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 72
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

Page 2 of 72
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: CYPERMETHRIN 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S ENVIRONMENTAL PROTECTION AGENCY

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CYPERMETHRIN 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...........................21 

IV. Committee’s Assessment of Weight of Evidence..........................................23 

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................24 

1. Tier 1 in vivo Assays.............................................................24 

2. OSRI...............................................................................24 

B. Estrogen Pathway...........................................................................26 

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

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

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

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

VI. References.......................................................................................29 

LIST OF TABLES: 

 Table 1. Tier 1 Assays for Cypermethrin..............................................................8 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Cypermethrin................................11 

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

 Table 4. Thyroid Pathway for Cypermethrin.......................................................22 

APPENDIX 1. EDSP Tier 1 Screening Assays.........................................................33 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)................................42 

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

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CYPERMETHRIN FINAL 

iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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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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CYPERMETHRIN FINAL 

vi 

Abbreviation Terminology 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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CYPERMETHRIN FINAL 

Page 1 of 64 

Executive Summary 

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

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

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

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

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) 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 cypermethrin interacts with E, A, or T hormone pathways, the number 

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

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

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

also resulted in general systemic toxicity or overt toxicity. 

On June 25, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) 

of the Office of Pesticide Programs and the Office of Science Coordination and Policy conducted 

a weight-of-evidence analysis of the potential interaction of cypermethrin 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). 

Cypermethrin is a member of the synthetic pyrethroid class of insecticide having similar toxicity 

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

conducted on isomeric mixtures (i.e., cypermethrin and β-cypermethrin, a resolved mix of the 

biologically-active isomers). 

There was no convincing evidence for potential interaction with the estrogen pathway. The in 

vitro studies yielded conflicting results; while some studies showed weak effects, other studies 

showed no effect on the estrogen pathway in ER binding or activation assays. The in vivo 

Uterotrophic and Female pubertal assays were negative, and no estrogen-related effects were 

seen in the mammalian Part 158 studies. In the fish short-term reproduction assay (FSTRA), 

there was a decrease in fecundity and some indication of mature oocyte atresia in females at the 

highest concentration tested (1.4 μg/L). These effects in female fish are further discussed under 

the androgen pathway section below. 

There was convincing evidence for potential interaction with the androgen pathway. The effects 

observed in the in vitro assays, along with evidence of androgen antagonist effects in some of the 

in vivo mammalian assays, shows potential anti-androgenic effects in both mammals and fish. 

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Page 2 of 64 

Positive responses were seen in the androgen binding and the androgen receptor (AR) binding 

and AR transactivation assays. Although no effects were observed in the Tier 1 mammalian 

assays, there were effects observed in mammalian literature studies of cypermethrin and βcypermethrin, including decreased sperm production at ≥ 15 mg/kg/day (Liu et al. 2010) and 

damage to the seminiferous tubules and spermatids at ≥ 6.25 mg/kg/day (Hu et al., 2013; Li et al. 

2013). 

In the FSTRA, significant increases in male GSI were observed (22-27%) at the mid and high 

concentration along with a marginally significant decrease (24%) in male tubercle score at the 

high concentration. While there appears to be limited complementary effects within a sex, 

collectively the effects observed in the FSTRA across both males and females appear to be 

consistent with reported effects for other chemicals (n=3) reported to be potential androgen 

antagonists (US EPA 2007) including decreased fecundity, increased female oocyte atresia, and 

decreased tubercle score. Effects on male or female gonadal staging index (GSI) were 

sometimes reported but the direction of effect (increased/decreased) was not consistent. It is 

noted that increases in female vitellogenin (VTG) were also reported for two of the three 

chemicals, and this response was not observed in the FSTRA for cypermethrin. 

There was no convincing evidence for potential interaction with the thyroid pathway. No 

thyroid-related responses were observed in mammalian Tier 1 assays, Part 158 studies, or other 

OSRI. Additionally, based on the lack of effects on developmental endpoints or alterations in 

thyroid histopathology, the amphibian metamorphosis assay (AMA) did not indicate a potential 

for a thyroid interaction in amphibians for cypermethrin. 

For cypermethrin, the current point of departure (POD) of 6.0 mg/kg/day used for human health 

risk assessment is based on clinical signs of neurotoxicity and mortality observed in male dogs 

and decreased body weight and body weight gain observed in female dog at 18.1 mg/kg/day 

(LOAEL) in the chronic toxicity study in the dogs. This POD is used to derive the chronic 

Reference Dose (RfD) used for chronic dietary exposure risk assessments and for calculating the 

Margins of Exposure (MOEs) for non-dietary exposure risk assessments.

 

In general, if the POD is lower or equal to the dose(s) that caused endocrine-related effects, there 

would be a concern that the POD may not be protective of potential estrogen, androgen or 

thyroid related effects. In the case of cypermethrin, the POD (6.0 mg/kg/day) is numerically 

identical to the doses (6.25- 7.25 mg/kg/day) that caused damage to the seminiferous tubules and 

spermatids in studies reported as OSRI (Hu et al., 2013 and Li et al. 2013). Additionally, 

decreased sperm production was seen at a dose (12 mg/kg/day) that is only approximately twofold higher than the POD. Therefore, the POD used in risk assessments does not address the 

concern for the adverse male reproductive effects 

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Page 3 of 64 

Although, adverse effects are seen, the EDSP Tier 2 study (post 1998 two-generation 

reproduction or the extended one-generation reproduction study) which evaluates male and 

female mammalian reproduction and fertility is not recommended since: 1) no estrogen-related 

effects were seen in the Tier 1 female pubertal assay; 2) no estrogen-related effects were reported 

in the OSRI; and 3) the concern is only for the androgen-related effects in the adult male 

reproductive system. 

Consequently, for mammals, a special study focused on evaluating the potential adverse effect of 

cypermethrin in the male reproductive system is recommended. The experimental design (i.e, 

protocol) for this study should be developed in consultation with the agency. 

For non-mammalian species, in the problem formulation for the registration review of 

cypermethrin, the chronic no observed adverse effect concentration (NOAEC) for fish was 

reported at 0.14 μg/L based on a 30-day exposure fathead minnow early life-stage study. 

Reproductive effects were observed in the FSTRA at measured concentrations of ≥0.12 μg/L. A 

fish life-cycle study for cypermethrin with fathead minnow is available with a reported NOAEC 

of 0.06 μg/L (MRID 40641701). However in this study, excessive analytical variation (high:low 

ratios of 3.0 to 7.8) was observed at all levels during the study, and the range of concentrations 

was so great that overlapping of concentrations occurred for all levels. Therefore, there is 

uncertainty with regards to the reported NOAEC in that study. Given the potential for antiandrogen effects observed in the Tier 1 weight of evidence analysis, the EDSP Tier 2 Medaka 

Extended One Generation Reproduction Toxicity (MEOGRT) study is recommended. With that 

recommendation, it is noted that given the physio-chemical properties of cypermethrin and the 

results in the fathead minnow life-cycle study, there may be difficulties in maintaining 

cypermethrin concentrations in aqueous systems over the duration of a long-term test such as the 

Tier 2 fish assay and careful attention and maintenance of the test system will likely be required. 

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

duck are available for cypermethrin. In both species, no effects on reproductive parameters were 

observed; an additional study with alpha cypermethrin did report potential reproductive effects 

but there are uncertainties in this study. 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 cypermethrin exposure. Relative to the EDSP, additional 

testing is not recommended. 

Given the lack of observed potential thyroid-related effects in the AMA, no additional testing 

with amphibians is recommended. 

 

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Page 4 of 64 

I. Introduction 

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

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

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

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

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

addition to the available Tier 1 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 cypermethrin interacts with E, A or T hormone pathways, the number 

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

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

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

resulted in general systemic toxicity or overt toxicity. 

On June 25, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) 

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

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

cypermethrin 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). 

Cypermethrin is a chiral compound with eight stereoisomers, each with an abundance of 

approximately 11-14%. Alpha(α)-cypermethrin consists of the (R)-alcohol (1S)-cis-acid and the 

(S)-alcohol (1R)-cis-acid; beta(β)-cypermethrin consists of the (R)-alcohol (1S)-cis and transacids and the (S)-alcohol (1R)-cis and trans-acids (alpha + theta forms); theta(θ)-cypermethrin 

consists of the (R)-alcohol (1S)-trans-acid and the (S)-alcohol (1R)-trans-acid; and zeta(ζ)-

cypermethrin is an S-enantiomer-enriched formulation of the four (S)-alcohol isomers of 

cypermethrin (24% each) with 1% each of the less active (R)-alcohol isomers. Cypermethrin has 

a vapor pressure of 1.7 (or 3.1) × 10−7 mm Hg at 20°C; cypermethrin will exist as a particulate if 

released to the atmosphere and is expected to be removed from the atmosphere by 

photodegradation and deposition. Cypermethrin is poorly soluble in water (4-8/7!qqc), and has 

an octanol/water partition coefficient (log Kow) of 6.6 with an estimated bioconcentration factor 

range from 420-468. Cypermethrin is expected to have low mobility in soil, with no 

volatilization, but will rapidly degrade in soil under aerobic conditions. 

The available information considered in determining the potential interaction of cypermethrin 

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

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Page 5 of 64 

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 studies recommendations are presented in Section V. 

II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

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

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

1. 

B. Other Scientifically Relevant Information (OSRI) 

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

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

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

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

Report of the Endocrine Disruptor Review Team on cypermethrin (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 include a battery of 11 assays 

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

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

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

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

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

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

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

or consistency (complementarity) of the responses in the individual study is evaluated. For the 

Tier 1 in vivo assays, often multiple endpoints are measured in each assay. 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

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

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

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

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

biological organization were the responses observed and whether organisms exhibit 

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

conditions did the responses occur including considerations 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 July 2013 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 non-endocrine organs or overall disruptions in homeostasis” (USEPA, 

2013). 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Section IV.A. 

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

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

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

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

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

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

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

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

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

Page 15 of 72
CYPERMETHRIN FINAL 

Page 8 of 64 

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 or not a substance has the potential to interact with the E, A or 

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

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

A. EDSP Tier 1 Screening Assays 

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

below in Table 1. 

Table 1. Tier 1 Screening Assay for Cypermethrin

Tier 1 Assays Test 

Guideline Test Order Status 

ER Binding Assay (Rat uterine cytosol) OCSPP 

890.1250 

Requirement Satisfied by OSRI 

(Saito et al., 2000; 

Chen et al., 2002) 

ERα Transcriptional Activation Assay (Human cell 

line HeLa 9903) 

OCSPP 

890.1300; 

OECD 455 

Requirement Satisfied 

(MRID 48670201) 

AR Binding Assay (Rat prostate cytosol) OCSPP 

890.1150 

Requirement Satisfied by OSRI 

(Zhang et al., 2008; 

 Xu et al., 2008) 

Steroidogenesis Assay (Human cell line H295R) 

OCSPP 

890.1550; 

OECD 456 

Requirement Satisfied 

(MRID 48670203) 

Aromatase Assay (human recombinant 

microsomes) 

OCSPP 

890.1200 

Requirement Satisfied 

(MRID 48670202) 

Uterotrophic Assay (Rat) 

OCSPP 

890.1600; 

OECD 440 

Requirement Satisfied 

(MRID 48615701) 

Hershberger Assay (Rat) 

OCSPP 

890.1400; 

OECD 441 

Requirement Satisfied by OSRI 

 (Zhang et al., 2008) 

Pubertal Female Assay (Rat) OCSPP 

890.1450 

Requirement Satisfied 

(MRID 48683002) 

Pubertal Male Assay (Rat) OCSPP 

890.1500 

Requirement Satisfied 

(MRID 48683003) 

Fish Short-term Reproduction Assay 

OCSPP 

890.1350; 

OECD 229 

Requirement Satisfied 

(MRID 48683001)

Amphibian Metamorphosis Assay (Frog) 

OCSPP 

890.1100; 

OECD 231 

Requirement Satisfied 

(MRID 49013801) 

Page 16 of 72
CYPERMETHRIN FINAL 

Page 9 of 64 

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

1. Effects on Estrogen Pathway 

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

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

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

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

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

considered further in the WOE assessment. 

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

results of Saito et al. (2000) and Chen et al. (2002). The ER competitive binding assay by Chen 

et al. (2002) was positive however the assay by Saito et al. (2000) and another OSRI assay by 

Kim et al. (2004) were negative. A yeast two-hybrid assay by Saito et al. (2000) was negative 

(using Nishikawa procedure) for ER interaction. In another published study, Tyler et al. (2000) 

did not demonstrate hER activation in a recombinant yeast expression system assay, but antiestrogenic activity was observed. 

Cypermethrin was classified as negative in the Tier 1 ERTA. A mammalian ERTA luciferase 

assay by Saito et al. (2000) was also negative, however Kjeldsen et al. (2013) showed weak 

induction by cypermethrin in an ERTA assay with a maximum response of 119% (at 10−6

 M); 

cypermethrin did not exert an ER-antagonistic effect in this assay. 

In pS2 (an ER responsive gene) gene expression assays, Chen et al. (2002) and Jin et al. (2010) 

observed an approximately 2-fold increase in pS2 expression at 10−6 M. Chen et al. (2002) and 

Jin et al. (2010) showed that cypermethrin yielded a weak agonistic response in an E-Screen 

(MCF-7 cell proliferation) assay. However Kim et al. (2004) reported negative responses to 

cypermethrin in an E-Screen (MCF-7 BUS cell proliferation). 

Cypermethrin was positive for estradiol induction in the Tier 1 steroidogenesis assay. The Tier 1 

aromatase assay and an OSRI aromatase assay by Kjeldsen et al. (2013) were negative for 

aromatase inhibition. 

Cypermethrin was negative in the Tier in vivo uterotrophic assay and no estrogen-related effects 

were seen in the female pubertal rat assay. 

In the FSTRA, there were no treatment-related effects on plasma VTG concentration, secondary 

sex characteristics, or behavior in females. Fecundity was decreased by 42% and 84% in the 

medium- and high-treatments, (0.12 and 1.4 μg a.i./L) respectively, but there were no treatmentrelated effects on fertilization success. An increased incidence of mature oocyte atresia was 

present at the low (0.013 μg a.i./L), medium, and high test concentration, respectively, (13%, 

Page 17 of 72
CYPERMETHRIN FINAL 

Page 10 of 64 

7%, and 50%), compared to control females which had no reported incidents. While this 

histopathological finding was associated with granulomatous inflammation and the presence of 

microsporidia in half of the cases across all treatment levels, the increased incidence of atresia 

was considered potentially treatment-related in the high dose. There were no differences 

observed in gonadal staging. 

There were no estrogen-related effects observed in the Part 158 mammalian toxicity studies. 

In Part 158 avian reproduction toxicity studies (one bobwhite quail and one mallard duck) for 

cypermethrin, no treatment-related effects were reported. In another Part 158 bobwhite quail 

reproduction toxicity study testing alpha cypermethrin, dietary administration resulted in 

significant decreases in the number of hatchlings/eggs set (%) at the low dose of 48 mg/kg-diet, 

the adult male body weight gain at the mid- and high treatment groups (149 and 452 ng/kg-diet), 

and 14-day old offspring survivors at the high treatment. However, in this study pen mate 

aggression was observed across all treatment levels and control groups, which likely resulted in 

the observed parental mortalities (one in control, 2 in low treatment group, and 3 in high 

treatment group), and therefore, as there may have been poor husbandry in this study it may 

potentially have impacted the results of this reproductive study. In a Part 158 freshwater fish 

early life-stage toxicity test with fathead minnow, larval survival was significantly reduced in the 

highest test concentration (0.33 μg a.i./L). A Part 158 fish life-cycle toxicity test with fathead 

minnow is available, for which reduction in fecundity at 0.32 μg/L were observed, along with 

effects on parental survival and growth at the same concentrations. However, excessive 

analytical variation (high:low ratios of 3.0 to 7.8) was observed at all levels during the study. 

The range of concentrations was so great that overlapping of concentrations occurred for all 

levels. Therefore, there is uncertainty in the reported NOAEC, and this study is not included in 

the table below.

