UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

PC Code: See table, Attachment A Decision No.: NA Petition No.: NA
Risk Assess ment T y pe: NA
TXR No.: See table

, Attachment A
MRIDNo.:NA DP Barcode: NA Registration No.: NA Regulatory Action: NA
Case No.: NA
CAS No.: NA
40CFR: NA

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

# Fat**~~ fec** **~~ ~n** **~** Office of Pesticide Program

THR O UG H: Je ss Rowland

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## ~~~ Deputy Divi s ion Director
Health Effects Division
Office of Pe s ticide Programs

TO: Jolene Trujillo Chemical Review Manager
Pe s ticide Re-Evaluation Division Office of Pesticide Programs Ver .A pr. 2010

EPA has completed its Weight of Evidence (WoE) assessment eval u ating results of the
Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. The WoE documents for the 52 chemicals are li sted in Attac hm ent A a l ong with the chemical
and report identifiers

.

Page I of2Page 2 of 2 Attachment A. EDSP List 1 Chemicals

Chemical Name PC Code TXR Number 2,4-D 030001 0057151
Abamectin 122804 0057152 Acephate 103301 0057153
Acetone 044101 0057154
Atrazine 080803 0057155
Benfluralin 084301 0057156
Bifenthrin 128825 0057157 Captan 081301 0057158 Carbaryl 056801 0057159
Carbofuran 090601 0057160
Chlorothalonil 081901 0057161 Chlorpyrifos 059101 0057162 Cyfluthrin 128831 0057163 Cypermethrin 109702 0057164
DCPA 078701 0057165
Diazinon 057801 0057166
Dichlobenil 027401 0057167
Dimethoate 035001 0057168
EPTC 041401 0057169
Esfenvalerate 109303 0057170 Ethoprop 041101 0057171
Fenbutatin-Oxide 104601 0057172
Flutolanil 128975 0057173 Folpet 081601 0057174 Glyphosate 417300 0057175 Imidacloprid 129099 0057176 Iprodione 109801 0057177 Isophorone 847401 0057178
Linuron 035506 0057179
Malathion 057701 0057180 Metalaxyl 113501 0057181 Methomyl 090301 0057182
Metolachlor 108801 0057183
Metribuzin 101101 0057184
MGK-264 057001 0057185 Myclobutanil 128857 0057186
Norflurazon 105801 0057150 o-Phenylphenol 064103 0057146 Oxamyl 103801 0057142
PCNB 056502 0057138
Permethrin 109701 0057149
Phosmet 059201 0057145 Piperonyl Butoxide 067501 0057141
Pronamide 101701 0057137 Propargite 097601 0057148 Propiconazole 122101 0057144 Pyriproxyfen 129032 0057140
Simazine 080807 0057136
Tebuconazole 128997 0057143 Tetrachlorvinphos (TCVP) 083701 0057147
Triadimefon 109901 0057139
Trifluralin 036101 0057135

### EDSP: WEIGHT OF EVIDEN CE ANALYSIS OF POTENTIAL
INTERACTION WITH THE ESTROG EN, ANDROGEN OR THYROID
PATHWAYS

CHEMICAL: PERMETHRIN

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYPERMETHRIN 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 Rele vant Information (OSRI)......................................5

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

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

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9
1. Effects on Estrogen Pathway.....................................................9
2. Effects on Androgen Pathway..................................................15

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

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

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

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

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

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

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

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

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

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

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

LIST OF TABLES: Table 1. Tier 1 Assays for Permethrin.................................................................8
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Permethrin..................................12
Table 3. Androgenic/Anti-Androge nic Pathway for Permethrin.................................17
Table 4. Thyroid Pathway for Permethrin..........................................................21

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................32
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................40

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

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

ANOVA Analysis of Variance
AOP Adverse Outcome Pathwa y A R Andro g en Rece p to r B max Bindin g at maximum BROD Benz y lox y resorufin-O-dealk y lase BUN Blood Urea Nitro g e n CA R Constitutive Androstane Rece p to r CFR Code of Federal Regulations

CG Cowper’s Gland

ChE Cholinesterase
ChEI Cholinesterase inhibition
CMC Carboxymethyl cellulose
CTA Comparative Thyroid Assay
CV Coefficient of Variation
CYP Cytochrome 450

DE R Data Evaluation Recor d DMSO Dimeth y l Sulfoxide DNA Deox y ribonucleic Aci d DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
E R Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase ) ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g enerationPERMETHRIN FINAL

iv Abbreviation Terminology

F2 Second filial g eneration
Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Intera g enc y Coordinatin g Committee on the Validation of Alternative K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

L Low dose LABC Levator Ani-Bulbocavernosus LAGDA Larval Am p hibian Growth and Develo p ment Assa y LC50 Lethal Concentration in 50% of test organisms

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

M Male
MDL Minimum Detection Level MEOGRT Medaka Extended One Generation Re p roduction Test
MH Medium hi g h
ML Medium low MoA Mode of Action MOE Mar g in of Ex p osure MRID Master Record Identifier

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated ConcentrationPERMETHRIN FINAL

v Abbreviation Terminology

MTD Maximum Tolerated Dose
N Ne g ative
NE Not examined/evaluate d
NF sta g e Nieuwkoo p and Faber’s Sta g in g Atlas NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level NS Not Statisticall y Si g nificant N R Not Re p orte d OCSPP Office of Chemical Safet y Pollution and Prevention OECD Organization for Economic Co-Operation and Development

OPP Office of Pesticide Programs

ORD Office of Research and Develo p ment OSCP Office of Science Coordination and Polic y OSRI Other Scientificall y Relevant Informatio n P Positive
P Parental g eneratio n PC Positive Control PC 10 Positive Control at 10% of response

PC 50 Positive Control at 50% of response

PND Post-Natal Da y POD Point of De p arture PPS Pre p utial Se p aration PROD Pentaox y resorufin-O-dealk y lase ( or de p ent y lase )
PX R Pre g nane X rece p to r QC Quality Control

RBA Relative Binding Affinity

RBC Red Blood Cells
RfD Reference Dose RPC max Relative to Positive Control at maximum

SAP Scientific Advisor y Panel SC Solvent Control

s.c Subcutaneous SDH Sorbitol deh y dro g enase SDWA Safe Drinking Water Act

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th SV Seminal Vesicles
T Th y roid ( hormonal p athwa y) T1WoERC EDSP Tier 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)PERMETHRIN FINAL

vi Abbreviation Terminology

TP Testosterone Pro p ionate
T R Th y roid Rece p to r TSH Thyroid Stimulating Hormone

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

VP Ventral Prostate VTG Vitello g eni n WoE Wei g ht-of-EvidencePERMETHRIN FINAL

Page 1 of 67 Executive Summary

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evalua te the potential of chemicals to interact with the estrogen (E), androge n (A) or thyroid (T) signali ng pathways. This interaction includes agonism and antagonism at the es trogen and androgen receptors, altered steroidogenesis, as well as hypothalami c-pituitary-gonadal (HPG) and hypothalamicpituitary thyroid (HPT) axes. In addition to the available Tier 1 assay data, other scientifically relevant information (OSRI) , including general toxicity data and open literature studies of sufficient quality were co nsidered in this weight of evidence (WoE)

assessment.

In determining whether permethrin interact s with E, A or T hormone pathways, the number and type of effects induced, the magnitude and pa ttern of responses observed across studies, taxa a nd sexes were considered. Additi onally, the condi tions under which effects occur were considered, in particular , whether or not endoc rine-related responses occurred at dose(s) that also resulted in ge neral systemic toxicity or overt toxicity.

On July 23, 2014, the EDS{ Tier 1 Assay Wei ght of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) an alysis of the potential interaction of permethrin with the E, A or T hormone path ways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

For the estrogen pathway, a number of OSRI in vitro assays evaluating estrogen receptor activation resulted in conflicting results, i.e . some showed activation of estrogen signaling while others did not. The OSRI assays included estrog en receptor (ER) binding, cellular proliferation of estrogen sensitive MCF-7 cells, expression of estrogen sensitive pS2 mRNA in the cancer cells, and interaction with either ER α or ER β in transfered HeLa cells. Permethrin was negative
in the Tier I in vitro aromatase assay, whereas in the steroidogenesis assay permethrin caused an increase (p<0.05) in estradiol production. In vivo , permethrin was negative in both the uterotrophic and female pubertal rat assays, with no effects on uterine weight, mean age at vaginal opening, percent cycling, or histopathological changes in the ovaries and uterus. In the fish short-term reproduction assay (FSTRA), all e ffects in females were se en only at a dose that caused overt toxicity (i.e, mort ality, clinical signs of toxic ity). With the exception of one reproductive study in mallard duck which had a slight reduction in the number of eggs laid by the hen (which has limited ability to inform a bout potential estrogen-rela ted effects by itself and decreases in food consumption al so observed at this concentra tion), no estrogen-related effects
were noted in the in vivo studies in mammals, fish, or birds th at occurred in the absence of overtPERMETHRIN FINAL

Page 2 of 67 and/or systemic toxicity. Th erefore, although several of the in vitro studies [estrogen receptor (ER) binding, ER transactivation assay (ERTA), a nd steroidogenesis] indi cated that permethrin had the potential to affect the E pathway at the cellular level, these eff ects are not supported by
the available in vivo studies.

For the androgen pathway, conflicting results were reported for AR interaction across the in vitro assays. In an in vitro assay, permethrin reduced sperm motility in a concentration-dependent manner (1-64 μmol/L). Multiple Hershberger assays were available for permethrin, in which the Tier 1 assays as well as an addi tional open literature study (Kunimatsu, et al ., 2002) were negative at doses up to 75 or 120 mg/kg/day, respectively. However, in another OSRI Hershberger assay (Kim et al ., 2005), significant decr eases in AST weights were reported at
doses of ≥ 10 mg/kg/day.

In the male rat pubertal assay, no androgen-relate d effects were observed. While there does not appear to be androgen-related eff ects in rats with an intact HPG axis (i.e. male pubertal and Part 158 rat data); published results in the literature (Jin et al ., 2012, Wang et al ., 2012) reported that permethrin caused testicular toxicity in mice following oral exposure at a dose of 35 or 100 mg/kg/day. In these studies, testicular toxicity manifested as changes in testosterone levels, decreased testicular and epididym al weights, changes to the epididymal duct, regression of sperm quality and histopathologi cal lesions in the testes. In the FSTRA, all effects in males were only observed at the high concentration in the presence of overt toxicity.

Therefore based on the available in vitro and mammalian in vivo data, there appears to be a potential interaction with the androgen pathway in mammals. There was no convincing evidence of a potential interaction with the androgen pathway in wildlife.

