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 

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Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

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EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH ESTROGEN, ANDROGEN, OR THYROID 

PATHWAYS 

CHEMICAL: CYFLUTHRIN 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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CYFLUTHRIN FINAL 

ii 

TABLE OF CONTENTS 

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

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

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

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

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

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

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

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

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

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

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

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

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

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................23 

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

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

B. Estrogen Pathway...........................................................................27 

C. Androgen Pathway.........................................................................27 

D. Thyroid Pathway...........................................................................27 

E. Conclusions.................................................................................28 

V. EDSP Tier 2 Testing Recommendations....................................................28 

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

LIST OF TABLES: 

 Table 1. Tier 1 Assays for Cyfluthrin..................................................................8 

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

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

 Table 4. Thyroid Pathway for Cyfluthrin ...........................................................21 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................34 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................44 

APPENDIX 3. References Not Utilized in the Cyfluthrin WoE Analysis............................68 

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iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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iv 

Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

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Abbreviation Terminology 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

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Abbreviation Terminology 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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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 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 cyfluthrin interacts with E, A or T hormone pathways, the number and 

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

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

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

general systemic toxicity or overt toxicity. 

On July 23, 2014, the 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 cyfluthrin 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).

Cyfluthrin 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., cyfluthrin and β-cyfluthrin, a resolved mix of the 

biologically-active isomers). 

For the estrogen pathway, there was no evidence of interaction either in the Tier 1 in vitro

[estrogen receptor (ER) binding, ER transactivation assay (ERTA), and aromatase assay] or in 

the Tier 1 in vivo [uterotrophic, female pubertal and fish short-term reproduction assay 

(FSTRA)] in the absence of overt toxicity. There were no estrogen-related effects were seen in 

the Part 158 studies [(effects on offspring survival were observed in the quail reproduction study, 

however, this endpoint is limited in its ability to inform potential interactions with the estrogen 

(and androgen) pathway)]. In the Tier 1 in vitro steroidogenesis assay, while a significant 

increase in estradiol production was observed at a mid-concentration, there was a lack of 

estrogen-related effects observed in the other in vitro and in vivo assays (in the absence of overt 

toxicity) . 

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There was no evidence of interaction of cyfluthrin with the androgen pathway in the Tier 1 in 

vitro assays [androgen receptor (AR) binding or steriodogenesis]. While potential antiandrogenic activity was reported in three published in vitro AR transactivation assay (ARTA) 

studies, limited evidence of potential anti-androgenic effects was observed in vivo in the absence 

of overt toxicity. While no androgen-related effects were observed in Tier 1 assays (Hershberger, 

male pubertal and FSTRA) in the absence of overt toxicity, positive results were reported in a 

non-guideline Hershberger study by Zhang et al, 2008. In that study, which evaluated antiandrogenic activity only, cyfluthrin and β-cyfluthrin caused significant decreases in multiple 

secondary sex tissues at the 18 and 54 mg/kg/day doses. The dose levels (6, 18 and 54 

mg/kg/day) in this study were comparable to the dose levels (5, 10 and 20 mg/kg/day) tested in 

the Tier 1 Hershberger assay. While Zhang et al. reported not observing clinical signs in their 

study, increased salivation, piloerection and/or lack of coordination, which are indicative of 

neurotoxicity, were seen at doses greater than 20 mg/kg/day in the Tier 1 and mammalian Part 

158 studies. Therefore, the androgen-related effects observed in the Zhang et al. study may have 

occurred in the presence of overt toxicity. Since the effects may have potentially occurred in the 

presence of overt toxicity in addition to the other mammalian studies indicating no androgenrelated effects up to overtly toxic doses, the confidence in the results of this study are reduced. 

Androgen-related effects were not observed in the avian or fish Part 158 studies. Based on the 

available data, there is no convincing evidence of potential interaction with the androgenpathway for cyfluthrin. 

There was no convincing evidence of potential interaction of cyfluthrin with the thyroid 

pathway. Thyroid parameters were not altered in Tier 1 mammalian in vivo studies. Additionally, 

there were no thyroid-related effects were seen in the Part 158 mammalian studies. In the 

amphibian metamorphosis assay (AMA), although effects on developmental stage and growth 

parameters were observed in the mid- and high-treatment groups compared to the negative 

control, similar effects (increased growth and development) were noted in the solvent control 

group. Therefore, it appears that the increased development and growth in the treatments and the 

absence of effects on thyroid histopathology may have been due to the presence of the solvent 

(may have resulted in increases in food resources) as oppose to a potential thyroid-related effect. 

Based on the results in the AMA, there appears to be no convincing evidence of a potential 

thyroid-related interaction at the concentrations tested (up to 0.066 μg a.i./L). In the AMA, the 

achieved high-concentration group (0.066 μg a.i./L) did not exhibit any signs of toxicity. The 

achieved high concentration from the definitive AMA study is in the range of the no effect 

concentration reported in the rangefinder; however, due to issues in the range finding study, there 

is uncertainty in the actual exposure concentration where toxicity was observed. Given the lack 

of observed treatment-related effects in the definitive study along with having a clear 

understanding of the concentration where clinical signs occurred in the rangefinder, there is 

uncertainty whether the maximum concentration achieved in the AMA was adequate to test for 

an effect. However, as there is a lack of redundancy across the available data with no evidence 

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for interaction in the AMA at the concentrations tested, overall there is a lack of convincing 

evidence of a potential interact with the thyroid pathway for cyfluthrin. 

Based on weight of evidence considerations, mammalian and wildlife EDSP Tier 2 studies are 

not recommended for cyfluthrin since there was no convincing evidence of potential interaction 

with the estrogen, androgen or thyroid pathways. 

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I. Introduction 

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

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

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

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

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

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

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

weight of evidence (WoE) assessment. 

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

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

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

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

general systemic toxicity or overt toxicity. 

On July 23, 2014, the Tire 1 Assay Weight of Evidence Review Committee (T1WoERC) of the 

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

conducted a weight-of-evidence (WoE) analysis of the potential interaction of cyfluthrin 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).

 Cyfluthrin and β-cyfluthrin (an enriched isomer of cyfluthrin) are non-systemic pyrethroid 

insecticides and both are mixtures of the same four diastereomers. Isomers I and II are in the cis 

configuration, and Isomers III and IV are in the trans configuration. Cyfluthrin is comprised of 

approximately equal parts of the four isomers: Isomer I is approximately 25%, Isomer II is 

approximately 19%, Isomer III is approximately 34% and Isomer IV is approximately 23%. 

β-Cyfluthrin is enriched in Isomer II (approximately 35%) and Isomer IV (approximately 62%), 

and contains only minor amounts of Isomer I and Isomer III (less than 3% of total). This 

assessment includes studies conducted on isomeric mixtures (i.e., cyfluthrin and β-cyfluthrin, a 

resolved mix of the biologically-active isomers). 

Cyfluthrin is poorly soluble in water (2.32 ppb), and has an octanol/water partition coefficient 

(log Kow) of 5.93.!Data suggest that the primary routes of dissipation are aqueous and soil 

photolysis (4.5 and 5.6 days, respectively) and hydrolysis in alkaline media (2.1 days). 

Cyfluthrin and beta-cyfluthrin are moderately persistent in the environment and immobile as 

cyfluthrin and beta-cyfluthrin bind strongly to soils (aerobic soil metabolism 73.5-94.8 days, 

aerobic aquatic metabolism 32.9-42 days). The bioconcentration factor for cyfluthrin in whole 

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rainbow trout was 854, whole fish. Residues were depurated at moderately rapid rates in 

untreated water (apparent depuration half-life of approximately ⊩3 days). 

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

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

relevant information (OSRI) such as general toxicity 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 initial review of the OSRI is provided in the 

Report of the Endocrine Disruptor Review Team (EDRT) on cyfluthrin (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 

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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. 

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 consistency 

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

endpoints are measured in each assay. 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

the whole organism level. The extent of 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 

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

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

biological organization were the responses observed and whether organisms exhibit 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

forms of toxicity (e.g., organ weights, 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 components discussed 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 

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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. 

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

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

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

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

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

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

A. EDSP Tier 1 Screening Assays

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

in Table 1. 

Table 1. Tier 1 Screening Assays for Cyfluthrin 

Tier 1 Assays Test Guideline Test Order Status 

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

(MRID 48615603) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement Satisfied by OSRI (Kojima 

et al. ,2004); however, a Tier 1 assay was 

submitted (MRID No. 48615604)

AR Binding Assay (Rat prostate 

cytosol) OCSPP 890.1150 Requirement Satisfied (MRID 48615601)

Steroidogenesis Assay (Human cell line 

H295R) 

OCSPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID 48615607) 

Aromatase Assay (human recombinant 

microsomes) OCSPP 890.1200 Requirement Satisfied (MRID 48615602) 

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID 48615608) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement satisfied by OSRI (Zhang et 

al.,2008); however a Tier 1 assay was 

submitted (MRID 48615605) 

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied 

(MRID 48615606) 

Pubertal Male Assay (Rat) OCSPP 890.1500 

Requirement Satisfied by OSRI (Du et 

al., 2010; Kojima et al., 2004; Zhang et 

al., 2008); however a Tier 1 assay was 

submitted (MRID 48615606) 

Page 16 of 76
CYFLUTHRIN FINAL 

Page 9 of 68 

Tier 1 Assays Test Guideline Test Order Status 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement Satisfied 

 (MRID 48670101)

Amphibian Metamorphosis Assay 

(Frog) 

OCSPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID 48682901) 

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

1. Effects on Estrogen Pathway 

The potential for cyfluthrin 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. 

Cyfluthrin was negative (or not interactive) in the Tier 1 ER Binding, ERTA and aromatase 

assays. In the Tier 1 steroidogenesis assay, cyfluthrin increased estradiol production (1.33 to 

1.88 fold-change) across all three runs at concentrations of 10−10 to 10−5

 M; however, the 

increases in estradiol were not concentration dependent and were highly variable, attaining 

significance only at a mid-concentration of 10−7 M (p≤0.05; 1.81 fold-change). 

In a published study, Kojima et al., (2004) evaluated cyfluthrin for its potential to activate two 

human estrogen receptor subtypes (hERα and hERβ) in luciferase-based transactivation assays in 

CHO-K1 cells. Cyfluthrin was evaluated for potential ER agonism and antagonism in this study. 

Cyfluthrin induced ERα (but not ERβ) activity with a 20% relative effective concentration 

(REC20) of 5.9 × 10-6 M, and relative luciferase activity (RLA) of 45% at 10-5 M (100% activity 

defined as the activity achieved with 10-10 M estradiol). Cyfluthrin was negative for both ERα

and ERβ antagonism in this test system. However, in another published study, Du et al. (2010) 

showed that cyfluthrin was negative for ER agonism and positive for ER antagonism using an 

ER-mediated luciferase reporter gene assay in CV-1 cells. In that study, cyfluthrin was found to 

have anti-estrogenic activity, with a 20% relative inhibitory concentration (RIC20) of 1.36 x 10-6

M. Additionally, cyfluthrin was negative for ER activity in a gene reporter assay in human 

ovarian carcinoma cells (BG1) (Kojima et al., 2005). 

In the Tier 1 in vivo mammalian assays, cyfluthrin was negative for estrogenic activity in the 

uterotrophic assay. In the Tier 1 female pubertal assay, at the high dose (20 mg/kg/day), the age 

at VO was increased by 2.7 days while body weight at VO was increased by 10%, compared to 

the control. Clinical signs of toxicity (increased salivation) were noted in 5/15 rats at the high 

Page 17 of 76
CYFLUTHRIN FINAL 

Page 10 of 68 

dose. No other estrogen-related effects were observed in the assay. There were no estrogenrelated effects observed in the Part 158 mammalian studies. 

In the FSTRA, effects were noted at the highest test concentration (0.421 μg/L) in female fish 

including changes in ovarian histopathology (a narrowing in the range of ovarian gonadal stage) 

and a 92% decrease in fecundity. However, survival in the highest concentration group was only 

50% in males and 18.8% in females, and the surviving fish showed clinical signs of toxicity and 

loss of typical reproductive behavior and color pattern. No effects were noted in the two lower 

test concentration groups. 

In a Part 158 one-generation reproduction study with mallard ducks effects at the mid and high 

dose (1000 and 4000 mg/kg-diet) included a reduction in the number of eggs laid, embryo 

viability, hatchlings, and 14-day survivors. A high percentage of eggs cracked were observed at 

the low dose (250 mg/kg-diet), but this effects was not observed at the higher treatments. The 

study author reported a significant reduction in male and female body weight during the first four 

weeks of the study at the high concentration. However, due to low control reproductive 

performance, including low embryo viability, hatchling number and 14-day survivorship, there is 

uncertainty associated with the results of this study. In another Part 158 one-generation 

reproductive study conducted on mallard ducks, there were no statistically-significant effects on 

reproductive parameters up to 250 mg/kg-diet. The reviewer noted that pathological results 

showed an increase in the incident of slight egg yolk peritonitis in the treatment-groups 

compared to the control (≥10 mg/kg-diet). In this study, there was also observed low control 

reproductive performance. Therefore, given these uncertainties in control performance, these two 

mallard reproduction studies were not reported in table below. Finally, in a mallard onegeneration reproduction study with beta-cyfluthrin, no effects on reproductive parameters were 

observed up to the highest dose tested, 250 mg/kg-diet. In a Part 158 one-generation 

reproduction study with bobwhite quail, two-week old chick survivors were significantly reduced 

compared to the control at the high test concentration (4000 mg/kg-diet). For males, but not 

females, body weight and body weight gains were also significantly reduced at the high test 

concentration. Significant reductions in eggshell thickness were detected at all test 

concentrations (250, 1000, and 4000 mg/kg-diet). However, in a follow-up study examining 

eggshell strength and thickness in bobwhite quail, there were no effects on eggshell thickness up 

to 4000 mg/kg-diet. 

Part 158 fish early life-cycle studies reported significant effects on growth at concentrations of 

≥0.018 μg/L for rainbow trout, and reduced juvenile survival of sheepshead minnow at 

concentrations of ≥0.084 μg/L. In a Part 158 fish life cycle study with fathead minnow, there was 

a significant decrease in hatchability of F1 eggs at 0.25 μg/L as well as significant decreases in 

post-hatch survival of the parent and F1 generations at the same test concentration. 

Page 18 of 76
CYFLUTHRIN FINAL 

Page 11 of 68 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Cyfluthrin

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Cyfluthrin1Study 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 (MRID 48615603) N ERTA (MRID 48615604) NAromatase (MRID 48615602) N Steroidogenesis (MRID 48615607) N Uterotrophic (MRID 48615608) N Female Pubertal Rat (MRID 48615606) N N N N N N FSTRA (MRID 48670101) NE N N N N N 

OSRI 

ER Transactivation (Kojima et al., 2004) PER Transactivation (Du et al., 2010) PER Transactivation (Kojima et al., 2004) N 
CYFLUTHRIN FINAL 

Page 12 of 68 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Cyfluthrin

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Cyfluthrin1Study 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) 

Subchronic toxicity (Rat; MRID 41244108) 

NE NE N NE 

Subchronic inhalation (Rat; MRIDs 00157793, 40082901, 40239301) 

NE N N NE 

Developmental toxicity (Rat gavage; MRID 44116501) 

NE N 

Developmental toxicity (Rabbit; MRID 42675401) 

NR N 

Developmental toxicity (Rat inhalation; MRID 40780401) 

NR N 

Developmental toxicity (Rat inhalation; MRID 43393401) 

NR N 

Developmental Neurotoxicity (Rat; MRID 46054101) 

NE N N 

Two-generation reproduction (Rat; MRIDs 44371401, 44371402) 

 NE N N NE N 
CYFLUTHRIN FINAL 

Page 13 of 68 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Cyfluthrin

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Cyfluthrin1Study 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) 

Chronic toxicity/ carcinogenicity (Rat; MRID 00137303) 

NE N N NE 

Chronic toxicity/ carcinogenicity (Rat; MRID 44459301) 

NE N N NE 

Carcinogenicity (Mouse; MRIDs 44589701, 45228101) 

NE N N NE 

Chronic toxicity (Dog; MRID 44435401) 

NE N N N 

Avian reproduction (Duck; MRID 48350615) 

N 

Avian reproduction (Quail; MRIDs 00145330, 00152829) 

P6 

Fish life-cycle toxicity (Fathead minnow, MRID 41450401) 

N 

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

treatment. Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evapplicable in this assay. Changes in weight are absolute unless otherwise indicated. NR: Not Reported; NE: Not Examined; B

Clinical signs. 

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 
CYFLUTHRIN FINAL 

Page 14 of 68 

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 

4. Clinical signs of toxicity (increased salivation) were noted in 5/15 rats at the high dose. 

5. The high dose of 0.421 μg a.i./L resulted in 50% mortality in males and 18.8% in females. 6. Two-week old chick survivors were significantly reduced compared to the control at the high treatment concentration (4000 m

the males, but not females, body weight and body weight gains were significantly reduced at the high treatment concentration. 