Page 18 of 72
CYPERMETHRIN FINAL 

Page 11 of 64 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Cypermethrin

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Cypermethrin1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin (VTG) 

EDSP Tier 1 Assay 

ER Binding Requirement satisfied by OSRI (Saito et al., 2000, MRID 48176003 and Chen et al., 2002, MRID 4814ERTA (MRID 48670201) N Aromatase (MRID 48670202) N Steroidogenesis (MRID 48670203) P4Uterotrophic (MRID 48615701) N NE Female Pubertal Rat (MRID 48683002) N N N N N N FSTRA (MRID 48683001) NE N P (H)6 N 

Frt: N; 

Fcd: 

↓42, 84% 

(M, H) 

N 

OSRI 

ER Binding Saito et al. (2000) 

MRID 48176003 

N N 

ER Binding Chen et al. (2002) MRID 48146201 

P P 
CYPERMETHRIN FINAL 

Page 12 of 64 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Cypermethrin

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Cypermethrin1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin (VTG) 

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

N N 

Cell Proliferation/pS2 Expression (Jin et al., 2010) 

P 

ER Binding (Tyler et al., 2000) P ERTA (Kjeldsen et al., 

2013) 

P 

Aromatase (Kjeldsen et al., 

2013) 

N 

Subchronic toxicity (Dog; MRID 44527002) 

NE NE N NE 

Developmental toxicity (Rat; MRID 00056804) 

NE N 

Developmental toxicity (Rabbit; MRID 43776302) 

NE N 

Developmental Neurotoxicity (Rat; MRID 46670402) 

NE N N 
CYPERMETHRIN FINAL 

Page 13 of 64 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Cypermethrin

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Cypermethrin1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin (VTG) 

Three-generation reproduction (Rat; MRID 00112912) 

 NR NR N NR N 

Three-generation reproduction (Rat; MRID 00090040) 

 NR NR N NR N 

Chronic toxicity/ carcinogenicity (Rat; MRID 00112910) 

NE N N N 

Carcinogenicity (Mouse; MRID 00112911) 

NE NE N NE 

Chronic toxicity (Dog; MRID 00112909) 

NE N N N 

Avian reproduction (Duck; MRID 00090073) 

 N 

Avian reproduction (Quail; MRID 00090074) 

 N 

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

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

Changes in weight are absolute unless otherwise indicated. 

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

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

3. 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 

For details see Section IV. A 
CYPERMETHRIN FINAL 

Page 14 of 64 

4. Estradiol induced at 10 μM in Runs 3 and 4, and estradiol concentrations in all three runs at 10 μM were 111-117% of the solv

5. Treatment-related clinical signs of toxicity included one or two animals with dried red material around nose, hypersensitivity t

upon handling, and salivation and/or wet clear material around mouth at 80 mg/kg/day (HTD). 

6. Increased incidence of mature oocyte atresia in highest test concentration (1.4 μg a.i./L) 

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

Page 15 of 64 

2. Effects on Androgen Pathway 

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

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

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

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

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

table and not considered further in the WOE assessment. 

The Agency determined that the requirement for an AR binding study was satisfied by the results 

of AR transactivation (ARTA) studies by Zhang et al. (2008) and Xu et al. (2008). Zhang et al.

(2008) examined the effects of cypermethrin and β-cypermethrin at a single concentration (1 

×10−5

 M) in an ARTA assay. The results indicated that cypermethrin was anti-androgenic with 

an approximate 45% decrease in activity relative to the control; β-cypermethrin was negative in 

this assay. Xu et al. (2008) tested cypermethrin at 10−7

, 10−6, and 10−5 M in an ARTA assay. 

Cypermethrin was reported to be an anti-androgen with an IC50 of 6.80±2.3×10−5 M, with 

inhibition at the highest concentration (10−5 M). No androgen agonism was detected by Xu et al.

(2008); Zhang et al. (2008) did not test androgen agonism. 

In other OSRI studies, Du et al. (2010) demonstrated similar results to the earlier studies with 

cypermethrin displaying anti-androgenic activity with an IC20 of 1.64×10−6

 M, and no androgen 

agonism. However, negative responses for both androgenicity and anti-androgenicity were 

determined by Kjeldsen et al. (2013) in ARTA assays. In addition, cypermethrin did not displace 

3

H-DHT in an AR biding study by Bauer et al. (2002). 

Cypermethrin was classified as negative in the Tier 1 steroidogenesis assay. The Agency 

determined that the requirement for Hershberger assay was satisfied by Zhang et al. (2008). In 

that study, cypermethrin demonstrated an androgen-antagonist effect; androgen agonism was not 

investigated. Treatment with cypermethrin at 50 mg/kg/day (only dose tested) resulted in 

decreased absolute weights for seminal vesicles, ventral prostate and dorsolateral prostate; 

however, there were no decreases in LABC muscle, Cowper’s glands, or glans penis weights. 

Treatment with β-cypermethrin at 50 mg/kg/day did not result in any effects on the accessory sex 

organ (ASO) weights. However, there were no androgen-related effects observed in the Tier 1 

male pubertal assay. 

In the FSTRA, increases in gonado-somatic index (GSI) of 22 and 27% in the medium- and 

high-treatment groups (0.12, and 1.4 μg a.i./L), respectively, were observed compared to the 

negative control. Additionally, there was a marginally significant (p=0.055) decrease of 24% in 

median tubercle score for males at the high treatment concentration. No other effects [e.g., body 

weight, plasma vitellogenin (VTG) concentration, gonadal stages, and histopathology] were 

observed. 

Page 23 of 72
CYPERMETHRIN FINAL 

Page 16 of 64 

No androgenic or anti-androgenic effects on reproductive parameters due to cypermethrin 

treatment were reported in existing mammalian Part 158 studies. However, several published 

mechanistic studies from the same laboratory (Hu et al. 2013, Li et al. 2013 and Liu et al. 2010) 

in orally dosed adult SD rats reported adverse effects of cypermethrin or β-cypermethrin on the 

reproductive system of male rats dosed for 15 days. At doses up to 50 mg/kg/day, Hu et al.

(2013) observed decreased prostate weights at the higher doses and decreased sperm production 

at the high dose; as well as damage to seminiferous tubules and spermatids, decreased 

testosterone concentration and increased FSH and LH concentrations at the high dose. In a 

similar study by Li et al. (2013) at doses up to 60 mg/kg/day, no effects on reproductive organ 

weights were noted, but decreased sperm production, gross and microscopic changes in 

seminiferous tubules and spermatids, decreased testosterone concentrations, and increased FSH 

concentration were noted. In addition, in a study of β-cypermethrin at doses up to 30 mg/kg/day 

(Liu et al., 2010), no organ weight effects were seen, but sperm counts and sperm production 

were decreased. 

In another study, Wang et al. (2009) addressed effects of β-cypermethrin exposure in mice at 0, 

1, 10 or 20 mg/kg/day for 35 days. Testes, epididymis, and seminal vesicles organ weights were 

dose-dependently decreased at 10 and 20 mg/kg, and prostrate organ weights were decreased at 

20 mg/kg only. Sperm counts, viability, motility, and testosterone concentrations were dosedependently decreased. However these effects were observed in the presence of decreased body weight 

gain of 52 and 81% at 10 and 20 mg/kg/day doses (body weight data were not reported). Histological 

and ultrastructural changes due to β-cypermethrin exposure at the two highest doses were similar 

to changes observed in the Hu et al. 2013, Li et al. 2013 and Liu et al. 2010 studies, with damage 

to seminiferous tubules and sperm development. There were no reported androgen-related 

endpoints affected in the ecotoxicology OSRI.

Page 24 of 72
CYPERMETHRIN FINAL 

Page 17 of 64 

Table 3. Androgenic/Anti-Androgenic Pathway for Cypermethrin

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Cypermethrin1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Age and Weight at 

PPS 

EDSP Tier 1 Assay 

AR Binding Requirement satisfied by OSRI (Zhang et al., 2008; MRID 48196302 and Xu et al., 2008; MRID 481Steroidogenesis (MRID 48670203) N Hershberger Requirement satisfied by OSRI (Zhang et al., 2008; MRID 48196302 see below) Male Pubertal Rat (MRID 48683003) N N N N N N N N FSTRA (MRID 48683001) NE 

↑22, 

27% 

(M, H) 

N 

Tubercle

: 

↓24% 

(H) 

N 

OSRI 

AR Binding (Zhang et al., 2002) MRID 48196302 

N 

AR Binding (Tyler et al., 2000) P ARTA Zhang et al. (2008) P ARTA Xu et al. (2008) MRID 48176006 

P 
CYPERMETHRIN FINAL 

Page 18 of 64 

Table 3. Androgenic/Anti-Androgenic Pathway for Cypermethrin

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Cypermethrin1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Age and Weight at 

PPS 

ARTA (Du et al., 2010) P ARTA (Kjeldsen et al., 

2013) 

 N 

Hershberger Zhang et al. (2008) MRID 48196302 

N ↓SV, VP, DP 

Subchronic toxicity (Dog; MRID 44527002) 

N N N N NE NE 

Developmental Neurotoxicity (Rat; MRID 46670402) 

N 

Three-generation reproduction (Rat; MRID 00112912) 

NR N NR N NR NR N 

Three-generation reproduction (Rat; MRID 00090040) 

NR N NR N NR NR N 

Chronic toxicity/ Carcinogenicity (Rat; MRID 00112910) 

N N NR N N NR 

Carcinogenicity (Mouse; MRID 00112911) 

 N N NE N NE NE 
CYPERMETHRIN FINAL 

Page 19 of 64 

Table 3. Androgenic/Anti-Androgenic Pathway for Cypermethrin

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Cypermethrin1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Chronic toxicity (Dog; MRID 00112909) 

N N NE N N NE 

Avian reproduction (Duck; MRID 00090073) 

N 

Avian reproduction (Quail; MRID 00090074) 

N 

Mechanistic Study: Male Reproductive System (Rat; Hu et al., 2013) 

 N N P (L, ML) N NR N 

Mechanistic Study: Male Reproductive System (Rat; Li et al., 2013) 

↓

(MH

,H) 

N 

P 

(L, 

ML, 

MH, 

H) 

N NR N 

Mechanistic Study: Male Reproductive System (Rat; Liu et al., 2010) 

NE N P (L, H) N NR N 
CYPERMETHRIN FINAL 

Page 20 of 64 

Table 3. Androgenic/Anti-Androgenic Pathway for Cypermethrin

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Cypermethrin1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Mechanistic Study: Male Reproductive System (Mouse; Wang et al., 2009)8

↓35, 

70% 

(M, 

H) 

↓21, 

39% 

(M, H) 

P 

(M, H) 

↓6, 

10% 

(M, H)

NR 

SV: 

↓28, 

58% 

(M, H); 

PR: 

↓11% 

(H) 

Avian reproduction (Duck; MRID 00090073) N Avian reproduction (Quail; MRID 00090074) 

N 

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

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

assay. Changes in weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesprostate (VP), Dorsal prostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland body weight gain (BWG). 

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

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 

details see Section IV. A 

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

 NE Not examined  NR Not reported
CYPERMETHRIN FINAL 

Page 21 of 64 

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 redundancy and have the potential to detect 

changes in the HPT axis, i.e., the pubertal female and male rat assays, and the AMA (frog). 

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

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

to facilitate determination of potential for thyroid or HPT axis effects. 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. 

 No effects on the thyroid pathway (thyroid weight, histopathology, or hormone concentrations) 

were observed in the in vivo male and female pubertal assays. 

In the AMA, cypermethrin did not delay or accelerate median Nieuwkoop-Faber (NF) 

developmental stage at Day 7 or 21 relative to the negative control. Although, more than half of 

all individuals developed beyond NF stage 60 by Day 21 (i.e., NF stage 61-62), with 53%, 59%, 

54%, and 52% of surviving frogs exhibiting development beyond NF60 (termed “late-stage”) in 

the control, low-, medium-, and high-concentration groups (0.0019, 0.109, and 0.0867 μg a.i./L, 

respectively) it did not appear to be treatment-related. No effects on thyroid gland 

histopathology were observed at any treatment concentration. There also was no effect on 7- or 

21-day hind-limb length (HLL). In terms of growth endpoints, there was a decrease in snout-tovent length (SVL; ↓4%) and body weight (↓; 11%) at the high concentration on Day 7 only. 

Spinal deformities were observed in all treatment groups including the negative control group 

(45% in the negative control and low treatment group and 37% in the medium and high treatment 

groups); these were determined to not be treatment related. 

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

Page 29 of 72
CYPERMETHRIN FINAL 

Page 22 of 64 

Table 4. Thyroid Pathway for Cypermethrin

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Cypermethrin1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

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

Overt Toxicity Observed 3 

EDSP Tier 1 Assay 

Female Pubertal Rat 

(MRID 48683002) N N N N N N N 

Male Pubertal Rat 

(MRID 48683003 ) N N N N N N N 

AMA (Frog; MRID 

49013801) N N 

Day 7: 

BW: ↓11% 

(H); SVL: 

↓4% (H) 

 N 

OSRI 

Subchronic toxicity 

(Dog; MRID 44527002) N N NE X 

(H) 

X 

(ML, 

MH, H) 

Developmental toxicity 

(Rat; MRID 00056804) 

X 

(M, H) N 

Developmental toxicity 

(Rabbit; MRID 

43776302) 

X 

(M, H) N 

Developmental 

Neurotoxicity (Rat; 

MRID 46670402) 

X 

(H) N 

Three-generation 

reproduction (Rat; 

MRID 00090040) 

NR NR NR X 

(H) 

X 

(H) 

Three-generation 

reproduction (Rat; 

MRID 00112912) 

NR NR NR N N 

Chronic toxicity/ 

carcinogenicity (Rat; 

MRID 00112910) 

NE N N X 

(H) N 

Carcinogenicity 

(Mouse; MRID 

00112911) 

NE N NE N N 

Chronic toxicity (Dog; 

MRID 00112909) N N N X 

(H) N 

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

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

indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are 

absolute unless otherwise indicated. 

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

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

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

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

Page 30 of 72
CYPERMETHRIN FINAL 

Page 23 of 64 

 P Positive findings 

N Negative findings 

NE Not examined 

NR Not reported 

 

IV. Committee’s Assessment of Weight of Evidence 

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

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

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

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

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

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

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

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

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

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

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

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

alternatively a sequelae of systemic effects. Endocrine modes of action may 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 Tire 1 assays stated that “ In summary, 

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

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

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

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

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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).

1. Tier 1 in vivo Assays 

Systemic or overt toxicity in the Tier 1 in vivo studies was minimal. In the female and male 

pubertal rat studies, one female in the 30 mg/kg/day group (HTD) was found dead on PND 25, 

and one male in the 15 mg/kg/day group was found dead approximately one hour after dosing on 

PND 45. Neither death was considered related to treatment. All other rats survived until 

scheduled sacrifice. There were no treatment-related clinical signs of toxicity at any dose. 

Findings noted in the treated groups, including red and/or clear material around the nose and/or 

mouth, occurred infrequently in single animals and/or in a manner that was not considered dose 

related. In the Uterotrophic assay, all animals survived until scheduled termination. Treatmentrelated clinical signs of toxicity included one or two animals with dried red material around nose, 

hypersensitivity to handling, vocalization upon handling, and salivation and/or wet clear material 

around mouth at 80 mg/kg/day (HTD). No treatment-related mortalities or clinical signs were 

present in the FSTRA study. No treatment-related overt toxicity or systemic signs of toxicity 

were observed in the AMA study at test termination. In the AMA, spinal deformities were 

observed in all treatment groups including the negative control group, and more than half of all 

individuals developed beyond NF stage 60 by Day 21 (i.e., NF stage 61-62), but these findings 

were determined to not be treatment-related. 

2. OSRI 

Systemic or overt toxicity in the OSRI in vivo studies was also minimal. No mortality or 

treatment-related clinical signs were noted in any of the groups in a Hershberger assay (Zhang et 

al., 2008). No deaths occurred during a subchronic oral toxicity study in dogs. In the high-dose 

group, two males and one female exhibited slight tremor activity beginning on Days 49 (female), 

60, or 71 through study termination. In addition, one male and one female exhibited moderate 

tremor activity either on Day 76/77 (male) or during the Week 8, 9, and 12 detailed physical 

examinations (female). 

In a prenatal development study in rats, dams in the high-dose group displayed neurological 

signs such as splayed limbs, spasms, hypersensitivity to noise, and convulsions. In a prenatal 

development study in rabbits, four deaths occurred during the study, but were not, or could not 

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conclusively be, attributed to treatment. Exacerbation of some clinical signs such as anorexia, 

abdominal/genital staining, decreased feces, and red or pink material in the pan occurred in the 

high-dose group and in a few does in the medium-dose group, but these effects were not 

definitely attributed to treatment. In a developmental neurotoxicity study in rats, one control 

female was found dead late in gestation; necropsy revealed a ruptured uterus and late resorptions; 

all remaining animals survived to scheduled sacrifice. No treatment-related clinical signs of 

toxicity were observed during the daily examinations. 

In a three-generation reproduction study in rats, one high-dose male (1000 pm) was sacrificed in 

extremis, and this death was considered to be treatment related. There were obvious clinical 

signs of neurotoxicity (i.e., ataxia, increased sensitivity to sound, and high-stepping gait) in the 

high-dose (1000 ppm) group that resulted in a dose reduction to 750 ppm after twelve weeks. No 

adverse health or behavioral effects were noted in a previous three-generation reproduction study 

in rats with a high dose of 500 ppm. 