There was no convincing evidence of an interacti on of permethrin with the thyroid pathway in
mammals or wildlife in the absence of overt toxic ity. The only thyroid-related effect observed in the absence of overt toxicity was a decrease in serum T4 levels in the male and female pubertal assays. No thyroid related effects were obser ved in the mammalian Part 158 studies. No treatment-related thyroid effects were seen in the AMA.

Overall, there is no convincing evidence of poten tial interaction with th e estrogen or thyroid pathways in mammals or wildlife. There is evid ence for potential intera ction with the androgen pathway in mammals. There is no convincing ev idence for a potential interaction with the androgen pathway in wildlife.

For permethrin, a point of departure (POD) of 25 mg/kg/day for human health risk assessment is
based on neurotoxic clinical signs (abnormal and/or decreas ed movement, increased body temperature, and aggression) observed at 75 mg/ kg/day (LOAEL) in an acute neurotoxicity study in rats. This POD is used for deriving the chro nic Reference Dose (RfD) for chronic dietary riskPERMETHRIN FINAL

Page 3 of 67 assessment. A second POD of 11 mg/kg/day ba sed on clinical signs of toxicity (body temperature and hypersensitivity to noise) s een at 154 mg/kg/day (LOAEL) in a subchronic inhalation toxicity study in rats. This POD is used for calculating the Margins of Exposure (MOEs) for non-dietary risk assessment. These P ODs are lower than the dose (100 mg/kg/day)
that caused the testicular effects in mice. Consequently, the current RfD and MOEs are protective of the androgen-mediate effects observed in the published literature study. Additionally, there was no evidence of interaction with the androgen pathway in the EDSP Tier 1 assays. Therefore, there is no concerns for the findings reported in the literature study.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not recommended for permethrin since additional testing is not expected to impact EPA’s current regulatory point of departur es and endpoints for human h ealth risk assessments.

Given the lack of evidence for a potential intera ction with the estrogen and androgen pathway in fish and thyroid pathway in amphibians, no additional testing is recommended.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for permethri n. In the bobwhite quail, no repr oductive effects were observed, but effects on reproductive parameters were obs erved in the mallard duck. The type of data
obtained from Part 158 avian re production studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from pe rmethrin exposure. Relative to the EDSP, additional testing is not recommended.PERMETHRIN FINAL

Page 4 of 67 I. Introduction

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evalua te the potential of chemicals to interact with the estrogen (E), androge n (A) or thyroid (T) signali ng pathways. This interaction includes agonism and antagonism at the es trogen and androgen receptors, altered steroidogenesis, as well as hypothalami c-pituitary-gonadal (HPG) and hypothalamicpituitary thyroid (HPT) axes. In addition to the available Tier 1 assay data, other scientifically relevant information (OSRI) , including general toxicity data and open literature studies of sufficient quality were co nsidered in this weight of evidence (WoE)

assessment.

In determining whether permethrin interact s with E, A or T hormone pathways, the number and type of effects induced, the magnitude and pa ttern of responses observed across studies, taxa, and sexes were consider ed. Additionally, th e conditions under which effects occur were considered, in particular , whether or not endoc rine-related responses occurred at dose(s) that also resulted in ge neral systemic toxicity or overt toxicity.

On July 23, the Tire 1 Assay Weight of Evidence Review Committee (T1W oERC) 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 pot ential interaction of permethrin with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in

vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited
OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

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

− 8 mm Hg (25°C). Permethrin was immobile in several so ils tested (Koc >5000). It is also slow to hydrolyze and biodegrade (anaerobi c aquatic study half-lives of 113-175 days). Based upon its Henry’s law constant (1.4x10

− 6 atm-m 3 /mol) and vapor pressure, permet hrin is expected to have a relatively low potential for volat ilization from soil and water surfaces. The potential of permethrin for volatilization is also reduced sign ificantly because it adsorb s strongly to soils and suspended solids or sediment in the water column.

The available information considered in determin ing the potential interacti on of permethrin with the E, A, or T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxi city studies and other pu blished articles. These data are summarized in Sections III.A through I II.C. An analysis of the data submitted to the agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The EDSP Tier 2 study recomme ndations are presented in Section V.PERMETHRIN FINAL

Page 5 of 67 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 believe d to be relevant to one or more of the Tier 1 Screening assays were submitted as OSRI by the Te st Order recipients and/or the public. This included studies published in the open literatu re and/or data submitted to support pesticide registration (e.g., Part 158 guideli ne studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disrupt or Review Team on permethrin (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any additional relevant info rmation. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/ studies considered but not uti lized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and approach used in the WoE determination on the potential of a
substance to interact with endocrine-related processes ( i.e., E, A, or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the indivi dual studies for their scientific quality and relevance in evaluating potential endocrine interaction(s). The second step is to integrate the data along different levels of biological organization while examining the extent of complementarity ( i.e ., the concordance of endpoints within an assay that measures multiple endpoints) and redundancy ( i.e ., the concordance of endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to
evaluate whether additional te sting is needed based on the evidence and conclusions described
above.PERMETHRIN FINAL

Page 6 of 67 As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data includes a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the 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 speci fic endpoint for discerni ng whether or not the chemical has interacted with the endocrine syst em, are considered. The co llective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indi cation of potential interaction as measured by the study.

The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data, as well as characterizing the extent to which the available data suppor t a hypothesis that a chemical has the potential to interact E, A or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, coherence, strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are
meant to evaluate whether or not a chemical can in teract with E, A, and T consisting of different levels of biological organization from a molecular initiating ev ent, such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment and fecundity at the whole organism level. The extent of expecta tion of responses at higher levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy, as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biological organization?. Finally, under what conditions did the responses occur including co nsideration of whether the responses were observed in the presence of overt or systemic t oxicity?. The presence of overt and/or systemicPERMETHRIN FINAL

Page 7 of 67 toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.

This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting in that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October, 2013 the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

For these WoE analyses, overt toxicity was gene rally defined in accordance with EPA’s current approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g ., organ weights/histopathology), biochemical ( e.g ., blood chemistry), and other clinical signs ( e.g ., lethargy) were also considered when evaluating overt toxicity, es pecially if the effects were extreme. In some instances, one parameter ( i.e ., death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g ., change in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

Therefore, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addressing the three main components described above is fundamental to the WoE process and in determ ining whether additional data are needed. In addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting data; 4) overall strength of eviden ce supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing th e need for additional data is based on a caseby-case analysis which took all availa ble toxicity data into account.PERMETHRIN FINAL

Page 8 of 67 The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

A. EDSP T1 Screening Assays

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

.

Table 1. Tier 1 Screening Assays for Permethrin

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied by OSRI (Saito et. al. , 2000)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300 Requirement Satisfied by OSRI (Tyler et. al., 2000)

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied by OSRI (Xu et. al. , 2008; Zhang et. al.

, 2008a; Tyler et. al. , 2000; Zhang et. al. , 2008b; Kim et. al. , 2005) Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550 Requirement Satisfied (MRID No. 48618603) Aromatase Assay (human recombinant microsomes) OSCPP 890.1200 Requirement Satisfied (MRID No. 48618601)

Uterotrophic Assay (Rat) OSCPP 890.1600 Requirement Satisfied by OSRI (Kim et. al. , 2005; Kunimatsu et. al. , 2002)

Hershberger Assay (Rat) OSCPP 890.1400 Requirement Satisfied (MRID No. 48618602)

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied (MRID No. 48672901)

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied (MRID No. 48687101)

Fish Short-term Reproduction Assay OSCPP 890.1350 Requirement Satisfied (MRID No. 48702301)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100 Requirement Satisfied (MRID No. 48988601)PERMETHRIN FINAL

Page 9 of 67 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential interactions of permethrin with the estrogen pathway are summarized in Table 2. The various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for estrogen ic, anti-estrogenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

The requirement for the Tier 1 ER binding and ERTA assays were satisfied by Saito et al ., 2000 and Tyler et al ., 2000, respectively. In the study by Saito et al ., permethrin was negative for
interaction with the ER ba sed on the results of three in vitro assays utilizing human ER (hER α ): 1) a mammalian cell-based luciferase reporter assay, 2) a yeast two-hybrid method, and 3) a competitive ligand-binding assay. Conversely, in the study by Tyler et al ., permethrin showed weak activity in a recombinant ye ast reporter assay with a half maximal effective concentration (EC 50 ) of 2 × 10

− 3 M. In other published studies considered as OSRI, the effects of permethrin in ER binding, ER transcriptional activation, cell prolif eration, and ER-responsive pS2 gene expression showed conflicting results regarding the estrogenic pot ential of permethrin. The findings from the additional in vitro OSRI considered to evaluate potential ER activity are
summarized below.

In an ER competitive-binding study using rat uterine cytosol (Chen et al ., 2002), permethrin exhibited equivocal evidence of specif ic binding to the ER. The percent [ 3 H]-E 2 binding to the

ER was ≥ 67% at all concentrations up to 10 -3 M permethrin. However, in another ER competitive binding assay also using rat uterine cytosol, Kim et al . (2004) reported that permethrin did not competitively bind to th e ER, while in a whole cell competitive binding study, Lemaire et al . (2006), reported that perm ethrin did not bind to ER α or ER β

.

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

. (2010), permethrin was tested for potential interaction with the ER α using an ER transactivation assay in African green monkey CV-1 cells. Perm ethrin exhibited weak agonist effects at low concentrations (10

− 8 to 10

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

− 6 to 10

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

ER β activity in transfected HeLa-derived (HELN) cel ls. In this study, the authors reported that permethrin showed no agonist or antagonist activity for ER α or ER β at the single concentration (10

− 5 M) tested.PERMETHRIN FINAL

Page 10 of 67 Published studies also showed conflicting results regarding the ability of permethrin to induce ER-responsive cell proliferation. In a MCF-7 cell proliferation assay by Chen et al . (2002), permethrin induced proliferation at and above 10

− 10 M, with maximal proliferation at 10

− 8 M. In a study by Go et al . (1999), permethrin induced modest in creases in MCF-7 proliferation at 10

− 4 M and in a study Jin et al . (2010), permethrin induced prol iferation of MCF-7 cells at
concentrations of 10

− 8 to 10

− 6 M. Conversely, in a study by Kim et al . (2004), permethrin did not induce proliferation in MCF-7 cells, but it did antagonize the prolif erative response of
estradiol at 10

− 6 M.