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

Page 15 of 68 

2. Effects on Androgen Pathway 

The potential for cyfluthrin 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. 

Cyfluthrin was negative (or not interactive) in the Tier 1 AR binding and steroidogenesis assay. 

In a published study, Kojima et al., (2004) evaluated cyfluthrin for its potential to interact with a 

human androgen receptor (hAR) in vitro in a transactivation assay using CHO-K1 cells. In this 

study, cyfluthrin tested positive for weak anti-androgenic activity, with a RIC20 of 8.4 × 10-6 M 

and a RLA of 74% at 10-5 M (100% activity defined as the activity achieved with 10-10 M DHT). 

Cyfluthrin did not demonstrate androgenic transcriptional activity of more than 20% of that 

induced by 10-9 M DHT. In another published study, Du et al., (2010) evaluated cyfluthrin for in 

vitro androgen receptor binding activity (both as agonist and antagonist) in a receptor-mediated 

luciferase reporter gene assay using a MDA-kb2 cell line. Cyfluthrin did not exhibit agonistic 

activity, but antagonistic activity was observed for cyfluthrin, with a RIC20 of 8.84 × 10-8 M. 

Finally, in another published in vitro study, Zhang et al., (2008) examined the potential antiandrogenic effects of cyfluthrin and β-cyfluthrin with reporter gene assays using a MDA-kb2 

breast cancer cell line. In this study, cyfluthrin inhibited (p<0.05) DHT-induced transcriptional 

activation at concentrations from 10−7 to 10−5 M. 

The Tier 1 Hershberger assay was negative, and no androgen-related effects were observed in the 

Tier 1 male pubertal assay. These studies were conducted at dose levels up to 20 mg/kg/day; a 

dose level which showed signs of systemic toxicity (piloerection and/or increased salivation). 

In the FSTRA, effects were noted only at the highest test concentration (0.421 μg/L) and 

included changes in GSI, testicular histopathology and secondary sex characteristics. However, 

overt toxicity was also noted at the highest test concentration (i.e., reduced survival and clinical 

signs of toxicity). There were no androgen-related effects in the mammalian or fish Part 158 

toxicity studies. In the Part 158 avian reproduction studies, the only treatment-related effect was 

a decrease on offspring survival in quail study. 

In a published, non-guideline Hershberger study, Zhang et al. (2008) also examined the potential 

anti-androgenic effects of cyfluthrin or β-cyfluthrin at doses up to 54 and 36 mg/kg/day, 

respectively. No mortality or treatment-related clinical signs were noted in any of the groups in 

the Hershberger assay. There were also no significant differences between initial or final body 

weights or weight gains of the cyfluthrin-treated groups compared to the control groups, and 

Page 23 of 76
CYFLUTHRIN FINAL 

Page 16 of 68 

serum testosterone concentrations were not affected. However, treatment with either cyfluthrin 

or β-cyfluthrin (in combination with TP) resulted in dose-dependent decreases at both the midand high-dose in the weights of the seminal vesicle, ventral prostate, dorsolateral prostate, LABC 

and Cowper’s glands. 

Page 24 of 76
CYFLUTHRIN FINAL 

Page 17 of 68 

Table 3. Androgenic/Anti-Androgenic Pathway for Cyfluthrin

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Cyfluthrin1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

EDSP Tier 1 Assay 

AR Binding (MRID 48615601) N Steroidogenesis (MRID 48615607) NHershberger (MRID 48615605) NE N Male Pubertal Rat (MRID 48615606) N N N N N NE N N FSTRA (MRID 48670101) NE N N N N N 

OSRI 

AR Binding & Transcriptional Activation (Kojima et al., 2004) 

P 

AR Binding & Transcriptional Activation (Du et al., 

2010) 

P 

 AR Transcriptional Activation (Zhang et al., 2008) 

P 

Hershberger (Zhang et al., 2008) 

P 

 (M,H) 
CYFLUTHRIN FINAL 

Page 18 of 68 

Table 3. Androgenic/Anti-Androgenic Pathway for Cyfluthrin

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Cyfluthrin1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

Subchronic toxicity (Rat; MRID 41244108) 

N N NE N NE N 

Subchronic inhalation toxicity (MRIDs 00157793, 40082901, 40239301) 

N N NE NE NE NE 

Developmental Neurotoxicity (Rat; MRID 46054101) 

N 

Two-generation reproduction (Rat; MRIDs 44371401, 44371402) 

N N NE N NE NE N 

Chronic toxicity/ carcinogenicity (Rat; MRID 00137303) 

N N NE NE NE N 

Chronic toxicity/ carcinogenicity (Rat; MRID, 44459301) 

N N NE N NE N 

Carcinogenicity (Mouse; MRIDs 44589701, 45228101) 

 N N NE N NE NE 

Chronic toxicity (Dog; MRID 44435401) 

NE N NE N NE N 
CYFLUTHRIN FINAL 

Page 19 of 68 

Table 3. Androgenic/Anti-Androgenic Pathway for Cyfluthrin

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Cyfluthrin1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

Avian reproduction (Duck; MRID 48350615) 

N 

Avian reproduction (Quail; MRIDs 00145330, 00152829) 

P6 

Fish life-cycle toxicity (Fathead minnow, MRID 41450401) 

 NE N N

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

treatment. Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evain this assay. Changes in weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Semiprostate (VP), Dorsal prostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland 

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 

4. A variety of clinical signs (increased salivation, piloerection, lack of grooming, uncoordinated movements, and/or tremors) we

animals on at least one occasion. 

 5. The high dose of 0.421 μg a.i./L resulted in 50% mortality in males and 18.8% in females.  6. Male, but not female, body weight and body weight gains were significantly reduced at the high treatment concentration. Sign

survival of 14-d old chick offspring observed at this high treatment. 

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

 NE Not examined 
CYFLUTHRIN FINAL 

Page 20 of 68 

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

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

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

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

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

The potential for cyfluthrin 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 hormone concentrations, weight, or histopathology) 

were observed in the in vivo male and female pubertal assays. No thyroid-related effects were 

observed in the Part 158 mammalian studies. 

In the AMA, a co-solvent (DMF) was used in the study and there were no significant differences 

between the negative and solvent control for any endpoint [(for Day 21 NF stage the significance 

was marginally close (p=0.058)], and as such, the reported results in Table 4 reflect comparisons 

to the negative control. The Day 21 median NF developmental stage was significantly increased 

(p<0.05) by 1 stage in the mid and high treatment groups (0.032 and 0.066 μg/L) compared to 

the negative control (NF stage 58 vs. 57 in negative control), but not when compared to the 

solvent control (NF stage 58). Cyfluthrin did not significantly affect 7- or 21-Day normalized 

hind limb length (HLL), and no effects on thyroid gland histopathology were observed at any 

treatment concentration. In regard to the growth parameters, Day-21 snout-vent length (SVL) 

was significantly increased by 12.6% in the 0.032 μg a.i./L group and decreased by 10.8% in the 

0.066 μg a.i./L group, compared to the negative control. Increases of 47.4 and 50.7% were also 

observed in wet body weight on Day 21 for the 0.032 and 0.066 μg a.i./L groups, respectively, 

and wet body weight was increased (p<0.05) for Day 7 at the high concentration (↑31%). 

 

Page 28 of 76
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Page 21 of 68 

Table 4. Thyroid Pathway for Cyfluthrin.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Cyfluthrin1

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 48615606) N N N N N N X (H)4

Male Pubertal Rat 

(MRID 48615606 ) N N N N N N X (H)4

AMA 

(Frog; MRID 

48682901) 

 N 

Day 21, 

+1 NF 

stage 

(M, H)5 

Day 7 BW: 

↑31% (H); 

Day 21 BW: 

↑47, 51% 

(M, H)5

; 

Day 21 SVL: 

↑13, ↓11% 

 (M ,H)5 

 N 

OSRI 

Subchronic oral toxicity 

(Rat; MRID 41244108) NR N NR X 

(H) N 

Subchronic oral toxicity 

(Dog; MRID 41267801) N N NR X 

(H) N 

Subchronic inhalation 

(Rat; MRIDs 00157793, 

40082901, 40239301) 

N N NR X 

(M, H) N 

Developmental toxicity 

(Rat gavage; MRID 

44116501) 

X 

(M, H)

X 

(H)

Developmental toxicity 

(Rabbit; MRID 

42675401) 

X 

(M, H) N 

Developmental toxicity 

(Rat inhalation; MRID 

43393401) 

X 

(ML, 

MH, H)

N 

Developmental 

Neurotoxicity (Rat; 

MRID 46054101) 

X 

(H) N 

Two-generation 

reproduction (Rat; 

MRID 44371401 and 

44371402) 

NE NE NR X 

(M, H) N 

Chronic toxicity/ 

carcinogenicity (Rat; 

MRID 00137303) 

NR N NR X 

(H) N 

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Page 22 of 68 

Table 4. Thyroid Pathway for Cyfluthrin.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Cyfluthrin1

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 

Chronic toxicity/ 

oncogenicity (Rat; 

MRID 44459301) 

NR N NR X 

(M, H)

X 

(H)

Carcinogenicity 

(Mouse; MRIDs 

44589701, 45228101) 

NR N NR X 

(H) N 

Chronic toxicity 

(Dog; MRID 44435401) N N N X 

(M, H) N 

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

4. Clinical signs were noted in 6/15 males and 5/15 females at the high-dose on at least one occasion. 

5. Treatment related effects noted at the mid and high concentrations were similar to the effects noted in the 

solvent control. 

 P Positive finding 

 N Negative findings 

NE Not examined 

NR Not reported 

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Page 23 of 68 

IV. Committee’s Assessment of Weight of Evidence 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

whether or not the responses are mediated through an endocrine pathway, therefore nonendocrine toxic responses (including but not limited to mortality or body weight changes) are 

also considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues 

associated with weight of evidence evaluation of the results of the Tier 1 assays stated that “In 

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

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

evaluated in terms of primary interactions with the endocrine system vs. secondary effects 

related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 

2013). 

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

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

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

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

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

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

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

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1. Tier 1 in vivo Assays 

In the Hershberger assay, sporadic clinical signs (increased salivation or piloerection) were 

observed in two high-dose (20 mg/kg/day) animals and one mid-dose (10 mg/kg/day) animal. In 

the female pubertal rat study, increased salivation was observed in 5/15 animals in the high-dose 

group (20 mg/kg/day). In the male pubertal rat study, increased salivation was noted in one middose (10 mg/kg/day) animal on one day, and a variety of clinical signs (increased salivation, 

piloerection, lack of grooming, uncoordinated movements and/or tremors) were noted in 6/15 

high-dose animals on at least one occasion. Similar clinical signs (piloerection, reduced or 

limited activity, uncoordinated movements and increased salivation) were also observed in one 

high-dose animal (20 mg/kg/day) in the uterotrophic assay. 

In the FSTRA study, overt toxicity was observed at the highest tested cyfluthrin concentration 

(0.421 μg/L) based on mortality in both the males (50%) and females (18.8%); clinical signs of 

toxicity were also evident in the survivors at this concentration (i.e., swimming at the surface, 

erratic behavior, loss of equilibrium, labored respiration and decreased food intake). No signs of 

toxicity were noted in fish at test concentrations ≤0.149 μg/L. No overt or systemic toxicity was 

noted in the AMA study at test concentrations up to 0.066 μg/L. 

2. OSRI 

Systemic and/or overt toxicity of cyfluthrin and β-cyfluthrin in the OSRI in vivo studies 

consisted primarily of effects on body weight and body weight gains and neurological effects. 

Treatment-related mortality was not observed in the oral subchronic toxicity studies on rats or 

dogs, but clinical signs of neurotoxicity were observed at the high doses in both studies. In rats, 

lack of coordination and poor general condition were noted at the high dose (39-42 mg/kg/day), 

and body weight gains were reduced in both sexes at the high dose. In the dog study, 

neurological signs (awkward, staggering gait and occasional buckling of the hind limbs) were 

noted in 4/8 dogs from both sexes at the high dose (14-15 mg/kg/day). In the subchronic 

inhalation study on rats, clinical signs (unthriftiness/ unkempt fur and lethargy) were observed in 

all mid-dose females (0.16 mg/kg/day) during the second half of the study, and similar signs 

were noted in all high-dose (0.91 mg/kg/day) males and females within 2 weeks of study 

initiation. Mid and high-dose males also had reductions in body weight and body weight gains 

compared to the control. 

In the rat oral developmental study, overt and systemic toxicity were noted in the high-dose dams 

(40 mg/kg/day); reductions were observed in maternal survival (↓10%), body weight gain 

(↓56%) and food consumption (↓20-49%). In addition, clinical signs of toxicity at the high dose 

included hypoactivity, locomotor incoordination, and salivation. Systemic toxicity was also 

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CYFLUTHRIN FINAL 

Page 25 of 68 

noted at the mid-dose (10 mg/kg/day) and consisted of reductions in food consumption and body 

weight gains during dosing. In the rabbit oral developmental study, there were no maternal 

deaths or clinical signs of toxicity during the study. However, food consumption and body 

weight gains were reduced in the mid- and high-dose groups (60 and 180 mg/kg/day) during 

dosing. In the developmental neurotoxicity study, no overt or systemic toxicity was noted in 

dams orally dosed with β-cyfluthrin at up to 17.8 mg/kg/day. 

 

In two rat inhalation developmental studies, there was no mortality, but clinical signs of toxicity 

(reduced motility, dyspnea, piloerection and ungroomed coats) were noted at doses ≥0.0047 

mg/L (≥1.28 mg/kg/day). However, food consumption and body weight gains were reduced in 

the mid- and high-dose groups (60 and 180 mg/kg/day) during dosing. In another rat inhalation 

developmental study, maternal body weights and food efficiency were reduced in all dose levels 

(0.125-3.48 mg/kg/day) during the dosing period. In addition, clinical signs (labored and 

irregular breathing, hypoactivity, high-stepping gait, salivation, and reduced stool) were 

observed at the high-dose (3.23 mg/kg/day), but the effects were attributed to bradypnea in the 

dams since clinical signs were not observed in dams dosed at 3.48 mg/kg/day and supplemented 

with O2. 

Two 2-generation reproductive toxicity studies on rats are available for cypermethrin In one 

study, no treatment related mortality was noted, but systemic toxicity was observed in adults in 

the mid- and high-dose groups ([M/F] 9/10 and 29/33 mg/kg/day). At the mid-dose, body 

weights were decreased in males and reduced food consumption was reduced in females during 

lactation. At the high-dose, there were reductions in body weight of both males and females as 

well as decreases in food consumption (relative to control) and clinical signs (splaying of hind 

limbs) in females during lactation. In the supplemental reproductive study, no overt or systemic 

toxicity was noted in adult animals at doses up to 3.8/4.2 mg/kg/day. 

In the Hershberger study from Zhang et al. (2008), no signs of overt or systemic toxicity were 

noted in castrated male rats following 7 days of dosing with cyfluthrin at up to 54 mg/kg/day or 

with β-cyfluthrin at up to 36 mg/kg/day. 

In a chronic toxicity study in rats, there were no treatment-related effects on survival or clinical 

signs at dietary levels of cyfluthrin up to 450 ppm, equivalent 22.8 and 28.3 mg/kg/day for males 

and females, respectively. At the mid-dose (11.6/14.4 mg/kg/day), mean body weights were 

reduced by ≤7% in both sexes, and body weight gains were decreased by ≤24%. At the highdose, mean body weights were decreased by ≤13% and≤14% for males and females, 

respectively, and mean body weight gains were decreased by ≤23% and ≤24%, respectively. 

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Page 26 of 68 

In a mouse carcinogenicity study, there were no treatment-related effect on mortality, food 

efficiency, hematology, or organ weights for both sexes at all doses. At the high dose (233/310 

mg/kg/day [M/F]), reductions in body weight were observed in both sexes (↓7-20%) throughout 

the study, and overall body weight gain was decreased in the males (↓25%) and females (↓54%). 

Mean absolute food consumption was also decreased in the females (↓6-25%) throughout the 

study. In addition, increased incidence of rough coat (both sexes) and hunched back (females 

only) were noted in the high-dose group of both sexes. 

In the chronic toxicity study in dogs, no overt or systemic toxicity was noted at the low and midlow doses (1.4/1.5 and 2.4/3.6 mg/kg/day [M/F]). At the mid-high dose (10.6/10.7 mg/kg/day 

[M/F]), clinical signs of neurotoxicity were noted in both sexes, including gait and posture 

abnormalities. At the high dose [(15.5/18.0 mg/kg/day (M/F)], the incidence and severity of 

posture and gait abnormalities increased. Decreases (NS) in male body weights and body weight 

gains were also noted at the high dose. 