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

study in rats. Transient clinical (behavioral) signs (i.e., frequent face washing, increased 

sensitivity to sound, hind limb incoordination) were noted during the first six days of dosing in 

six (6/52) high-dose males. Similarly, in the carcinogenicity study in mice, there were no 

treatment-related differences in survival and no clinical signs were present. In a chronic oral 

toxicity/carcinogenicity study in dogs, no mortality occurred during the study. Treated males 

and females displayed increases in vomiting during Week 1, and high-dose animals displayed 

signs of nervous system stimulation in the form of body tremors, gait abnormalities, 

incoordination, disorientation, and hyper-sensitivity to noise during Week 1. 

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

reproduction studies with mallard ducks and bobwhite quail for cypermethrin. Although no 

reproductive effects were observed, numerous mortalities were observed, and prior to the onset 

of death birds were observed being “bullied”. In addition, alpha cypermethrin reproductive 

effects were observed in the presence of pen mate aggression, and the presence of aggressive 

behavior, was a sign that the animals were stressed, which could have potentially biased the 

results of the reproductive study. 

An early life-stage study in fathead minnow was submitted, and after 30-day exposure to 

cypermethrin, survival was significantly reduced compared to controls at 0.33 μg a.i./L. The 

resulting NOAEC value for survival was 0.14 μg a.i./L; and no treatment-related effects were 

observed for hatchability or growth. 

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B. Estrogen Pathway 

For cypermethrin the in vitro studies yielded conflicting results; while some studies showed 

weak effects, other studies showed no effect on the estrogen pathway in ER binding or activation 

assays. The in vivo Uterotrophic and Female pubertal assays were negative, and no estrogen 

related effects were seen in the mammalian Part 158 studies. In the FSTRA, there was a 

decrease in fecundity and some indication of mature oocyte atresia in females at the highest 

concentration tested (1.4 μg/L). These effects in female fish are further discussed under the 

androgen pathway section below. 

C. Androgen Pathway 

For the androgen pathway, positive responses were seen for cypermethrin in AR Binding and 

ARTA studies. Although no effects were observed in the Tier 1 mammalian assays, there were 

effects observed in mammalian literature studies of cypermethrin and β-cypermethrin, including 

decreased sperm production at ≥ 15 mg/kg/day (Liu et al. 2010) and damage to the seminiferous 

tubules and spermatids at ≥ 6.25 mg/kg/day (Hu et al., 2013; Li et al. 2013). 

In the FSTRA, significant increases in male GSI was observed (22-27%) at the mid and high 

concentration along with a marginally significant decrease (24%) in male tubercle score at the 

high concentration. While there appears to be limited complementary effects within a sex, 

collectively the effects observed in the FSTRA across both males and females appear to be 

consistent with reported effects for other chemicals (n=3) reported to be potential androgen 

antagonists (US EPA 2007) including decreased fecundity, increased female oocyte atresia, and 

decreased tubercle score. Effects on male or female GSI were sometimes reported but the 

direction of effect (increased/decreased) was not consistent. It is noted that increases in female 

VTG were also reported for two of the three chemicals, and this response was not observed in the 

FSTRA for cypermethrin. 

However, on a weight of evidence basis, given the effects observed in the in vitro assays along 

with evidence of potential androgen antagonist effects in some of the in vivo mammalian assays, 

there appears to be evidence for potential anti-androgenic effects in both mammals and fish. 

D. Thyroid Pathway 

No thyroid-related responses were observed in mammalian Tier 1 assays, Part 158 studies, or 

other OSRI. Additionally, based on the lack of effects on developmental endpoints or alterations 

in thyroid histopathology, the AMA did not support a potential for a thyroid interaction in 

amphibians for cypermethrin. 

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E. Conclusions 

Cypermethrin demonstrates no convincing evidence of potential interaction with the estrogen or 

thyroid pathways. However there appears to be a potential for interaction with the androgen 

(anti-androgenic) pathway in mammals and fish. 

V. EDSP Tier 2 Testing Recommendations 

For cypermethrin, the current point of departure (POD) of 6.0 mg/kg/day used for human health 

risk assessment is based on clinical signs of neurotoxicity and mortality observed in male dogs 

and decreased body weight and body weight gain observed in female dog at 18.1 mg/kg/day 

(LOAEL) in the chronic toxicity study in the dogs. This POD is used to derive the chronic 

Reference Dose (RfD) used for chronic dietary exposure risk assessments and for calculating the 

Margins of Exposure (MOEs) for non-dietary exposure risk assessments.

 

In general, if the POD is lower or equal to the dose(s) that caused endocrine-related effects, there 

would be a concern that the POD may not be protective of potential estrogen, androgen or 

thyroid related effects. In the case of cypermethrin, the POD (6.0 mg/kg/day) is numerically 

identical to the doses (6.25- 7.25 mg/kg/day) that caused damage to the seminiferous tubules and 

spermatids in studies reported as OSRI (Hu et al., 2013 and Li et al. 2013). Additionally, 

decreased sperm production was seen at a dose (12 mg/kg/day) that is only approximately twofold higher than the POD. Therefore, the POD used in risk assessments does not address the 

concern for the adverse male reproductive effects Therefore, the POD used in risk assessments 

does not address the concern for the adverse male reproductive effects 

Although, adverse effects are seen, the EDSP Tier 2 study (post 1998 two-generation 

reproduction or the extended one-generation reproduction study) which evaluates male and 

female mammalian reproduction and fertility is not recommended since: 1) no estrogen-related 

effects were seen in the Tier 1 female pubertal assay; 2) no estrogen-related effects were reported 

in the OSRI; and 3) the concern is only for the androgen-related effects in the adult male 

reproductive system.. 

For non-mammalian species, in the problem formulation for the registration review of 

cypermethrin, the chronic no observed adverse effect concentration (NOAEC) for fish was 

reported at 0.14 μg/L based on a 30-day exposure fathead minnow early life-stage study. 

Reproductive effects were observed in the FSTRA at measured concentrations of ≥0.12 μg/L. A 

fish life-cycle study for cypermethrin with fathead minnow is available with a reported NOAEC 

of 0.06 μg/L (MRID 40641701). However in this study, excessive analytical variation (high:low 

ratios of 3.0 to 7.8) was observed at all levels during the study, and the range of concentrations 

was so great that overlapping of concentrations occurred for all levels. Therefore, there is 

uncertainty with regards to the reported NOAEC in that study. Given the potential for antiPage 35 of 72
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Page 28 of 64 

androgen effects observed in the Tier 1 weight of evidence analysis, the EDSP Tier 2 Medaka 

Extended One Generation Reproduction Toxicity (MEOGRT) study is recommended. With that 

recommendation, it is noted that given the physio-chemical properties of cypermethrin and the 

results in the fathead minnow life-cycle study, there may be difficulties in maintaining 

cypermethrin concentrations in aqueous systems over the duration of a long-term test such as the 

Tier 2 fish assay and careful attention and maintenance of the test system will likely be required. 

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

duck are available for cypermethrin. In both species, no effects on reproductive parameters were 

observed; an additional study with alpha cypermethrin did report potential reproductive effects 

but there are uncertainties in this study. 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 cypermethrin exposure. Relative to the EDSP, additional 

testing is not recommended. 

Given the lack of observed potential thyroid-related effects in the AMA, no additional testing 

with amphibians is recommended. 

 

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VI. References 

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an immune-immobilized androgen receptor assay (IRA) and its application for the 

characterization of the receptor binding affinity of different pesticides. Chemosphere 46:1107-

1115. 

Chen, H., Xiao, J., Hu, G., Zhou, J., Xiao, H., and Wang, X. (2002) Estrogenicity of 

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Part A 65:1419-1435. 

Daly, I.W. (1994) A Subchronic (3-Month) Oral Toxicity Study of FMC 30980 Technical in the 

Dog via Dietary Administration. Unpublished study performed by Pharmaco LSR Inc., 

Toxicology Services North America, Mettlers Road, East Millstone, NJ. Study No. 92-3114. 

FMC No. A92-3706. March 31, 1994. (MRID 44527002) 

Du, G., Shen, O., Sun, H., Fei, J., Lu, C., Song, L., Xia, Y., Wang, S., and Wang, X. (2010) 

Assessing hormone receptor activities of pyrethroid insecticides and their metabolites in reporter 

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Freeman, C. (1994) Cypermethrin Technical Oral Teratology Study in Rabbits. Unpublished 

study performed by FMC Corporation Toxicology Laboratory, Study No.: A93-3822, October 

28, 1994. (MRID 43776302) 

Hend, R.W., Hendy, R., and Fleming, D.J. (1979) Toxicity studies on the insecticide WL 43467: 

A three generation reproduction study in rats. Unpublished study performed by Shell Toxicology 

Laboratory, Study No.: TLGR.0188.78, February, 1979. (MRID 00090040) 

Herberth, M.T. (2011) Cypermethrin-Uterotrophic Assay in Ovariectomized Rats. Unpublished 

study performed by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Report No.: 

WIL-639150, November 10, 2011. (MRID 48615701) 

Herberth, M.T. (2012) Cypermethrin – Pubertal Development and Thyroid Function in Intact 

Juvenile/Peripubertal Female Rats. Unpublished study performed by WIL Research 

Laboratories, LLC, Ashland, OH. Laboratory Project ID Number: WIL-639152, April 9, 2012. 

(MRID 48683002) 

Herberth, M.T. (2012) Cypermethrin – Pubertal Development and Thyroid Function in Intact 

Juvenile/Peripubertal Male Rats. Unpublished study performed by WIL Research Laboratories, 

LLC, Ashland, OH. Laboratory Project ID Number: WIL-639151, April 9, 2012. (MRID 

48683003) 

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Hu, J.X., Li, Y.F., Li, J., Pan, C., He, Z., Dong, H.Y., and Xu, L.C. (2013) Toxic effects of 

cypermethrin on the male reproductive system: with emphasis on the androgen receptor. J Appl 

Toxicol. 33(7):576-585. 

Jin, M., Li, L., Xu, C., Wen, Y., and Zhao, M. (2010) Estrogenic activities of two synthetic 

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

Kalinowski, A.E., Banham, P.B., Chart, I.S., Cook, S.K., Gore, C.W., and Moreland, S.F. (1982) 

Cypermethrin: One Year Oral Dosing Study in Dogs, Unpublished study performed by ICI, 

Report: CTUP/703, FMC study number A82-763. (MRIDs 00112909, 42068503, and 92027037)

Kim, I.Y., Shin, J.H., Kim, H.S., Lee, S.J., Kang, I.H., Kim, T.S., Moon, H.J., Choi, K.S., Moon, 

A., and Han, S.Y. (2004) Assessing estrogenic activity of pyrethroids insecticides using in vitro 

combination assays. Journal of Reproduction and Development 50(2): 245-255. 

Kjeldsen, L. S., Ghisari, M., and Bonefeld-Jørgensen, E.C. (2013) Currently used pesticides and 

their mixtures affect the function of sex hormone receptors and aromatase enzyme activity. 

Toxicol Appl Pharmacol. 272(2): 453-464.

Lee, M.R. (2012) Cypermethrin: Amphibian Metamorphosis Assay with African Clawed Frog 

(Xenopus laevis). Unpublished study performed by Smithers Viscient, Inc., Wareham, MA. 

Laboratory report number 1781-6765. Study sponsored by Syngenta Crop Protection, LLC, 

Greensboro, NC. Study completed November 28, 2012. (MRID 49013801)

Lindsay, S., Banham, P.B., Chart, I.S., Chalmers, D.T., Godley, M.J., Taylor, K. (1982) Lifetime 

Feeding Study in Mice, Unpublished study performed by ICI, Report: CTUP/687, FMC study 

number A82-762. (MRIDs 00112911 and 92027038) 

Li, Y.F., Pan, C., Hu, J.X., Li, J., and Xu, L.C. (2013) Effects of cypermethrin on male 

reproductive system in adult rats. Biomed Environ Sci. 26(3):201-208.

Liu, L., Hu, J.X., Wang, H., Chen, B.J., He, Z., and Xu, L.C. (2010) Effects of beta-cypermethrin 

on male rat reproductive system. Environ Toxicol Pharmacol. 30(3):251-256.

Milburn, G.M., Forbes, D., Banham, P.B., Chart, I.S., Godley, M.J., Gore, C.W., Pratt, I., Scales, 

M.D.C., Stonard, M.D., and Woollen, B.H. (1982) Cypermethrin: 2 Year Feeding Study in Rats. 

CTL report number: CTUP/669. FMC study number A82-761. (MRID 00112910) 

Milburn, G.M., Banham, P.B., Birtly, R.O.N., Godley, M.J., and Moreland, S.F. (1982) 

Cypermethrin: Three-Generation Reproduction Study in the Rat. Unpublished study performed 

by ICI Central Toxicology Laboratory, Study No.: RR0143, July 9, 1982. (MRIDs 00112912, 

42068504, 92027040) 

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Nemec, M.D. (2005) A dietary developmental neurotoxicity study of zeta-cypermethrin technical 

in rats. Unpublished study performed by WIL Research Laboratories, LLC, Ashland, Ohio. 

Study Number WIL-105018; August 24, 2005. (MRID 46670402) 

Roberts, N.L., Fairley, C., Chanter, D., McAllister, A., and Almond, R. (1981) The effect of 

dietary inclusion of cypermethrin on reproduction in the bobwhite quail. ICI 342/81341 (FMC 

no. Cy 10.1.2/1). Unpublished study performed by The Huntingdon Research Centre, 

Cambridgeshire, UK; submitted by ICI Americas Inc., Wilmington, Delaware. (MRID 

00090074)

Roberts, N.L., Fairley, C. (1981) The effect of dietary inclusion of cypermethrin on reproduction 

in the Mallard duck. Unpublished study performed by Huntingdon Research Centre, 

Cambridgeshire, UK. Report no.: ICI 34118164 (FMC no. Cy 10.1.2/2). (MRID 00090073) 

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

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

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

Stevens, J (2013). Cypermethrin - Weight of Evidence Evaluation of Tier 1 Endocrine 

Disruptor Screening Program Results Assessment. Report Number: CCWOE-001 

FMC Corporation, Philadelphia, PA 19103. Syngenta Crop Protection, LLC, Greensboro, NC 

27419-8300 and United Phosphorus, Inc.,King of Prussia, PA 19046. MRID No. 49068201 

Tapp, J.F. et al. 1998. Cypermethrin: determination of chronic toxicity to fathead minnow 

(Pimephales promelas) full lifecycle. ICI PLC, Wilmington DE, Brixham Laboratory, Deveon 

England. Laboratory Report EL/B/3173. MRID 40641701. 

Tesh, J.M., Tesh, S.A., and Davies, W. (1978) WL 43467: Effect upon the progress and outcome 

of pregnancy in the rat. Unpublished study performed by Life Science Research, Study No.: 

78/SHL2/364, October 4, 1978. (MRID 00056804, 92027039 or 92027061) 

Tyler, C.R., Beresford, N., Van der Woning, M., Sumpter, J.P., and Thorpe, K. (2000) 

Metabolism and environmental degradation of pyrethroid insecticides produce compounds with 

endocrine activities. Environ. Toxicol. and Chem. 19(4):801-809. 

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

EDSP-109702-75, 76, and 78. December 14, 2010. Office of Chemical Safety and Pollution 

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

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

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

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

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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. (2012) Cypermethrin - H295R Steroidogenesis Assay. Unpublished study performed 

by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9047V-100353STER, January 17, 

2012. (MRID 48670203) 

Wang, X.Z., Liu, S.S., Sun, Y., Wu, J.Y., Zhou, Y.L., and Zhang, J.H. (2009) Beta-cypermethrin 

impairs reproductive function in male mice by inducing oxidative stress. Theriogenology

72(5):599-611. 

Wilga, P. (2012) Cypermethrin: Human Recombinant Aromatase Assay. Final Report. 

Unpublished study performed by CeeTox, Inc., Kalamazoo, MI. Laboratory Report No.: 9047V100353AROM, January 17, 2012. (MRID 48670202) 

Willoughby, J.A. (2012) Cypermethrin: Estrogen Receptor Transcriptional Activation (Human 

Cell Line (HeLa-9903)) Final Report. Unpublished study performed by CeeTox, Inc, 

Kalamazoo, MI. Laboratory Report No.: 9047V-100353ERTA, January 16, 2012. (MRID 

48670201) 

Xu, L.C., Liu, L., Ren, X.M., Zhang, M.R., Cong, N., Xu, A.Q., and Shao, J.H. (2008) 

Evaluation of androgen receptor transcriptional activities of some pesticides in vitro. Toxicology

14; 243(1-2):59-65. 