Several of the published studies also measured pS2 mRNA levels as a biomarker for estrogenic activity in MCF-7 cells. The pS2 gene is strongly expressed in response to estrogen or estrogenlike compounds. In a study by Chen et al . (2002), pS2 mRNA expre ssion increased 1.3-fold
after treatment with 10

− 7 M permethrin. Go et al . (1999), reported permet hrin weakly induced pS2 expression at 10

− 4 M and Jin et al . (2010) reported that permet hrin induced pS2 expression
2.6-fold and decreased ER α mRNA expression levels at 10

− 6 M.

In another in vitro study, Garey and Wolf (1998) evaluated the potential for permethrin to act as an estrogen agonist or antagonist using the Is hikawa Var-1 human endom etrial adenocarcinoma cell line. This cell line pr oduces a dose-dependent increase in al kaline phosphatase in response to estrogens. Permethrin did not show estrogen agonist or antagonist activity in this test system.

Permethrin was negative in the Tier 1 aromatase assay; while in the Tier 1 steroidogenesis assay, permethrin produced an increase (p<0.05) in estr adiol production in two runs at 1 and 10 μM (average 1.16-fold for 1 μM and 1.51-fold for 10 μM).

The test order requirement for the uterotrophic assay was satisfied by the results of two literature
studies submitted as OSRI: Kim et al ., 2005; and Kunimatsu et al ., 2002. In Kim et al

., permethrin (administered s.c.) increased relative (to body) uterus weight s at a dose level of 800 mg/kg/day. Permethrin also enhanced E 2 -induced uterine weight incr eases at a lower dose (200 mg/kg); permethrin-induced uterine weight increas es were inhibited by co-administration of the anti-estrogen ICI 182,780, but inhibition was only statistically different at a lower dose (50 mg/kg). Kim also reported that permethrin treatment had no effects on relative vagina weights.
In Kunimatsu et al ., no estrogenic or anti-estrogenic eff ects were observed on uterine weights with oral administration up to 150 mg/kg/day permethrin which also caused clinical signs of toxicity (3-6/6 rats disp laying tremor). No estrogen-related ef fects were observed in the Tier 1 female pubertal assay.

In the Tier 1 FSTRA, increased GSI and eff ects on gonadal histopathology along with a 42% decrease in fecundity were observed at the highest concentration tested. However, overt toxicity ( i.e., mortality, clinical signs of toxic ity) was noted at th is dose level.PERMETHRIN FINAL

Page 11 of 67 In a Part 158 developmental toxicity study in rabbits, post-implantation loss was increased (p<0.05) at the mid (1200 ppm) and high (1800 pp m) dose levels; however , rabbits in all dose groups (including 600 ppm) had decreases in body weight ga ins of 50-91% during GD 0-18. There were no estrogen-related effects noted in the other Part 158 studies.

In a Part 158 avian reproduction toxicity study w ith quail, no treatment-related systemic or reproductive effects were noted at dietary levels up to 500 ppm. In the mallard duck reproduction study, there appeared to be a slight reduction (NS, but considered biologically significant) in the number of eggs laid by hens at the high dose (500 ppm) during the last two weeks of egg production, which was correlated with an increase in the number of hens with a regressing ovary. However, feed consumption at th is dose was observed to be slightly decreased throughout the study, with a signif icant difference from controls during Weeks 6, 12, 14, and 19.

In a Part 158 fish life-cycle toxicity study with fathead minnows, at the mean measured concentration 0.41 ppb, percent surviv al of fry were reduced in th e first and second generations after 30 days. In a study by Nillos et al . (2010), permethrin stereoisomers were examined for estrogenic activity in vivo using VTG protein measurements in male medaka. After exposure of medaka for 8 days to the indivi dual permethrin stereoisomers a nd permethrin mixture at 10 μg/L, hepatic VTG production was increased (p<0.05) comp ared to controls. Additionally, significant differences in the relative estrog enic potential of the enantiomers were reported: 1S-cis- and 1Strans-permethrin had responses 2.5 and 1.3 times greater than their resp ective R enantiomers. However, this study was conducted under static conditions and as such, given the physical properties of permethrin (low solubility, propens ity to bind), there is c oncern about the actual exposure concentrations (study not included in table below).

In Brander et al ., 2012, silverside fish ( Menidia beryllina ) exposed for 14 days to 0.1 ng/L of bifenthrin exhibited sign ificantly greater chorioge nin (precursor to chori on, which is an estrogen dependent protein that forms the layer between the embryo and the outer layer of the egg) expression than 1 ng/L of the positive control, ethi nylestradiol. It is wo rth noting that this study did not have a negative control but rather comparisons were made to a methanol solvent control. Additionally, the measured test c oncentrations reported appear to reflect results from a single 24 hour renewal cycl e under abiotic ( i.e. without fish) conditions. Since, these results are confounded by several uncertainties including the absence of a negative control and limited exposure measurements, and this study is not reported in the table below.

In Jin et al. 2009, zerbrafish embryo -larvae were exposed to permet hrin for 7 days under staticrenewal conditions exhibited up regulation of several estrogen -responsive genes (e.g., vtg1, esr α , cyp19b) at 250 ng/L and above. In this study, as in other studies, only a solvent control was used and given the static-renewal test design without reported measured test concentra tions, there is uncertainty in the actual exposure concentrations . As such, this study is not reported in table
below.PERMETHRINFINALPage 12 of 67Table 2: Estrogenic/Anti-Estrogenic Pathway for Permethrin.Lines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Permethrin1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER BindingRequirement satisfied by OSRI (Saitoet al., 2000); see below.ERTARequirement satisfied by OSRI (Tyleret al., 2000); see below.Aromatase(MRID 48618601)NSteroidogenesis(MRID 48618603)PUterotrophicRequirement satisfied by OSRI (Kimet al., 2005; Kunimatsuet al., 2002); see below.Female Pubertal Rat(MRID 48672901)N N N N N N↑LW(H)X(H)FSTRA(MRID 48702301)NEN NN N NX(H)OSRIER Binding andTransactivation(Saitoet al., 2000)N NRecombinant YeastTransactivation Assay(Tyleret al., 2000)PER Binding/Proliferation/PS2(Chenet al., 2002)E P4ER Binding/Proliferation/PS2(Kimet al., 2004)N P4PERMETHRINFINALPage 13 of 67Table 2: Estrogenic/Anti-Estrogenic Pathway for Permethrin.Lines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Permethrin1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3ER Binding/ER Transactivation(Lemaireet al., 2006)N N5ER Transactivation (Duet al., 2010)P6Proliferation/PS2 (Jinetal., 2010)P4Proliferation/PS2 (Goetal., 1999)P4Estrogenic Potential(Garey and Wolff, 1998)N7Uterotrophic(Kimet al., 2005)NN NUterotrophic(Kunimatsuet al., 2002)NX(H)NThree-GenerationReproduction (Rat;MRID 00120271)NENENNENENX(H)X(H)Developmental Toxicity(Rat; MRID 40943603)NX(H)X(H)Developmental Toxicity(Rabbit; MRID40943602)P(M)X(L,M, H)X(H)Chronic Toxicity/Carcinogenicity(Rat; MRID 00069701)NE N N NX(H)X(H)Chronic Toxicity(Dog; MRID 00129600)NE N N NX(M,H)X(H)PERMETHRINFINALPage 14 of 67Table 2: Estrogenic/Anti-Estrogenic Pathway for Permethrin.Lines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Permethrin1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 328-Day Feeding (Rat;MRID 00120267)NE N N NX(MH,H)X(H)Avian Reproduction(Quail; MRID 2322901)NN NAvian Reproduction(Duck; MRID 2322902)P8NN9Fish Full Life Cycle(Minnow; MRID00102096)NX(MH,H)1.Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatmentArrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changesin weight are absolute unless otherwise indicated. LW= Liver weight2. 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 thiscolumn. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For details seeSection IV. A4.Reported increased MCF-7 proliferation and induction of PS2 expression.5.Negative at the single permethrin concentration tested (10-5M)6.Permethrin exhibited weak agonisteffects at low concentrations (10−8to 10−5M), and exhibited weak antagonist effects at higher concentrations (10−6to 10−5M) inCV-1 cells.7.Negative for estrogen agonist or antagonist using the IshikawaVar-1 human endometrial adenocarcinoma cell line test system.8.Slight reduction (NS, but considered biologically significant) in the number of eggs laid by hens at the high dose (500 ppm) during the last two weeks of eggproduction, which was correlated with an increase in the number of hens with a regressing ovary.9.Feed consumption at this dose was slightly decreased throughout the study, with a significant difference from controls during Weeks 6, 12, 14, and 19.E EquivocalP Positive findingsN Negative findings (in vitro)/ No effects (in vivo)NE Not ExaminedPERMETHRIN FINAL

Page 15 of 67 2. Effects on Androgen Pathway

The potential interactions of permethrin with the androgen path way are summarized in Table 3. The various targets of the androgen pathway across the relevant Ti er 1 assays are delineated so as to facilitate determination of potential for a ndrogenic, anti-androgenic, or HPG axis effects. This table also includes HPG-relevant findings from data evaluated as OSRI. 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 reporte d in the table and not considered further in the WOE assessment.

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

as OSRI: Xu et al., 2008; Zhang et al., 2008; Tyler et al.

, 2000; and Kim et al., 2005. In an in

vitro AR transcriptional activation assay Xu et al ., reported that permethrin showed AR antagonist activity by attenuating dihydrotestosterone (DHT)-related chloramphenicol transferase (reporter gene) expression with an IC 50 value of 5.86 × 10

− 5 M. In Zhang et al ., 2008, permethrin significantly inhibited DHT- induced hAR transcriptional activation in vitro at a
concentration of 10

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

− 3 M) induced slight increases in activity above baseline values, thus suggesting very weak agonist activity.
Permethrin also exhibited weak anti-andr ogenic activity in this assay with an IC 50 of 7.3 × 10

− 4 M. Additionally, two published studies examin ing the effects of permethrin on AR binding
and/or AR transactivation were consid ered as OSRI. In a study by Bauer et al . (2002), permethrin did not displace DHT from immobilized recombinant hAR. Conversely, in a receptor transactivation study by Du et al . (2010), permethrin showed AR antagonist activity with a
calculated 20% relative inhi bitory concentration (RIC 20 ) of 1.88 x 10

− 6 M. In the Tier 1 in vitro steroidogenesis assay, permethrin induced a sl ight, but significant (p<0.05) increase in testosterone production at 10 μM in two runs (average 1.1-fold).