No overt or systemic toxicity was observed in the mallard duck reproductive study up to 250 

mg/kg-diet for cyfluthrin (MRID 00158782). In another mallard duck reproductive study testing 

up to 4000 mg/kg-diet, there was no treatment-related effect on adult mortality or clinical signs 

in any treatment group, and no significant difference in food consumption (MRID 00145331). 

Significant reductions in body weight gains occurred at the high concentration from Weeks 0-4, 

but no significant differences in body weight or body weight gain were noted from Week 8 until 

study termination. The mallard duck reproduction study with beta-cyfluthrin reported no overt 

or systemic toxicity up to 250 mg/kg-diet (MRID 48350615). 

In a bobwhite quail reproductive study, there was no treatment-related mortality at dietary 

concentrations up to 4000 mg/kg-diet (MRID 00145330). There was no significant difference in 

food consumption, and no treatment-related effects were noted on body weight or body weight 

gain for the females. However, body weight and body weight gains for males were significantly 

reduced at the high concentration (4000 mg/kg-diet) in this study. 

In the fish life-cycle study, no treatment-related effects were noted on percent hatch in the 

parental or F1 generations at test concentrations up to 0.14 μg/L (MRID 41450401). Standard 

length and wet weight as measures of growth were also not affected at any treatment level in 

either the parental or F1 generations. However, there was a significant decrease in hatchability of 

F1 eggs at the highest test concentration, 0.25 μg/L, and significant decreases in post-hatch 

survival of the parent and F1 generations at the same test concentration. Part 158 fish early lifecycle studies reported significant effects on growth at concentrations of ≥0.018 μg/L for rainbow 

trout (MRID 00155898), and reduced juvenile survival of sheepshead minnow at concentrations 

of ≥0.084 μg/L (MRID 00158781). 

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CYFLUTHRIN FINAL 

Page 27 of 68 

B. Estrogen Pathway 

There was no evidence of interaction of cyfluthrin with the estrogen pathway either in the Tier 1 

in vitro (ER binding, ERTA and aromatase) or in the Tier 1 in vivo (uterotrophic, female pubertal 

and FSTRA) assays in the absence of overt toxicity. There were no estrogen-related effects were 

seen in the Part 158 studies [(effects on offspring survival were observed in the quail 

reproduction study, however, this endpoint is limited in its ability to inform potential interactions 

with the estrogen (and androgen) pathway)]. In the Tier 1 in vitro steroidogenesis assay, while a 

significant increase in estradiol production was observed at a mid-concentration, there was a lack 

of estrogen-related effects observed in the other in vitro and in vivo assays (in the absence of 

overt toxicity) . 

C. Androgen Pathway 

There was no evidence of interaction of cyfluthrin with the androgen pathway in the Tier 1 in 

vitro assays (AR binding or steriodogenesis). While potential anti-androgenic activity was 

reported in three published in vitro ARTA studies, limited evidence of potential anti-androgenic 

effects was observed in vivo in the absence of overt toxicity. While no androgen-related effects 

were observed in Tier 1 assays (Hershberger, male pubertal and FSTRA) in the absence of overt 

toxicity, positive results were reported in a non-guideline Hershberger study by Zhang et al, 

2008. In that study, which evaluated anti-androgenic activity only, cyfluthrin and β-cyfluthrin 

caused significant decreases in multiple secondary sex tissues at the 18 and 54 mg/kg/day doses. 

The dose levels (6, 18 and 54 mg/kg/day) in this study were comparable to the dose levels (5, 10 

and 20 mg/kg/day) tested in the Tier 1 Hershberger assay. While Zhang et al. reported not 

observing clinical signs in their study, increased salivation, piloerection and/or lack of 

coordination, which are indicative of neurotoxicity, were seen at doses greater than 20 

mg/kg/day in the Tier 1 and mammalian Part 158 studies. Therefore, the androgen-related effects 

observed in the Zhang et al. study may have occurred in the presence of overt toxicity. Since the 

effects may have potentially occurred in the presence of overt toxicity, in addition to the other 

mammalian studies indicating no androgen-related effects up to overtly toxic doses, the 

confidence in the results of this study are reduced. No androgen-related effects were observed in 

the avian and fish Part 158 studies. Based on the available data, there is no convincing evidence 

of potential interaction with the androgen-pathway for cyfluthrin. 

D. Thyroid Pathway 

There was no convincing evidence of potential interaction of cyfluthrin with the thyroid 

pathway. Thyroid parameters were not altered in Tier 1 mammalian in vivo studies. Additionally, 

there were no thyroid-related effects were seen in the Part 158 mammalian studies. In the AMA, 

although effects on developmental stage and growth parameters were observed in the mid- and 

Page 35 of 76
CYFLUTHRIN FINAL 

Page 28 of 68 

high-treatment groups compared to the negative control, similar effects (increased growth and 

development) were noted in the solvent control group. Therefore, it appears that the increased 

development and growth in the treatments and the absence of effects on thyroid histopathology 

may have been due to the presence of the solvent (may have resulted in increases in food 

resources) as oppose to a potential thyroid-related effect. Based on the results in the AMA, there 

appears to be no convincing evidence of a potential thyroid-related interaction at the 

concentrations tested (up to 0.066 μg a.i./L). In the AMA, the achieved high-concentration 

group (0.066 μg a.i./L) did not exhibit any signs of toxicity. The achieved high concentration 

from the definitive AMA study is in the range of the no effect concentration reported in the 

rangefinder; however, due to issues in dosing in the range-finding study, there is uncertainty in 

the actual exposure concentration where the toxicity was observed. Given the lack of observed 

treatment-related effects in the definitive study along with having a clear understanding of the 

concentration where clinical signs occurred in the rangefinder, there is uncertainty whether the 

maximum concentration achieved in the AMA was adequate to test for an effect. However, as 

there is a lack of redundancy across the available data with no evidence for interaction in the 

AMA at the concentrations tested, overall there is a lack of convincing evidence of a potential 

interact with the thyroid pathway for cyfluthrin. 

E. Conclusions 

Cyfluthrin demonstrates no convincing evidence of potential interaction with the estrogen, 

androgen or thyroid pathways. 

V. EDSP Tier 2 Testing Recommendations 

Based on weight of evidence considerations, mammalian and wildlife EDSP Tier 2 studies are 

not recommended for cyfluthrin since there was no convincing evidence of potential interaction 

with the estrogen, androgen or thyroid pathways. 

Page 36 of 76
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Page 29 of 68 

VI. References 

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Banman, C.S.; Matlock, D.; and Lam, C.V.,(2012). Short-term Reproduction Assay with Fathead 

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Banman, C.S.; Alexander, T.M.; Lam, C.V.; and Wolf, J.C., (2012). Amphibian Metamorphosis 

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Beavers, J. (1986) Baythroid Technical: A One-generation Reproduction Study with the Mallard 

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Blanck, O. (2011a) Cyfluthrin: Evaluation in the in vitro (HeLa-9903) estrogen receptor 

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48615605). 

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juvenile/peripubertal male and female rats. Unpublished study prepared by Bayer S.A.S., Bayer 

CropScience, Sophia Antipolis Cedex, France. Laboratory Study Number SA 10206, June 27, 

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Carlisle, J. (1984a) Effect of Cyfluthrin (Baythroid Technical) on Bobwhite Quail Reproduction: 

Study Number 83-675-08. Unpublished study prepared by Mobay Chemical Corp. 94 p. (MRID 

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Carlisle, J. (1984b) Effect of Cyfluthrin (Technical Baythroid) on Mallard Duck Reproduction: 

Study Number 83-675-06. Unpublished study prepared by Mobay Chemical Corp. 85 p. (MRID 

00145331) 

Carlisle, J. (1985c) Effects of Cyfluthrin (Baythroid Technical) on Bobwhite Quail Eggshells: 

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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 Gene Assays. Toxicological Sciences 116(1):58-66. (MRID 48196301). 

Eigenberg, D.; Elcock, L. (1996) A Two-Generation Reproduction Study in Rats Using 

Technical Grade Cyfluthrin Administered Via the Diet: Lab Project Number: 93-672-UZ: 

107408: 93-672UZ1. Unpublished study prepared by Bayer Corp. 973 p. (MRID 44371401) 

Eigenberg, D. (1997) A Supplementary Two-Generation Dietary Reproduction Study in Rats 

Using Technical Grade Cyfluthrin: Lab Project Number: 94-672-CK: 107474: 94-672-CKl. 

Unpublished study prepared by Bayer Corp. 771 p. (MRID 44371402) 

Holzum, B.; Schmidt, U.; Pauluhn, J.; et al. (1994) FCR 1275: Explanatory Report on Results 

and Mechanistic Studies for Embryotoxic Effects in Rats after Inhalation: Lab Project Number: 

06369: 23219: 22581. Unpublished study prepared by Bayer Ag. (MRID 43393401) 

Jones, R.D. and Hastings, T.F. (1997) Technical Grade Cyfluthrin (FCR 1272): A Chronic 

Toxicity Feeding Study in the Beagle Dog. Laboratory Study No.: 94-276-ZR. Unpublished 

study prepared by Bayer Corp. (MRID 44435401) 

Jones, R and Hastings, T. (2000) Technical Grade Cyfluthrin (FCR 1272): A Chronic Toxicity 

Feeding Study in the Beagle Dog: Lab Project Number: 94-276-ZR: 8365: 108007. Unpublished 

study prepared by Bayer Corporation. 14 p. Supplemental to MRID 44435401. (MRID 

45189201) 

Kojima, H., Katsura, E., Takeuchi, S., Niiyama, K., and Kobayashi, K. (2004). Screening for 

estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using 

Chinese Hamster Ovary cells. Environ. Health Perspect. 112: 524-531. (MRID 48033008) 

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Kojima, M., K Fukunaga, M. Sasaki, M. Nakamura, M. Tsuji, T. Nishiyama. (2005) Evaluation 

of estrogenic activities of pesticides using an in vitro reporter gene assay. Int. J. Environ. Health 

Res. 15:271-280. 

Pauluhn, J. (1983) Study of the Subchronic Inhalation Toxicity in Accordance with OECD 

Guideline No. 413: Baythroid: Supplemental Submission: Second Addendum to Bayer No. 

12436.Unpublished study prepared by Bayer AG. 24 p. (MRID 40239301) 

Pauluhn, J. (1984) Study for Subchronic Inhalation Toxicity to the Rat for 13 Weeks: FCR 1272 

(Cyfluthrin): Rept. No. 12436. Unpublished Mobay study no. 86443 prepared by Bayer AG 

Institute of Toxicology and Hanover Medical University. 170 p. (MRID 00157793) 

Pauluhn, J. (1987) Study of the Subchronic Inhalation Toxicity in Accordance with OECD 

Guideline No. 413: FCR 1272: Cyfluthrin: Addendum to Bayer Report No. 12436, Dated Feb. 1, 

1984: Study No. T9015085. Unpublished Mobay Report No. 86443 prepared by Bayer AG. 157 

p. (MRID 40082901) 

Pauluhn, J. (1988) Addendum to MRID 40780401: FCR 1272: Cyfluthrin: Study for 

Embryotoxic Effects on Rats after Inhalation: Report No. 97403; 16391-A. Unpublished study 

prepared by Bayer Ag. 12 p. (MRID 40968501) 

Pauluhn, J. (1989) FCR: 4545 (C.N. Beta-cyfluthrin, Proposed) Subacute Inhalation Toxicity 

Study in the Rat: Lab Project No.: 146:100571; Report No.: 18146. Unpublished study prepared 

by Bayer Ag. 949p. (MRID 41783001)

Renhof, M.; Pauluhn, J. (1988a) FCR 1272: Common Name Cyfluthrin: Study for Embryotoxic 

Effects on Rats after Inhalation: Report No. 16391: Report No. 97403. Unpublished study 

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Renhof, M.; Pauluhn, J. (1988b) FCR 1272 (Common Name Cyfluthrin): Study for Embryotoxic 

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Unpublished study prepared by Bayer Ag, Wuppertal. 5 p. (MRID 42332001) 

Rhodes, J.E.; McAllister, WA.; Leak, T.; Stuerman, L. (1990) Full Life-Cycle Toxicity of 14CCyfluthrin (Baythroid") to the Fathead Minnow (Pimephales promelas) under flow-through 

conditions. Report N0.1 00097; 33821. Unpublished study prepared by ABC Labs. 905 p. 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100) 

The 21-day assay (MRID 48682901) of cyfluthrin (94.04% purity) on amphibian metamorphosis 

of the South African clawed-frog (Xenopus laevis) was conducted under flow-through conditions 

in an amphibian metamorphosis assay (AMA). Amphibian larva at developmental NieuwkoopFaber Stage (NF) 51 (80/control and treatment group; 20/replicate) were exposed to a negative 

control, a solvent control (dimethylformamide, DMF; 0.1 mL/L), and cyfluthrin at nominal 

concentrations of 0.000024, 0.000060, and 0.00015 mg a.i./L. Mean-measured concentrations 

were 0.000013, 0.000032, and 0.000066 mg a.i./L, respectively. The test system was maintained 

within a temperature range of 20.86 to 22.25oC and a pH of 7.5 to 7.9. 

There were no significant differences between the negative and solvent control for any endpoint; 

however, for Day 21 median NF stage, the difference of one NF stage (negative control: NF 

stage 57; solvent control: NF stage 58) was marginally significant (p=0.058). Unless otherwise 

indicated, all effects are reported based on comparison to the negative control. 

By Day 21, two mortalities each were observed in the negative control, mid, and high treatment 

groups. No mortalities were observed in the solvent control or low treatment group. Bent tail was 

observed in 13-23 tadpoles in negative and solvent controls and each treatment group. 

Day 7 snout-vent length (SVL) was marginally significantly increased (p=0.056) by 7.7% in the 

high treatment group compared to the negative control. Day 21 SVL was significantly increased 

(p<0.05) by 12.6% in the mid treatment group and significantly decreased (p<0.05) by 10.8% in 

the high treatment group compared to the negative control. Significant increases (p<0.05) of 47.4 

and 50.7% were also observed in Day 21 body weight for the mid and high treatment groups, 

respectively, and Day 7 body weight was significantly increased by 31% in the high treatment 

group (p<0.05) compared to the negative control. 

Acceleration of median NF developmental stage and asynchronous development were not 

observed at Day 7. Day 21 median NF stage was significantly increased (p<0.05) by one stage in 

the mid and high treatment groups (NF stage 58) compared to the negative control but was 

comparable to the solvent control. Cyfluthrin did not significantly affect (p>0.05) Day 7 or 21 

normalized (for snout-vent length) hind limb length (HLL). There were no treatment-related 

effects on thyroid gland histopathology observed at any treatment level. 

The coefficient of variation (CV) for mean-measured concentrations ranged from 40 to 60% (due 

to an error in the initial stock solution), exceeding the guideline performance criterion of ≤20%. 

This deficiency introduces uncertainty into the interpretation of results; however, appropriate 

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analytical measurements were provided to document the exposure, and the study is otherwise 

scientifically sound. 

Although significant increases in NF developmental stage as well as growth parameters, 

especially wet weight, were observed at the mid and high treatment levels compared to the 

negative control, treatment-related alterations in thyroid histopathology were not reported. 

Though not significant, increases in Day 21 developmental stage and growth were also observed 

in the solvent control compared to the negative control, and these increases in the solvent control 

were comparable to the increases observed in the treatment groups. Therefore, it appears that the 

increased development and growth in the treatments may have been due to increases in food 

resources as a result of the presence of the solvent with the solvent concentration being 

consistent between the solvent control and treatments groups. 

Androgen Receptor (AR) Binding Assay (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48615601), ventral prostate cytosol from 

male Sprague Dawley rats was used as the source of AR to conduct a saturation binding 

experiment and a competitive binding experiment. The saturation binding experiment was 

conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonable 

numbers and was functioning with appropriate affinity for the radio-labeled reference androgen 

(R1881) prior to routinely conducting AR competitive binding experiments. The competitive 

binding assay was conducted to measure the binding of a single concentration of [3H]-R1881 (1 

nM) in the presence of increasing concentrations of cyfluthrin (logarithmic increase from 10-10 to 

10-4 M). DMSO was used as the vehicle at a final assay concentration of ≤3.3%. The assay 

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

 

In the saturation binding experiment, the mean maximum binding capacity (Bmax) was 14 

fmol/100 μg protein and the mean dissociation constant (Kd) was 1.01 nM. The Kd obtained 

from Saturation Assay #2 was slightly low (0.59 nM), but the Kds from the other two runs were 

within the recommended range (0.685 to 1.57 nM) and the Scatchard plots for the three 

saturation assays were linear. The specific binding reached a plateau, and non-specific binding 

(NSB) was less than 20% of total binding (4.6-12.6%). The saturation binding experiment 

demonstrated that the androgen receptor was present in a reasonable concentration and 

functioning with appropriate affinity for the R1881 ligand. 

