM), permethrin, or permethrin plus 1 nM E2; permethrin was tested at 0.1-1,000 μg/L. 

Mean survival in all in vivo treatments after 14 days was ≥80%, with the exception of 70% mean 

survival observed in the 10 μg/L permethrin group. Juvenile M. beryllina exposed to all three 

concentrations of permethrin and ethinylestradiol had higher (p<0.05) relative concentrations of 

Chg expressed in whole-body homogenates. None of the permethrin 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 permethrin was apparent. 

Permethrin treatments were not significantly different from positive control concentrations of 1 

and 10 ng/L; however, all permethrin 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. All study concentrations of permethrin induced the 

expression of Chg in juvenile M. beryllina relative to control, and 1 ng/L permethrin induction 

yielded greater Chg expression compared to 1 ng/L ethinylestradiol. 

For the CALUX assay, ER agonism was not observed for any of the permethrin test 

concentrations. For permethrin, some reduction in the ability of E2 to induce ER-dependent 

reported gene activity was observed (~30-40% of maximal estradiol activity), but this inhibitory 

effect was not concentration-dependent (data presented graphically). It was concluded that 

permethrin was estrogenic in the in vivo test, but anti-estrogenic in cells in vitro, and noted that 

the difference observed between in vivo and in vitro results may be attributable to the lack of 

appropriate metabolism in the CALUX cell line. 

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Estrogen Effects (Embryo-Larval Zebrafish) 

In a published study by Y. Jin et al. (2009), the estrogenic effects of permethrin enantiomers 

were evaluated by detecting the expression levels of estrogen-responsive genes in embryo-larval 

zebrafish. Permethrin (99.2%) was dissolved in acetone and diluted to 100, 250, 500, and 1000 

ng/L in rearing water containing 0.1% acetone (v/v). The four permethrin enantiomers, (+)-cis-, 

(−)-cis-, (+)-trans-, and (−)-trans-permethrin, were also diluted to 250 or 1000 ng/L in water 

containing 0.1% acetone. Embryo-larval zebrafish were exposed to the above testing solutions 

under static renewal conditions for 7 days (0-7 days post-fertilization). Control embryos and 

larvae were exposed in water containing 0.1% acetone (v/v) only. The exposure solutions were 

renewed daily. No difference in survival rate was found during the study. Separate groups of 

embryo-larval zebrafish were exposed for 7 days to estradiol (E2) at concentrations of 25, 50, 

100, 250, 500, 1000 ng/L in water containing 0.1% acetone. Samples were homogenized and 

total RNA was isolated and reverse transcribed for real-time quantitative PCR analyses to 

determine the expression of vtg1, vtg2, esrα, cyp19a, and cyp19b genes. 

The expression levels of vtg1, vtg2, esrα, cyp19a, and cyp19b mRNA in the zebrafish larvae 

were induced effectively when exposed to E2 for 7 days. The lowest concentration of E2 to 

significantly induce transcripts of these estrogenic-responsive genes was 50 ng/L, and the 

induction in mRNA levels of vtg1, esrα, cyp19a, and cyp19b were 5.0-, 2.6-, 2.3-, and 5.4-fold 

respectively, at this concentration compared with those in the control; vtg2 was the only one of 

these transcripts which was significantly induced at 25 ng/L E2. In addition, the E2-induced 

increase in the mRNA level of the five genes was concentration-dependent. The sensitivity of 

these genes (in terms of the concentration needed to elicit a significant response) was the same, 

with the exception of vtg2 which was slightly more sensitive than the others. Thus, the 

expression of vtg1, vtg2, esrα, cyp19a, and cyp19b genes were adopted as biomarkers, and the 

50 ng/L E2 group was selected as the positive control in subsequent experiments. 

Gene-specific responses to exposure to racemic permethrin were observed at concentrations 

≥250 ng/L. The mRNA levels of three genes, vtg1, esrα, and cyp19b were higher (p<0.05) than 

those of the control group at exposure concentrations of 250, 500, and 1000 ng/L. In addition, 

permethrin induced the expression of the esrα in a concentration-dependent manner. Significant 

induction was observed for cyp19a only in the 500 and 1000 ng/L permethrin treatment groups. 

No significant difference was observed between the control and treatment groups for vtg2 

expression, even at 1000 ng/L. Maximum induction in the transcript levels of vtg1, esrα, 

cyp19a, and cyp19b occurred in the highest exposure group, being about 3.8-, 5.0-, 4.8- and 3.0-

fold higher than the control group, respectively. In the case of esrα and cyp19a, the induction 

folds in the highest permethrin treatment group were higher than that of the positive control 

group of 50 ng/L E2. From these results, embryo-larval expressions of vtg1, esrα, cyp19a, and 

cyp19b genes were adopted as biomarkers, and the 250 and 1000 ng/L racemic permethrin 

groups were selected as the positive control in the enantiomer exposure experiments. 

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Generally, at 250 ng/L each permethrin enantiomer exhibited a similar effect on the mRNA 

levels of the four selected genes as the racemate did. The only significant difference between 

enantiomers was found in cyp19b mRNA induction between the (+)-trans and (−)-trans 

enantiomer treatment groups. The mRNA levels of cyp19b in larval zebrafish receiving (−)-trans 

treatment were about 1.6-fold higher than those in the group treated with (+)-trans enantiomer. 