Permethrin was negative in the Tier 1 Hershberg er assay and no androgen related effects were noted in the Tier 1 male pubertal assay. Additio nally, in a published 5-day Hershberger assay by
Kunimatsu et al . submitted as OSRI, no effects were noted on the accessory sex tissues at dose levels that caused tremors (75 mg/kg/day). In another published 10-da y Hershberger assay, Kim
et al . (2005) reported statistically si gnificant reductions in the fi ve sex accessory tissue weights at all doses tested (10, 50, and 100 mg/kg/day), although the decreases were not dose dependent. In addition, permethrin caused significant decr eases in seminal vesicle and ventral prostate weights in a modified Hershbe rger assay conducted by Zhang et al . (2008) using a single dose level (50 mg/kg/day) of permethrin; however, no chemical purity information was provided which reduces confidence in the study and ther efore the results are not included in the table
below.PERMETHRIN FINAL

Page 16 of 67 In the Tier 1 FSTRA, effects were noted in male s at the highest concentration tested (2.6 μg a.i./L), including an increase in the GSI and decreased nuptial t ubercle score. However, only
63% of the males survived at the hi gh dose, and clinical sings of t oxicity were noted at this dose, including erratic swimming, loss of equilibrium, surfacing, and excess feed noted in the test chambers. There were no androgen-related effects determined in the Part 158 ecotoxicity studies.

The only androgen-related effect in the Part 158 mammalian studies was decreased testicular weight seen in a rat 28-day feeding toxicity study at doses that al so caused neurotoxicity (hypersensitivity).

In addition to the Part 158 toxicity studies, publ ished studies were revi ewed that specifically examined effects of permethrin on testes and/or sperm parameters in rodents. In a testicular toxicity study by Jin et al ., (2012), (+)-cis, (

− )-cis, and (

− )-trans permethrin exposure at 100 mg/kg significantly reduced serum testosterone le vels, and decreased both absolute and relative weights of testes in the (+)-cis, (

− )-cis and (

− )-trans-permethrin treated groups. A decreased number of spermatogenic cells and large interstiti al spaces in the seminiferous tubules were also observed in the 100 mg/kg (+)-cis, (

− )-cis, and (

− )-trans-permethrin tr eated groups. In a testicular toxicity study by Wang et al . (2012), the percentage of se miniferous tubules in stage group VII-VIII was significantly de creased in the cis-permethr in group (oral dosing at ~35 mg/kg/day), and in the caput epididymis, cytoplas mic vacuolation was observed more frequently in the cis-permethrin group, and median vacuolation scores were signif icantly higher. Caudal epididymal sperm count was significantly reduced in the cis-permethrin group ( ↓ 60%), and sperm motility was significantly decreased.PERMETHRINFINALPage 17 of 67Table 3: Androgenic/Anti-Androgenic Pathway for Permethrin.Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Permethrin1Study Type/ LiteratureCitation AR Binding AR Activation Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histo p atholo gy Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR BindingRequirement satisfied by OSRI (Xuet al.,2008; Zhanget al.,2008a; Tyleret al.,2000; Zhanget al.,2008b; Kimet al.,2005)Steroidogenesis (MRID48618603)PHershberger(MRID 48618602)NENN NMale Pubertal Rat (MRID486687101)N N N N N N NN↑LW(L, H)X(H)FSTRA(MRID 48702301)NE N NN NNX(H)OSRIAR Binding(Baueret al., 2002)NRecombinant Yeast ARAssay(Tyleret al., 2000)PReceptor Transactivation(Duet al., 2010)PAR Transactivation(Xuet al., 2008)PHershberger(Kimet al., 2005)NEP(L, M, H)N NHershberger(Kunimatsuet al., 2002)NENNX(H)PERMETHRINFINALPage 18 of 67Table 3: Androgenic/Anti-Androgenic Pathway for Permethrin.Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Permethrin1Study Type/ LiteratureCitation AR Binding AR Activation Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histo p atholo gy Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Three-GenerationReproduction (Rat;MRID 00120271)NE N NE N NE NE NX(H)X(H)Chronic Toxicity /Carcinogenicity (Rat;MRID 00069701)N N NE NNX(H)NChronic Toxicity (Dog;MRID 00129600)N N NE NNX(M, H)X(H)28-Day Feeding (Rat;MRID 00120267)↓10%(MH)NE NE NENX(MH,H)X(H)Testicular Toxicity(Mouse; Jinet al., 2012)P(H)P(H)P(H)N NTesticular Toxicity(Mouse; Wanget al.,2012)N N P N PN NAvian Reproduction(Quail; MRID 42322901)NN NAvian Reproduction(Duck; MRID 42322902)NNN4Fish Full Life Cycle(Minnow; MRID00102096)NX(MH,H)PERMETHRINFINALPage 19 of 671. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑)indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight areabsolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate(PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP).2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in thiscolumn. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For details seeSection IV. A4. Feed consumption at this dose was slightly decreased (NS) throughout the study, with a significant difference from controls during Weeks 6, 12, 14, and 19.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedPERMETHRIN FINAL

Page 20 of 67 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it
does include three in vivo assays that provide data to de tect changes in the HPT axis, i.e ., the pubertal male and female (rat) assays, and the AMA (frog).

The potential interactions of permethrin with thyroid regulation are summarized in Table 4. The various targets of the th yroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. This table also includes HPT-relevant findings from data evaluated as OSRI. This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the pr esence of overt toxicity are
discussed in the text but are not reported in the table and not considered further in the WOE

assessment.

In the Tier 1 male pubertal assay, serum T 4 levels were decreased by 28 and 42% at the low (75 mg/kg/day) and high (120 mg/kg/ day) doses, respectively, and seru m TSH levels were increased by 77% at the high dose. Howeve r, the thyroid hormone changes at the high dose occurred in the presence of overt toxicity. No ch anges were seen in thyroid weight or histopathology. Similarly, in the female pubertal assay, serum T 4 levels were decreased by 26 and 42% at the low (75 mg/kg/day) and high (150 mg/kg/ day) doses, respectively, and seru m TSH levels were increased by 40% at the high dose. In addi tion, thyroid weights in females we re also decreased by 22% at the high dose, but without a change in thyroid histopathology. It was determined that the high dose in the female pubert al was overtly toxic.

In the AMA, Day-21 normalized hind-limb length (HLL) was increased by 21-23% compared to negative control, however, it was not dose-depend ent. Additionally, when Day-21 normalized HLL results were compared to the solvent cont rol, no significant diffe rences were observed. There was no significant acceleration or delay of median Nieuwkoop-Faber (NF) developmental stage at Day 7 or 21 for any treatment level. Further, no asynchronous development was observed. A statistically significant increase in Day 21 wet weight a nd snout-to-vent length (SVL) was observed at the lowest concentration tested (0.054 μg a.i./L). Finally, effects on thyroid gland histopathology were observed in both controls and all treatment groups. Histopathological effects included thyroid gla nd atrophy and hypertrophy, follicular cell height increases and decreases, and follicular cell asymmetry. These effects were of low frequency and did not exhibit a concentration-dependent response ; therefore, they are not considered to be
treatment-related.

In a receptor transact ivation study by Du et al . (2010), permethrin was observed to have antagonist effects at the thyroid receptor in a transfected Afri can green monkey kidney CV-1 cell
line.PERMETHRIN FINAL

Page 21 of 67 In a Part 158 chronic toxici ty study in dogs, thyroid weights were increased in a non-dose dependent manner in the low (5 mg/kg/day; ↑ 27%), mid (100 mg/kg/day; ↑ 28%) and high (1000 mg/kg/day; ↑ 17%) dose males, and in the mid ( ↑ 27%) and high ( ↑ 33%) dose females; additionally, no corroborating histopat hological changes were observed.

Table 4. Thyroid Pathway for Permethrin.

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

Study Type/ Literature CitationTR BindingThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth(BW, SVL)Systemic Toxicity2Overt ToxicityObserved3

EDSP Tier 1 Assay

Male Pubertal Rat (MRID 48687101) N N ↓ 28% (L) N N ↑ LW (L, H) X (H)
Female Pubertal Rat (MRID 48672901) N N ↓ 26% (L) N N ↑ LW (H) X (H)

AMA (MRID 48988601) N N 4 Day 21: ↑ BW, SVL (L) N

OSRI Receptor Transactivation (Du et al. , 2010) P

Chronic Toxicity/ Carcinogenicity (Rat; MRID 00069701) N N ↑ LW; LE ( H) X (H)

28-Day Feeding (Rat; MRID 00120267) N N X (MH, H) X (H)

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

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 4. Day-21 normalized hind-limb length (HLL) was increased by 21-23%, however, it was not dose-dependent. Additionally, when Day-21 normalized HLL results were comp ared to the solvent control, no significant differences were observed P Positive findings N Negative findings NE Not examinedPERMETHRIN FINAL

Page 22 of 67 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of cyfluthrin to interact with endocrine related processes ( i.e., E, A, or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated an alysis of those line s of evidence.

The WoE evaluation includes consid erations of biological plausibility of the findings from the different lines of evidence by examining the cons istency, coherence and in terrelationships among the measured endpoints within and across studi es. The available findings from standard toxicology studies on the substance may contribute to the WoE evaluati on in helping el ucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively sequelae of systemic effects. E ndocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence eval uation of the results of the Tier 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues shou ld be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l disruptions in homeostasis” (USEPA, 2013).

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. Generally, one parameter (i .e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concen tration to be considered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were ge nerally less severe forms of toxicity (e.g., changes in orga n weights or blood chemistry).PERMETHRIN FINAL

Page 23 of 67 1. Tier 1 in vivo Assays

Overt and/or systemic toxicity was not observe d in the AMA at concentrations up to 0.68 μg a.i./L or in the Hershberger assay at doses up to 120 mg/kg/day. In the FSTRA, overt toxicity was noted at the highest tested concentration (2.6 μg a.i./L) as survival was reduced to 63% for males and 50% for females. Clinical signs of toxici ty were also noted in the surviving fish at the high concentration, including erra tic swimming, loss of equilibriu m and surfacing. Excess feed was also noted in the test chambers on Day 10 and Days 17-19.

In the female pubertal rat assay, systemic toxici ty was evident at the hi gh dose (150 mg/kg/day) based the 10% increase (p<0.05) in absolute liv er weights (both adjusted and unadjusted). Additionally, overt toxicity was noted at the high dose in the fe male and male pubertal assays. For females, tremors were observed in 4 animals at the low dose (75 mg/kg/ d) and in all animals at the high dose. In the male pubertal, three high dose (150 mg/kg/day) rats were found dead after 2 days of dosing; therefor e, the high dose was reduced to 120 mg/kg/day. At this reduced dose, one rat was considered uncoordinated on one day. Tremors were observed in 3 animals at the low dose (75 mg/kg/day) and mild to severe tremors in all animals at the high (120 mg/kg/day) at 6 h post-dosing. Additionally, absolute and relativ e (to body) liver weights were increased (p<0.05) by 6 and 11% at the low and high dose, respectively.