In the competitive binding experiment, cyfluthrin was not fully soluble at the highest 

concentration (10-3 M), therefore the highest concentration used in data interpretation was 10-4 M 

cyfluthrin. Cyfluthrin is classified as non-binding as specific [3

H]-R1881 binding was >75% at 

all concentrations of cyfluthrin tested in all three runs. An IC50 and relative binding affinity 

(RBA) could not be calculated for cyfluthrin. 

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The mean log IC50s for R1881 and the weak positive control (dexamethasone) were -9.1 and -4.4 

M, respectively, and the mean RBA for the weak positive control was 0.0021%. Confidence in 

these numbers is high due to the small variation. The solvent control responses indicated no drift 

in the study assay. All performance criteria were met, with the exception that the ligand 

depletion and NSB were not reported, but the IC50s for R1881 and the weak positive control, 

dexamethasone, were within the expected ranges demonstrating that the ligand was available at 

an appropriate level. 

Based on the results from the three runs, cyfluthrin is classified as a Non-Binder in the Androgen 

Receptor Binding Assay. 

Aromatase Assay (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48615602), cyfluthrin (95.8% a.i., Batch # AE 

F057122-01-05) was incubated with human recombinant aromatase and tritiated androstenedione 

(1-β [3

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

increasing logarithmic concentrations from 10–10 M to 10−3

 M for 15 minutes to assess the 

potential of cyfluthrin to inhibit aromatase activity. 

Aromatase activity was determined by measuring the amount of tritiated water produced at the 

end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified 

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

full activity control, a background activity control, a positive control series (10–10 M to 10–5 M) 

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

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

Aromatase activity in the full activity controls ranged from 0.143 to 0.670 nmol/mg-protein/min 

for the three test runs, with a mean and standard deviation of 0.353±0.183 nmol/mg-protein/min. 

Activity in the background controls ranged from 1.8 to 5.2 and averaged 3.4% of the full control 

activity. The % change for the background controls at the beginning and end of each run fell 

between the guideline recommendations of −5% and +6% of the overall average full activity. 

However, for the full activity data, a slight assay drift was observed in the first and third runs, as 

the % changes at the beginning and end of each run were between 88.2 and 111.8% for the first 

run and between 87.4 and 112.6% for the second instead of the recommended guideline range of 

between 90 and 110%. Overall, the response of the full activity controls and background 

controls was acceptable for each run. 

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.341±0.182 

nmol/mg-protein/min at the lowest tested concentration (10−10 M) and 0.004±0.001 

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nmol/mg-protein/min at the highest tested concentration (10−5 M). For cyfluthrin, aromatase 

activity averaged 0.313±0.161 nmol/mg-protein/min at the lowest tested concentration (10−10 M) 

and 0.224±0.096 nmol/mg-protein/min at the highest tested concentration (10−3 M). Analyses of 

the cyfluthrin data indicated they did not fit the 4-parameter regression model used for data 

interpretation. The mean aromatase activity of 10–3 M cyfluthrin was determined to be 66% of 

control activity and the best fit mean inhibition response curve was a straight line (essentially no 

inhibition was observed at other doses). For 4-OH ASDN, the estimated Log IC50 averaged 

−7.09 M, and the slope was −0.98. Confidence in these values is very high due to the small 

variations (<6% CV). 

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

aromatase activity in this assay. 

Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48615603), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct saturation and competitive binding 

experiments. A saturation binding experiment was conducted to demonstrate that the ER in the 

rat uterine cytosol was present in reasonable numbers and was functioning with appropriate 

affinity for the radio-labeled reference estrogen prior to conducting ER competitive binding 

experiments. The competitive binding experiment was conducted to measure the binding of a 

single concentration of [3

H]-17β-estradiol (1 nM) in the presence of increasing concentrations of 

cyfluthrin (logarithmic increase from 10–10 to 10–3 M). DMSO was used as the vehicle at a final 

concentration of 2%. The assay included 19-norethindrone as a weak positive control, 

octyltriethoxysilane as a negative control, and the natural ligand 17β-estradiol. 

For the saturation binding experiment, the appropriate protein concentration for the saturation 

binding assay was determined to be 0.5 mg/mL as it bound 25-35% of the [3

H]-17β-estradiol. 

The Kd for [3

H]-17β-estradiol was 0.070 nM, and the estimated Bmax was 0.034 nM for the 

prepared rat uterine cytosol. The Kd for each run was within the expected range of 0.03 to 1.5 

nM. 

For the competitive binding experiment, the estimated mean IC50 was not calculated for 

cyfluthrin as it did not achieve 50% binding. The mean IC50 for the positive control was 9 × 10–6

M, with a mean relative binding affinity (RBA) of 0.019% compared to the natural ligand; 

however, the Hill Slope fell outside the recommended range, at −2.0 to −1.7. Cyfluthrin was 

tested over a concentration range that fully defined the top of the curve. The maximum percent 

binding 108.8-117.4% was within approximately 25 percentage points of the lowest 

concentration of the estradiol standard 83.5-116.2%. The mean specific ER binding was >75% 

at cyfluthrin concentrations up to 10–4 M in all three independent runs. 

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Based on the results from the three runs, cyfluthrin is classified as Not Interactive in the Estrogen 

Receptor Binding Assay. 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48615604), hERα-HeLa-9903 

cells cultured in vitro were exposed to cyfluthrin (95.8% a.i., Batch # FHER904218) at 

logarithmically increasing concentrations from 10−11 to 10−5

 M in DMSO (0.1%) for 24 hours. 

The experiments were performed using 96-well plates and each cyfluthrin concentration was 

tested in triplicate (3 wells/plate). Cells were exposed to the test agent for 23±1 hr to induce 

reporter (luciferase) gene products. Luciferase expression in response to activation of the 

estrogen receptor by cyfluthrin was measured upon addition of a luciferase substrate and 

detection with a luminometer with acceptable sensitivity. 

Cyfluthrin was tested up to the limit of cytotoxicity, 10−5

 M. The laboratory proficiency assays 

using the required reference compounds adequately demonstrated that the assay was functioning 

properly. The only deviation was a weak positive response in one run in response to the negative 

control, corticosterone. Performance criteria were met for 17β-estradiol and corticosterone, and 

the deviations from expected performance criteria values for 17α-estradiol and 17αmethyltestosterone, indicate an increased sensitivity to weak agonists. The fold-induction was 

slightly lower than the required four-fold induction in the first run. 

Cyfluthrin was only able to reach a maximum of 3.2-6.5% PC when tested up to the highest 

concentration possible based on cytotoxicity. Because the RPCMax < PC10 in both assay runs, 

cyfluthrin was considered negative for estrogen receptor transcriptional activation in this test 

system. 

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350) 

The 21-day fish short-term reproduction assay (MRID 48670101) of cyfluthrin (94.1% purity) 

with fathead minnows (Pimephales promelas) was conducted under flow-through conditions. 

Adult fish (16 spawning groups; 2 males and 4 females per group; ca. 4.5 months of age) were 

exposed to cyfluthrin at nominal concentrations of 0 (negative control), 0.000108, 0.000325, and 

0.000976 mg a.i./L, which resulted in mean-measured concentrations of <0.0000066 (<LOQ, 

negative control), 0.000063, 0.000149, and 0.000421 mg a.i./L, respectively (59%, 46%, and 

43% of nominal, respectively). The test system was maintained at 24.5 to 25.6o

C and a pH of 7.6 

to 8.1. 

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Overall survival was 96% in the negative control and 92% in the low and mid treatment groups; 

high mortality was observed in the high treatment group, with overall survival of 29% (7 out of 

24 fish surviving; 50% survival in males and 19% survival in females). Effects on secondary sex 

characteristics and clinical signs (i.e., behavioral and other sublethal effects) were observed in all 

surviving fish at the high treatment level; effects included swimming at the surface, erratic 

behavior, loss of equilibrium, labored respiration, decreased food intake, and loss of typical 

reproductive behavior and color pattern. Body weight and length in males and females were not 

significantly different (p>0.05; Dunnett) at any treatment level. 

In the negative control, spawning frequency and fecundity were, respectively, at least every 4 

days and 22.5 eggs/female/day/replicate; fertilization success in the negative control was 98%. 

Fecundity was significantly reduced (p<0.05; Jonckheere-Terpstra) by 92% at the high treatment 

level compared to the negative control. Fertilization success in the treatment groups was not 

significantly different (p>0.05; Dunnett) from the negative control. Only two of the four 

exposure replicates in the high treatment group produced eggs; fertilization success for these 

replicates was 99%. 

No significant difference (p>0.05; Mann Whitney U) in gonado-somatic index (GSI) was 

observed in females; however, male GSI was significantly reduced (p<0.05; JonckheereTerpstra) by 46% in the high treatment group. Nuptial tubercle score was not significantly 

different (p>0.05; Dunnett) in males at any treatment level; no nuptial tubercles were present in 

females. Plasma vitellogenin (VTG) concentrations were not significantly different (p>0.05; 

Mann Whitney U) in males or females. Testosterone and 17β-estradiol concentrations were not 

determined. Treatment-related effects on gonadal histopathology were observed in male and 

female fish at the high treatment level. Histopathological effects included a narrowing in the 

range of testicular stage scores in the high treatment group in males (Stage 2.5 only), with an 

absence of stages that indicate currently active or recent spawning, and a similar narrowing in the 

range of ovarian stage scores in females (Stages 3 to 3.5). 

Hershberger Assay (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48615605) screening for androgenic activity, cyfluthrin (95.8% 

purity, batch # FHER904218) in corn oil was administered daily via oral gavage to 58-day old, 

castrated male Sprague Dawley rats (6/dose) at dose levels of 0 (vehicle), 10 or 20 mg/kg/day. 

An androgenic positive control group consisted of 6 castrated rats exposed to 0.4 mg/kg/day of 

testosterone propionate (TP) by subcutaneous (s.c.) injection. 

To screen for potential anti-androgenic activity, cyfluthrin (95.8% purity, batch # FHER904218) 

in corn oil was administered daily via oral gavage to 58-day old, castrated male Sprague Dawley 

rats (6/dose) at dose levels of 0 (vehicle), 5, 10 or 20 mg/kg/day in conjunction with a daily dose 

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of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The anti-androgenic positive 

control group consisted of 6 castrated rats exposed to 0.4 mg/kg/day TP and 3 mg/kg/day 

flutamide (FT). TP alone was used as the anti-androgenic negative control. 

For both components of the assay, body weights were determined daily. The animals were dosed 

for 10 consecutive days and terminated approximately 24 hours after the final dose 

administration. At necropsy, the five androgen-dependent tissues were collected and weighed. 

All animals survived until scheduled termination. Clinical signs of toxicity (piloerection or 

increased salivation) were observed in one animal treated with cyfluthrin at the highest 

concentration (20 mg/kg/day) during the androgen agonist assay, and increased salivation was 

observed in one animal treated with cyfluthrin (+TP) at 20 and 10 mg/kg/day in the anti-agonist 

assay. No clinical signs of toxicity were observed at 10 mg/kg/day cyfluthrin or at 5 mg/kg/day 

cyfluthrin with TP. 

In the androgen agonist assay, no adverse treatment-related effects on body weights or overall 

body weight gains were observed in either cyfluthrin treated group compared to controls. In the 

0.4 mg/kg/day TP group, overall body weight gain was increased (p<0.05) by 34% compared to 

the controls. Rats in the positive control (TP) group had accessory sex organ weight increases 

(p<0.01) as follows: 1002% in seminal vesicles; 867% in ventral prostate; 199% in levator anibulbocavernosus (LABC) muscle complex; 474% in Cowper’s glands; and 108% in glans penis. 

The maximum guideline-specified %CV was slightly exceeded for the glans penis %CV in the 

control group (22.5% vs. 22%), but all other %CV values were less than the maximum 

permissible values. 

In the anti-androgen assay, body weights and body weights gains for all cyfluthrin (+TP)-treated 

groups and the positive control (FT +TP) group were comparable to the control (TP only) 

throughout the duration of the study. Rats in the positive control (FT) group had accessory sex 

organ weight decreases (p<0.01) as follows: 77% in seminal vesicles; 70% in ventral prostate; 

48% in LABC; 63% in Cowper’s glands; and 29% in glans penis. All %CV values were less 

than the maximum permissible values. 

The dosing for this study (high-dose of 20 mg/kg/day) was considered adequate based on the 

clinical signs of toxicity observed. 

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

sensitive tissues. Cyfluthrin was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

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Female Pubertal Assay (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48615606), 15 Sprague Dawley [Crl:CD (SD)] rats/dose 

group were treated daily via oral gavage (dose volume 5 mL/kg) with cyfluthrin (95.8% a.i., 

Batch# FHER904218) in corn oil at doses of 0 (corn oil), 10, or 20 mg/kg/day from post-natal 

day (PND) 22 to 42. 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, total 

thyroxine (T4) and thyroid stimulating hormone (TSH) levels were determined using an enzymelinked immunosorbent assay (ELISA). Weights were recorded for liver, kidneys, urogenital 

organs, pituitary, thyroid, and adrenal glands and microscopic examinations of the ovaries, 

uterus, thyroid, and kidneys were performed. 

There were no effects of treatment on mortality, final body weights, body weight gains, mean 

age at first vaginal estrus, mean cycle length, percent cycling, percent regularly cycling, clinical 

chemistry parameters, or microscopic pathology. 

At 20 mg/kg/day, increased salivation was observed in 5 of the 15 rats. Additionally at this dose, 

the age at VO was delayed (p<0.01; 35.73 days treated vs. 33.00 days controls), the body weight 

at VO was increased (p<0.05) by 10%, and the mean age at first vaginal estrus was delayed (not 

significant; 36.36 days vs. 34.00 days) as a result of the delay in complete vaginal opening. 

The doses tested were adequate based on clinical signs of toxicity (increased salivation) at the 

high dose and the results of a dose range-finding study. 

Male Pubertal Assay (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48615606), 15 Sprague Dawley [Crl:CD (SD)] rats/dose group 

were treated daily via oral gavage (dose volume 5 mL/kg) with cyfluthrin (95.8% a.i.; Batch # 

FHER904218) in corn oil at doses of 0 (corn oil), 10, or 20 mg/kg/day from post-natal day 

(PND) 23 to 53. 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, total serum 

testosterone levels were determined using enzyme immunoassay (EIA), and thyroxine (T4) and 

thyroid stimulating hormone (TSH) levels were determined using enzyme-linked immunosorbent 

assay (ELISA). Weights were recorded for the liver, kidneys, urogenital organs, pituitary, 

thyroid, and adrenal glands, and microscopic examinations of the testes, epididymides, thyroid, 

and kidneys were performed. 

No treatment-related effects were observed on mortality, final body weights, body weight gains, 

age or weight at attainment of PPS, organ weights, hormone (serum T4, TSH, or testosterone) 

levels, or histopathology parameters at any dose. 

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In the 10 mg/kg/day dose group, increased salivation was observed in 1 of the 15 rats on Day 8. 

In the 20 mg/kg/day dose group, increased salivation, piloerection, lack of grooming, wasted 

appearance, uncoordinated movements, and/or tremors were observed in 6 of 15 animals on at 

least one occasion. At 20 mg/kg/day, urea levels were decreased (p<0.01) by 28%. This 

decrease was not considered a biologically adverse effect due to only 2 of 13 urea values in this 

group were lower than the lowest urea value in the control group. Serum TSH levels were 

decreased by 37% compared to controls, although the decrease was not statistically significant. 

The dosing levels utilized in the current study are considered appropriate based on clinical signs 

of toxicity (increased salivation and/or tremors) at the high dose and the results of a dose rangefinding study. 

Steroidogenesis Assay (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48615607), H295R cells cultured in vitro in 24-well plates 

were incubated with cyfluthrin (>95.8% purity, Batch # AE F057122-01-05) at logarithmic 

concentrations from 10−10 to 10−4

 M for 48 hours in triplicate for three independent experiments. 

Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.1%. 

Testosterone and estradiol levels were measured using specific EIA kits. 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. The positive controls included a known inhibitor (prochloraz) and a known inducer 

(forskolin) of estradiol and testosterone production. 

Guideline acceptability recommendations and requirements were generally met, including lack 

of cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low 

%CV), and appropriate induction and inhibition with positive controls. The exceptions included 

that testosterone was only induced by 1.8-fold rather than the Guideline requirement of ≥2-fold, 

and the between plate %CV for the SC in the test and QC plates was 33-34% for estradiol, which 

is above the Guideline recommendation (≤30%). 

Treatment with cyfluthrin at 10−9

 to 10−4 M had no significant effect on testosterone production, 

although there was a marginal reduction (0.70-fold) in testosterone observed at 10−4

 M. The 

only statistically significant change in testosterone production was a 0.65-fold change (p≤0.05) at 

the lowest concentration tested (10−10 M); however, this reduction was not concentration 

dependent. Cyfluthrin generally increased estradiol production (1.33- to 1.88-fold) across all 

three runs at concentrations of 10−10 to 10−5

 M. However, the increases in estradiol were not 

concentration dependent, and were highly variable with the increases attaining significance only 

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at 10−7 M (p≤0.05; 1.81-fold). A reduction in estradiol production (0.77-fold) was also observed 

at the highest concentration tested (10−4 M), but the change was not significant. 