York, D.O. (2012) Cypermethrin – Fish Short-term Reproduction Assay with the Fathead 

Minnow (Pimephales promelas). Performed by Smithers Viscient, Wareham, Massachusetts, 

Laboratory Project No. 1781.6766. Submitted by Syngenta Ltd, Jealott’s Hill International 

Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom; FMC Corporation, 

Philadelphia, Pennsylvania; United Phosphorus, Inc., King of Prussia, Pennsylvania. Completion 

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pyrethroid pesticides cyfluthrin and β-cyfluthrin. Reproductive Toxicology 25:491-496. 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA): (OCSPP 890.1100) 

The 21-day assay (MRID 49013801) of cypermethrin (95.2% purity) on amphibian 

metamorphosis of African Clawed Frog (Xenopus laevis) was conducted under flow-through 

conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 larvae per group) were 

exposed to a negative control and test chemical nominal concentrations of 0.0000030, 0.000030, 

and 0.00030 mg a.i./L. The time-weighted average (TWA) concentrations were <0.0000010 

(<LOQ, control), 0.0000019, 0.0000109, and 0.0000867 mg a.i/L; the %CV exceeded 20% for 

all treated levels. The test system was maintained at 22 to 23°C and a pH of 7.2 to 8.2. 

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

water) control. Two mortalities occurred at the low treatment level and one mortality occurred at 

the mid treatment level. No other mortalities were observed. Clinical signs including spinal 

deformities were observed in 27 tadpoles at the negative control and low treatment levels and in 

22 tadpoles at the mid and high treatment levels. 

Cypermethrin caused no significant increase or decrease (p>0.05) in 7-day hind-limb length 

(HLL) at any treatment level. There was no significant acceleration or delay (p>0.05) of median 

NF developmental stage observed at Day 7 or 21 at any treatment level relative to the negative 

control. Further, no asynchronous development was observed. More than half of all individuals 

developed beyond NF stage 60 by Day 21 (i.e., NF stage 61-62), with 53%, 59%, 54%, and 52% 

of living frogs exhibiting late stage (NF>60) development in the control, low, mid, and high 

groups, respectively. There was no indication of a dose response pattern for occurrence of latestage development and both the reviewer and study author conducted separate analysis of only 

late-stage individuals. The reviewer’s analysis indicated a significant dose-dependent reduction 

(p<0.05; Jonckheere-Terpstra) in both Day 7 snout-vent length (SVL) (4%) and wet weight 

(12%) of tadpoles exposed to the high treatment group, relative to the control. No other growth 

or developmental effects were noted in this study, including for the additional analysis of latestage frogs on Day 21. No effects on thyroid gland histopathology were observed at any 

treatment level. 

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

Requirement satisfied by OSRI (Zhang et al., 2008; and Xu et al., 2008); see Appendix 2.

Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP19) assay (MRID 48670202), cypermethrin (95.2% a.i. Lot # 

PL10-0492) was incubated with human recombinant aromatase and tritiated androstenedione (1-

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

concentrations of 10-10 to 10-4 M for 15 minutes to assess the potential of cypermethrin 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 independent runs of the aromatase assay were 

conducted and each test concentration of cypermethrin was tested in triplicate. 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). The highest 

concentrations of cypermethrin tested in Run 1 (10-3 M) and in Run 2 (10-3.5 M) showed marked 

precipitation during the 15-minute incubation period and these data were not evaluated. The 

highest concentration for all three runs used for data interpretation was 10-4 M because it did not 

result in marked precipitation. 

Aromatase activity in the full activity controls averaged 0.222 ± 0.049 nmol/mg-protein/min. 

The response of each full activity control within a run was between 90 to 110% of the average 

full activity. Activity in the background controls ranged from 0.45 to 0.78% and averaged 

0.64% of the full activity control. 

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 -7.17 M and the slope was -0.94. 

For cypermethrin, aromatase activity averaged 0.170 ± 0.018 nmol/mg-protein/min at the lowest 

tested concentration 10−10 M and 0.193 ± 0.042 nmol/mg-protein/min at the highest tested 

concentration, 10−4

 M, used for data interpretation. The average dose-response curve indicated 

that the aromatase activity at all concentrations of cypermethrin tested was essentially equivalent 

to the activity observed in the full activity control (>75% activity= non-inhibitor). 

Based on the data from the average response curve, cypermethrin 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; Chen et al., 2002); see Appendix 2.

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

In an estrogen receptor transcriptional activation assay (MRID 48670201), hERα-HeLa-9903 

cells cultured in vitro were exposed to cypermethrin (95.2% a.i., Batch PL10-0492) at 

logarithmically increasing concentrations from 10−10.3 to 10−3.3 M in DMSO (0.1%) for 

approximately 24 hours in three independent runs. A total of 8 runs were conducted, but only 

the data from three runs were reported. The experiments were performed using 96-well plates 

and each cypermethrin concentration was tested in replicates of 6 wells/plate in each run. Cells 

were exposed to the test agent for 24±2 hours at 37±1°C to induce reporter (luciferase) gene 

products. Luciferase expression in response to activation of the estrogen receptor by 

cypermethrin was measured using a proprietary luciferase assay. 

Cypermethrin was tested up to the limit of solubility, 10-4 M. The RPCMax was 16.3% at 10-4 M 

and the PC10 was 10-4.6 M in Run 1, indicating a positive response to the assay. However, in 

Runs 2 and 3 the RPCMax for cypermethrin was ≤0.9% at concentrations up to 10-3.3 M, indicating 

that cypermethrin was negative for estrogenic activity under the conditions of the assay. Review 

of the performance criteria parameters for Run 1 (Table 3) shows that Run 1 was unusually 

sensitive, with all parameters for all reference chemicals at lower levels than expected according 

to the guidance. 

In all runs, cypermethrin response was similar to that of the negative control (corticosterone), 

and in no run did it reach levels similar to the weak estrogen 17α-methyltestosterone. For Run 2, 

Log PC50 values could not be determined for 17α-methyltestosterone as it did not reach 50% 

activation at concentrations up to 10-5 M. This indicates a decreased sensitivity of cells. 

However in that same run, responses were close to zero at all cypermethrin concentrations. 

Although the conditions of this assay were not optimal, in general cypermethrin responses were 

similar to those of the negative control corticosterone and not comparable to the responses of 

17α-methyltestosterone, which was able to reach maximums of 124.3, 16.5, and 52.8% PC in 

Runs 1, 2, and 3, respectively. Cypermethrin was only able to reach maximums of 16.3, 0.9, and 

0.6% PC in Runs 1, 2, and 3, respectively, when tested up to the highest concentration possible 

based on cytotoxicity. Because the RPCMax < PC10 in both assay runs, cypermethrin was 

considered negative for estrogen receptor transcriptional activation in this test system. 

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

The 21-day short-term reproduction assay (MRID 48683001) of cypermethrin with fathead 

minnows (Pimephales promelas) was conducted under flow-through conditions. Adult fish (16 

spawning groups, 2 males and 4 females in each group; 5 months old) were exposed to 

cypermethrin (95.2% purity) at nominal concentrations of 0 (control), 0.000030, 0.00030, and 

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0.003 mg a.i./L; mean-measured concentrations were <LOQ (<0.000001), 0.000013, 0.00012, 

and 0.0014 mg a.i./L. The test system was maintained at 25 to 26°C and a pH of 7.0 to 7.7. 

There were no significant differences in either male or female survival at any treatment levels 

compared to the negative control. Percent survival was 100% for all groups of males and was 

100, 94, 94, and 88% for the negative control and low, mid, and high treatment concentrations, 

respectively, for females. During the in-life exposure, there were no notable observations that 

occurred with regards to behavior, coloration/banding, changes in ovipositor appearance, or size 

of the dorsal nape pad, and no clinical signs of toxicity were observed. There were no treatmentrelated effects on male and female body weight and length. 

Spawning occurred at least every 4 days in thee of the four control replicates and mean fecundity 

in control averaged 14 eggs/female/day/replicate (range: 13-18 eggs/female/day among the 

replicates); fertilization success in the control group averaged 96%. Fecundity was decreased 

(p<0.05) by 42% and 84% in the mid- and high-treatment females, respectively. There were no 

treatment-related effects (p>0.05) on fertilization success. 

Male gonadal somatic index (GSI) was increased (p<0.05) by 22 and 27% at the mid and high 

treatment concentrations, respectively, compared to the negative control. There were no 

treatment-related effects on female GSI (p>0.05) or male and female plasma vitellogenin (VTG). 

Additionally, there were no significant effects on median male tubercle score, and no tubercles 

were observed on any females. 

Although not analyzed statistically, in male fish, there were only background or sporadic gonadal 

histopathological findings noted that were not considered related to treatment. In female fish, 

there was an increased incidence of mature oocyte atresia at the high treatment concentration, 

which was associated with granulomatous inflammation and the presence of microsporidia in 

half the observed cases across all treatment levels. There were no significant findings observed 

in the gonadal stage for either males or females, and no notable observations in secondary sex 

characteristics for any treatment group compared to the controls. 

The performance criteria for fecundity was not met. There were one replicate in the control 

group (Replicate C) where there was a period of 4 days in which no spawning occurred and 

fecundity was 13 eggs/female/day. Therefore, this replicate did not meet the criteria of either at 

least 15 eggs/female/day or spawning at least every 4 days. However, it is noted that the overall 

control mean fecundity value (14) was just slightly below 15 eggs/female/day, and the other two 

replicates did achieve 15 or greater eggs/female/day and the remaining replicate spawned at least 

every 4 days (11 eggs/female/day). Also, the %CV values for the mean recoveries at the two 

highest treatment concentrations were 45.6 and 51.2%, which did not meet the validity criteria of 

<20% over the 21-day test. There was no pattern of decline during the study period and no undissolved test substance was observed in the dilution system, as such, the recoveries indicate the 

test material was generally poorly recovered in solution under the test conditions. Given the 

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physicochemical properties of cypermethrin, these results may be considered consistent with 

expectations for the chemical and previous experience with the test item. The remaining validity 

criteria for OCSPP 890.1350 were met. Therefore, while these deviations are noted, they did not 

sufficiently impact the interpretation of the assay results. 

Hershberger Assay; (OCSPP 890.1400) 

Requirement satisfied by OSRI (Zhang et al., 2008; MRID 48196302); see Appendix 2. 

Female Pubertal Assay; (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48683002), 15 Sprague Dawley rats/dose group were treated 

daily via oral gavage with cypermethrin (95.2% a.i.; cis/trans ratio: 49.8/50.2; Batch #PL10-

0492) in corn oil at doses of 0 (corn oil), 15 or 30 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 

weight at day of attainment was recorded. Following sacrifice on PND 42/43, total thyroxine 

(T4) levels were determined using an electrochemi-luminescent immunoassay and thyroid 

stimulating hormone (TSH) levels were determined using a radioimmunoassay. Adrenal, liver, 

thyroid, pituitary, and urogenital organ weights were recorded, and microscopic examinations of 

the uterus, ovaries, thyroid, and kidney were performed. 

One animal in the 30 mg/kg/day group was found dead on PND 25. No internal findings were 

noted for this animal during gross examination at necropsy. All other rats survived until 

scheduled sacrifice. As there was no other evidence of toxicity in this study, this single death 

was considered incidental. There were no treatment-related clinical signs of toxicity at any dose 

level; findings noted in the treated groups occurred infrequently in single animals and/or in a 

manner that was not considered dose related. Mean body weights, daily body weight gains, 

cumulative mean body weight gain, and food consumption in the treated groups were similar to 

the control group throughout the study. 

No treatment-related effects were observed on mean age or weight at vaginal opening (VO), and 

mean age at first estrus. The cycle status at necropsy (diestrus, proestrus, estrus, or not cycling) 

appeared similar across all groups. There were no treatment-related changes in serum hormone 

or clinical chemistry levels. In addition, there were no effects of treatment observed in gross or 

microscopic pathology. 

There was no evidence of direct test substance-related effects on pubertal development or thyroid 

function in the juvenile/peripubertal female rat at cypermethrin dose levels of 15 and 30 

mg/kg/day. 

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No evidence of toxicity was observed in the study up to the highest dose tested; however, 

treatment-related clinical signs were seen in the preliminary range-finding study at 30 and 65 

mg/kg/day. In the preliminary study, tremors and clear material around the mouth were seen at 

30 mg/kg/day, 4 hours after administration. At 65 mg/kg/day (after 4 hours), tremors, splayed 

limbs, and clear material around the mouth, ventral neck and thoracic area, and forelimbs were 

seen. It is possible that the effects observed in the preliminary study were missed in the 

definitive study since clinical signs were measured 1 hour after chemical administration. 

Clinical signs were measured 4 hours after administration in the preliminary study, which is 

closer to the time-to-peak effect for cypermethrin. Therefore, dosing levels tested in this assay 

are considered appropriate. 

Male Pubertal Assay; (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48683003), 15 Sprague Dawley rats/dose group were treated 

daily via oral gavage with cypermethrin (95.2% a.i.; cis/trans ratio: 49.8/50.2; Batch #PL10-

0492) in corn oil at doses of 0 (corn oil), 15, or 30 mg/kg/day from post-natal day (PND) 23 to 

53/54. Animals were examined for preputial separation (PPS) daily beginning on PND 30 and 

weight at day of attainment was recorded. Following sacrifice on PND 53 or 54, total serum 

testosterone and thyroxine (T4) levels were determined using an electrochemiluminescent 

immunoassay, and thyroid stimulating hormone (TSH) levels were determined using a 

radioimmunoassay. Adrenal, liver, thyroid, pituitary, and urogenital organ weights were 

recorded, and microscopic examinations of the testis, epididymis, thyroid, and kidney were 

performed. 

One male in the 15 mg/kg/day group was found dead approximately one hour following dosing 

on PND 45, which was likely due to dosing error. All other rats survived until scheduled 

sacrifice. There were no treatment-related clinical signs of toxicity at any dose level. Findings 

noted in the treated groups, including red and/or clear material around the nose and/or mouth, 

occurred infrequently in single animals and/or in a manner that was not considered dose related. 

Mean body weights, daily body weight gains, cumulative mean body weight gain, food 

consumption, mean age of attainment of complete PPS, and mean body weight at the age of 

attainment in the 15 and 30 mg/kg/day groups were similar to the control group throughout the 

study. 

No treatment-related gross observations were noted at necropsy and no significant effects of 

treatment were observed on the weights of any of the organs and tissues collected. 

There were no test substance-related changes in serum T4, TSH and testosterone values at any 

dose level. Urea nitrogen (↓17%) and bile acid (↓52%) were decreased (p<0.05) in the 30 

mg/kg/day group compared to controls. However, decreases in bile acids have not been 

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associated with toxicological effects. The slight decrease in blood urea nitrogen was within the 

historical control reference range and considered an incidental finding. There were no other 

serum chemistry changes in the cypermethrin-dosed groups compared to the control group. 

There were no test substance-related histologic changes in the thyroid, testes, epididymides, and 

kidneys. The amount of colloid and follicular height of thyroid epithelial cells were similar 

between control and test substance-treated animals. 

No evidence of toxicity was observed in the study up to the highest dose tested; however, 

treatment-related clinical signs were seen in the preliminary range-finding study at 30 and 65 

mg/kg/day. In the preliminary study, tremors and clear material around the mouth were seen at 

30 mg/kg/day, 4 hours after administration. At 65 mg/kg/day (after 4 hours), tremors, splayed 

limbs, and clear material around the mouth, ventral neck and thoracic area, and forelimbs were 

seen. It is possible that the effects observed in the preliminary study were missed in the 

definitive study since clinical signs were measured 1 hour after chemical administration. 

Clinical signs were measured 4 hours after administration in the preliminary study, which is 

closer to the time to peak effect for cypermethrin. The dose levels used in the study are 

considered adequate based on the results of the range-finding study. 

Steroidogenesis Assay; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48670203), H295R cells cultured in vitro in 24-well plates 

were incubated with cypermethrin (95.2% purity, Lot # PL10-0492) at concentrations of 100, 10, 

1, 0.1, 0.01, 0.001, and 0.0001 μM for 48 hours in triplicate for four independent experiments. 

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

precipitation was observed at 100 μM, this group was not evaluated statistically. Run 1 was not 

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

(3.3 μM was used instead of 1 μM). 

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

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

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

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

testosterone production. 

Guideline recommendations and requirements were met, including adequate production of 

testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction and 

inhibition with positive controls. Cytotoxicity greater than 20% was not observed. 

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Statistically significant increases in estradiol were noted at 10 μM in Runs 3 and 4, and estradiol 

levels in all three runs at 10 μM were 111-117% of the solvent control (SC). All other values of 

testosterone and estradiol in the treated cells were similar to the SC. 

Based on the hormone responses in each of the three independent runs, cypermethrin treatment 

resulted in statistically significant and reproducible increases in estradiol production, but 

cypermethrin treatment did not result in statistically significant and reproducible alterations in 

testosterone production. 