2. OSRI

In the three-generation reproduction toxicity study in rats, treatm ent-related clinical signs in high-dose parental animals were limited to w hole body tremors, occurring in all parental generations (exception: tremors we re not observed in the P males) during the first few days of the premating period. In the 2500 ppm groups, the incidence rates for the tremors were 20/24 P females, 11/12 and 24/24 F 1 males and females, respectively, and 12/12 and 24/24 F 2 males and females, respectively. Tremors were also observed in pregnant and lactating females exposed to 2500 ppm permethrin. Micros copic examination of F 3b weanlings revealed slight to moderate centrilobular hypertrophy, ranging from 0 to 80% for the males and from 10 to 100% for the
females.

In a developmental toxicity study in rats, clin ical signs of toxicity seen between GD 8-19
included tremors in 21/24 rats and head flic king in 6/24 rats in the high-dose group (150 mg/kg/day). Body weight gains and food c onsumption by the high-dose dams were less (p<0.05) than that of the c ontrols throughout the dosing interval. For GD 7-10, 10-13, and 13- 16, body weight gains were decreased by 88%, 32% , and 18%, respectively. Mean fetal body weight of the high-dose group was 3.2% (p ≤ 0.05) less than that of the controls.

In a developmental toxicity study in rabbits, a total of 0, 5, 5, or 4 does died or were sacrificedmoribund in the control, low-, mid- or high-dose groups, respectively. Due to the lack of a doseresponse, the deaths could not be definitively attributed to test article administration . Clinical signsPERMETHRIN FINAL

Page 24 of 67 of toxicity included body tremors observed in 5 of the high-dose (1800 ppm) animals. Body weight gains by the low- (600 ppm), mid- (1200 ppm) and high-dose groups were decreased by 79%, 50% and 91%, respectively, during GD 0-18 w ith significance (p<0.05) attained for the low- and high-dose groups. During the post-dosi ng interval, recovery of body weights was noted for the low- and mid-dose groups, but not for the high-dose group. Post-implantation loss was increased (p<0.05) in the mid- and high-dose groups to 155% and 248% of the control level. Correspondingly, the number of early and late resorptions was higher in these groups as compared to the control group values. Mean fetal body weights in the high-dose group were slightly ( ↓ 9%; NS) less than that of the controls and attributed to maternal body weight
decreases.

In a chronic toxicity/carcinogenic ity study in rats, treatment-rela ted tremors and hypersensitivity were observed in the high-dose (2500 ppm) male s and females during the first two weeks of the study. Liver changes suggestive of adaptiv e hypertrophy included increased aminopyrine- N

demethylase activity in all male treatment groups in the mid- (1000 ppm) and high-dose female at 52 weeks, and in the high-dose male and female groups at 104 weeks. This was coupled with modestly increased absolute and relative liver weights in the high-dose males and high and lowdose females at 52 weeks and in all male treatme nt groups and mid-dose females at 104 weeks. Further evidence for adaptive cha nges included hypert rophy of centrilobular hepatocytes with increased cytoplasmic eosinophili a in the mid- and high-dose male and females at 104 weeks’ exposure and increased smooth endoplasmic reti culum proliferation in all treatment groups except low-dose males at 52 weeks and high-dose groups at 104 weeks. Liver changes also included fatty vacuoles confirme d by electron microscopy in th e mid- and high-dose males at both 52 and 104 weeks and in the high-dose females at 104 weeks.

In a chronic toxicity study in dogs, neurological clinical sign s (tremors, uncoordinated gait, nervousness and convulsions, also excessive saliva tion and vomiting) were observed in the highdose group (1000 mg/kg/day). Other findings of systemic toxicity at the high-dose included: decreased body weight gains ( ↓ 37% males; ↓ 33% females), decreased food consumption (increased food left uneaten), increased liver weight ( ↑ 30% males; ↑ 36% females) and alkaline phosphatase levels ( ↑ 377% males; ↑ 220% females). At mid-dose (100 mg/kg/day), increased liver weight ( ↑ 25% both sexes) and alkali ne phosphatase levels ( ↑ 134% males; ↑ 99% females) were observed. Microscopic evaluation of the ad renals showed focal degeneration and necrosis in the cortex with variable inflammatory cell in filtration along with swel ling and vacuolization of the cells in the inner cortex at high-dose males and females (5/5 males and 4/6 females) and at mid-dose males (1/5). The liver also showed hepatic cellular swelling in the mid- and high-dose
males and females.

In a 28-day feeding toxicity study in rats, one fe male and four males died by Day 18 at the high dose (5000 ppm). Surviving rats receiving 2500 ppm and above became hypersensitive during
the first week of treatment and remained so throughout the study. Body weight of the 5000 ppmPERMETHRIN FINAL

Page 25 of 67 males was 16-22% below that of controls throughout the treatmen t period, and overall body weight gain of that group was decreased by 31% . Liver weight was increased 28-33% in the 2500 and 5000 ppm females.

In an avian reproduction toxicity study in quail, systemic toxicity was not observed at the highest dose tested (500 ppm). However, in an avia n reproduction toxicity study in ducks, feed consumption was slightly decreased throughout the study, with a signif icant difference from controls during Weeks 6, 12, 14 and 19 at the high dose (500 ppm).

In a fish full life-cycle toxici ty study on fathead minnows, mean measured concentrations of 0.41 ppb significantly reduced the percen t survival of fry during the ini tial 30 days of exposure in the first and second generations. However, minnow s which survived the initial exposure period demonstrated normal ranges of measured parame ters of survival, growth, reproduction and egg hatchability.

In the published uterotrophic and He rshberger studies in rats by Kim et al . (2005), systemic toxicity was not mentioned despite dosing the females at up to 800 mg/kg/day via s.c. injection and the males at up to 100 mg/kg/day via oral gavage. However in a similar study by Kunimatsu
et al . (2002), tremors were observed in 3-6/6 female rats at th e highest dose tested (150 mg/kg/day) and in 2-6/6 male rats at the highest dose tested (75 mg/kg/day). Systemic toxicity was also not observed in the publishe d Hershberger study on rats by Zhang et al . (2008) at a single dose level of 50 mg/kg/day (oral dosing). In two other published testic ular toxicity studies (Y. Jin et al ., 2012, and Wang et al ., 2012), systemic toxicity was not noted in mice dose orally at up to 100 and 35 mg/kg/day, respectively. In a published study by Nillos et al . (2010) in medaka, a slight decrease in survival wa s observed at a concentration of 10 μg/L.

B. Estrogen Pathway

A number of OSRI in vitro assays evaluating estrogen receptor activation resulted in conflicting results, i.e . some showed activation of estrogen signali ng while others did not. The OSRI assays included estrogen receptor (ER) bi nding, cellular prolifer ation of estrogen sensitive MCF-7 cells, expression of estrogen sensitive pS2 mRNA in th e cancer cells, and interaction with either ER α or ER β in transfered HeLa cells. Permethrin was negative in the Tier I in vitro aromatase assay, whereas in the steroidogenesis assay permethrin caused an increase (p<0.05) in estradiol production. In vivo , permethrin was negative in both the uterotrophic and female pubertal rat assays, with no effects on uterine weight, mean age at vaginal opening, percent cycling, or histopathological changes in th e ovaries and uterus. In the fi sh short-term reproduction assay (FSTRA), all effects in females were seen only at a dose that caused overt toxicity (i.e, mortality, clinical signs of toxici ty). With the exception of one reproductive study in mallard duck which had a slight reduction in the number of eggs laid by the hen (which has lim ited ability to inform about potential estrogen-related effects by itself and decrea ses in food consumption also observed at this concentration), no estr ogen-related effects were noted in the in vivo studies inPERMETHRIN FINAL

Page 26 of 67 mammals, fish, or birds that occurred in the ab sence of overt and/or systemic toxicity. Therefore, although several of the in vitro studies [estrogen re ceptor (ER) binding, ER transactivation assay (ERTA), and steroidogenesis] indicated that permethrin had the potential to affect the E pathway at the cellular level, these effects are not s upported by the available in vivo
studies.

C. Androgen Pathway

For AR binding, conflicting resu lts were reported across the in vitro assays with different cell
lines. In an in vitro assay, permethrin reduced sperm motility in a concentration-dependent manner (1-64 μmol/L). Multiple Hershberger assays were available for permethrin, in which the Tier 1 assays as well as an addi tional open literature study (Kunimatsu, et al ., 2002) were negative at doses up to 75 or 120 mg/kg/day. However, in another OSRI Hershberger assay (Kim et al ., 2005), significant decreases in AST weights were reported at doses of ≥ 10 mg/kg/day.

In the male rat pubertal assay, no androgen-relate d effects were observed. While there does not appear to be androgen-related eff ects in rats with an intact HPG axis (i.e. male pubertal and Part 158 rat data); published results in the literature (Jin et al ., 2012, Wang et al ., 2012) reported that permethrin caused testicular toxicity in mice following oral exposure at a dose of 35 or 100 mg/kg/day. In these studies, testicular toxicity manifested as changes in testosterone levels, decreased testicular and epididym al weights, changes to the epididymal duct, regression of sperm quality and histopathologi cal lesions in the testes. In the FSTRA, all effects in males were only observed at the high concentration in the presence of overt toxicity.

Therefore based on the available in vitro and mammalian in vivo data, there appears to be a potential interaction with the androgen pathway in mammals. There was no convincing evidence of a potential interaction with the androgen pathway in wildlife.

D. Thyroid Pathway

There was no convincing evidence of an interacti on of permethrin with the thyroid pathway in
mammals or wildlife in the absence of overt toxic ity. The only thyroid-related effect observed in the absence of overt toxicity was a decrease in serum T4 levels in the male and female pubertal assays. No thyroid related effects were obser ved in the mammalian Part 158 studies. No treatment-related thyroid effects were seen in the AMA.PERMETHRIN FINAL

Page 27 of 67 E. Conclusions

There is no convincing evidence of potential interaction with the estrogen or thyroid pathways in mammals or wildlife. There is evidence for poten tial interaction with the androgen pathway in mammals. There is no convincing evidence for a potential interaction with the androgen pathway in wildlife.