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

statistically significant and reproducible alterations in testosterone or estradiol production. 

Uterotrophic Assay (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48615608) conducted to screen for potential estrogenic activity, 

cyfluthrin (95.8%% purity, batch #FHER904218) in corn oil was administered daily via oral 

gavage to groups of 6 immature female, Sprague-Dawley rats at dose levels of 0 (vehicle), 5, 10, 

or 20 mg/kg/day on post-natal days (PND) 19-21. A positive control group was treated with 

17α-ethynyl estradiol (EE) in corn oil by gavage at a dose level of 1 μg/kg/day. All animals 

were terminated and necropsied on PND 22 approximately 24 hours after the final dose to 

determine wet and blotted uterine weights. 

All animals survived until scheduled termination. Clinical signs of toxicity were observed in one 

animal treated with cyfluthrin at the highest concentration (20 mg/kg/day); no treatment related 

effects were observed at the lower dosing levels. No precocious vaginal opening was observed 

in treated females. Body weights and overall body weight gains (Days 1-4) in the cyfluthrin 

treated groups were comparable to the controls throughout the study. Uterine weights in the 

cyfluthrin treated groups were also comparable to the controls. Absolute wet and blotted uterus 

weights for the positive EE group were increased (p<0.01) by 195% and 201%, respectively, as 

expected. 

The high dose tested in this study (20 mg/kg/day) is adequate based on the results of the rangefinding study which showed mortality, severe clinical signs and reduced body weight gain at 40 

mg/kg/day. 

No statistically significant changes were seen in uterine weight in this assay. Cyfluthrin is 

negative in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Receptor Transcriptional Activation 

The purpose of this study was to screen and evaluate 200 pesticides, covering nine chemical 

classes, for their potential to interact with two human estrogen receptor subtypes (hERα and 

hERβ) and one human androgen receptor (hAR) in transactivation assays with Chinese hamster 

ovary cells (CHO-K1 cells) (Kojima et.al., 2004). Cyfluthrin was dissolved in 

dimethylsulfoxide to a final concentration of 10 mM and diluted to the desired concentrations in 

Dulbecco’s modified Eagle medium plus Ham’s F-12 nutrient mixture. The final solvent 

concentration in the culture medium did not exceed 0.1% and did not affect cell yields. The 

human estrogen receptor (hERα) and androgen receptor (hAR) expression vectors (pcDNAERα

and pZeoSV2AR, respectively) were used in the study. Estrogen or androgen responsive 

elements containing reporter plasmid (pGL3-tkERE and pIND-ARE, respectively) were used for 

the binding experiments. The pRL-SV40 containing the Renilla luciferase gene was used as an 

internal control for transfection efficiency. 

Cyfluthrin tested positive for both estrogenic and anti-androgenic activities with the in vitro

screening assays, indicating pleiotropic effects on hER and hAR via ERα and AR. Cyfluthrin 

was found to induce estrogenic activity in the ERα transactivation assay, with a REC20 of 5.9 x 

10-6 M, and relative luciferase activity (RLA) of 45% at 10-5 M (100% activity defined as the 

activity achieved with 10-10 M E2). Cyfluthrin also had weak anti-androgenic activity in the AR 

transcriptional assay, with a RIC20 of 8.4 x 10-6 M and RLA of 74% at 10-5 M (100% activity 

defined as the activity achieved with 10-10 M DHT). Cyfluthrin did not demonstrate androgenic 

transcriptional activity of more than 20% of that induced by 10-9 M DHT (cyfluthrin 

concentration tested was not reported). 

In a published study by Kojima et al. (2005), the response of an in vitro ER–related reporter 

gene assay was investigated after exposure to thirty two pesticides either alone or in 

combination. The source of the test compounds was not reported; however, cyfluthrin had a 

reported purity of >96%. The test compounds were prepared in DMSO at 10−2 M. 

The E-CALUX Assay (Xenobiotic Detection Systems, Inc., NC, USA) uses a human ovarian 

carcinoma cell line (BG1) transfected with a luciferase reporter gene under the control of a 

mouse mammary tumor virus (MMTV) promoter. Estrogen-responsive concatemers (repetitive 

DNA sequences), inserted directly above the promoter, confer estrogen responsiveness to the 

viral promoter and reporter gene. The transfected cells are made stable by an antibiotic resistance 

gene resulting in the cell line used in the assay (BG1Luc4E2). Compounds that interact with 

estrogen-responsive DNA are identified by the luciferase gene response and quantified in terms 

of Relative Light Units (RLU). 

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The BG1Luc4E2 cells were grown in medium for four days, then the assay medium was 

replaced, and cells were transferred (2 × 105 cells/mL) into 96-well plates and incubated at 37 ̊C 

with 5% CO2 for 24 hours. The medium was removed, and the test compound or estrogen 

standard (E2) was added. After incubation at 37 ̊C with 5% CO2 for another 24-hour period, the 

medium was removed and the cells were lysed. Luciferase activity was measured with a 

luminometer in order to compare treatment RLU to the E2 standard. For comparative purposes, 

the EC10 (Effective Concentration at which 10% of the maximum response was observed) was 

used. The data were not sufficient to allow calculation of an EC50. Although the article does not 

specifically identify the E2 standard, the reviewers assumed that E2 referred to 17β-estradiol. 

Cyfluthrin did not induce an estrogenic response in this assay system. 

In a non-guideline study by Du et al., 2010, nine pyrethroid insecticides, including cyfluthrin, 

were evaluated for estrogen, androgen, and thyroid hormone receptor activity (both as agonist 

and antagonist) using receptor-mediated luciferase reporter gene assays. Two cell lines (CV-1 

and MDA-kb2) were employed; both were stably transfected to express either the estrogen 

receptor α (ERα), androgen receptor (AR), or thyroid hormone receptor β (TRβ) genes. 

Cyfluthrin did not exhibit agonistic activity for estrogen, androgen, or thyroid hormone. The 

following antagonistic activities were presented for cyfluthrin: anti-estrogenic RIC20 = 1.36 x 

10-6 M; anti-androgenic RIC20 = 8.84 x 10-8 M; and thyroid hormone RIC20 = 5.39 x 10-7 M. 

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). 

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%. 

Cyfluthrin and β-cyfluthrin were each tested at concentrations ranging from 10−8

 to 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, cyfluthrin inhibited (p<0.05) DHT-induced transcriptional 

activation at concentrations from 10−7

 to 10−5 M. The positive control performed as expected 

within the same dose range. 

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Prenatal Development in Rodents (Rat; OSCPP 870.3700) 

In a developmental toxicity study (MRID 44116501, 1996), β-cyfluthrin (96.5-97.3% purity) 

was administered by gavage to 30 female Wistar rats/dose in aqueous 1% Cremophor solution at 

dose levels of 0, 3, 10, or 40 mg/kg/day from gestation days (GD) 6 through 15, inclusive. Dams 

were sacrificed on GD 20. 

Maternal toxicity was noted at 10 mg/kg/day by reduction in mean food consumption (10-15%) 

with recovery of appetite following treatment. Mean body weights and mean body weight gains 

were not significantly reduced, however, mean adjusted terminal weights were significantly 

reduced (14%) at 10 mg/kg/day. Clinical signs of toxicity were absent. Maternal toxicity at 40 

mg/kg/day included reduced survival (10%), reduced mean body weight gains (56%, Days 6-16 

and 16%, Days 0-20), reduced weight gain (32%, p≤0.01) corrected for gravid uterine weights, 

and reduced mean food consumption during treatment (20-49%). Clinical signs of toxicity at the 

high dose included hypoactivity, locomotor incoordination, and salivation. 

No treatment-related changes were seen in pregnancy rate, number of corpora lutea, implantation 

sites, the extent of resorptions /implantations, number of live fetuses/litter, fetal sex ratio, or soft 

tissue abnormalities at any dose. 

The maternal LOAEL was 10 mg/kg/day, based on reduced body weight gain and reduced food 

consumption with post-treatment recovery. The maternal NOAEL was 3 mg/kg/day. The 

developmental LOAEL was 40 mg/kg/day, based on reduced fetal body weights and increased 

skeletal variations. The developmental NOAEL was 10 mg/kg/day. 

Prenatal Development in Non-Rodents (Rabbit; OCSPP 870.3700) 

In a developmental study (MRID 42675401, 1992), cyfluthrin (96% purity) in corn oil was 

administered via gavage to pregnant female Chinchilla (CHbb: CH, Hybrids, SPF Quality) 

rabbits (25/dose) during GD 6-18 of gestation at dose levels of 0, 20, 60, or 180 mg/kg/day. 

There were no maternal deaths or clinical signs of toxicity during the study, and no treatmentrelated gross pathology was observed. All groups lost weight during the dosing period. The midand high dose groups lost a significantly (p≤0.01) greater amount of weight than the control 

group: a loss of 40 grams in the control group versus losses of 189 and 233 grams in the midand high-dose groups, respectively. Increases in body weight (NS) were observed in the two 

highest dose groups during the post-dosing period. At 180 mg/kg/day, food consumption was 

decreased during days 6-15 (↓41-48%, p≤0.01) and Days 15-19 (↓27%). At 60 mg/kg/day, food 

consumption was decreased during days 6-19 (↓24-29%). Food consumption in the mid- and 

high-dose groups also increased during the post-dosing period. 

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There were no treatment-related effects on the number of live litters, fetal weights, implantations, 

external, visceral or skeletal malformations and variations. Post-implantation loss (expressed as 

a percentage of the number of implantation sites) was increased at 60 mg/kg/day (p≤0.05) and 

180 mg/kg/day (p≤0.01). “Embryonic resorptions” (early resorptions, when expressed as a 

percentage of the number of implantation sites: 3.6, 6.3, 11.5 and 15.1for the 0, 20, 60 and 180 

mg/kg/day groups, respectively, were increased (p≤0.01) at 60 and 180 mg/kg/day. The total 

number of fetuses (also expressed as a percentage of the number of implantation sites) was 

significantly decreased at 60 and 180 mg/kg/day: (89.1, 89.1, 80.3 and 71.5 for the 0, 20, 60 and 

180 mg/kg/day groups, respectively). Upon examination of the data, it appears that the increases 

in resorptions over the control group are not likely to be toxicologically significant because the 

number of live pups/litter are comparable across the dose groups and although the control group 

has slightly more, there is no indication of a dose-response across a dose range of 1 order of 

magnitude. In addition, the individual animal data indicate that the does with a higher number of 

implantation sites generally have a higher number of resorptions. This is probably because the 

does cannot support a higher number of fetuses and thus, some are resorbed. The highest dose 

group had five does with 15-19 implantation sites. They each resorbed 4-9 embryos/fetuses. 

The control group had two does with 15 implantation sites. These 2 does resorbed 2 and 5 

embryos/fetuses, respectively. The mid-dose group had one doe with 18 implantation sites. This 

doe resorbed 7 embryos/fetuses. 

The maternal LOAEL was 60 mg/kg/day, based on decreased body weight gain and food 

consumption during the dosing period; the maternal NOAEL was 20 mg/kg/day. The 

developmental NOAEL was 180 mg/kg/day, and a developmental LOAEL was not observed. 

Inhalation Developmental Toxicity in Rodents (Rat; OCSPP 870.3600)

Two developmental toxicity studies via inhalation (MRIDs 40780401, 40968501 and 42332001, 

1988) were conducted. In the first study, 4 groups of 30 female Bor:WISW (SPF Cpb) rats were 

inseminated by being housed overnight with males. The presence of sperm in the vaginal smears 

following mating established GD 0. The dams were dynamically exposed head-only to 

cyfluthrin dissolved in a 1:1 mixture of Lutrol and ethanol at analytical concentrations of 0, 

0.0011, 0.0047 or 0.0237 mg/L/day for 6 hours/day on GD 6 through 15. A second study was 

conducted in order to establish a NOAEL for offspring toxicity. In that study, the dams were 

exposed to analytical concentrations of 0, 0.00009, 0.00025, 0.00059 or 0.0042 mg/L of the test 

material. The MMAD was 1.23 - 1.45 μm in the first study and 1.29 - 1.53 μm in the second 

study. An oxygen enriched atmosphere (30%) was provided for the 0.0042 mg/L group to see if 

the embryotoxic effects seen in the first study at this concentration could be lessened. The rats 

were observed several times on the exposure days except during the exposures (because of 

restraint for head-only exposure). They were weighed on GDs 0, 6, 9, 12 and 20. The dams 

were sacrificed on day 20 and their pups removed by caesarian section. Their ovaries and uteri 

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were examined for implantations, live young, embryonic and fetal deaths, fetal sex and weights, 

and external fetal abnormalities. 

Combining the results of the two studies, maternal effects were observed at 0.0047 mg/L and 

above, and clinical signs included reduced motility, dyspnea, piloerection, ungroomed coats and 

eye irritation. The symptoms were mainly observed after the end of exposure in the 0.0047 mg/L 

concentration group and were largely gone by the next exposure day. At 0.0237 mg/L 

concentration, the symptoms were observed at greater intensity over the entire exposure period. 

No treatment-related changes were seen in pregnancy rate, implantation sites, the mean number 

of resorptions /implantations, number of live fetuses/litter, fetal sex ratio, or soft tissue 

abnormalities at doses of ≤0.0047 mg/L. However, effects were observed on the pups at ≥0.0011 

mg/L. At 0.0011 mg/L and above, a dose-related increase in the incidence of runts and skeletal 

anomalies in the sternum were observed. At 0.0047 mg/L and above, biologically significant 

decreases in pup weights were observed. At 0.0237 mg/L, increases in post-implantation loss, 

late embryonic deaths and in skeletal anomalies in the extremities were observed as well as 

microphthalmia. 

The maternal NOAEL was 0.0011 mg/L and the maternal LOAEL was 0.0047 mg/L (reduced 

motility, dyspnea, piloerection, ungroomed coats and eye irritation). The developmental 

NOAEL was 0.00059 mg/L and the developmental LOAEL was 0.0011 mg/L (increases in the 

incidence of runts and skeletal anomalies in the sternum). 

An ad hoc committee met on 4/22/93 to discuss the developmental toxicity data base for 

cyfluthrin. At that time, the Committee recommended that due to deficiencies that were 

mentioned in the review of the study, this study should be re-examined if it is to be used as a 

regulatory endpoint. Although the study had been graded Acceptable and NOAELs and 

LOAELs had been established for maternal and developmental toxicity, comments had also been 

made that developmental anomalies in the study had not been adequately reported. The dams in 

this study had reflex apnea, although it was poorly characterized. 

Inhalation Developmental Toxicity in Rodents (Rat; OCSPP 870.3700)

Cyfluthrin (96.2% purity) (MRID 43393401, 1994) was administered by inhalation to 25 

pregnant female Wistar rats/group at the following analytically determined concentrations: air 

control, vehicle control (polyethylene glycol-400: 50% ethanol), 0.00046, 0.00255, 0.0119 or 

0.0128 mg/L plus 39% oxygen for GD 6 through 15 in a dynamic nose-only inhalation chamber. 

The rats were exposed to the test material 6 hours/day, 7 days/week. The MMAD ± geometric 

standard deviation was 1.1 ± 1.5 μm for all the groups. Additional satellite rats (5/group) were 

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exposed similarly from GD 6 through day 13 to determine the effect of cyfluthrin exposure on 

body temperature, ventilation rate and plasma cyfluthrin levels. 

 

Maternal body weight was statistically significantly decreased at ≥ 0.00046 mg/L for the interval 

GD 6 to 15 (≥ 26% from the vehicle control) and GD 0 to 20 (≥ 16% from the vehicle control). 

The relative efficiency of food utilization appeared to be decreased at ≥ 0.00046 mg/L (≥ 9% of 

the vehicle control). Increases in untidy and ruffled fur and retarded breathing were observed at 

0.0119 and 0.0128 (with O2) mg/L. Labored and irregular breathing, hypoactivity, high-stepping 

gait, salivation and reduced stool were observed only at 0.0119 mg/L. The satellite animals 

showed a decreased (but relatively flat dose-response) breathing rate/minute in the treated 

animals versus the air and vehicle control groups. Minimum volume (ml/min, ml/min/kg and as 

% of vehicle control) were decreased in a dose-related manner (bradypnea). Rectal temperatures 

were slightly decreased in the treated animals. Placental weights (≥ 7% from the vehicle control) 

and fetal weights (≥11% from the vehicle control) were decreased at ≥ 0.00255 mg/L. 