When the exposure concentration of permethrin enantiomers was increased to 1000 ng/L, the 

enantioselectivity in the induction of embryo-larval estrogenic-responsive gene expression 

became more obvious. The maximum induction in the mRNA levels of all four genes was 

observed in the group treated with (−)-trans enantiomer, being much higher even when compared 

with that of the racemate group. Moreover, a significant difference in the mRNA levels of vtg1, 

cyp19a, and cyp19b existed between the (+)-trans and (−)-trans enantiomer treatments. The 

mRNA levels of vtg1, cyp19a, and cyp19b of (−)-trans treatment were about 3.2-, 1.8- and 1.5-

fold higher than that in the group treated with (+)-trans enantiomer, respectively. There was also 

a significant difference in mRNA levels of the cyp19b gene between the two cis-enantiomer 

treatment groups; the mRNA level of cyp19b in (+)-cis treatment group was about 1.5-fold 

higher than that in the group treated with (−)-cis-enantiomer. No significant difference was 

observed in expression of vtg1 or cyp19a between the two cis-enantiomers. For esrα, no 

significant difference was found between (+)-trans and (−)-trans or (+)-cis and (−)-cisenantiomers, although the inductive tendency was similar to vtg1, cyp19a, and cyp19b. The 

study authors concluded that the results of these experiments strongly indicate the occurrence of 

significant enantioselectivity in estrogenic activity of permethrin enantiomers exposed to 

embryo-larval zebrafish 

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APPENDIX 3: References Not Utilized in the Permethrin 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 Permethrin 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. 

Aït-Aïssa, S, Laskowski, S, Laville, N, et al. (2010) Anti-androgenic activities of environmental 

pesticides in the MDA-kb2 reporter cell line. Toxicol. In Vitro 24: 1979-1985. 

Boone, M (2008) Examining the single and interactive effects of three insecticides on amphibian 

metamorphosis. Environ. Toxicol. Chem. 27 (7): 1561-1568.

DeLorenzo, M, Serrano, L, Chung, K, et al. (2006) Effects of the insecticide permethrin on three 

life stages of the grass shrimp, Palaemontes pugio. Ecotoxicol. Environ. Saf. 64: 122-127. 

DeMicco, A, Cooper, K, Richardson, J, et al. (2010) Developmental neurotoxicity of pyrethroid 

insecticides in zebrafish embryos. Toxicol. Sci. 113 (1): 177-186. 

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Hinfray, N, Porcher, J, and Brion, F (2006) Inhibition of rainbow trout (Oncorhynchus mykiss) 

P450 aromatase activities in brain and ovarian microsomes by various environmental substances. 

MRID 48203005. Published: Comp. Biochem. Physiol. Part C. 144: 252-262. 

Issam, C, Zohra, H, Monia, Z, et al. (2011) Effects of dermal sub-chronic exposure of pubescent 

male rats to permethrin (PRMT) on the histological structures of genital tract, testosterone and 

lipoperoxidation. Exp. Toxicol. Pathol. 63 (4): 393-400. 

Kojima, H, Katsura, E, Takeuchi, S, et al. (2004) Screening for estrogen and androgen receptor 

activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. 

Environ. Health Perspect. 112 (5): 524-531. 

Laville, N, Balaguer, P, Brion, F, et al. (2006) Modulation of aromatase activity and mRNA by 

various selected pesticides in the human choriocarcinoma JEG-3 cell line. MRID 48074115. 

Published: Toxicology 228 (1): 98-108. 

Manikkam, M, Guerrero-Bosagna, C, Tracey, R, et al. (2012a) Transgenerational actions of 

environmental compounds on reproductive disease and identification of epigenetic biomarkers of 

ancestral exposures. PLoS ONE 7 (2): 1-12. 

Manikkam, M, Tracey, R, Guerrero-Bosagna, C, et al. (2012b) Pesticide and insect repellent 

misture (permethrin and DEET) induces epigenetic transgenerational inheritance of disease and 

sperm epimutations. Reprod. Toxicol. 34: 708-719. 

Nishihara, T, Nishikawa, J, Kanayama, T, et al. (2000) Estrogenic activities of 517 chemicals by 

yeast two-hybrid assay. Journal of Health Science 46 (4): 282-298. 

Perobelli, J, Martinez, M, da Silva Franchi, C, et al. (2010) Decreased sperm motility in rats 

orally exposed to single or mixed pesticides. J. Toxicol. Environ. Health, Part A 73 (13-14): 

991-1002. 

Schimmel, S, Garnas, R, Patrick, J, et al. (1983) Acute toxicity, bioconcentration, and 

persistence of AC 222,705, benthiocarb, chlorpyrifos, fenvalerate, methyl parathion, and 

permethrin in the estuarine environment. J. Agric. Food Chem. 31: 104-113. 

Taylor, J. S., Thomson, B. M., Lang, C. N., Sin, F. Y., and Podivinsky, E. (2010). Estrogenic 

pyrethroid pesticides regulate expression of estrogen receptor transcripts in mouse Sertoli cells 

differently from 17beta-estradiol. J Toxicol Environ Health, Part A. 73(16):1075-1089. 

Tyler, C, Jobling, S, and Sumpter, J (1998) Endocrine disruption in wildlife: a critical review of 

the evidence. Crit. Rev. Toxicol. 28: 319-361. 

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Wang S. et al. 2002. "Effects of pyrethroids on the concentrations of thyroid hormones in rat 

serum and brain". Chinese Journal of Industrial Hygiene. 20 (3): 173-176. 

Yuan, C, Wang, C, Gao, S, et al. (2010) Effects of permethrin, cypermethrin and 3-

phenoxybenzoic acid on rat sperm motility in vitro evaluated with computer-assisted sperm 

analysis. Toxicol. In Vitro 24 (2): 382-386. 

Zhang S. et al (2007) Permethrin may disrupt testosterone biosynthesis via mitochondrial 

membrane damage of Leydig cells in adult male mouse. Endocrinology. 148(8):3941-3949. 

Zhang, S, Ueyama, J, Ito, Y, et al. (2008) Permethrin may induce adult male mouse reproductive 

toxicity due to cis isomer not trans isomer. Toxicology 248: 136-141. 

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