V. EDSP Tier 2 Testing Recommendations

For permethrin, a point of departure (POD) of 25 mg/kg/day for human health risk assessment is
based on neurotoxic clinical signs (abnormal and/or decreas ed movement, increased body temperature, and aggression) observed at 75 mg/ kg/day (LOAEL) in an acute neurotoxicity study in rats. This POD is used for deriving the chro nic Reference Dose (RfD) for chronic dietary risk assessment. A second POD of 11 mg/kg/day ba sed on clinical signs of toxicity (body temperature and hypersensitivity to noise) s een at 154 mg/kg/day (LOAEL) in a subchronic inhalation toxicity study in rats. This POD is used for calculating the Margins of Exposure (MOEs) for non-dietary risk assessment. These P ODs are lower than the dose (100 mg/kg/day)
that caused the testicular effects in mice. Consequently, the current RfD and MOEs are protective of the androgen-mediate effects observed in the published literature study. Additionally, there was no evidence of interaction with the androgen pathway in the EDSP Tier 1 assays. Therefore, there is no concerns for the findings reported in the literature study.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not recommended for permethrin since additional testing is not expected to impact EPA’s current regulatory point of departur es and endpoints for human h ealth risk assessments.

Given the lack of evidence for a potential intera ction with the estrogen and androgen pathway in fish and thyroid pathway in amphibians, no additional testing is recommended.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for permethri n. In the bobwhite quail, no repr oductive effects were observed, but effects on reproductive parameters were obs erved in the mallard duck. The type of data
obtained from Part 158 avian re production studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from pe rmethrin exposure. Relative to the EDSP, additional testing is not recommended.PERMETHRIN FINAL

Page 28 of 67 VI. References

(Authors not provided) (1977a) Chronic toxi city of FMC 33297 to the fathead minnow ( Pimephales promelas ). E. G. and G. Bionomics, Aqua tic Toxicology Laboratory, Wareham, MA. Sponsored by FMC Corporaton, Prince ton, NJ. FMC Study No. ACT092.72, October 1977. MRID 00102096. Unpublished.

Bauer, E, Bitsch, N, Brunn, H, et al . (2002) Development of an immuno-immobilized androgen receptor assay (IRA) and its application for the characterization of recep tor binding affinity of different pesticides. MRID 48176001. Published: Chemosphere 46 : 1107 + 1115.

Beavers, J, Foster, J, Lynn, S, et al . (1992) Permethrin: A on e generation reproduction study with the northern bobwhite ( Colinus virginianus ). Wildlife lnternat ional Ltd., Easton, MD. Sponsored by FMC Corporaton, Princeton, NJ . Laboratory Project No. 104166, May 20, 1992. MRID 42322901. Unpublished.

Beavers, J, Foster, J, Lynn, S, et al . (1992). Permethrin: A one generation reproduction study with the Mallard ( Anas platyrhynchos ) Wildlife lnternational Lt d., Easton, MD. Sponsored by FMC Corporaton, Princeton, NJ. Laborat ory Project No. 104166, May 20, 1992. MRID 42322902. Unpublished.

Brander, S, He, G, Smalling, K, et al . (2012) The in vivo estrogenic and in vitro anti-estrogenic activity of permethrin and bifenthrin. Environ. Toxicol. Chem. 31 (12) : 2848-2855.

Chen, H, Xiao, J, Hu, G, et al . (2002) Estrogenicity of organophosphorous and pyrethroid pesticides. MRID 48160705. Published: J. Toxicol. Environ. Health, Part A. 65: 1419-1435.

Clapp, M, Banham, P, Chart, I, et al . (1977) PP557: 28-Day feeding study in rats. Imperial Chemical Industries, Ltd., Central Toxicology Laboratory (location no t given). Laboratory Report No. CTL/P/355, December 20, 1977. MRID 00120267. Unpublished.

Du, G, Shen, O, Sun, H, et al . (2010) Assessing hormone recep tor activities of pyrethroid
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Garey, J, and Wolff, M (1998) Estrogenic a nd antiprogestagenic activities of pyrethroid
insecticides. MRID 48176005. Published: Biochem. and Biophys. Res. Comm. 251 : 855-859.

Go, V, Garey, J, Wolff, M, et al . (1999) Estrogenic pot ential of certain pyrethroid compounds in
the MCF-7 human breast carcinoma cell line. MRID 48176004. Published: Environ. Health Perspect. 107 (3) : 173-177.PERMETHRIN FINAL

Page 29 of 67 Hodge, M (1988) Permethrin: teratogenicity study in the rat. IC I Central Toxicology Laboratory, Alderley Park Macclesfield, Ches hire, UK. Laboratory Study No. CTL/P/2269, September 20, 1988. MRID 40943603. Unpublished.

Hodge, M, Banham, P, Glaister, J, et al. (1977) Permethrin (PP557) : 3-generation reproduction study in rats. ICI Central Toxicology Laborator y, Alderley Park, Maccle sfield, Cheshire, UK. CTL Study Number: RR0015, December 1977. MRID 00120271. Unpublished.

Jin, M, Li, L, Xu, C, et al . (2010) Estrogenic activities of tw o synthetic pyrethroids and their
metabolites. J. Environ. Sci. (China) 22 (2) : 290-296.

Jin, Y, Chen, R, Sun, L, et al . (2009) Enantioselective induction of estrogen-responsive gene expression by permethrin enantiomers in embryo-larval zebrafish. Chemosphere 74 (9) : 1238-

1244.

Jin, Y, Liu, J, Wang, L, et al . (2012) Permethrin exposure duri ng puberty has the potential to enantioselectively induce reprodu ctive toxicity in mice. Environ. Int. 42 : 144-151.

Kalinowski, A.E, et al . (1982) Permethrin: one year oral dosing study in dogs . ICI Central Toxicology Laboratory Alderley Park, Macclesfi eld, Cheshire, UK. Laboratory Study No. CTL/P/647, February 24, 1982. MRID 00129600. Unpublished.

Kim, S, Lee, R, Lim, K, et al . (2005) Potential estrogenic and a ndrogenic effects of permethrin
in rats. MRID 48176002. Published: J. Reprod. Dev. 51 (2) : 201-210.

Kim, Y, Shin, J, Kim, H, et al . (2004) Assessing estrogenic activity of pyrethroid insecticides using in vitro combination assays. MRID 48176007. Published: J. Reprod. Dev. 50 (2): 245-

255.

Kunimatsu, T, Yamada, T, Ose, K, et al . (2002) Lack of (anti-) andr ogenic or estrogenic effects of the three pyrethroids (esfe nvalerate, fenvalerate, and permethrin) in the Hershberger and uterotrophic assays. MRID 48176008. Published: Regul. Toxicol. Pharm

. 35: 227-237.

Lemaire, G, Minf, W, Mauvais, P, et al . (2006) Activation of α - and β -estrogen receptors by persistent pesticides in reporter cell lines. MRID 48074114. Published Life Sciences 79 : 1160-
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Nillos, M, Chajkowski, S, Rimoldi, J, et al . (2010) Stereoselectiv e biotransformation of permethrin to estrogenic metabolites in fish. Chem. Res. Toxicol. 23 (10) : 1568-1575.

Richards, D, Banham , P, Chart, I, et al . (1977) PP557: Two year feeding study in rats. Imperial Chemical Industries Limited, Central Toxicol ogy Laboratory. Sponsored by FMC Corporaton,PERMETHRIN FINAL

Page 30 of 67 Saito, K, Tomigahara, Y, Ohe, N, et al . (2000) Lack of significant es trogenic or anti-estrogenic activity of pyrethroid insecticides in three in vitro assays based on clas sic estrogen receptor α

related mechanisms. MRID 48176003. Published: Toxicol. Sci. 57 : 54-60.

Schneider, S, Kendall, T, and Kruger, H (2012) Permethrin: Amphibian Metamorphosis Assay for the Detection of Thyroid Active Substan ces. Wildlife Internati onal, Ltd., Easton, MD. Sponsored by Consumer Specialty Products Associ ation, For the Permethrin Data Group II Task Force Steering Committee/Joint Venture, Wash ington, DC. Laboratory Report No. 701A-109A, November 6, 2012. MRID 48988601. Unpublished.

Schneider, S, Martin, K, Kendall, T et al . (2012) Permethrin: fish short-term reproduction assay with the fathead minnow ( Pimephales promelas ). Wildlife In ternational, Ltd., Easton, MD. Sponsored by Consumer Specialty Products Associ ation for the Permethrin Data Group II Task Force Steering Committee/Joint Venture, Wa shington, DC. Laboratory report No. 701A-108, September 11, 2012. MRID 48702301 Unpublished

Tyler, C, Beresford, N, Van der Woning, M, et al . (2000) Metabolism and environmental degradation of pyrethroid inse cticides produce compounds with endocrine activities. MRID
48213601. Published: Environ. Toxicol. Chem. 19 (4) : 801–809.

USEPA (2010) Permethrin: Report of the E ndocrine Disruptor Review Team; Test Order # EDSP- 109701-83, 84, 85, 87, 89, 90 and 91, December 6, 2010. Office of Chemical Safety and
Pollution Prevention. U.S. Environmen tal Protection Agency, Washington, DC.

USEPA (2011) Endocrine Disrupt or Screening Program – Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention Science Coor dination and Policy. U.S. Environmental Protection Agency, Washington, DC.

USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
held July 30 - August 1, 2013 on Weight-of

- Evidence: Evaluati ng Results of EDS P Tier 1 Screening. Office of Chemical Safety and Pollution Pr evention. U.S. Environmental Protection Agency, Washington, DC.

Wagner, H (2011) Permethrin: H295R steroi dogenesis assay. CeeTox, Kalamazoo, MI. Sponsored by Consumer Specialty Products Associ ation for the Permethrin Data Group II Task Force Steering Committee/Joint Venture, Wash ington, DC. Laboratory Report No.: 9146V100383STER, November 17, 2011. MRID 48618603. Unpublished.

Wang, D, Kamijima, M, Okamura, A, et al . (2012) Evidence for diazinon-related inhibition of cis-permethrin metabolism and its effects on reproductive toxicity in adult male mice. Reprod.
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Page 31 of 67 Wilga, P (2011) Permethrin: human recombinant aromatase assay. CeeTox, Inc., Kalamazoo, MI. Laboratory Report No. 9146V-100383A ROM, November 17, 2011. MRID 48618601. Unpublished.

Xu L, Liu L, Ren X, et al . (2008) Evaluation of androgen recept or transcriptiona l activities of some pesticides in vitro . MRID 48176006. Published: Toxicology 243 (l-2) : 59-65.

Zhang, J, Zhu, W, Zheng, Y, et al . (2008) The anti-androgenic activ ity of pyrethroid pesticides cyfluthrin and β -cyfluthrin. MRID 48196302. Published: Reprod. Toxicol. 25: 491-496.

Zorrilla, L (2011) The Hershberger bioassay for pe rmethrin. Integrated Laboratory Systems, Inc., Durham, NC. Sponsored by Consumer Specia lty Products Associati on for the Permethrin Data Group II Task Force Steering Committee/Jo int Venture, Washington, DC. Laboratory Project Study ID: C195-200, November 15, 2011. MRID 48618602. Unpublished.