Developmental toxicity was also expressed in the form of retarded skeletal ossification in the 

phalanx, metacarpals, cervical vertebrae, sacral and caudal arches at ≥ 0.00255 mg/L. Doserelated total fetal malformations such as microphthalmia and skeletal dysplasia were evident at 

0.0119 mg/L (8.8% in fetuses/43% in letters vs. vehicle controls with 1.0% in fetuses/14% in 

litters). 

The evidence suggests that the cyfluthrin exposure at 0.0119 and 0.0128 mg/L caused the 

developmental toxicity partly through the bradypnea in dams. While the bradypnea in dams at 

0.00255 mg/L may have caused the reduced fetal and placental weight and retarded ossification, 

the data presented were insufficient to draw this conclusion. 

The maternal LOAEL is 0.00046 mg/L based on decreased body weight gain and reduced 

relative food efficiency. The NOAEL is not determined. The developmental NOAEL is 0.00046 

mg/L and the developmental LOAEL is 0.00255 mg/L based on reduced fetal and placental 

weights and reduced ossification in the phalanx, metacarpals and vertebrae. 

Developmental Neurotoxicity (Rat; OCSPP 870.6300)

In a developmental neurotoxicity study (MRID 46054101, 2003), β-Cyfluthrin (95.1-97.6% a.i.) 

was administered to 30 parent female Wistar rats/dose in the diet at nominal concentrations of 0, 

30, 125, or 200 ppm from GD 0 through postnatal day 21. The average daily intake of βCyfluthrin was 0, 2.4, 11.0, and 17.8 mg/kg/day during gestation and 0, 5.9, 25.4, and 40.9 

mg/kg/day during lactation for the 0, 30, 125, and 200 ppm groups, respectively. Pup physical 

development was assessed by body weight, and sexual maturation of females was assessed by 

age at VO. Maturation of males was assessed by age at completion of PPS. 

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In dams, no treatment-related effects were seen on mortality, clinical signs, body weight, body 

weight gain, or reproductive performances (mating, fertility, gestation, live birth, viability or 

lactation indices). The maternal NOAEL was 17.8 mg/kg/day; a maternal LOAEL was not 

established. 

In offspring, treatment had no adverse effects on survival, clinical signs, birth weight, 

developmental landmarks, FOB parameters, motor or locomotor activity, auditory startle reflex, 

learning and memory, brain morphology or neuropathology. Body weight and body weight gain 

were comparable across all dose groups at birth and on PND 4. However, pup body weight was 

decreased by 8-11 % in the high-dose group (17.8 mg/kg/day) on subsequent days through 

weaning. Weight gain of high-dose pups was decreased 8.5%-13% from PND 4-21. After 

weaning, pups received only untreated diet, and the only compound-related effects were noted in 

high-dose males and females and were associated with the decreased body weight that developed 

during lactation. High-dose males weighed 10% less than controls during the first week postweaning and 7% less during the last week of the study. High-dose females weighed 7.5% less 

than controls during the first week post-weaning and had weight comparable to controls by the 

last two weeks of the study. At termination, absolute brain weight was decreased 5.8% (p≤0.05) 

in high-dose females compared to controls. Acquisition of VO and PPS in F1 offspring were 

unaffected by treatment even in the presence of up to 10% decrease in body weight and body 

weight gain. 

The offspring LOAEL was 17.8 mg/kg/day based on decreased body weight and body weight 

gain and decreased brain weights in females at termination. The offspring NOAEL was 11.0 

mg/kg/day. 

Multi-Generation Reproduction in Rodents (Rat; OCSPP 870.3800) 

In a 2-generation reproduction study (MRID 44371401, 1996) cyfluthrin (95.4% purity) was 

administered in the diet to 30 Sprague Dawley rats/sex/dose at dose levels of 0, 50, 125, or 400 

ppm (equivalent to doses of 0, 3/4, 9/10, or 29/33 (M/F) mg/kg/day during premating and 

gestation and 0, 7, 19, or 59 mg/kg/day during the first two weeks of lactation). Exposure to P 

animals (30/sex/dose) began at 7 weeks of age and lasted for 10 weeks prior to mating to 

produce F1 pups. Upon weaning, F1 pups (30/sex/dose) selected to become parents of the F2 

generation were fed cyfluthrin in test diets at the same concentration their dam received. F1 

animals were given test diets for 11 weeks prior to mating. All animals were mated on a 1:1 

ratio. 

Parental toxicity of cyfluthrin was evident at 125 and 400 ppm. At 125 ppm, marginal treatmentrelated reductions in mean body weight (↓6-8%) were observed during the premating period and 

terminal body weights were similarly reduced (↓6%) in F1 males. During lactation there was a 

treatment related decrease in food consumption (↓9-11%, p<0.05 or 0.01) for F1 females at this 

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dose. At 400 ppm, treatment-related splaying of hind limbs was observed in P and F1 females 

during lactation (15/30 P females, 9/30 F1 females vs. 0/30 in both P and F1 controls). 

Treatment-related reductions in body weights (↓7-14%) and food consumption (↓11-22%) 

compared to lactating controls were also noted in lactating females of both generations. 

Marginal treatment-related decreases in mean body weights (↓7-8%) were observed in F1 males 

during the premating period. The reduction of terminal body weights (↓6-8%) in F1 males and 

females was also considered treatment-related. Food consumption for all dams (treated and 

controls) was markedly increased during lactation when compared to the premating and gestation 

periods. Although a decrease in food consumption was observed in the mid- and high-dose 

groups during lactation, this was in comparison to the control group. Food consumption in the 

treated groups was still increased in comparison to food consumption in these same groups 

during the premating and gestation periods. As a result, cyfluthrin intake by dams was 

approximately doubled during lactation compared to premating and gestation periods. 

Therefore, since most of the treatment-related effects of cyfluthrin in females (decreases in food 

consumption and body weights and splayed hind limbs) were observed during this period, they 

were likely associated with the increase in dose during lactation. In contrast, during the 

premating period most of the observed parental treatment related changes consisted of marginal 

reductions in body weights. There were no treatment related mortalities or treatment-related 

effects on reproductive parameters or function. 

No treatment-related effects were observed in the reproductive performance (male or female 

fertility indices, gestation index, gestation length, live birth index, viability index, lactation 

index, parturition, or fetal sex ratio) at any dose. No changes were seen in absolute or relative 

testicular weights, but absolute ovarian weights were significantly decreased (↓25%) in the F0 

animals. However, this isolated finding was not attributed to treatment due to lack of a dose 

response, no corresponding decrease in relative weight, no corroborative ovarian lesions, and the 

change was not seen across generations in this study nor in the follow-up study. No treatmentrelated histopathological changes were seen in the testes, epididymides, seminal vesicle, prostate, 

coagulating gland, ovaries, uterus, vagina, cervix or the pituitary glands at any dose in either 

generation. 

The LOAEL for parental toxicity was 125 ppm (9/10 mg/kg/day, M/F) based on reductions in 

body weight and food consumption. The parental NOAEL was 50 ppm (3/4 mg/kg/day, M/F). 

Offspring toxicity of cyfluthrin was manifested at 125 and 400 ppm. At 125 ppm, treatment 

related coarse tremors were observed in 16% of the F1 pups and 73% of the F2 pups during 

lactation days 5-17. Treatment-related decreases (decreased 6-14%) in mean body weight were 

observed between 4-21 days in F1 and F2 pups. At 400 ppm, treatment-related coarse tremors 

were observed in F1 and F2 pups between days 5 and 17 of lactation. These tremors were 

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observed in 54% of the F1 pups and 36% of the F2 pups. A treatment-related reduction 

(decreased 6-26%) in mean litter weights was observed in F1 and F2 pups between days 0 and 21 

of lactation. The offspring LOAEL was 125 ppm (19 mg/kg/day) based on coarse tremors 

during lactation and decreases in mean litter weights. The offspring NOAEL was 50 ppm (7 

mg/kg/day). 

Multi-Generation Reproduction in Rodents (Rat; OCSPP 870.3800) 

In a supplemental 2-generation reproduction study (MRID 44371402, 1997) cyfluthrin (95.5% 

purity) was administered to 30 Sprague Dawley rats/sex/dose at dose levels of 0, 25, or 50 ppm 

in the diet (equivalent to doses of 0, 1.9 or 3.8 mg/kg/day in males; 0, 2.1, or 4.2 mg/kg/day in 

females during premating; and 0, 2.0 or 3.9 mg/kg/day during gestation in females). Exposure to 

P animals (30/sex/dose) began at 7 weeks of age and lasted for 10 weeks prior to mating to 

produce F1 pups. Upon weaning, F1 pups (30/sex/dose) selected to become parents of the F2 

generation were fed cyfluthrin in test diets at the same concentration their dam received. The F1 

animals were given test diets for 11 weeks prior to mating. All animals were mated on a 1:1 

ratio. 

The objectives of the present study were to determine the reproducibility of the effect on pup 

weight and to verify and/or establish a reproductive NOAEL for cyfluthrin at 50 ppm. There 

was no evidence of parental toxicity of cyfluthrin at 25 or 50 ppm. There were no treatmentrelated clinical findings or increases in mortality. There were no significant differences in body 

weights between the controls, treated P males, P females, F1 males, or F1 females during the 

premating interval. There were no treatment-related changes in food consumption in males or 

females. During gestation and lactation there were no treatment-related effects on body weights, 

body weight gains, or food consumption of dams. No treatment-related effects were observed in 

the reproductive performance (male or female fertility indices, gestation index, gestation length, 

live birth index, viability index, lactation index, parturition, or fetal sex ratio) at any dose tested. 

No changes were seen in absolute or relative ovarian weights in either generation. The absolute 

and relative testicular weights were significantly (p<0.05) decreased (↓6-8%) in the F1 males, 

but this decrease was not considered to be biologically significant due to lack of a dose response, 

and no corresponding decrease in relative testes weight. In addition, the change was not seen 

across generations either in this study or in the previous study (discussed above). No treatmentrelated histopathological changes were seen in the testes, epididymides, seminal vesicle, prostate, 

coagulating gland, ovaries, uterus, vagina, cervix or the pituitary glands at any dose in either 

generation. 

The LOAEL for parental toxicity was not observed. The parental NOAEL is 50 ppm (3.8/4.2 

mg/kg/day). The reduction in pup weights observed at 50 ppm in the previous two-generation 

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reproductive study (MRID 44371401) could not be reproduced in this study. The LOAEL for 

offspring toxicity was not observed. The offspring NOAEL is 50 ppm (3.8/4.2 mg/kg/day). 

Hershberger Assay Rat 

In a study examining the potential anti-androgenic effect of several pyrethroid pesticides (Zhang 

et al., 2008), cyfluthrin or β-cyfluthrin (purities not reported; cis/trans ratio is not stated) were 

assessed in an in vivo Hershberger assay. 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 anti-androgenic Hershberger 

assay included ten treatment groups: a sham control (uncastrated); a vehicle control group 

(peanut oil only); a control group dosed with peanut oil (orally) and testosterone propionate (TP) 

by s.c. injection at 0.5 mg/kg/day ; a positive control group dosed orally with flutamide (50 

mg/kg/day) plus TP; three groups dosed orally cyfluthrin at 16, 18 or 54 mg/kg/day plus TP; and 

three groups dosed orally with β-cyfluthrin at 4, 12 or 36 mg/kg/day plus TP. All groups were 

dosed for seven days. After the final dose, rats were anaesthetized and sacrificed by 

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

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

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

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

fluorescence with commercially available FIA kits. 

No mortality or treatment-related clinical signs were noted in any of the groups in the 

Hershberger assay. There were no significant differences between initial or final body weights, 

or weight gains of the cyfluthrin-treated groups compared to the control groups. Serum 

testosterone concentrations were not shown for the cyfluthrin-treated rats, but it was reported that 

no treatment-related effects were observed. Compared with the control group (TP plus peanut 

oil), there was no treatment-related effect on weights of liver, kidney and spleen in the flutamide 

and cyfluthrin groups. Pairs of adrenal glands weights of the 36 mg/kg/day β-cyfluthrin plus TP 

group were significantly higher than those of peanut oil plus TP control group 

As expected, dosing castrated rats with TP increased the accessory sex organ weights to normal 

levels compared to hormone deficient peanut oil control group, and flutamide, a known antiandrogen, significantly (p<0.05) inhibited the TP-induced growth of accessory sex organs. Both 

cyfluthrin and β-cyfluthrin demonstrated anti-androgenic activity. With the exception of the 

glans penis, treatment with either cyfluthrin or β-cyfluthrin (in combination with TP), resulted in 

dose-dependent decreases in the weights the seminal vesicle, ventral prostate, dorsolateral 

prostate, LABC, and Cowper’s glands, when compared to the TP+peanut oil control, These 

weight decreases attained significant (p<0.05) at both the mid- and high-dose for each chemical. 

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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 cyfluthrin and β-cyfluthrin (no androgenic portion), and the castrated rats 

were dosed with TP at 0.5 mg/kg instead of 0.2-0.4 mg/kg/day. However, because the control 

group (castrated + TP) exhibited similar growth of accessory sex tissues to the sham-operated 

group (uncastrated), the 0.5 mg/kg dose of TP was acceptable. 

Subchronic Oral Toxicity in Rodents (Rat; OCSPP 870.3050) 

In an subchronic oral toxicity study (MRID 41244108, 1986), Wistar (BOR:WJSW strain) rats 

(15/sex/group) were treated with beta-cyfluthrin (99.7% purity) in the diet at 0. 30, 125 or 500 

ppm (0. 2.3/2.5. 9.5/10.9 or 38.9/42.4 mg/kg/day, males/females. respectively). In addition. 

15/sex were treated with beta-cyfluthrin in the diet at 0 or 500 ppm (0 or 37.0/43.0 mg/kg/day, 

males/females. respectively) and then allowed to recover on control diet for an additional period 

of 4 weeks The dietary mixtures were prepared weekly and based on stability analyses were 

considered to be stable. 

Three deaths at 30 ppm and 1 death at 125 ppm were observed and were believed to have been 

due to improper blood collection techniques. At 500 ppm, 2 deaths occurred, 1 each in the main 

study and in the recovery study. The cause of death at this dose level was not determined and 

may have been due to the test material. Lack of coordination and poor general condition were 

also observed at this dose level during the first week of treatment (not reported by day). 

Necrosis in the head and neck regions was seen in 6 males and l female. Two males also had a 

"sore". In males, the mean body weight gain was lower throughout the study (weekly range of 8-

80% of the control value). In females, the mean body weight gain was lower during weeks l, 6 

and 10 (range of 16-67% of the control value). In addition, water consumption was reduced by 

19% when compared to the control groups. No treatment-related changes were seen in the

absolute or relative weights of the liver, testes or adrenal glands, and no treatment-related 

histopathological lesions were seen in the testes, uterus, mammary, thyroid, adrenal, or pituitary 

glands.

The NOAEL for the 3-month rat feeding study was 125 ppm (9.5/10.9 mg/kg/day for males/ 

females, respectively). The LOAEL was 500 ppm (37.0/43.0 mg/kg/day for males/females, 

respectively) based on lack of coordination, poor general condition, decreased body weight gain 

and water consumption, necrosis and sores in the head and neck regions and mortality. 

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Subchronic Oral Toxicity in Non-Rodents (Dog; OCSPP 870.3050) 

In subchronic oral toxicity study (MRID 41267801, 1987), beta-cyfluthrin (99% purity) was 

administered to beagle dogs (4/sex/dose) at dose levels of 0, 10, 60, or 360 ppm in the diet for 13 

weeks; actual achieved doses were 0, 0.39/0.39, 2.36/2.5 or 13.9/15.4 mg/kg/day [M/F], 

respectively. The following observations/measurements were conducted: clinical signs, body 

weights, food consumption, ophthalmological examinations, body temperature, femoral pulse, 

neurological examinations, hematology, clinical chemistry, urinalysis, organ weights, gross 

examinations and microscopic examinations. 

No treatment-related effects were observed at the low and mid-doses. At the high dose, the 

following clinical signs were noted: awkward, staggering gait and occasional buckling of the hind 

limbs (both sexes, 4/8 dogs) and an increase in incidence of vomiting (both sexes). In 3/8 dogs, 

the gait abnormalities began 6-8 hours after feeding during Week 1 (not reported in terms of days; 

in the fourth dog, the gait abnormalities did not begin until after week 1). In addition, a suggestive 

decrease in body weight gain was observed in high-dose females (0.35 kg versus 1.0 kg in 

controls). No treatment-related changes were seen in absolute or relative weights of the testes, 

prostate, ovaries, thyroid or adrenal glands. No treatment-related histopathological lesions were 

seen in the testes, epididymides, seminal vesicle, prostate ovaries, uterus, vagina, thyroid, adrenal 

or pituitary glands.

The NOAEL is 2.36/2.5 mg/kg/day for males/females, respectively. The LOAEL is 13.9/15.4 

mg/kg/day for males/females, respectively, based on gait abnormalities, increased incidence of 

vomiting, and suggestive decreased body weight gain. 