Uterotrophic; (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48615701) conducted to screen for potential estrogenic activity, 

cypermethrin (technical, 95.2% a.i., Batch # PL10-0492) in corn oil was administered daily via 

oral gavage (dosing volume 5 mL/kg bw) to groups of six ovariectomized female Sprague 

Dawley rats/dose at dose levels of 0 (vehicle), 25, or 80 mg/kg/day on post-natal days (PND) 66 

to 68. The positive control group was treated with a daily dose of 17α-ethynyl estradiol (EE) in 

corn oil at 0.3 mg/kg/day by oral gavage (dosing volume 5 mL/kg bw). The selected dose levels 

were based on toxicity observed in the dose range-finding study (MRID 48615702), in which 

clinical signs and body weight gain effects were observed at 80 and 100 mg/kg/day, and 

marginal effects at 65 mg/kg/day. Body weights and food consumption were determined daily. 

All animals were terminated and necropsied on PND 69 approximately 24 hours after the final 

dose administration to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. Treatment-related clinical signs of toxicity 

included one or two animals with dried red material around nose, hypersensitivity to handling, 

vocalization upon handling, salivation and/or wet clear material around mouth at 80 mg/kg/day. 

Body weights in the treatment groups were comparable to vehicle controls throughout the 

duration of the study. At 80 mg/kg/day, the overall body weight gain was significantly 

decreased (p<0.05; ↓51%). The overall average food consumption (Days 1-4) was decreased 

(p<0.05) by 15% at 80 mg/kg/day. Overall average food consumption in the 25 mg//kg/day 

group was comparable to the vehicle control. Uterine weights in the cypermethrin treatment 

groups were comparable to the vehicle controls. 

In the positive control (EE) group, body weights were decreased (p<0.05) by 11% on Day 4 

compared to the vehicle control, leading to an overall body weight loss (p<0.05) of –14.9 g 

compared to a gain of 14.6 g in the vehicle controls. Overall average food consumption in the 

positive control (EE) group was decreased (p<0.05) by 46%. Absolute wet and blotted uterus 

weights for the positive control (EE) group were increased (p<0.05) by 370% and 171%, 

respectively, as expected. 

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The clinical signs and 51% decrease in overall body weight gains in the high-dose group 

(together with consistent findings in the range-finding study at doses of 65, 80 and 100 

mg/kg/day) indicated that the tested dose levels were adequate. 

No statistically significant changes were seen in uterine weight in this assay. Cypermethrin is 

negative in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

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

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

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

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

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

development studies, chronic toxicity studies, etc.). 

AR Binding

In a published study, Bauer et al. (2002) assessed cypermethrin (91.0% purity; cis/trans ratio is 

not stated) binding to recombinant human AR (hAR) with an immuno-immobilized AR binding 

assay. 

The cDNA of the hAR was inserted into the transfer plasmid pAcSG-His NT-C. The 

recombinant Baculovirus was obtained by transfection of insect cells (Sf9) with linearized 

Baculo gold virus DNA and the recombinant plasmid. After 40 hours, the cells were collected 

by centrifugation, combined with disruption buffer, and disrupted by three freezing and thawing 

cycles. Supernatant was collected after a 100,000×g centrifugation for 15 minutes at 4°C. 

The receptor preparation was diluted 1:60 in assay buffer and a 100 μL aliquot/well was 

incubated for 16 hours at 4°C with 0.44 nM 3

H-DHT in the presence or absence of increasing 

concentrations of the pesticides or DHT. The receptor was fixed on the microtiter plate with 

50 μL of the 1:500-diluted specific hAR antibody. Receptor binding was determined by LSC. 

Specific binding represents the difference of total binding and non-specific binding in presence 

of a 200-fold surplus of unlabelled DHT. Cypermethrin was tested at multiple concentrations up 

to the maximal soluble concentration (concentration range not reported). Cypermethrin did not 

displace 3

H-DHT; therefore, a Ki was not determined and a relative binding affinity of <0.01 was 

assigned. 

In another published study, Tyler et al. (2000) assessed cypermethrin (95.8% purity; cis/trans

ratio is not stated) binding to hAR in a recombinant yeast expression system (PGKhAR) assay. 

In the presence of an active ligand, such as the positive control (DHT), the androgen receptor 

binds to an androgen-responsive element on a plasmid, thereby initiating transcription of the 

reporter gene lacZ, which encodes the enzyme β-galactosidase. This produces a change in color 

from yellow to red that is determined by measuring the absorbance at 540 nm. A stock solution 

of cypermethrin was serially diluted in ethanol, and 10-μL portions of the appropriate dilutions 

were added to sterile incubation plates and evaporated to dryness. The androgenic positive 

control was DHT, the anti-androgenic positive control was flutamide, and the negative control 

was ethanol. For the anti-androgenic assay, DHT was added to the medium at a concentration of 

2.5×10−9

 M. Portions (200 μL) of the assay medium that contained the recombinant yeast were 

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added to the plates; plates were sealed and shaken for two minutes prior to incubation at 28°C for 

24 hours followed by room temperature incubation until optimal color development had occurred 

(details not reported). The plates were read at 540 and 620 nm (turbidity correction for yeast 

growth). 

Cypermethrin was assessed for androgenic and anti-androgenic activity across a concentration 

range of 10−6 to approximately 10−3 (possibly 10−2.8). Cypermethrin did not demonstrate any 

androgenic activity; therefore, EC50 and LOEC50 estimates could not be determined. Antiandrogenic activity was observed at cypermethrin concentrations approximately >10−3

 M, 

yielding an LOIC value of 1×10−3

 M (1000-fold less potent compared to flutamide). 

AR Transcriptional Activation Assays 

In a published study, Zhang et al. (2008) examined the potential anti-androgenic effects of 

cyfluthrin and β-cyfluthrin with reporter gene assays in vitro. A comparison of anti-androgenic 

activities was also made to four structurally-related pyrethroids (permethrin, cypermethrin, 

β-cypermethrin, and bifenthrin). This study was evaluated with specific attention to cypermethrin 

and β-cypermethrin (purities not reported; cis/trans ratio is not stated). 

For the in vitro reporter gene assay, the MDA-kb2 breast cancer cell line, which was stably 

transfected with an androgen-responsive reporter gene (pMMTV.neo.luc), was treated when the 

cell reached 80% of confluence with the various pyrethroids or the anti-androgen 

hydroxyflutamide (OHF), both with and without dihydrotestosterone (DHT; 1 nM). Each test 

substance was prepared in ethanol with a final ethanol concentration in the medium of 0.1%. 

Cypermethrin and β-cypermethrin were each tested at a concentration of 1 × 10−5 M. Cells were 

incubated at 37°C with CO2 overnight for 24 hours, the dosing medium was removed, cells were 

washed once with phosphate-buffered saline, and 25 μL of lysis buffer was added per well. 

After incubation for 25 minutes, the cells were lysed, 100 μL of luciferase reaction agent was 

added, and luciferase activity was determined. The results of the assay were expressed as the 

percent of androgenic activity for each compound relative to the DHT control. 

In the in vitro reporter gene assay, cypermethrin inhibited (p<0.05) DHT-induced transcriptional 

activation at a concentration of 1×10−5

 M (the only concentration tested), but β-cypermethrin had 

no effect. Although the data were only presented graphically, treatment with cypermethrin 

resulted in approximately a 45% decrease in androgenic activity compared to control (1 nM 

DHT). The positive control (OHF) resulted in an approximate 65% decrease in androgenic 

activity. 

The authors concluded that cypermethrin has anti-androgenic effects in vitro; however, 

β-cypermethrin did not demonstrate anti-androgenic activity. 

In another published study, Xu et al. (2008) conducted transcriptional activation assays to detect 

AR agonists and antagonists using an African monkey kidney cell line (CV-1 cells) transfected 

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with the reported plasmid pCAT3-Basic (containing mouse mammary tumor virus sequences for 

four androgen receptor elements) and a pSV-Gal reporter. Cells were transfected, and incubated 

for an additional 24-hour period prior to addition of DHT (1nM) and test chemicals. 

Cypermethrin (>98% purity; cis/trans ratio is not stated) was tested at 10−7

, 10−6, and 10−5 M (in 

ethanol, 0.1%) with and without DHT against a vehicle control (medium with 0.1% ethanol). 

The highest concentration (10−5 M) was the limit of cytotoxicity. Cypermethrin was reported to 

be an anti-androgen with an IC50 of 6.80 ± 2.3×10−5

 M, with a decrease (p<0.01) in androgenic 

activity compared to DHT at 10−5 M cypermethrin. No androgen agonism was detected.

In a published study, Du et al. (2010) examined the potential androgenic and anti-androgenic 

effects of pyrethroids (including cypermethrin, purity 92%; cis/trans ratio is not stated). For the 

in vitro reporter gene assay, the MDA-kb2 breast cancer cell line was stably transfected with the 

androgen-responsive reporter gene (pMMTV.neo.luc) as used by Zhang et al. (2008). The cells 

were plated, allowed to attach (4-6 hours), followed by treatment with cypermethrin (10−9

 to 10−5

M) with or without DHT, 5α-DHT (10−12 to 10−7 M; positive control), or 0.1% DMSO (negative 

control). Cells were treated with the cypermethrin alone and with 1×10−6 M flutamide, a potent 

antagonist of AR, to distinguish between AR- and GR-related activity. Determination of 

luciferase activity was similar to that described in Zhang et al. (2008). No AR agonist activity 

was observed for cypermethrin; however, an anti-androgenic effect was observed with a 

calculated relative IC20 of 1.64×10−6

 M. 

In a published study, Kjeldsen et al. (2013) determined the androgenic and anti-androgenic 

properties of cypermethrin (and other pesticides) with in vitro tests including an ARTA assay 

that used the Chinese hamster ovary cell line (CHO-K1). Cypermethrin (>95% purity) was 

dissolved in DMSO, with final DMSO concentrations of 0.1%. Cypermethrin was tested in 

various concentrations within the range of 1×10−10 to 1×10−5

 M, without removal of the 

transfection reagent and cDNA. Cypermethrin was tested alone and with 25 pM of one of the 

AR agonists (R1881 or DHT, corresponding to approximately half of the maximal effect 

concentration). Cypermethrin did not display cytotoxicity at the highest tested concentration 

(1×10−5

 M), and it did not demonstrate an agonistic or antagonistic AR effect up to the highest 

cocentration.

Aromatase

In another published study, Kjeldsen et al. (2013) also assessed effects of cypermethrin on 

aromatase enzyme activity with the human choriocarcinoma JEG-3 cell line. Cypermethrin 

(>95% purity) was dissolved in DMSO, with final DMSO concentrations of 0.1%. 

Cypermethrin was tested at various concentrations within the range of 1×10−9

 to 1×10−4 M. 

Cypermethrin was found to be cytotoxic at concentrations ≥5×10−5

 M, but did not inhibit 

aromatase activity at any tested concentration ≤5×10−5 M.

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ERTA/ ER Binding/Cell Proliferation 

In a published study, Saito et al. (2000) investigated the activity of several pyrethroids 

(including cypermethrin, purity >93%; cis/trans ratio is not stated) on the ERα pathway with 

three different assay systems: a mammalian ERTA assay with HeLa cells and a luciferase 

reporter, a yeast two-hybrid system (Nishikawa procedure), and hER competitive ligand-binding 

with fluorescence polarization (Pan Vera). Positive controls were run for agonism (17β-estradiol 

and 4-nonylphenol) and antagonism (4-hydroxytamoxifen). 17β-estradiol displayed the expected 

responses in all three assays, with dose-dependent induction in the ERTA and the yeast twohybrid assay, and dose-dependent inhibition (displacement of fluorescence standard) in the 

competitive ligand-binding assay. Cypermethrin was tested at 10−7, 10−6, and 10−5 M in the 

competitive binding assay, at 1 and 10 μM in the HeLa cell assay, and 10 μM in the yeast twohybrid assay; tested dose(s) were determined on the basis of solubility. The results were 

displayed graphically; negative results for all pyrethroids (including cypermethrin) were 

observed in all assays. 

In a published study, Chen et al. (2002) tested organophosphorus and pyrethroid pesticides in an 

E-Screen (MCF-7 cell proliferation) assay and an ER Competitive-Binding assay. MCF-7 cells 

were further analyzed with the pS2 mRNA gene expression assay as assessed by qRT-PCR. 

MCF-7 cells were plated with medium at an initial concentration of 103

 cells per well and 

allowed to attach for 24 hours. The medium was removed and replaced by medium containing 

cypermethrin at a concentration range of 10−11 to 10−6

 M. The bioassay was terminated on Day 6 

(late exponential phase) by removal of the medium and addition of a thiazolyl blue solution 

(5 mg/mL PBS buffer). Absorbance was determined, after 4-hour incubation, at 490 nm. Fold 

proliferation of each exposure group was determined as the ratio of group absorbance to control 

absorbance. Uterine cytosol from female SD rats was prepared for isolation of ER. The 

Competitive Binding assay was conducted similarly to the Guideline ER Binding assay with 

[

3

H]-estradiol and 17β-estradiol. 

Cypermethrin (>90% purity; cis/trans ratio is not stated) was tested in all three assays. The PE 

(proliferation effect) value for cypermethrin was 1.46. In comparison with the PE of 17βestradiol (3.46), cypermethrin showed a partial agonistic response. Cypermethrin inhibited the 

binding of [3

H]-17β-estradiol to ER with an IC50 of 0.562 mM. These results indicate an 

equivocal or very weak positive response with percent bound of approximately 50%. In the pS2 

gene expression assay (10−10 to 10−6 M cypermethrin), cypermethrin was reported to be positive 

at 10−6

 M with an approximately 2-fold response relative to β-actin mRNA. 

Kim et al. (2004) conducted a similar investigation on seven pyrethroid pesticides (including 

cypermethrin) in an E-Screen (MCF-7 BUS cell proliferation) assay and an ER CompetitiveBinding assay. The E-Screen and ER Competitive-Binding assays were conducted in a similar 

manner as described previously for Chen et al. (2002). In addition, anti-estrogenic activity in the 

E-Screen assay was examined by combining cypermethrin (10−6

 M) and 17β-estradiol (10−10 or 

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10−11 M) in MCF-7 BUS for six days. Anti-estrogenic activity was assessed relative to the 

negative control, ICI 182,780 (10−9 M). 

Cypermethrin (98% purity; cis/trans ratio is not stated) was tested in both assays. Cypermethrin 

did not induce MCF-7 BUS cell proliferation at any concentration, and also did not demonstrate 

anti-estrogenic activity against cell proliferation. Cypermethrin was also negative in the 

Competitive Binding assay against [

3

H]-estradiol. 

In a published study, another similar investigation was conducted by Jin et al. (2010) with 

cypermethrin and permethrin in an E-Screen (MCF-7 cell proliferation) assay. MCF-7 cells were 

further analyzed with the pS2 mRNA gene expression assay as assessed by qRT-PCR. MCF-7 

cells were plated with medium at an initial concentration of 103

 cells per well and allowed to 

attach for 24 hours. The medium was removed and replaced by medium containing 

cypermethrin at a concentration range of 10−9 to 10−5 M, 17β-estradiol at 10−9 mol/L (positive 

control), or ethanol at 0.1% (v/v; negative control). The bioassay was terminated on Day 6, and 

absorbance was determined at 490 nm. Fold proliferation of each exposure group was 

determined as the ratio of group absorbance to control absorbance. 

Cypermethrin (98% purity; cis/trans ratio is not stated) was tested in both assays. Cypermethrin 

induced (p<0.05) proliferation of MCF-7 cells with a PE of 1.63, and a RPE of 56.3% at a 

cypermethrin concentration of 10−7

 M. In comparison with the PE of 17β-estradiol (2.12), 

cypermethrin showed a partial agonistic response. It was noted that the full dose response of 

cypermethrin was an inverted U-shaped dose-response curve. In the pS2 gene expression assay 

(10−9 to 10−5 M cypermethrin), cypermethrin was reported to be positive at 10−6 M with a 

2.15-fold response relative to GAPDH mRNA. 

In a published study, Tyler et al. (2000) assessed cypermethrin (95.8% purity; cis/trans ratio is 

not stated) binding to hER in a recombinant yeast expression system assay with Saccharomyces 

cerevisiae yeast. In the presence of an active ligand, such as the positive control (17β-estradiol), 

the estrogen receptor binds to an estrogen-responsive element on a plasmid, thereby initiating 

transcription of the reporter gene lacZ, which encodes the enzyme β-galactosidase. This 

produces a change in color from yellow to red that is determined by measuring the absorbance at 

540 nm. A stock solution of cypermethrin was serially diluted in ethanol, and 10-μL portions of 

the appropriate dilutions were added to sterile incubation plates and evaporated to dryness. The 

estrogenic positive control was 17β-estradiol, the anti-estrogenic positive control was 4-

hydroxytamoxifen, and the negative control was ethanol. For the anti-estrogenic assay, 17βestradiol was added to the medium at a concentration of 2.5 × 10−10 M. Portions (200 μL) of the 

assay medium that contained the recombinant yeast were added to the plates; plates were sealed 

and shaken for two minutes prior to incubation at 28°C for 24 hours followed by room 

temperature incubation until optimal color development had occurred (details not reported). The 

plates were read at 540 and 620 nm (turbidity correction for yeast growth). 