Zorilla, L (2012) Pubertal development and thyr oid function in intact juvenile/peripubertal female rats; permethrin. Integrated Laborat ory Systems, Inc., Durham NC. Sponsored by Consumer Specialty Products A ssociation for the Permethrin Data Group II Task Force Steering Committee/Joint Venture, Washi ngton, DC. Laboratory Report No.: C195-300, April 13, 2012. MRID 48672901. Unpublished.

Zorrilla, L (2012) Pubertal development and thyroid function in intact juve nile/peripubertal male rats with permethrin. Integrated Laborato ry Systems, Inc., Durham, NC. Sponsored by Consumer Specialty Products A ssociation for the Permethrin Data Group II Task Force Steering Committee/Joint Venture, Wash ington, DC. Laboratory Study No.: C195-800, September 5, 2012. MRID 48687101. Unpublished.PERMETHRIN FINAL

Page 32 of 67 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis A ssay; (AMA, OCSPP 890.1100)

The 21-day assay (MRID 48988601) of permethrin (97.7% purity) on amphibian metamorphosis of African clawed frogs ( Xenopus laevis ) was conducted under fl ow-through conditions. Amphibian larvae at developmental Nieuwkoop-Fa ber Stage (NF) 51 (80; 20/replicate) were exposed to control (water only; negative), a solvent control (0.020 mL/L dimethylformamide; DMF), and permethrin at nominal con centrations of 0.00011, 0.00033, and 0.0010 mg a.i./L;
mean-measured concentrations were <0.0000600 (<LOQ; controls), 0.000052, 0.00022, and 0.00063 mg a.i./L. The reviewer-calculated time- weighted average concentrations were <0.0000600 (<LOQ; controls), 0.000054, 0.00022, and 0.00068 mg a.i./L. The test system was maintained at 21.5 to 23.0°C and a pH of 7.9 to 8.2.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. Two non-incidental mortalities were observed in the test, one each in the low and high treatment groups. Clinical signs included ta il curvature, which was observe d in both controls and all treatment groups (12-28% of individuals), with no indication of a dos e-dependent pattern.

Compared to the negative control, permethr in significantly promoted Day 21 normalized hindlimb length (HLL) at the low and mid treatment groups (by 21% and 23% respectively). There were no effects on normalized HLL on Day 7. However, there was an apparent solvent effect (21% stimulation of normalized HLL, p=0.01) rela tive to the negative control at Day 21. When Day 21 normalized HLL results were compared to th e solvent control, no significant differences were observed. There was no significant acceleration or delay of median NF developmental stage at Day 7 or 21 for any treatment level. Furthe r, no asynchronous development was observed. Compared to the negative control, there was a statistically significant increase in Day 21 wet weight and snout-to-vent length (S VL) at the low treatment group. No solvent effect was seen for

SVL.

Thyroid gland histopathology obs ervations included thyroid gland atrophy and hypertrophy, follicular cell height increases and decreases, a nd/or follicular cell asymmetry in both controls and all treatment groups. These effects were of low frequency and did not exhibit a concentration-dependent response; therefore, they are not considered to be treatment-related.

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

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

, 2000; Zhang et al., 2008b; Kim et al., 2005); see Appendix 2.PERMETHRIN FINAL

Page 33 of 67 Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48618601), permethrin (97.7% a.i., Lot # PMPS000014) was incubated with human recombinant aromatase and tritiated androstenedione (1- β [ 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) in dimethyl su lfoxide (DMSO) at log
concentrations of 10

− 10 to 10

− 3 M for 15 minutes to assess the poten tial of permethrin to inhibit aromatase activity.

Aromatase activity was determined by measuring the amount of tritiated water produced at the
end of a 15-minute incubation for each concentration of chemical. Tritia ted water was quantified using liquid scintillation counting (LSC). Th ree runs were conducted and each run included a full activity control, a bac kground activity control, a pos itive control series (10

− 10 to 10

− 5 M) using a known inhibitor (4-hydroxyandrostenedi one; 4-OH ASDN), and the permethrin series (10

− 10 to 10

− 3 M) with three repetitions per concentration.

Aromatase activity in the full activity controls was 0.588 ± 0.079 nmol·mg-protein

− 1 ·min

− 1 . The response of each full activity c ontrol within a run was between 90 to 110% of the average full activity, and the activity in the background c ontrols ranged from 0.21 to 0.31% and averaged 0.28% of the full activity. For the positive cont rol substance (4-OH ASDN), aromatase activity results were within the recommended ranges for th e performance criteria. The estimated log IC 50 for 4-OH ASDN averaged

− 7.3 M and the Hill slope was

− 1.0.

Precipitation was noted at the two highest c oncentrations of permethrin tested (10

− 4 and 10

− 3 M); therefore, the maximum concentratio n used for data analysis was 10

− 4.5 M. The lowest portion of the response curve was greater than 75% activity at all soluble concentrations of permethrin. Aromatase activity averaged 101.3% and 101.9% activity at concentrations of 10 -10 and 10

− 4.5 M permethrin, respectively, indicati ng that permethrin did not inhi bit aromatase activity in this

assay.

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

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

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

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

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

Page 34 of 67 Fish Short Term Reproduction Assay; (FSTRA, OCSPP 890.1350)

The 21-day short-term reproduction assay (M RID 48702301) of permethrin with fathead minnow ( Pimephales promelas ) was conducted under flow-through conditions. Adult fish (6 months old, 2 males and 4 females per replicate) were exposed to permethrin (97.7% purity) at
nominal concentrations of 0 (negative and solvent c ontrols), 0.00033, 0.00099, and 0.0030 mg a.i./L. Time-weighted average (TWA) con centrations were <0.000150 (<LOQ; controls), 0.00022, 0.00075, and 0.0026 mg a.i./L. The solvent cont rol consisted of dimethylformamide (DMF) at a final concentration of 0.02 mL/L. The test system was maintained at 24.6 to 25.8°C and a pH of 7.9 to 8.1.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. Survival in the negative cont rol and the low and mid treatment groups was 100%, but there was 63% survival for males (5 of 8) an d 50% survival for females (8 of 16) at the high
treatment concentration. A dditionally, clinical signs of toxic ity occurring in the high treatment group included erratic swimming (5 fish), loss of equilibrium (2 fish), and surfacing (2 fish).
There were also observations of excess feed noted in the test chambers of the high treatment group on Day 10 and Days 17-19. These observations indicate that overt to xicity was present during the course of this study at the high treatment concentration.

Fecundity in the negative contro l averaged 19.8 eggs/female/reproduc tive day with all replicates having at least 15 eggs/female/ reproductive day; spawning occurred at least every 4 days. Fertilization success in the negative control av eraged 95.4%. Permethrin had no significant effect on fecundity or fertility in any treatment group relative to the negative control (p>0.05). While not statistically si gnificant (p>0.05), there wa s a 42% decrease on fecundity at the high treatment
concentration.

Plasma vitellogenin (VTG) concentrations were comparable between the negative control and treatment groups for both sexes. While not statis tically significant (p> 0.05), there was a 37.8% reduction in VTG as compared to the negative control at the hi gh treatment concentration. The data from this treatment group were based on thr ee replicates and not four (as the rest of the treatment groups) as 3 of 4 female fish from Re plicate A died during the course of the study.

Effects on gonadal histopathology were observed in female fish at the high treatment level, including increased post-atretic follicles, decrea sed post-ovulatory follicles, and granulomatous inflammation. Microsporidia were observed in one fish each of the control and treatment
concentrations that showed a minimal to mild severity of granulomatous inflammation. There were no effects of treatment on gona dal histopathology in male fish.PERMETHRIN FINAL

Page 35 of 67 A significant increase (p<0.05) in gonado-somatic index (GSI) was detected at the high treatment level for both sexes ( ↑ 48.9% for males and 28.4% for female s). Nuptial tubercle score was significantly decreased in males at the high concentration (<0.05) . Permethrin exposure was not associated with any significant changes (p>0.05) in body weight or le ngth for either sex.

Other clinical signs ( i.e. , behavioral and other sublethal e ffects) including bloody lower jaw and weakness were observed in 1 to 3 of the 8 surviving females at the high treatment level.

Hershberger Assay; (OCSPP 890.1400)

In a Hershberger assay (MRID 48618602) screeni ng for androgenic activity, permethrin (97.7% a.i., Lot# PMPS000014) in corn oil was administered daily via oral gavage to groups of 59 to 60- day old, castrated male Sprague Dawley rats (8/g roup) at dose levels of 0 (vehicle), 37.5, or 120 mg/kg/day. An androgenic positive control gr oup consisting of eight cas trated rats exposed to 0.4 mg/kg/day testosterone propionate (TP) by s ubcutaneous (s.c.) injec tion was also included as a positive control.

To screen for potential anti-andr ogenic activity, permethrin in corn oil was also administered daily via oral gavage to 59 to 60-day old, castrated male Sprague Dawley rats (8/group) at dose levels of 12, 37.5, 120 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. Th e control group consisted of ei ght castrated male rats dosed daily with TP (0.4 mg/kg/day) by s.c. injecti on in corn oil, and the anti-androgenic positive control group consisted of eight cas trated male rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg/day in conjunction with a daily s.c. dose of TP (0.4 mg/kg/day).

For both components of the assay, the animal s were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration to determine weights of the five androgen-dependent tissues.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in the permethrin dose groups of the androgen agon ist or antagonist assa ys. Body weights and overall body weight gains in th e treated groups were similar to controls in both the androgen agonist and anti-androgen assays.

In the androgen agonist assay, animals dosed with TP had increased (p<0.05) body weights ( ↑ 12%) on Day 11, and increased (p< 0.05) overall body weight gains ( ↑ 110%) compared to controls. No significant increa ses in the organ weights were observed in any of the five accessory sex tissues of animals dosed with perm ethrin. Animals in the TP group had increases (p ≤ 0.05) in accessory sex organ weights as follows: 1002% in seminal vesicles; 898% in ventral prostate; 187% in levator ani- bulbocavernosus (LABC); 486% in Cowper’s gland; and 51% inPERMETHRIN FINAL

Page 36 of 67 glans penis. For the vehicle control, the ma ximum guideline-specified %CV was exceeded for the LABC (45% vs. 30%). All other %CV values were less than the maximum permissible
values.