4-Week Inhalation Toxicity in Rodents (Rat; Non-guideline)

In a subchronic inhalation toxicity study (MRID 41783001, 1989), Bor:WISW (SPF-Cpb) rats 

(10/sex/concentration) were dynamically exposed by head/nose only inhalation to β-cyfluthrin 

(97.9% purity) in ethanol/polyethylene glycol 400 (1: 1) at concentrations of 0 (air control, 0 

(vehicle control), 0.00026, 0.0027 or 0.023 mg/L for 6 hours/day, 5 days/week for 4 weeks 

(equivalent to 0, 0.07, 0.73 or 6.3 mg/kg/day). 

At 0.0002 mg/L, no treatment-related effects were observed. Although the decrease in mean 

body weight gain at this dose level appears to be biologically significant (↓67-80%) compared to 

the vehicle control group, it was not considered to be toxicologically significant, as mean body 

weights were 96% of the vehicle control values. At 0.0027 mg/L, statistically significant 

decreases in body weight were observed in both sexes (p≤0.01, ≤10%) as well as decreased 

urinary pH in males. In addition, there was a decrease in mean respiratory rate in males. At the 

highest concentration tested (0.023 mg/L), additional observations include clinical signs in both 

sexes (10/10/sex: ungroomed fur and piloerection; increased activity with slightly reduced 

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motility at all times); decrease in absolute and relative spleen and thymus weights in both sexes 

(p≤0.01); increase in alkaline phosphatase (p≤0.01, 21% in females); decrease in cholesterol 

(p≤0.01, females) and slight decreases in protein, potassium, calcium, phosphorus and 

lymphocytes in females (p≤0.01 for all). 

With regard to specific endocrine endpoints, no treatment-related changes were seen in absolute 

or relative weights of the testes, ovaries, thyroid or adrenal glands. In addition, no treatmentrelated histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, 

ductus deferens, ovaries, uterus, mammary, thyroid, adrenal or pituitary glands.

The LOAEL is 0.0027 mg/L (0.73 mg/kg/day), based on decreases in body weight in both sexes 

and decreased urinary pH in males. The NOAEL is 0.00026 mg/L (0.07 mg/kg/day). 

90-Day Inhalation Toxicity (Rat; OCSPP 870.3465)

In a subchronic inhalation toxicity study (MRIDs 00157793, 40082901, 40239301; 1983-1987), 

Bor:WISW (SPF-Cpb) rats (10/sex/dose) were dynamically exposed by head-only inhalation to 

cyfluthrin (94.9% purity) in ethanol/polyethylene glycol 400 (1:1) at concentrations of 0 (air 

control), 0 (vehicle control), 0.00009, 0.00071, or 0.00451 mg/L for 6 hours/day, 5 consecutive 

days/week for 13 weeks (estimated to be equivalent to 0, 0.02, 0.16 or 0.91 mg/kg/day). 

All animals survived the 13-week study, and no treatment-related changes were observed in 

organ weight, gross pathology, and histopathology. At 0.00009 mg/L, total decreases in mean 

body weight (6%, p<0.05) and mean body weight gain (21%) were observed in males by study 

termination, when compared to the control group. Because there only appeared to be a slight 

effect on body weights in males and there were no other effects at this dose level, the apparent 

decrease in body weight gain is not considered to be toxicologically significant. At 0.00071 

mg/L, total decreases in mean body weight (8%, p<0.01) and mean body weight gain (21%) were 

observed in males by study termination when compared to the control group. Ten of 10 females 

were observed to display unthriftiness/unkempt fur and lethargy following exposure on Days 42 

to 86. These signs were not present on days where no exposure took place. In addition, 

increased urinary protein was noted in mid- and high-dose males at 6 and 12 weeks. At 0.00451 

mg/L, 10/10 of both sexes were observed to display the same clinical signs as the mid-dose 

group on Days 13-88 for males and 9-86 for females. Agitation and “erect tails” were also 

observed in these animals, starting at Week 5. On days when no exposure took place, these signs 

were still present, although only slightly. Mean body weights in high-dose males were decreased 

by 14% at study termination and mean body weight gain (weeks 0-13) was decreased by 46% 

when compared to the control group. Decreased urinary pH was also noted; however, no 

corresponding lesions were found to verify any possible nephritic damage. 

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With regard to specific endocrine endpoints, no treatment-related changes were seen in absolute 

or relative weights of the testes, ovaries, thyroid or adrenal glands. In addition, no treatmentrelated histopathological lesions were seen in the testes, ovaries, thyroid or adrenal glands.

The LOAEL is 0.00071 mg/L (0.16 mg/kg/day), based on decreases in body weight and body 

weight gain in males and clinical signs in females. The NOAEL is 0.00009 mg/L (0.02 

mg/kg/day). 

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

In a chronic/carcinogenicity study (MRID 00137303, 1983), cyfluthrin (49.7-51.0% purity in a 

premix concentrate in Wessalon S) was administered for 24 months in the diet to Wistar SPF rats 

(65/sex/dose) at dose levels of 0, 50, 150 or 450 ppm (equivalent to 0, 2.02, 6.19 or 19.20 

mg/kg/day in males and 0, 2.71, 8.15 or 25.47 mg/kg/day in females). 

There were no treatment-related deaths and no treatment-related changes noted in the clinical 

observations, food consumption, hematology, urinalysis, and the gross or microscopic data. No 

ophthalmologic examinations were performed. Mean body weights of the high-dose males and 

females were lower (↓5-10%) than the controls throughout the study. Mean body weights of the 

mid-dose males were also lower than the controls during the first year of the study (↓4-5%), with 

a body weight gain 6% less than controls), but the animals recovered thereafter. 

No treatment-related changes were seen in absolute or relative weights of the testes, ovaries or 

adrenal glands at any dose level, and no treatment-related histopathological lesions were seen in 

the testes, seminal vesicle, prostate, ovaries, uterus, thyroid, adrenal or pituitary glands at any 

dose level. Significant (p<0.05) increases were observed in the incidences of inflammatory foci 

of the kidneys in the mid-dose females (7/50 treated versus 1/50 controls) and the high-dose 

females (7/49 treated) and in hyperplastic nodules of the adrenals in the high-dose animals 

(males: treated = 20/50, control = 10/48; females: treated = 18/49, control = 4/50). Medullary 

hyperplasia was also observed in high-dose males (14/50 treated vs 4/48 controls). In addition, 

compared to concurrent controls, the following were observed: (i) increased (75%; p<0.01) liver 

N-demethylase activity in the high-dose females after 7 days of dosing; and (ii) increased 

fluoride levels in the bones of the mid- and high-dose males (11-21%; p<0.05) and the high-dose 

females (19%; p<0.01), as well as in the teeth of the high-dose males (20%; p<0.01). 

The chronic LOAEL was 450 ppm (equivalent to 19.20 mg/kg/day in males and 25.47 

mg/kg/day in females) based on decreased body weights (up to 10%) in both sexes. The chronic 

NOAEL was 150 ppm (equivalent to 6.19 mg/kg/day in males and 8.15 mg/kg/day in females). 

There was no evidence of carcinogenicity in the study. 

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

In another combined chronic toxicity/carcinogenicity study (MRID 44459301, 1997), cyfluthrin

(94.7% purity), was administered to separate 1- and 2-yr sacrifice groups of Fischer 344 rats. 

The 1-year sacrifice group consisted of 40 animals (20 males and 20 females) in both the control 

and high dose groups and 20 animals (10 males and 10 females) in both the low and intermediate 

dose levels for a total of 120 animals. The two-year sacrifice group consisted of 100 animals 

(50males and 50 females) in all 4 dose groups for a total of 400 animals. Nominal dietary 

concentrations of 0, 50, 225, 450 ppm corresponded to mean daily cyfluthrin intakes of 

approximately 0, 2.6, 11.6, 22.8 mg/kg/day for males and 0, 3.3, 14.4, 28.3 mg/kg/day for 

females. 

At 225 ppm, mean body weights were reduced by ≤7% in both sexes and mean body weight 

gains were decreased by ≤12% and ≤10% for males and females, respectively, compared to the 

control group. At 450 ppm, mean body weights were decreased by ≤13% and≤14% and mean 

body weight gains were decreased by ≤23% and ≤24% for males and females, respectively. An 

increased frequency of alopecia was also observed in 450-ppm males and females (M: 17, 16, 17, 

30; F: 30, 35, 34, 43). There were treatment-related effects on mortality, food consumption, 

hematology, urinalysis, or gross pathology. In addition, there were no treatment-related changes 

in absolute or relative testes and ovary weights. No treatment-related histopathological lesions 

were seen in the testes, epididymides, seminal vesicle, prostate, preputial gland, ovaries, uterus, 

vagina, cervix, clitoral gland, thyroid, adrenal or pituitary glands at any dose level. There was a 

statistically significant increase in the incidences of mammary gland adenocarcinomas in females 

at the high dose (4/50; 8%) compared to controls (1150; 2%). However, with the exception of 

one animal that died on Study Day 668 with a fibroadenoma, all the tumors were seen in animals 

that were sacrificed at the end of the study.

The LOAEL was 225 ppm (11.6 and 14.4 mg/kg/day for male and female rats, respectively), 

based on decreases in body weight gain of 12% and 10% in males and females, respectively. 

The NOAEL was 50 ppm (2.6 and 3.3 mg/kg/day [M/F]). 

Carcinogenicity in Rodents (Mouse; OCSPP 870.4200) 

In a carcinogenicity study (MRID 00137304, 1983), cyfluthrin (49.7-51.0% purity as a premix 

concentrate in Wessalon S) was administered in the diet for 23 months to SPF mice 

(50/sex/dose) at dose levels of 0, 50,200 or 800 ppm (equivalent to 0, 11.6, 45.8 or 194.5 

mg/kg/day in males and 0, 15.3, 63.0 or 259.9 mg/kg/day in females). 

There were no treatment related changes noted in the clinical observations, food consumption, 

hematology, gross observations, organ weights and microscopic pathology data. No 

ophthalmologic examinations or urinalyses were performed. The mortality data were equivocal 

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with respect to treatment. The incidence of mortality in the mid- and high-dose females was 

somewhat higher than the controls throughout the study. However, only the mid-dose at 

termination was significantly (p≤0.05) different from controls. At study termination, mortality in 

the females was 52, 60, 74 and 68% for the control, low-, mid- and high-dose groups, 

respectively. Mortality in the males was 80, 78, 82, and 88% for the control, low, mid- and highdose groups, respectively at study termination. Mean body weights relative to controls were no 

more than 10% less for mid- and high-dose males and 9% less for the mid- and high-dose 

females throughout the study. The mean body weights of the treated groups, however, were not 

significantly different at termination, possibly due to the mortality differences in the dosed 

groups compared to the concurrent controls. 

In the clinical chemistry data, alkaline phosphatase activity was elevated in treated males at all 

dose levels when compared to controls at each time point except termination where there was 

evidence that the samples were hemolyzed, leading to spurious results. A dose-response was 

evident at months 6 (143-230%; p≤0.05 or 0.01) and 12 (137-73%; p≤0.05 or 0.01); however, it 

was somewhat flat for the low- and mid-dose groups at Month 12. At Month 18, there was no 

dose-response. The low-dose group had the highest elevation in alkaline phosphatase ( 1 114%, 

p≤0.05) while the mid- and high-dose groups remained somewhat flat (161 and 68%, 

respectively (p<0.05 and 0.01: the odd significance values indicate some wide-ranging individual 

values in at least one dose group). The histopathology data did not confirm the liver as a target 

organ. Under the conditions of this study, there was no evidence of carcinogenic potential. 

With regards to specific endocrine endpoints, there were no treatment-related changes in 

absolute or relative weights of the testes or ovaries at any dose level, and no treatment-related 

histopathological lesions were seen in the testes, seminal vesicle, prostate, ovaries, uterus, 

mammary glands, thyroid, adrenal or pituitary glands at any dose level. 

Carcinogenicity in Rodents (Mouse; OCSPP 870.4200) 

In a mouse oncogenicity study (MRID 44589701, 1998), cyfluthrin (≥ 93.9% purity) was 

administered in the diet to CD-1 mice (50/sex/group) for up to 80 weeks at 0, 200,750, or 

1400/1600 ppm (equivalent to 0/0, 31.9/38.4, 114.8/140.6, and 232.7/309.7 mg/kg/day [M/F], 

respectively). A supplementary submission was made (MRID 45228101, 2000) containing the 

neoplastic incidence summary. The high-doses were chosen based on the results of a 6-week 

range-finding study, and the selected doses include the estimated maximum tolerated doses for 

each sex for a lifetime study in the mouse. Mortality, food efficiency, hematology, and organ 

weights for both sexes at all doses were unaffected by treatment with cyfluthrin. There were no 

observations of toxicological concern in either sex at 200 ppm or in the females at 750 ppm. 

At 750 ppm in males, a toxic effect on the ears was observed and considered to be treatmentrelated based on macroscopic and microscopic pathology findings. Macroscopic findings 

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included crusty zone of the skin of the ear in the males (11150 treated vs 5/50 controls). 

Microscopic findings involving the skin of the ear in the males included: acanthosis (16/50 

treated vs 9/50 controls); chronic active inflammation (11150 treated vs 4/50 controls); 

inflammation, all types (13/50 treated vs 6/50 controls); ulcer (12/50 treated vs 5/50 controls); 

and debris (13/50 treated vs 4/50 controls). Monthly body weight gain was reduced at the first 

month in the males (↓ 21 %), but not for the overall study. 

At 1400/1600 ppm [M/F], a toxic effect on the ears was also observed and considered to be 

treatment-related based on clinical signs and macroscopic and microscopic findings. The 

following were increased in the females at clinical examination: ear lesion redness (5/50 treated 

vs 0/50 controls) and ear lesion scab (22/50 treated vs 2/50 controls). Related gross necropsy 

findings included crusty zone of the skin of the ear in the males (12/50 treated vs 5/50 controls) 

and females (13/50 treated vs 1/50 controls). Related microscopic findings observed in the skin 

of the ear both sexes, including: acanthosis (16-17/50 treated vs 2-9/50 controls); chronic active 

inflammation (8-10/50 treated vs 1-4/50 controls); inflammation, all types (9-16/50 treated vs 1-

6/50 controls); ulcer (5-1150 treated vs1-5/50 controls); and debris (8-11150 treated vs 2-4/50 

controls). In addition, at clinical examination, an increased incidence of rough coat was 

observed in the males (30/50 treated vs 9/50 controls) and females (20/50 treated vs 9/50 

controls) and hunched back was observed in the females (5/50 treated vs 0/50 controls). 

Additional gross necropsy findings included rough coat in the males (22/50 treated vs 7/50 

controls) and females (16/50 treated vs 8/50 controls) and wet/stained ventrum in the males (7/50 

treated vs 0/50 controls). Reductions in mean body weight were observed in the males and 

females (↓7-20%) throughout the study. Monthly body weight gain was reduced for the first 

month in the males and females (↓60-69%) as compared to controls. Overall body weight gain, 

as calculated by the reviewers, was decreased in the males (↓25%) and females (↓54%). Mean 

absolute food consumption was decreased in the females (↓6-25%) throughout the study. 

With regards to specific endocrine endpoints, no treatment-related changes were seen in absolute 

or relative weights of the testes, ovaries, or adrenal glands at any dose level. In addition, no 

treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, 

prostate, ovaries, uterus, vagina, cervix, clitoral gland, mammary glands, thyroid, adrenal or 

pituitary glands at any dose level.

The LOAEL was 750 ppm for males (equivalent to 114.8 mg/kg/day) based on macroscopic and 

microscopic ear skin lesions and reduced body weight gains, and 1600 ppm in the females 

(equivalent to 309.7 mg/kg/day) based on clinical signs, macroscopic and microscopic pathology 

findings, and reduced body weights, body weight gains, and food consumption. The NOAEL for 

males was 200 ppm (equivalent to 31.9 mg/kg/day). The NOAEL for females was 750 ppm 

(equivalent to 140.6 mg/kg/day). There was no evidence of carcinogenic potential under the 

conditions of this study. 

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

In a chronic toxicity study (MRIDs 44435401, 1997; and 45189201, 2000), cyfluthrin (94.8-

95.1% purity) in corn oil was administered to beagle dogs (4/sex/dose) in the diet at dose levels 

of 0, 50, 100, 360, or 500 ppm for 12 months; actual achieved doses were of 0/0, 1.36/1.46, 

2.43/3.61, 10.64/10.74, or 15.47/17.99 mg/kg/day [M/F], respectively. High-dose animals 

initially received 640 ppm, but the dose was reduced to 500 ppm at Week 8 due to excessive 

toxicity. 

One high-dose female was sacrificed on Day 56 after exhibiting gait (including incoordination 

and increased stride width), postural, and behavioral abnormalities, convulsions, tremors, and 

cranial and peripheral nerve deficits. The high-dose was reduced to 500 ppm for the remainder 

of the study. One control male died on Day 318 and one control female died on Day 210 from 

asymptomatic idiopathic epilepsy. 