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Cypermethrin was assessed for estrogenic and anti-estrogenic activity across a concentration 

range of 10−6 to approximately 10−3 M (possibly 10−2.8 M). Cypermethrin did not demonstrate 

any estrogenic activity; therefore, EC50 and LOEC50 estimates could not be determined. Antiestrogenic activity was observed at cypermethrin concentrations approximately >10−4

 M, 

yielding an LOIC value of 7±3×10−4

 M (between 1000- and 10,000-fold less potent compared to 

4-hydroxytamoxifen). 

In another published study, Kjeldsen et al. (2013) determined the estrogenic and anti-estrogenic 

properties of various pesticides (including cypermethrin) with in vitro tests including an ERTA 

assay with the MVLN cell line. Cypermethrin (>95% purity) was dissolved in DMSO, with final 

DMSO concentrations of 0.1%. Cypermethrin was assessed alone in various concentrations 

within the range of 1×10−10 to 1×10−4

 M, and 17β-estradiol co-exposure experiments were 

carried out with pesticide concentrations ranging from 1×10−10 to 1×10−5 M. In the ERTA assay, 

cytotoxicity was observed with cypermethrin (≥5×10-5 M). Cypermethrin weakly induced the 

luciferase activity with a maximum response of 119% (at 10−6 M) compared to the solvent 

control (set to 100%). Co-exposure with 25 pM 17β-estradiol (E2-EC65; 65% of maximum 

response) did not enhance the 17β-estradiol-induced response of cypermethrin; cypermethrin did 

not exert an ER-antagonistic effect in this assay.

Hershberger 

In a study examining the potential anti-androgenic effect of several pyrethroid pesticides (Zhang 

et al., 2008), cypermethrin or β-cypermethrin (purities not reported; cis/trans ratio is not stated) 

were assessed in an in vivo Hershberger assay. The study evaluated the androgenic (cyfluthrin 

and β-cyfluthrin only) and anti-androgenic activities of cyfluthrin, β-cyfluthrin, and four 

structurally-related pyrethroids (permethrin, cypermethrin, β-cypermethrin, and bifenthrin). All 

test substances were administered in peanut oil. 

For the in vivo portion, three-week old male SD rats were maintained under standard laboratory 

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

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

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

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

conducted with a sham-operated control group, but this assessment did not include cypermethrin 

or β-cypermethrin. The anti-androgenic portion of the Hershberger assay included eight 

treatment groups: a vehicle control group (peanut oil only); a positive control group dosed orally 

with flutamide (50 mg/kg/day); and six groups each dosed orally with testosterone propionate 

(TP) and a pyrethroid. Cypermethrin and β-cypermethrin were each dosed orally at 

50 mg/kg/day for seven days in addition to a daily s.c. injection of TP at 0.5 mg/kg/day. After 

the final dose, rats were anaesthetized and sacrificed by exsanguination. Blood from each rat 

was collected for serum analysis of testosterone concentration. The seminal vesicles, ventral 

prostate, dorsolateral prostate, LABC, Cowper’s glands, glans penis, liver, paired adrenal glands, 

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spleen, and paired kidneys of each rat were dissected and weighed. Serum testosterone 

concentrations were analyzed by time-resolved fluorescence with commercially available FIA 

kits. 

No mortality or treatment-related clinical signs were noted in any of the groups in the 

Hershberger assays. The text states there were no significant differences between initial or final 

body weights, or weight gains of the pyrethroid-treated groups compared to the control groups, 

but the actual data for the cypermethrin-treated groups were not presented. Serum testosterone 

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

treatment-related effects were observed. Treatment with cypermethrin at 50 mg/kg/day resulted 

in a decrease (p<0.05) in absolute weights of the seminal vesicles, ventral prostate, and 

dorsolateral prostate compared to the castrated control + TP; however, there were no significant 

decreases in the weights of the LABC muscle, Cowper’s glands, or glans penis. However, the 

exact percentage decreases, relative to control, could not be ascertained as the data were only 

reported graphically. Treatment with β-cypermethrin at 50 mg/kg/day had no effect on any of 

the androgen-sensitive accessory sex tissues compared to the castrated control + TP. The 

weights of all accessory sex tissues for the castrated control +TP were similar to the uncastrated 

males, indicating that the dosage of TP (0.5 mg/kg/day) was appropriate, and the positive control 

(castrated + TP + flutamide) displayed a greater decrease in tissue weights compared to 

cypermethrin and β-cypermethrin. The authors concluded that cypermethrin has anti-androgenic 

effects in the in vivo assay; however, β-cypermethrin does not have anti-androgenic activity. 

Although the study was conducted prior to the establishment of the OPPTS 890 Test Guideline 

for the Hershberger (890.1400) assay, the in vivo portion of the study generally conforms to the 

Guideline. Differences from the Guideline were that the rats were castrated at PND 28 instead of 

PND 42, as recommended by the Guideline, and dosing was conducted for seven days instead of 

the recommended ten days. In addition, the Hershberger assay investigated only the antiandrogenic activity of cypermethrin and β-cypermethrin (no androgenic portion), and the 

castrated rats were dosed with TP at 0.5 mg/kg instead of 0.2-0.4 mg/kg/day. However, because 

the control group (castrated + TP) exhibited similar growth of accessory sex tissues to the shamoperated group (uncastrated), the 0.5 mg/kg dose of TP was acceptable. The study is considered 

acceptable for use in a WoE evaluation. 

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

In a 13-week, subchronic toxicity study in dogs (MRID 44527002, 1994), cypermethrin (95.7% 

purity; cis/trans ratio is not stated) was administered to Beagle dogs (4/sex/group) at 

concentrations of 0, 300, 600, 800, or 1100 ppm in the diet (achieved doses of 0/0, 10.4/12.2, 

20.7/25.4, 24.6/34.3, and 37.0/45.2 mg/kg/day [M/F], respectively) for three months. Organs 

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

Gross and microscopic pathological examinations were conducted on these tissues as well as the 

pituitary, adrenals, ovaries, prostate, mammary gland, and uterus. 

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No deaths occurred during the study. In the high-dose group, two males and one female 

exhibited slight tremor activity beginning on Days 49 (female), 60, or 71 through study 

termination. In addition, one male and one female exhibited moderate tremor activity either on 

Day 76/77 (male) or during the Week 8, 9, and 12 detailed physical examinations (female). 

Males and females displayed decreases in body weights throughout the study in the high-dose 

males and females (↓≤13 and 19%, respectively); decreases in medium-low (↓≤14%) and 

medium-high (↓≤11%) females were observed, but were generally <10%. Reduced body weight 

gains were generally noted throughout the study in both sexes, with negative or no body weight 

gains observed in high-dose males (Weeks 0-9), medium-high females (Weeks 0-7), and highdose females (Weeks 0-13, except between Weeks 2 and 3). 

No treatment-related differences in food consumption, hematological, clinical chemistry, or 

urinalysis parameters, organ weight, or gross or microscopic pathology were observed. No 

neoplastic tissue was observed. The LOAEL is 1100 ppm (equivalent to 37.0/45.2 mg/kg/day 

[M/F]), based on tremor activity in both sexes and decreased body weight and body weight gains 

in the males and females. The NOAEL is 800 ppm (equivalent to 24.6/34.3 mg/kg/day [M/F]). 

Prenatal Development in Rodents (Rat; OSCPP 870.3700) 

In a developmental toxicity study in rats (MRIDs 00056804, 92027039 and 92027061; 1978), 

cypermethrin (98.2% purity; cis/trans ratio is not stated) was administered by gavage to mated 

CD rats (25/dose group) at doses of 0 (corn oil), 17.5, 35, or 70 mg/kg/day on GDs 6-15, 

inclusive. The rats were sacrificed at GD 21 of gestation and subjected to gross necropsy. All 

fetuses were weighed, sexed and examined for gross external alterations. One-third of the 

fetuses in each litter were examined for visceral soft tissue alterations by a free-hand serial 

sectioning technique. The remaining two-thirds of the fetuses in each litter were examined for 

skeletal alterations. 

Dams in the high-dose group displayed neurological signs such as splayed limbs, spasms, 

hypersensitivity to noise, and convulsions. Decreased (p<0.05 and 0.001, respectively) body 

weight gains of 12 and 28% were observed in the medium- and high-dose groups during GDs 

6-15. Overall body weight gain was decreased (p<0.05) in the high-dose dams by 8%. 

Reproductive parameters were not affected by treatment with cypermethrin; pregnancy rates 

ranged from 88-100%. No effects on either skeletal or visceral structures were reported. The 

maternal LOAEL is 35 mg/kg/day, based on body weight decreases, and the maternal NOAEL is 

17.5 mg/kg/day. The developmental LOAEL is not determined (>70 mg/kg/day), and the 

developmental NOAEL is ≥70 mg/kg/day. 

Prenatal Development in Non-Rodents (Rabbit; OCSPP 870.3700) 

In a developmental toxicity study in rabbits (MRID 43776302, 1994), cypermethrin (94-96% 

purity, cis/trans ratio approximately 1:1) was administered to female New Zealand White rabbits 

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Page 50 of 64 

(20/dose group) by gavage at doses of 0 (corn oil), 100, 450 or 700 mg/kg/day from GDs 7-19, 

inclusive. Cypermethrin was administered as a 50% w/v solution in corn oil at varying volumes. 

On GD 29, all surviving does were sacrificed, and all fetuses were weighed and examined for 

external malformation/variations. Each fetus was examined viscerally by fresh dissection and 

the sex determined. All carcasses were eviscerated and processed for skeletal examination. 

Four deaths occurred during the study, but were not, or could not conclusively be, attributed to 

treatment. Exacerbation of some clinical signs such as anorexia, abdominal/genital staining, 

decreased feces, and red or pink material in the pan occurred in the high-dose group and in a few 

does in the medium-dose group. Due to the infrequency of occurrences, these effects were not 

definitely attributed to treatment. Body weight gain was decreased during the dosing period 

(GDs 7-19) at the medium (NS; ↓25%) and high (p<0.05; ↓30%) doses, with compensatory 

increases during GDs 19-29. There were no indications of reproductive or developmental 

toxicity; pregnancy rate ranged from 85-100%. The maternal LOAEL is 450 mg/kg/day, based 

on body weight gain, and the maternal NOAEL is 100 mg/kg/day. The LOAEL for 

developmental toxicity is not determined (>700 mg/kg/day), and the NOAEL is ≥700 mg/kg/day. 

Developmental Neurotoxicity in Rodents (Rat; OCSPP 870.6300) 

In a developmental neurotoxicity study in rats (MRID 46670402, 2005), zeta-cypermethrin 

(81.8% purity) was administered in the diet to mated female Crl:CD®(SD)IGS BR rats 

(25/group) at nominal concentrations of 0, 50, 125, or 300 ppm from GD 6 through LD 21. 

Mean-measured doses to the animals, adjusted for purity, were 0, 2.9, 7.4, or 17.3 mg/kg/day, 

respectively, during gestation and 0, 7.1, 17.5, or 39.8 mg/kg/day, respectively, during lactation. 

Functional Observations (FO) were conducted on 25 dams/group on GDs 10 and 15, and on all 

surviving dams that delivered offspring on LDs 10 and 21. On PND 4, litters were culled to 

eight offspring, with at least three males and three females per litter. Offspring were allocated 

for detailed clinical observations (FO) and assessment of motor activity, auditory startle 

response, and learning and memory, as well as for neuropathology at study termination 

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

and subjected to micropathologic examination and morphometric analysis. Pup physical 

development was evaluated by body weight. The age of sexual maturation (VO in females and 

PPS in males) was assessed. 

One control female was found dead late in gestation; necropsy revealed a ruptured uterus and 

late resorptions; all remaining animals survived to scheduled sacrifice. No treatment-related 

clinical signs of toxicity were observed during the daily examinations. 

Absolute body weight of dams was similar between the treated and control groups throughout 

gestation. However, body weight gain in the high-dose group was slightly less (NS) than that of 

control for each interval, resulting in an overall gestational weight gain (GDs 0-20) in the highdose animals that was decreased (p<0.05; ↓10%). Food consumption by the high-dose group 

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was decreased (p≤0.05 or 0.01) compared to control during GDs 6-9 (↓13%) and GDs 6-20 

(↓4%). Throughout lactation, mean body weight of the high-dose group was decreased (p≤0.05 

or 0.01; ↓5-8%) compared to control; overall body weight gain (LDs 1-21) was also decreased 

(NS; ↓15%). Food consumption by the high-dose group was decreased (p≤0.01; ↓11%) 

throughout lactation. The observed changes in body weight, body weight gain, and food 

consumption were not considered to be adverse due to the small magnitude of the change. No 

treatment-related effects were observed in reproductive parameters, and gross necropsy was 

unremarkable. 

No treatment-related effect on the mean number of pups born, mean live litter size, percentage of 

males per litter, pup survival, or clinical signs was observed. Pup body weights were similar 

between the treated and control groups on PNDs 1-11. On PNDs 13-21, mean body weight was 

decreased (p≤0.05 or 0.01) in the high-dose female offspring (↓8-10%) and was slightly 

decreased (NS) in the high-dose male offspring (↓6-7%). Mean body weight gain was decreased 

(p≤0.05 or 0.01) in the high-dose females (↓11-14%) for all intervals during PNDs 7-17 and for 

PND 4-21 (↓13%). Mean weight gain was decreased (p≤0.01) in the high-dose males (↓14%) 

during the PND 11-13 interval. Post-weaning body weight and body weight gain were similar 

between the treated and control groups. The average age and body weight at attainment of 

sexual maturation (VO) was not affected by treatment. 

No significant difference in total motor activity was found between the treated and control 

groups on any testing day. Auditory startle response, learning and memory, and gross and 

qualitative microscopic findings were not affected by treatment. 

A slight, non-dose-related increase (NS) in male brain weights was noted at the high dose on 

PND 21 (↑4%). An increase (p≤0.05) in the vertical thickness of the cortex was observed in high 

dose PND 21 females (↑6%). This sole brain morphometric change was determined to occur in 

isolation, only in female pups on PND 21, and was not considered biologically significant. 

The maternal NOAEL is 300 ppm (17.3 mg/kg/day, HDT); a LOAEL is not established. The 

offspring LOAEL is 300 ppm (17.3 mg/kg/day to dams), based on decreased body weight and 

body weight gain in females. The offspring NOAEL is 125 ppm (7.4 mg/kg/day to dams).

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Multi-Generation Reproduction in Rodents (Rat; OCSPP 870.3800) 

In a three-generation reproduction study in rats (MRIDs 00112912, 42068504, and 92027040; 

1982), cypermethrin (90.6 to 93.1%, cis/trans ratio: 53.4/46.6%) was administered to Wistarderived SPF strain rats (15 males and 30 females/dose group) at nominal doses of 0, 50, 150 or 

1000/750 ppm (high dose was reduced to 750 ppm after twelve weeks because of severe 

neurological symptoms). These doses correspond to estimated intakes of 0, 2.5, 7.5, or 50/37.5 

mg/kg/day. Three successive generations were produced, each consisting of two separate 

breedings yielding six sets of litters. 

One high-dose male (1000 pm) was sacrificed in extremis, and this death was considered to be 

treatment-related. There were obvious clinical signs of neurotoxicity (i.e., ataxia, increased 

sensitivity to sound, and high-stepping gait) in the high-dose (1000 ppm) group. Parental weight 

gain was decreased in the medium- and high-dose groups throughout the study; selected 

generations include F2 males (↓7 and 9%, respectively) and F0 and F2 females (↓5 and 10%, and 

↓9 and 11%, respectively). Food consumption was also decreased (significance not reported) in 

the medium- and high-dose groups. There were no treatment-related effects on the fertility, 

gestation, viability, or lactation indices [a non-dose-dependent decrease (p<0.05) in fertility in 

the F1a low-dose females was not considered a treatment effect]. There were also no treatmentrelated effects on litter parameters, with the exception of decreased mean litter weight gains 

during lactation (i.e., ↓12 to 21% and ↓12 to 17% for F1b males and females, respectively, for 

PNDs 10 to 28 and ↓8 to 11% and ↓9 to 13% for F2b males and females, respectively, for PNDs 

21 to 28). There were no consistent, treatment-related histopathological changes in pups at birth 

or in parental rats. 

The parental/offspring LOAEL is 750 ppm (50/37.5 mg/kg/day) based on consistent decreased 

body weight gain in both sexes in the parents and decreased mean litter weight gain during 

lactation. The parental/offspring NOAEL is 150 ppm (7.5 mg/kg/day; decreases in mean body 

weight gain at the medium dose are not considered to be toxicologically significant). 

In a previous three-generation reproduction study in rats (MRID 00090040, 1979), cypermethrin 

(98% purity) was administered in the diet to Wistar SPF strain rats (30/sex /dose group) at 

nominal doses of 0, 10, 100 or 500 ppm (estimated intakes of 0, 0.5, 5, or 25 mg/kg/day). The 

first parental group (F0) produced two litters (F1a and F1b), the F1b litter was culled to produce 

the F2a and F2b litters, and the F2b litter was culled to produce the F3a and F3b litters. 

No adverse effects were noted for the general health or behavior of rats in any treatment group. 

In the high-dose group, there was decreased parental weight gain (↓3% for males and 7% for 

females) for the F1 generation. There were no treatment-related effects on the fertility, gestation, 

viability, or lactation indices. There were also no treatment-related effects on litter parameters, 

with the exception of decreased (p<0.01) pup weight at PND 21 in the medium- (↓2-3%) and 

high-dose pups (↓4%). This parameter was only significant for the combined weights from all 

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litters; these differences were not apparent for individual generation weights. The small 

decrement at 100 ppm is not considered of sufficient magnitude to be included in the LOAEL. 

There were no consistent, treatment-related histopathological changes in pups at birth or in 

parental rats. The LOAEL is 500 ppm (25 mg/kg/day) based on decreased weight gain. The 

NOAEL is 100 ppm (5 mg/kg/day). 

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

In a chronic toxicity/carcinogenicity study in rats (MRID 00112910, 1982), cypermethrin (88-

93% purity, cis/trans ratio: 55/45%) was administered in the diet to Wistar-derived SPF strain 

rats (52/sex/dose group) at nominal doses of 0 (two vehicle control groups), 20, 150 or 1500 ppm 

(estimated intakes of 0, 0, 1, 7.5, or 75 mg/kg/day) for 2 years. The high-dose animals received 

diets containing nominal doses of 1000 ppm instead of 1500 ppm for the first six weeks of the 

study, but no explanation for this difference was provided. Satellite groups of 12/sex/dose group 

were sacrificed after one year of dosing. At study termination, surviving rats in all groups were 

sacrificed for gross and histopathological evaluation. Select organs weighed at Week 52 and at 

termination included the liver, kidneys, testes, ovaries, adrenals, pituitary gland, and brain. 

Gross and microscopic pathological examinations were conducted on 42 tissues (complete list of 

organs was not included). 

There were no treatment-related differences in survival. Transient clinical (behavioral) signs 

(i.e., frequent face washing, increased sensitivity to sound, hind limb uncoordination) were noted 

during the first six days of dosing in six (6/52) high-dose males. Body weight gain was 

decreased throughout the study in the high-dose animals (i.e., ↓10% for males and 13% for 

females at Week 13 and ↓12% for males and 18% for females at study termination). Lesser 

decreases in body weight gain (<10%) were occasionally noted for the medium-dose groups. 

Relative liver weight was increased (significance not reported; ↑21%) in high-dose females at the 

interim sacrifice (Week 52). Absolute and relative kidney weights were decreased (significance 

not reported; ↓17%) in high-dose females at study termination. No treatment-related effects on 

gross and microscopic pathology were observed in examined organs, including the testes, 

epididymides, seminal vesicle, prostate, ovaries, uterus, mammary, thyroid, pituitary or adrenal 

glands. Cypermethrin was not considered to be oncogenic in this study. A possible treatment 

association with increased testicular interstitial tumors was not considered definite. Frequent 

occurrences of adenomas and occasional carcinomas of the pituitary were noted in all test 

animals (females>males), but no dose response was evident. The LOAEL is 1500 ppm (75 

mg/kg/day) based on body weight gain decreases, and the NOAEL is 150 ppm (7.5 mg/kg/day).

Carcinogenicity in Rodents (Mouse; OCSPP 870.4200) 

In a chronic toxicity/carcinogenicity study in mice (MRIDs 00112911 and 92027038, 1982), 

cypermethrin (91.5% and 94-94.2% purity, cis/trans ratio: 53-54/46-47%) was administered to 

Swiss-derived SPF mice (70/sex/dose group) at doses of 0 (two vehicle control groups), 100, 

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400, and 1600 ppm (estimated intakes of 0, 0, 14, 57 or 229 mg/kg/day) for 97 weeks for males 

and 101 weeks for females. Interim sacrifices were conducted after one year of dosing with 

9-10/sex/dose group. At study termination, all surviving mice in all groups were sacrificed for 

gross and histopathological evaluation. Select organs weighed at Week 52 and at termination 

included the liver, kidneys, testes, and brain. Gross and microscopic pathological examinations 

were conducted on 45 tissues (complete list of organs was not included). 

There were no treatment-related differences in survival, and clinical signs were not present. 

Decreased body weight gains were observed in the high-dose animals, with significant decreases 

(significance not reported) noted only during the first year; no treatment-related differences were 

noted in the low- and medium-dose animals. Liver weights were affected at the interim and 

terminal sacrifices in both sexes. At the interim sacrifice, absolute liver weights were increased 

(p<0.05) by 19 and 20% in the medium- and high-dose males (with 28 and 16% increases in 

relative liver weight in the high dose males and females, respectively). Liver weight effects were 

not observed at the terminal sacrifice, but increased liver weights were noted in one of the two 

vehicle control groups for both males and females; this liver weight variability apparently 

masked increased absolute and relative liver weights in the high-dose males at the terminal 

sacrifice. Absolute and relative testis weight was decreased in high-dose males (significance not 

reported; ↓18%) at the interim sacrifice only. No treatment-related effects on histopathology 

were noted in the testes, epididymides, seminal vesicle, prostate, ovaries, uterus, cervix, 

mammary glands, thyroid, pituitary or adrenal glands at any dose level. 

The LOAEL is 400 ppm (57 mg/kg/day) based on liver weight decreases, and the NOAEL is 100 

ppm (14 mg/kg/day). This study was determined to be positive for the induction of benign 

alveologenic neoplasms, with a treatment-related increase (p<0.05) in high-dose females (14 vs. 

5 control). 

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

In a chronic toxicity/carcinogenicity study in dogs (MRIDs 00112909, 42068503, and 

92027037; 1982), cypermethrin (90.6% purity, cis/trans ratio: 53.9/46.1%) dissolved in corn oil 

was administered to Beagle dogs (6/sex/dose group) in gelatin capsules at doses of 0, 1, 5, or 15 

mg/kg/day (adjusted for purity) for 52 weeks. Organ weights determined included adrenals, 

kidneys, liver, ovaries, testes, pituitary, and thyroid; histopathological examinations were 

conducted on 37 tissues (complete list of organs was not included). 

No mortality occurred during the study. Treated males and females displayed increases in 

vomiting during Week 1, and high-dose animals displayed signs of nervous system stimulation in 

the form of body tremors, gait abnormalities, uncoordination, disorientation, and hypersensitivity to noise during Week 1. Males and females in the medium-dose group had increased 

incidences of passage of liquid stools throughout the study period (approximately 5- and 10-fold, 

respectively), and the incidence of this condition was increased approximately 30-fold at the 

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high-dose. Decreased body weight/body weight gain and food consumption was noted in the 

high-dose animals (extent and significance not reported). Non-significant increases in liver 

weight were noted (only high-dose females were reported), and a potential increase (NS) in 

adrenal weight in high-dose males (↑19 or 11%). There were no treatment-related changes in the 

absolute or relative weights of the testes, ovaries, thyroid or pituitary gland. There were also no 

treatment-related effects on gross or microscopic pathology, including in the testes, 

epididymides, prostate, ovaries, uterus, mammary glands, thyroid, pituitary or adrenal glands at 

any dose level. There was no evidence for cypermethrin being oncogenic in the dog. 

The LOAEL is 15 mg/kg/day based on neurological symptoms (body tremors, gait abnormalities, 

uncoordination, disorientation, and hypersensitivity to noise) and decreased body weight, and the 

NOAEL is 5 mg/kg/day.

Avian Reproduction (OCSPP 850.2300) 

In an avian reproduction study (MRID 00090073, 1981), Mallard ducks (Anas platyrhynchos) 

were administered cypermethrin (purity and cis/trans ratio not reported) in the feed. No 

treatment-related effects on reproduction or any other endpoint were seen up to 50 ppm. In the 

study report, although no reproductive effects were observed, numerous mortalities were 

observed, and prior to the onset of death birds were observed being “bullied”. 

In an avian reproduction study (MRID 00090074, 1981), young Bobwhite quail approaching 

their first breeding season (Colinus virginianus) were administered cypermethrin (87.8% purity, 

cis/trans ratio: 53/47%) in the feed at nominal concentrations of 0, 5, or 50 ppm (adjustment for 

purity was not reported) for 31 weeks (nineteen weeks prior to the start of egg production and 

during twelve weeks of egg production). 

There were increased (NS) proportions of eggs cracked or broken in the control (↑19.2%) and 

high-dose (↑17.6%) groups compared to the low-dose group (10.9%), and all were above the 

Guideline upper limit of 2%; no differences in mean egg weight or egg shell thickness were 

noted. There were no treatment-related differences in the numbers of viable embryos/eggs set, 

eggs laid/hen, and normal hatchlings. Finally, mean body weights at time of hatching and at Day 

14, showed no treatment-related difference, as well as no differences in survival of chicks to 14 

days. 

The reproductive NOAEC for bobwhite quail exposed to cypermethrin in the diet for 31 weeks 

was 50 ppm (50 mg/kg diet). In the study report, although no reproductive effects were 

observed, numerous mortalities were observed, and prior to the onset of death birds were 

observed being “bullied”. 

In an avian reproduction study (MRID 47944018, 2001), bobwhite quail (20 pairs per level of 

ca. 12-month old) were administered alpha cypermethrin in diet at nominal concentrations of 

(negative control), 50, 150, and 450 mg ai.kg diet. Mean-measured concentrations were <1.125 

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(LOQ, control), 8, 149, and 452 mg ai/kg diet, respectively. Six adult mortalities occurred during 

the study one from the control group, two from the 48 mg ai/kg diet group, and three from the 

452 mg ai/kg diet group. Most findings were reported to be related to the long-term housing of 

birds in cages. Several chicks at the 452 mg ai/kg diet level were found dead without prior 

clinical signs. Post mortem examination showed no evidence of treatment-related abnormalities.

Significant reductions (p<0.05) were detected in adult male body weight gain at the two highest 

treatment levels. At the 452 mg ai/kg diet level, treatment-related effects included 14-day old 

survivors of number hatched. There was a significant difference observed at the 48 mg ai/kg dose 

for # hatchlings/egg set (%). Parents were confined to very small cages (0.31 x 0.39 x 0.29 m) 

for a protracted period of time before the study even began (10 week acclimation period). These 

conditions may have been responsible for the bird aggression observed across all treatment and 

control groups, which likely resulted in the reported mortalities. Fighting between birds is likely 

a sign that the animals were stressed, which may have biased the results of the reproductive 

study. This can be seen in the reproductive success of the control group, where one pair of birds 

did not lay any eggs and another pair of birds laid 48 eggs but had zero viable embryos. Due to 

poor husbandry of test birds, this study can be used for purposes of characterization only. 

Fish Early Life-Stage Toxicity; (OPPTS 850.1400) 

In a freshwater fish early life-stage toxicity test (MRID 00089039, 1981), Fathead minnow 

(Pimephales promelas) embryos and larvae were exposed to cypermethrin (91.5% purity) at 

nominal concentrations of water (negative) control, solvent control, 0.031, 0.062, 0.12, 0.25, and 

0.50 μg/L (mean-measured concentrations of negative control, solvent control, 0.048, 0.045, 

0.063, 0.14, and 0.33 μg a.i./L in a flow-through system for 30 days. Observations were made on 

embryo hatching (%), and larval survival and growth (length and weight). Each test chamber 

contained 60 embryos, 40 larvae were selected from the surviving embryos for each 

concentration. First day of hatch was not reported. Test were run at 25°C, rather than 22 or 

27°C. Data were analyzed by analysis of variance (ANOVA), parameters examined were 

percentage of hatch and the survival, length, and weight of larvae after 30 days of exposure. If 

treatment effects were indicated, the means of the test parameters were compared to those of the 

controls using Dunnett’s procedure. No treatment-related effects were observed on hatchability, 

or larval growth. Larval survival was significantly reduced in the highest test concentration 

(mean-measured concentration 0.33 μg a.i./L), p<0.05 compared to the controls. The NOAEC 

for fathead minnow exposed to cypermethrin was 0.14 μg a.i./L, based on reduced survival. 

Fish Life-Cycle Toxicity; (OCSPP 850.1500) 

A life-cycle toxicity test with fathead minnow (Pimephales promelas) was conducted using 

cypermethrin (92.9-93.2% purity). Newly fertilized embryos (<48 hours) were exposed to 

cypermethrin at nominal concentrations of 0.06, 0.12, 0.25, 0.5, and 1.0 μg/L (mean measured 

concentrations of 0.051, 0.077, 0.15, 0.32, and 0.65 μg/L) (MRID 40641701, 1998). F0 fish 

were monitored for survival and growth, and once fish attained sexual maturation, reproduction 

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was monitored, and F1 generation was exposed for 30 days. The study duration was 300 days.

The reviewer's analysis detected significant (p<0.05) reductions in % survival of F0 fish 30 and 60 

days post hatch at 0.32 μg/L, with the most sensitive endpoint being % survival of fish after 30 days 

post-hatch (based on the number of larvae released) with a LOAEC of 0.15 μg/L (NOEAC 0.077 

μg/L). The reviewer's analysis additionally detected significant treatment-related effects on length 

and weight of F0 female fish 300 days posthatch at 0.32μ/L, and on reproduction (i.e., # eggs, # 

eggs/batch, # eggs/spawn) at the highest treatment level (0.32μ/L) evaluated for those endpoints. No 

effects on F1 hatch, survival or growth was observed. However, this study is supplemental, as 

excessive analytical variation (high:low ratios of 3.0 to 7.8) was observed at all levels during the 

study. The range of concentrations was so great that overlapping of concentrations occurred for all 

levels.

Mechanistic Studies: Male Reproductive System (Rat) 

In a published study, Hu et al., (2013) investigated the reproductive toxicity of cypermethrin in 

intact adult male rats. Adult male SD rats (80-90 days old) were randomly separated, by weight, 

into 5 groups (12 rats/group) and orally dosed by gavage with cypermethrin (97.53% purity; 

cis/trans ratio is not stated) in corn oil at 0, 6.25, 12.5, 25, or 50 mg/kg/day for 15 days. Rats 

were weighed and observed for signs of treatment-related effects daily. At the conclusion of the 

treatment period, rats were sacrificed by decapitation, and the livers, kidneys, testes, 

epididymides, seminal vesicles, and prostates were excised and weighed. 

One testis from each rat was frozen (−20°C) to be used for testicular sperm head counts and daily 

sperm production determinations. The frozen testis was thawed and decapsulated prior weighing 

and homogenization in ice-cold 0.9% sodium chloride solution containing 0.01% Triton X-100. 

The spermatozoa in the sample were counted with a hemocytometer. The other excised testes 

were used for histological evaluations. The testis was fixed in Bouin’s fixative 10% neutral 

buffered formalin, embedded in paraffin, sectioned, and stained using the standard hematoxylineosin double staining procedure for microscopic examination. Additionally, fragments of the 

testis from fresh tissue were prepared for electron microscope examination. 

Immunohistochemical investigation of the AR were conducted, but will not be discussed further. 

Serum was collected (procedure not described), and concentrations of testosterone, folliclestimulating hormone (FSH) and luteinizing hormone (LH) were measured with commercial 

radioimmunoassay kits. 

Food consumption for all treatment groups was not different compared to control throughout the 

treatment period, and no mortalities, signs of toxicity, or abnormalities in behavior or appearance 

were observed. Body weight gains (Treatment Days 11-15) were decreased (p<0.05) in rats 

dosed with cypermethrin at 12.5, 25, and 50 mg/kg/day. A downward trend (NS) in liver and 

kidney weights was observed in treated rats. Prostate absolute weights were decreased by 24 and 

22% (p<0.01) in rats treated with cypermethrin at 25 and 50 mg/kg/day, respectively. No 

differences were noted for the testes, epididymides, and seminal vesicles weights in any of the 

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treatment groups. Daily sperm production was decreased (p<0.01) in rats treated with 

cypermethrin only at 50 mg/kg/day. 