In the 360 ppm animals, clinical signs (males and females combined) included abnormal posture 

(1/8 treated vs 0/8 controls, starting on day 183) and a higher incidence of vomiting (8/8 treated 

vs 4/8 controls, starting on day 1). Gait abnormalities, including hypermetria, abnormal stride 

width, and reluctance to walk, were found at the 6 month (2/4 males, 1/4 females) and 12 month 

(1/4 males, 2/4 females) neurological examinations. Abnormal postural reactions, including 

“wheelbarrowing” with head position abnormal or dorsally flexed, abnormal front foot 

placement during lateral hopping, hemistanding, and abnormal foot placement or weight support 

during backward stepping, were also found at the 6 month (4/4 males, 3/4 females) and 12 month 

(4/4 males, 3/4 females) examinations. No postural or gait abnormalities were observed in 

controls. 

At 500 ppm, clinical signs (males and females combined) included gait abnormalities such as 

reluctance/inability to walk, stiff-leggedness, ataxia, and abnormal stride width (1-2/8 treated vs 

0/8 controls); seizures (2/8 treated vs 0/8 controls); convulsions and tremors (2/8 treated vs 0/8 

controls); and abnormal posture (1/8 treated vs 0/8 controls). Vomiting (8/8 treated vs 4/8 

controls), diarrhea (4/8 treated vs 0/8 controls) and soft feces (2/8 treated vs 0/8 controls) were 

also observed. Gait abnormalities similar to those observed in the 360 ppm group were found at 

the 6 month (3/4 males; 1/3 females) and 12 month (2/4 males; 1/3 females) examinations. 

Abnormal postural reactions similar to those observed in the 360 ppm group, but also including 

abnormal time to initiate front leg lateral hopping, were found at the 6 month (4/4 males; 3/3 

females) and 12 month (4/4 males; 3/3 females) examinations. Again, no postural or gait 

abnormalities were observed in controls. Non-statistically significant decreases in body weights 

were observed in the males throughout the study (decreased 6-20%). Mean body weight gains, 

as calculated by the reviewers, were decreased at the end of treatment in the males (↓53%). 

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Hepatic N−demethylase activity was increased in males at termination, the only interval tested 

(increased 45%). 

There were no differences of toxicological concern observed in rectal body temperature, food 

consumption, electrocardiography, blood pressure, hematological, clinical chemistry or 

urinalysis parameters, ophthalmoscopic, necropsy, or histopathological findings, absolute or 

relative organ weights, and plasma cholinesterase, hepatic O−demethylase or cytochrome P450 

activities. No neoplastic tissue was observed in dogs from any test group. 

With regard to specific endocrine endpoints, no treatment-related changes were seen in absolute 

or relative weights of the testes, ovaries, thyroid, adrenal or pituitary glands at any dose level. In 

addition, no treatment-related histopathological lesions were seen in the testes, epididymides, 

prostate, ovaries, uterus, cervix, mammary glands, thyroid, adrenal or pituitary glands at any 

dose level.

The LOAEL was 360 ppm (equivalent to 10.64/10.74 mg/kg/day [M/F]), based on clinical signs, 

gait abnormalities, and abnormal postural reactions in males and females. The NOAEL was 100 

ppm (equivalent to 2.43/3.61 mg/kg/day [M/F]). 

Avian Reproduction (Duck; OCSPP 850.2300)

In an avian reproductive study, (MRID 00158782, 1986) 16-week old mallard ducks (Anas 

platyrhynchos ) were administered cyfluthrin (94% purity) in the feed at nominal concentrations 

of 0 (acetone/corn oil, 0.06/0.02% v/w), 10, 50 or 250 ppm. Actual mean measured 

concentrations in the diets were 9.87, 42.7 and 262 ppm, respectively. The ducks were dosed for 

a total of 21 weeks, beginning 9 weeks prior to the start of egg production and then through 12 

weeks of egg production. Each treatment group was comprised of 16 mating pairs, with one 

mating pair per pen. A progressive photo period was employed in this study: 8 hours/day 

through Week 9 and 17 hours/day until the end of the study. Temperature was maintained at 

74±7°F throughout the study and humidity was maintained at ca. 82%. Individual adult body 

weights were recorded at Week 0, 4, 6, 8 and study termination, and food consumption in each 

replicate was measured weekly throughout the study. Eggs were collected, assessed for cracks, 

and incubated; eggs were checked for viability on days 14 and 21. On day 24 the set eggs were 

transferred to a hatchling compartment. Individual chick body weights were recorded after 

hatching and on Day 14 after hatching; egg shell thickness was measured every two weeks 

during egg production. Adult birds and chicks were observed routinely for mortality and clinical 

signs. 

No mortality or clinical signs of toxicity were noted at any dose level. At dietary concentrations 

up to 250 ppm, the study author reported there was no treatment-related effect on adult body 

weight, body weight gain, or food consumption. There were also no significant treatment-related 

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effects on any reproductive parameters. In this study, there was also observed low control 

reproductive performance, as indicated by a low number of eggs laid, embryo viability, and 

hatching rate compared to guideline criteria. The reviewer did note that the treated males lost 

more weight than control males from week 0 to 8 and gained less weight than control males by 

study termination. Additionally, the reviewer noted that pathological results showed an increase 

in the incident of slight egg yolk peritonitis in the treatment-groups compared to the control (≥10 

mg/kg-diet). However, these reported observations by the reviewer did not impact the overall 

NOAEC. The NOAEC for the study is 250 ppm, the highest dose tested, and a LOAEC was not 

observed. 

In another duck reproduction study (MRID 00145331, 1984), 16-week old mallard ducks (Anas 

platyrhynchos ) were administered cyfluthrin (90.5 % purity) in the feed at nominal 

concentrations of 0 (acetone/corn oil, 0.03/0.01% v/w), 250, 1000, or 4000 ppm for 24 weeks (10 

weeks prior to the start of egg production and 14 weeks of egg production). Each treatment 

group was comprised of 12 mating pairs, with one pair per pen. A progressive photo period was 

employed in this study: 7 hours/day through Week 9 and 17 hours/day until the end of the study. 

Temperature was maintained at 64-73°F throughout the study and humidity was maintained at 

30-78%. Individual adult body weights were recorded at Week 0, 4, 8 and 24, and body weight 

changes were determined for each interim period. Food consumption in each replicate was 

measured weekly throughout the pre-treatment and treatment periods of the adult phase. Eggs 

were collected beginning on Week 10, and egg handling and hatching procedures, measurements 

and observations were as per guidelines. Eggshell thickness and strength measurement were 

made on Weeks 1, 3, 5, 7 and 9 of laying. Individual chick body weights were recorded after 

hatching and on Day 14 after hatching; egg shell thickness was measured every two weeks 

during egg production. Adult birds and chicks were observed routinely for mortality and clinical 

signs. 

There was no reported treatment-related effect on adult mortality or clinical sign in any dose 

group, and no significant difference in food consumption. The study author’s reported a 

significant reduction in male and female body weight during the first four weeks of the study at 

the high concentration. 

With regards to reproductive effects, there was a dose-dependent reduction in the number of eggs 

laid/hen at the mid and high doses, which attained significance at the high dose. At both the mid 

and high doses, there were also significant reductions in the number of eggs laid, eggs set, 

fertilized eggs, 3-week viable embryos, hatchlings and 14-day survivors. A high percentage of 

eggs cracked were observed at the low dose (250 mg/kg-diet), but this effects was not observed 

at the higher treatments. However, due to low control reproductive performance, including low 

embryo viability, hatchling number, and 14-day survivorship, there is uncertainty associated with 

the results of this study. 

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A strong trend of significant reproductive impairment is evident at > 250 ppm. The impairment 

at 250 ppm is tentative but not resolvable by this study because of poor control response and 

because of too many cracked eggs at 250 ppm.- A definitive NOEL is not established. 

The one-generation reproductive toxicity of beta cyfluthrin to 16 pairs per level of 22-week old 

mallard duck (Anas platyrhynchos) was assessed over ca. 21 weeks (MRID 48350615, 1990). 

Beta cyfluthrin was administered to the birds in the diet at nominal concentrations of 0 (negative 

control), 10, 50, and 250 mg ai/kg diet. Mean-measured concentrations were <0.5 (LOQ, 

control), 10.5, 53.1, and 269 mg ai/kg diet. No mortalities or signs of toxicity were observed in 

the control or any of the treatment groups during the 21-week study. Lesions or abnormal 

behavior noted during the study were attributed to pen wear and tear, and in some instances, 

excessive interaction between pen mates. No treatment-related effects were observed for feed 

consumption, changes in body weight, or reproductive parameters. No statistically significant 

differences upon egg shell thickness were observed at any treatment level. The NOEL was 250 

mg ai/kg diet. 

Avian Reproduction (Quail; OCSPP 850.2300)

In an avian reproduction study (MRID 00145330, 1984), 20-week old bobwhite quails (Colinus 

virginianus) were administered cyfluthrin (90.5 % purity) in the feed at nominal concentrations 

of 0 (acetone/corn oil, 0.03/0.01% v/w), 250, 1000, or 4000 ppm for 23 weeks (8 weeks prior to 

the start of egg production and 15 weeks of egg production). Mean measured concentrations 

were 275, 1010 and 3825 ppm, respectively. Each treatment group was comprised of 24 mating 

pairs, with one pair per pen. A progressive photo period was employed in this study: 7 hours/day 

through Week 8 and 17 hours/day until the end of the study. Temperature was maintained at 64-

73°F throughout the study and humidity was maintained at 30-78%. Individual adult body 

weights were recorded at Week 0, 4, 8 and study termination, and body weight changes were 

determined for each interim period. Food consumption in each replicate was measured weekly 

throughout the pre-treatment and treatment periods of the adult phase. Eggs were collected 

beginning on Week 11, and egg handling and hatching procedures, measurements and 

observations were as per guidelines. For eggshell thickness and strength measurements were 

made every 2 weeks beginning on Week 14. Individual chick body weights were recorded after 

hatching and on Day 14 after hatching. Adult birds and chicks were observed routinely for 

mortality and clinical signs. 

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One control female, and one male and one female in the high-dose died on test, but no clinical 

signs for toxicity were reported sign in any dose group. There was no significant difference in 

food consumption, and no treatment-related effects were noted on body weight or body weight 

gain for the females. For the males, body weight and body weight gains were significantly 

reduced at the high dose (4000 ppm). No reproductive effects were noted in the low- and middose groups. The high-dose group had fewer eggs laid/hen, a lower hatching percentage, and 

fewer 2-week old chicks, compared to the control; however, only 2-week old chick survivors 

were significantly different from the control. Significant reductions in egg shell thickness were 

observed on Weeks 16, 18 and 20 at the low dose, on Weeks 16 and 18 at the mid-dose, and on 

Weeks 16, 18, 20 and 22 at high dose. 

In a follow-up study examining eggshell strength and thickness (MRID 00152829, 1985), 

bobwhite quail were administered cyfluthrin at nominal concentrations of 12.5, 49.9, 228, 926 or 

4298 ppm in the diet. Adult birds were observed daily for clinical signs and mortality. Body 

weights were recorded at weeks 0, 5, and 14. The first egg was laid at the end of week 9; 

thereafter, eggs were collected daily through the first two days of week 13. Al1 eggs laid were 

tested for shell strength and thickness. Thickness and strength were compared between groups 

for the study as a whole and for each day on which eggs were laid. The NOAEC for eggshell 

thinness was determined to be >4000 ppm based on a 31⁄2 week egg laying period. 

Fish Life-cycle Toxicity (Fathead Minnow; OCSPP 850.1500)

In a fish life-cycle study (MRID 41450401, 1990), newly fertilized eggs of Fathead minnows 

(less than 24 hours post-fertilization) were obtained from U.S. Fish and Wildlife Service, 

Columbia, MO. The embryos resulting from these fertilized eggs formed the stocks for two 

generations of testing for survival, growth and reproduction. Mean measured water 

concentrations of cyfluthrin were 0.018, 0.033, 0.065, 0.14 and 0.25 μg/L representing 100 to 

116% of nominal. 

In the F0 phase of the study, percent hatch ranged from a low of 75.7% at 0.033 μg/L to a high of 

92.1% at 0.018 μg/L, with no statistical significance found for any of the groups when compared 

to the controls. For the F1 generation, hatching ranged between 72.9% at 0.25 μg/L to 94.3% at 

0.14 μg/L. Within replicates success ranged between 46% at the highest concentration to 100% 

at 0.14 μg/L. 

The F1 generation hatching ranged between 72.9% for 0.25 μg/L to 94.3% in 0.14 μg/L. Within 

replicates success ranged between 46% in 0.25 μg/L and 100% in 0.14 μg/L. Frequency analysis 

gave a significant reduction in hatch in the F1 generation at the highest test concentration. 

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Parental survival showed significant differences from the controls only at the highest 

concentration of 0.25 ug/L (53%) for the Day-7 to 60 post-hatch group. The to 60 to 120 posthatch groups as far as Day-301 showed no significant differences from the controls although 

mortality started to increase in the period between Day 153 to termination at 301. 

For the F1 generation the controls were pooled because frequency analysis and the Fisher's Exact 

test showed no significant differences. Further, only at the highest concentration (0.25 μg/L) 

significant differences in survival were found when compared to the controls after approximately 

60 days of post-hatch exposure. 

Standard length and wet weight as measures of growth were not affected at any treatment level in 

either the parental or F1 generations. Five parameters were used to indicate reproductive 

success: spawns/pair; number of eggs/pair; eggs/spawn; number of reproductive days and 

number of eggs/pair/ reproductive day. These parameters were tracked from study Day 159 to 

study day 307. The total level of spawns for the parental generation showed a significant 

decrease at 0.033 μg/L, when compared to controls. At 0.14 μg/L. no overall statistical 

significant difference with the controls was observed. The control had the largest number of 

eggs/spawn. 

There were no significant differences between the pooled controls and any of the test levels for 

the mean number of eggs/spawn in either generation, but the number of spawns and eggs per 

adult or pair of adults, as well as, the number of reproductive days, were lowest for the 0.033 and 

0.25 μg/L. The solvent control and the 0.25 μg/l treatment level had the lowest eggs/pair/ 

reproductive day. The greatest number of reproductive days (days from the first to the last 

spawn) was observed at 0.018 μg/L. None of the test concentrations showed a significant 

decrease in this parameter. No significant differences were observed, at any test level, with the 

controls for the mean number of eggs/pair/reproductive day parameter. For the parental 

generation, mean values for this parameter ranged between a high value of 38.2 for the blank 

control and a low value of 13.6 at 0.25 μg/L. For mean percent hatch, survival, standard length 

and wet weight results in the F1, no significant reduction from the pooled controls was observed. 

Fish Early-Life Stage Toxicity (OCSPP 850.1400) 

In a sheepshead minnow (Cyprinodon variegatus) early life-cycle study (MRID 00155898, 

1986) using cyfluthrin (90.5%), fish were exposed to cyfluthrin concentrations of 50, 100, 200, 

400, 800, and 1600 ng a.i./L for 39 days. Additionally, a negative and solvent control were used. 

Mean measured concentrations ranged from 12.2 to 527 ng a.i./L, and high variability within a 

treatment was observed. Endpoints analyzed included: embryo mortality and time to hatch, 

larval survival and growth (dry weight). Reduced juvenile survival of sheepshead minnow was 

observed at concentrations ≥0.084 μg/L. The no-effect concentration was 24.7 ng a.i./L. 

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CYFLUTHRIN FINAL 

Page 67 of 68 

In a rainbow trout (Oncorhynchus mykiss) early life-cycle study (MRID 00158781, 1985) using 

cyfluthrin (96%), fish (embryos/larvae) were exposed to cyfluthrin concentrations of 25, 50, 100, 

200, and 400 ng a.i./L for 58 days. Additionally, a solvent control, but not negative control was 

used. Mean measured concentrations ranged from 10 to 160 ng a.i./L, and high variability within 

a treatment was observed. Significant effects on growth were reported at concentrations of 

≥0.018 μg/L for rainbow trout. A number of fish showed behavioral signs at concentrations 

of 50 ng/L (nominal concentrations) and higher. Based on mean measured concentrations, the 

NOAEC was 10 ng a.i./L. 

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CYFLUTHRIN FINAL 

Page 68 of 68 

APPENDIX 3: References Not Utilized in the Cyfluthrin 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 Cyfluthrin 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. 

Deanovic, L.A., et al. (2013). Comparing the Effectiveness of Chronic Water Column Tests with 

the Crustaceans Hyalella azteca (Order: Amphipoda) and Ceriodaphnia dubia (Order: 

Cladocera) in Detecting Toxicity of Current-Use Insecticides. Environmental Toxicology and 

Chemistry. 32(3):707-712. 

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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