Slight distortion of seminiferous tubules in rats treated at 6.25 and 12.5 mg/kg/day was 

associated with changes in the lumen of the seminiferous tubules and decreased spermatids. In 

rats treated at 25 and 50 mg/kg/day, atrophy and distorted seminiferous tubules, reduction and 

deformation of germ cells, and disordered arrangement of spermatoblasts were observed. The 

number of seminiferous tubules was decreased (p<0.01) in males treated with cypermethrin at 

6.25 and 12.5 mg/kg/day; it was observed that the development of spermatogonia was obstructed 

and the arrangement of spermatids was disordered. The perimeter of the seminiferous tubule 

was decreased (p<0.05) and the number of tubules exhibiting degeneration was increased 

(p<0.01) in all treatment groups compared to control. 

The diameters of the seminiferous lumen also were decreased (p<0.05) at different 

spermatogenesis stages in rats treated with cypermethrin at 6.25, 12.5, and 25 mg/kg/day, and the 

number of cell layers of the seminiferous tubules was decreased in rats treated at 12.5, 25, and 

50 mg/kg/day. Significant ultrastructural changes of the seminiferous epithelium were observed 

with cypermethrin treatment. In addition, the number of AR positive Sertoli cells, Leydig cells, 

and peritubular myoid cells were decreased (p<0.01) in rats treated at 12.5, 25, and 50 

mg/kg/day. 

Finally, serum testosterone concentrations were decreased by 51% (p<0.05) in rats treated at 50 

mg/kg, and serum FSH and LH concentrations were increased by 35 and 50%, respectively 

(p<0.05 and 0.01, respectively) at 50 mg/kg/day. 

In a similar published study from the same group, Li et al. (2013), also investigated the 

reproductive toxicity of cypermethrin in intact adult male rats. Adult male SD rats (9 weeks old) 

were randomly separated, by weight, into 5 groups (12 rats/group) and orally dosed by gavage 

with cypermethrin (98% purity; cis/trans ratio is not stated) in corn oil at 0, 7.5, 15, 30, or 

60 mg/kg/day for 15 days. Rats were weighed and observed for signs of treatment-related effects 

daily. At the conclusion of the treatment period, rats were sacrificed by decapitation, and the 

livers, kidneys, testes, epididymides, seminal vesicles, and prostates were excised and weighed. 

The right testis from each rat was frozen (-80°C) to be used for testicular sperm head counts and 

daily sperm production determinations. The frozen testis was thawed and decapsulated prior 

weighing and homogenization in ice-cold 0.9% sodium chloride solution containing 0.01% 

Triton X-100. The spermatozoa in the sample were counted with a hemocytometer. The left 

testes were used for histological evaluations. The testis was fixed in modified Davidson’s fluid, 

embedded in paraffin, sectioned, and stained using the standard hematoxylin-eosin double 

staining procedure for microscopic examination. 

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Serum was collected (procedure not described), and concentrations of testosterone, FSH and LH 

were measured with commercial radioimmunoassay kits. 

Food consumption for all treatment groups was not different compared to control throughout the 

treatment period, and no mortalities, signs of toxicity, or abnormalities in behavior or appearance 

were observed. Body weights and absolute organ weights for mice treated with cypermethrin 

were similar to control. Daily sperm production was significantly (p<0.05) decreased in rats 

treated with cypermethrin at 30 and 60 mg/kg/day. 

Histopathological examination of the testes identified a slight distortion of the seminiferous 

tubules in rats dosed with cypermethrin at 7.5 and 15 mg/kg/day. In rats dosed with 

cypermethrin at 30 and 60 mg/kg/day, atrophic and distorted seminiferous tubules, and deformed 

and disordered arrangement of germ cells were observed in the testes. Additionally, the numbers 

of germ cells, Sertoli cells, and Leydig cells were reduced, and the number of cell layers of the 

seminiferous tubules was decreased. High-magnification dark-field microscopy (100×) showed 

that the arrangement of germ cells was in disorder. Intraepithelial vacuolization and 

multinucleated formation of spermatocyte were observed in the treatment groups. In addition, 

impairments of Sertoli cells and Leydig cells were seen in cypermethrin-treated rats; 

vacuolization was found in Sertoli cells, and deformed nucleus was noted in Leydig cells. The 

perimeters of the seminiferous tubule were decreased (p<0.05) in all cypermethrin-treated rats; 

the diameters of the seminiferous lumen were decreased (p<0.01) in rats treated at 7.5, 15, and 

30 mg/kg/day; and the number of cell layers of seminiferous tubules was decreased (p<0.01) in 

rats treated at 15, 30, and 60 mg/kg/day. 

Serum testosterone concentrations were decreased (p<0.05) in rats treated with cypermethrin at 

30 and 60 mg/kg/day, and serum FSH concentrations were increased (p<0.01) in rats treated at 

60 mg/kg/day compared to control. An upward trend in serum LH concentrations was observed 

in cypermethrin-treated rats, but increases were not significantly different from the control 

group. 

An earlier published study from the same group, Liu et al. (2010), investigated the reproductive 

toxicity of β-cypermethrin in intact adult male rats. Adult male SD rats were randomly assigned 

to three groups (10 rats/group), and orally dosed by gavage with β-cypermethrin (>95.0% purity) 

in corn oil at 0 (vehicle only), 15, or 30 mg/kg for 15 days. 

Following 15 days of treatment, rats were sacrificed by decapitation and the testes, epididymides, 

and seminal vesicles were excised and weighed; relative weights were calculated. After 

weighing, one testis was frozen (−20 °C) for testicular sperm head counts and daily sperm 

production determinations; the other testis was fixed with 10% formalin for 

immunohistochemistry testing of the AR (not discussed further). Frozen testes were thawed, 

decapsulated, and homogenized with ice-cold 0.9% sodium chloride solution containing 0.01% 

Triton X-100. The number of sperm heads in the sample was counted with a hemocytometer. 

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There were no treatment-related effects on body or organ weights. Sperm head counts and daily 

sperm production were decreased (p<0.05) in a dose-dependent manner in rats treated with 

β-cypermethrin at both 15 and 30 mg/kg compared to control. 

Finally in another published study from a different group (Wang et al., 2009), reproductive 

toxicity of β-cypermethrin was investigated in intact adult male mice. Adult male Kunbai mice 

were randomly assigned to six groups (10 mice/group), and orally dosed by gavage with βcypermethrin (>99.0% purity) in corn oil at 0 (vehicle only); 1, 10, or 20 mg/kg of βcypermethrin; 20 mg/kg of β-cypermethrin plus 20 mg/kg Vitamin E; or 20 mg/kg Vitamin E for 

35 days. 

After 35 days of treatment, rats were sacrificed by decapitation and blood was collected for 

analysis of serum testosterone concentration with a commercial radioimmunoassay kit. The 

testes, epididymides, seminal vesicles, and prostate were excised and weighed; overall body 

weight gain was also recorded. For histological examinations, samples of the testes and 

epididymides were fixed in Bouin’s solution, embedded in paraffin, sectioned, and then stained 

with hematoxylin and eosin for examination by light microscopy. Some testes samples were 

sectioned and prepared for ultrastructure analysis. 

The caudal epididymis was cleared of blood and adipose tissue, and opened to release sperm. 

The sperm were collected by centrifugation with saline-merthiolate-Triton, and the number of 

sperm was determined with a hemocytometer. Sperm viability was evaluated with eosin Y 

staining which determines whether the membrane is physically intact or if the membrane is 

damaged or broken; the percentage of viable sperm was calculated. Sperm malformation was 

assayed by the motility of the sperm and intact nature of the acrosome under a light microscope. 

Vitamin E treatment alone had no effect on any parameter values; all responses were similar to 

control. Body weight gains and testes, epididymis, and seminal vesicles organ weights were 

dose-dependently decreased (p<0.05) in mice treated with β-cypermethrin at 10 and 20 mg/kg, 

and prostrate organ weights were decreased (p<0.05) in mice treated at 20 mg/kg only. In mice 

treated with β-cypermethrin and vitamin E, body weight gain and testosterone-sensitive organ 

weights were increased compared to weights in animals dosed only with 20 mg/kg 

β-cypermethrin. Sperm count, viability, and motility were also dose-dependently decreased 

(p<0.05) in mice treated with β-cypermethrin at 10 and 20 mg/kg. At both 10 and 20 mg/kg, the 

rate of acrosome deformity was increased by approximately 183% and 296%, respectively. In 

mice dosed with β-cypermethrin and vitamin E, these sperm parameters were improved 

compared to those dosed only with 20 mg/kg β-cypermethrin. Finally, serum testosterone 

concentrations were dose-dependently decreased (p<0.05) after β-cypermethrin treatment at 

10 and 20 mg/kg (↓approximately 35 and 70%, respectively). Histological and ultrastructural 

changes due to β-cypermethrin exposure at the two highest doses were similar to changes 

observed in the previously discussed studies, with effects on seminiferous tubules and sperm 

development. 

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APPENDIX 3: References Not Utilized in the Cypermethrin 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 Cypermethrin 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 

Al-Hamdani, N.M. and Yajurvedi, H.N. (2010) Cypermethrin reversibly alters sperm count 

without altering fertility in mice. Ecotoxicol. Environ. Saf. 73(5):1092-1097. 

Aït-Aïssa, S., Laskowski, S., Laville, N., Porcher, J. M., and Brion, F. (2010) Anti-androgenic 

activities of environmental pesticides in the MDA-kb2 reporter cell line. Toxicol. In Vitro. 24: 

1979-1985.

Boone, M.D. (2008) Examining the single and interactive effects of three insecticides on 

amphibian metamorphosis. Environmental Toxicology and Chemistry Vol. 27, No. 7, pp. 1561–

1568. 

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Bossi, R., Vinggaard, A.M., Taxvig, C., Boberg, J., and Bonefeld-Jørgensen, E.C. (2013) Levels 

of pesticides and their metabolites in Wistar rat amniotic fluids and maternal urine upon 

gestational exposure. Int. J. Environ. Res. Public Health 10(6):2271-2281. 

Carriquiriborde, P., Díaz, J., López, G.C., Ronco, A.E., and Somoza, G.M. (2009) Effects of 

cypermethrin chronic exposure and water temperature on survival, growth, sex differentiation, 

and gonadal developmental stages of Odontesthes bonariensis (Teleostei). Chemosphere

76(3):374-380. 

Dahamna, S., Bencheikh, F., Harzallah, D., Boussahel, S., Belgeit, A., Merghem, M., and 

Bouriche, H. (2010) Cypermethrin toxic effects on spermatogenesis and male mouse 

reproductive organs. Commun. Appl. Biol. Sci. 75(2):209-216. 

Garey, J. and Wolff, M.S. (1998) Estrogenic and Antiprogestagenic Activities of Pyrethroid 

Insecticides. Biochemical and Biophysical Research Communications 251, 855–859. 

Ghodageri, M.G. and Katti, P. (2013) In vitro induction/inhibition of germinal vesicle 

breakdown (GVBD) in frog (Euphlyctis cyanophlyctis) oocytes by endocrine active compounds. 

Drug and Chem. Toxicol. 36(2), 217-223. 

Glaister, J.R., Gore, C.W., Marsat, G.J., Phillips, C., and Pratt, I. (1980) PP383: 90 Day Feeding 

Study in Rats. CTL report number: CTUP/327, ICI signed on 12-14-79. FMC study number 

A81-563. (MRID 00056802)

Go, V., Garey, J., Wolff, M.S., and Pogo, B.G.T. (1999) Estrogenic Potential of Certain 

Pyrethroid Compounds in the MCF-7 Human Breast Carcinoma Cell Line. Environmental 

Health Perspectives, Volume 107, Number 3, March. 

Hinfray, N., Porcher, J.M., and Brion, F. (2006) Inhibition of rainbow trout (Oncorhynchus mykiss) 

P450 aromatase activities in brain and ovarian microsomes by various environmental 

substances. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 144(3):252-62. 

Hu, J.X., Li, Y.F., Pan, C., Zhang, J.P., Wang, H.M., Li, J., and Xu, L.C. (2012) Anti-androgen 

effects of cypermethrin on the amino- and carboxyl-terminal interaction of the androgen 

receptor. Toxicology 292(2-3):99-104.

Jin, Y., Wang, L., Ruan, M., Liu, J., Yang, Y., Zhou, C., Xu, B., and Fu, Z. (2011) Cypermethrin 

exposure during puberty induces oxidative stress and endocrine disruption in male mice. 

Chemosphere 84(1):124-130.

Kim, S.-S., Lee, R.-D., Lim, K.-J., Kwack, S.-J., Rhee, G.-S., Seok, J.-H., Lee, G.-S., An, B.-S., 

Jeung, E.-B., and Park, K.-L. (2005) Potential Estrogenic and Antiandrogenic Effects of 

Permethrin in Rats. Journal of Reproduction and Development Vol. 51, No. 2. 

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

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

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

Regulatory Toxicology and Pharmacology 35, 227–237. 

Laville, N., Balaguer, P., Brion, F., Hinfray, N., Casellas, C., Porche,r J.M., and Ait-Aissa, S. 

(2006) Modulation of aromatase activity and mRNA by various selected pesticides in the human 

choriocarcinoma JEG-3 cell line. Toxicology 228(1):98-108. 

Lemaire, G., Mnif, W., Mauvais, P., Balaguer, P., and Rahmani, R. (2006) Activation of α- and 

β-estrogen receptors by persistent pesticides in reporter cell lines. Life Sciences 79:1160- 

1169. 

Morinaga, H., Yanase, T., Nomura, M., Okabe, T., Goto, K., Harada, N., and Nawata, H. (2004) 

A benzimidazole fungicide, benomyl, and its metabolite, carbendazim, induce aromatase activity 

in a human ovarian granulose-like tumor cell line (KGN). Endocrinology 145(4): 1860-9. 

Nataraj, M.B. and Krishnamurthy, S.V. (2012) Effects of Combinations of Malathion and 

Cypermethrin on Survivability and Time of Metamorphosis of Tadpoles of Indian Cricket Frog 

(Fejervarya limnocharis). Journal of Environmental Science and Health 47:67-73. 

Pan, C., Liu, Y.P., Li, Y.F., Hu, J.X., Zhang, J.P., Wang, H.M., Li, J., and Xu, L.C. (2012) 

Effects of cypermethrin on the ligand-independent interaction between androgen receptor and 

steroid receptor coactivator-1. Toxicology 299(2-3):160-164. 

Pan, C., Wang, Q., Liu, Y.P., Xu, L.F., Li, Y.F., Hu, J.X., Jiang, M., Zhang, J.P., Zhang, M.R., 

Yu, H.M., Zhou, J.L., Zhou, X.L., and Xu, L.C. (2013) Anti-androgen effects of the pyrethroid 

pesticide cypermethrin on interactions of androgen receptor with corepressors. Toxicology

311(3):178-183. 

Singh, A., Yadav, S., Srivastava, V., Kumar, R., Singh, D., Sethumadhavan, R., and Parmar, D. 

(2013) Imprinting of cerebral and hepatic cytochrome P450s in rat offsprings exposed prenatally 

to low doses of cypermethrin. Mol Neurobiol. 48(1):128-140.

Sumida, K., Ooe, N., Nagahori, H., Saito, K., Isobe, N., Kaneko, H., and Nakatsuka, I. (2001) An 

in vitro reporter gene assay method incorporating metabolic activation with human and rat S9 or 

liver microsomes. Biochem. Biophys. Res. Commun. 280(1):85-91. 

Taxvig, C., Hadrup, N., Boberg, J., Axelstad, M., Bossi, R., Bonefeld-Jorgensen, E.C., 

Vinggaard, A.M. (2013). In vitro – in vivo correlations for endocrine activity of a mixture of 

currently used pesticides. Toxicol Appl Pharmacol. 272: 757-766. 

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. 

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Wang, H., Wang, Q., Zhao, X.F., Liu, P., Meng, X.H., Yu, T., Ji, Y.L., Zhang, H., Zhang, C., 

Zhang, Y., and Xu, D.X. (2010) Cypermethrin exposure during puberty disrupts testosterone 

synthesis via down regulating StAR in mouse testes. Arch. Toxicol. 84(1):53-61. 

Wang, H., Wang, S.F., Ning, H., Ji, Y.L., Zhang, C., Zhang, Y., Yu, T., Ma, X.H., Zhao, X.F., 

Wang, Q., Liu, P., Meng, X.H., and Xu, D.X. (2011) Maternal cypermethrin exposure during 

lactation impairs testicular development and spermatogenesis in male mouse offspring. Environ. 

Toxicol. 26(4):382-394. 

Yuan, C., Wang, C., Gao, S.Q., Kong, T.T., Chen, L., Li, X.F., Song, L., and Wang, Y.B. (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. 

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