In the anti-androgen assay positive control group (TP + FT), overall body weight gains were decreased (p ≤ 0.05) by 35%. No significant decreases in the organ weights were observed in any of the five accessory sex tissues of animals dosed with permethrin+TP. Animals dosed with TP + FT (positive control) had decreased (p ≤ 0.05) accessory sex organ weights as follows: 84% in seminal vesicles; 77% in ventra l prostate; 60% in LABC; 62% in Cowper’s gland; and 20% in glans penis. The maximum guideline-specified %CV was exceeded for the Cowper’s gland in the 120 mg/kg/day permethrin group (22% vs. 20%). All other %CV values were less than the maximum permissible values.

The dosing for this study (high- dose of 120 mg/kg/day) was consid ered adequate based on data
from two earlier studies showi ng either mortality at doses ≥ 200 mg/kg/day or clinical signs of neurotoxicity at 150 mg/kg/day.

Statistically significant changes were not seen in two or more of the five androgen sensitive tissue weights. Permethrin was negative fo r androgenicity and anti-androgenicity in the Hershberger assay.

Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID48672901), 16 Sprague-Dawley (Crl:CD ® [SD] IGS) female rats/dose group were treated daily via oral gavage with permethrin (Lot # PMPS00014, 97.7% a.i.) at doses of 0 (corn oil) 75, or 150 mg/kg/ day from post-natal day (PND) 22 to 42/43. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at attainment of VO were recorde d. Following sacrifice on PND 42/43, blood was collected for clinical chemistry analyses, including analysis of total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) using a radioimmunoassay (RIA). Adrena l, liver, thyroid, pituitary and urogenital organ weights were recorded, a nd microscopic examinations were performed on the ovaries, uterus, t hyroid, and kidneys.

All rats administered permethrin survived until schedule termination. From PND 34 onward,
tremors were observed in 4 animals administered 75 mg/kg/day and all an imals administered 150 mg/kg/day at 4-6 hours post-dosing on at least one day. These findings did not persist to the daily examinations, which were conducted just prior to dosing.PERMETHRIN FINAL

Page 37 of 67 No treatment-related effects were observed on body weights, body weight gains, age of attainment of VO, or weight at day of attainme nt at any dose. All su rviving animals attained complete VO.

Absolute liver weights (adjus ted and unadjusted) were increased (p<0.05) by 10% at 150 mg/kg/day. Relative (to body weight ) liver weights were increase d (p<0.05) by 4 and 8% at 75 and 150 mg/kg/day. Both unadjusted and adjusted thyroid weights were decreased (p<0.05) by 22% at 150 mg/kg/day.

Administration of permethrin did not change the day of first estrus compared to the vehicle control group. Animals exhibited similar cycle lengths and regular ity. The majority of females in the 150 mg/kg/day group were in diestrus at necropsy.

Serum T 4 levels were decreased (p<0.05) by 26 and 42% at 75 and 150 mg/ kg/day, respectively, and, serum TSH levels were increased (p<0.05) by 40% at 150 mg/kg/day. Additionally, at 150 mg/kg/day, increases (p<0.05) we re observed in sodium ( ↑ 3%), chloride ( ↑ 4%), and sorbitol dehydrogenase (SDH; ↑ 20%), alkaline phosphatase (ALP) was decreased ( ↓ 16%).

There were no effects of treatment in the thyroid on either colloid area or follicular cell height in any treated group. No effects of treatment were observed microscopically in the ovaries, uterus or kidney.

The dose levels tested were determined to be adeq uate since they were based on the results of the range finding study where mortality was seen at doses greater than 200 mg/kg/day and tremors at 150 mg/kg/day.

Male Pubertal A ssay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48687101), 16 Sp rague-Dawley [Crl:CD( SD) IGS] rats/dose group were treated daily via oral gavage with permethrin (97.7% a.i., Lot # PMPS00014) in corn oil at doses of 0, 75, or 150 mg/kg/day from postnatal day (PND) 23 to 53/54. However, three 150 mg/kg/day rats were found dead after two da ys of dosing. Therefore, the high dose was reduced to 120 mg/kg/day on PND 25. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and age and weight on day of attainme nt were recorded. Following sacrifice on PND 53/54, blood was collected for clin ical chemistry and hormone analyses. Total serum testosterone, thyroxine (T 4 ) and thyroid stimulating ho rmone (TSH) levels were determined using radioimmunoassays. Adrenal, liv er, pituitary, thyroid, and urogenital organs were weighed and examined macroscopically. The left testes, left epidid ymides, left kidney, and thyroid were examined microscopically.PERMETHRIN FINAL

Page 38 of 67 No treatment-related related effects were obser ved on body weights, body weight gains, age or weight at attainment of PPS, organ weights (except liver), cl inical chemistry, and gross or histopathology parameters.

After reduction of the high dose to 120 mg/kg/ day, all remaining animals survived to study termination. At 6 hours post-dose, tremors we re observed at 75 and 120 mg/kg/day in a dosedependent manner, with incidence, frequency, and severity increasing with dose. At 75 mg/kg/day, three animals exhibited mild tremor s on one day and one animal exhibited mild tremors on two days. At 120 mg/kg/day, very m ild to severe tremors were observed in all surviving animals on several days. Additio nally, one 120 mg/kg/day rat was considered uncoordinated on one day.

Serum testosterone levels in the treated groups were similar to controls. Mean serum T 4 levels were decreased (p<0.001) by 28 and 42% at 75 and 120 mg/kg/day, respec tively, and serum TSH level was increased (p<0.05) by 77% at 120 mg/kg/day. The T 4 and TSH values were all within or just below their respective reference ranges. Additionally, there were no concomitant effects observed on thyroid weights or thyroid follicular cell height or colloid area. Absolute and relative liver weight were incr eased (p<0.05) by 6-11% at 75 a nd 120 mg/kg/day. There were no changes in clinical chemistry parameters that suggested hepatotoxicity due to permethrin administration. The increased liver weights were considered to be the result of hypertrophy and associated microsomal enzyme inducti on, a common finding with permethrin.

The 150 mg/kg/day dose was an excessive dose le vel as three rats were found dead on Day 2. Therefore, the dose was lowered to 120 mg/kg/ day which was an adequate high dose based on the clinical signs (tremor and in coordination) seen in these animals. Tremors were also noted at 75 mg/kg/day.

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48618603) H295R cells cultured in vitro in 24-well plates were incubated with permethrin (97.7% a.i. ; Batch PMPS000014) at concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.05%.

Testosterone and estradiol le vels were measured using HPLC/MS-MS. Three independent experiments were performed (Run #s 2-4). A Qu ality 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 concen tration levels; positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol and testos terone production.PERMETHRIN FINAL

Page 39 of 67 Guideline acceptability recommendations and requirements were met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptabl e reproducibility (low %CV) and appropriate induction and inhibition with pos itive controls.

No cytotoxicity was observed over the range of tested permethrin concentrations. However, precipitation of the permethr in was observed at the 100 μ M concentration in each run and at

10 μ M in Run #2. Accordingly, the data from thes e concentrations were not included in the analysis of the respective runs. For permethr in, there was a statisti cally significant (p<0.05) increase in testosterone pr oduction at 10 μM in Runs #3 a nd #4 (average 1.14-fold), and a statistically significant increase in estradiol production at 1 and 10 μM in Runs #3 and #4 (average 1.16-fold for 1 μM and 1.51-fold for 10 μM).

Based on hormone responses in two of the three independent runs, permethrin treatment resulted in statistically significant and re producible increases in testoste rone and estradiol production.

Uterotrophic Assay; (OCSPP 890.1600)

Requirement satisfied by OSRI (Kim et al ., 2005; Kunimatsu et al ., 2002); see Appendix 2

.PERMETHRIN FINAL

Page 40 of 67 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

ER Transactivation and Binding Assays

In a published study by Saito et al. ( MRID 48176003 ), the estrogenic and anti-estrogenic activity of permethrin (>93% pur ity) was evaluated using three in vitro assays based on hER α

related mechanisms: 1) a mammalian cell-based lu ciferase reporter assay, 2) a yeast two-hybrid method, and 3) a competitive ligand-binding assa y. For the luciferase reporter assay, HeLa cells were transiently transfected with an e xpression vector, a reporter plasmid for h ER α , and a luciferase control plasmid. Permethrin was added to the medium at concentrations of 100 nM to 10 μM. For anti-estrogenic activ ity measurements, 100 pM of E 2 was incubated along with permethrin. For the yeast two-hybrid assay, yeas t transformants were grown overnight and then treated with permethrin at 10 μM (maximum so lubility). For antagonist assays, 100 pM of E 2 was incubated with permethri n. After incubation, induced β -galactosidase was measured by chemiluminescent detection. Competitive binding to h ER α was assayed by a fluorescence polarization method. Test chemical-dependent displacement of a labeling ligand, fluoromone ES1, from ER α was measured by changes in fluorescence anisotropy. Dose-dependent displacement of fluormone ES1 from h ER α was detected with the positive controls DES, p

nonylphenol, and HTM.

In the luciferase reporter assay, permethrin was tested at a maximum dose of 30 μM and no statistically significant estrogeni c or anti-estrogenic activity wa s detected. No statistically significant estrogenic or antiestrogenic activity fo r permethrin was detected in the yeast twohybrid assay or the receptor binding assay. The lack of statistically significant effects at concentrations of 100 nM-10 μM in dicates that permethrin is not estrogenic or anti-estrogenic by classic ER-related pathways in vitro

.

Recombinant Yeast Assay

In a published study by Tyler et al. (MRID 48213601) , permethrin was tested for estrogen and androgen receptor binding activity (both agonist and antagonist) using a recombinant yeast assay. Two yeast strains were st ably transfected to express eith er the human ER (hER) or the AR (hAR) genes along with the lacZ reporter gene. In addition to permethrin, three major derivatives of permethrin resulting from meta bolism and/or environmental degradation were similarly tested: cyclopropane permethrin aci d, 3-PBOH, and 3-phenoxybenzoic acid (3-PBA). Permethrin from four separate chemical supplie rs was tested (purities 97.3-99.4%). In screens for agonistic activity, E 2 and 5 α -dihydrotestosterone (DHT) were used as positive controls. The positive controls in the anti-estrogen and an ti-androgen screens were hydroxytamoxifen and flutamide, respectively. EC 50 s and IC 50 s were calculated relative to the reference standards, and for all chemicals inducing a response, LOEC (agonist) and/or LOIC (antagonists) were
determined relative to the reference standards.PERMETHRIN FINAL

Page 41 of 67 Permethrin was a very weak estrogen agonist w ith potencies between seve n and eight orders of magnitude less than E 2 . In the anti-estrogen screen, increasing concentr ations of permethrin produced a progressive increase in the E 2 -stimulated background absorbance, indicating an additive estrogenic response. In the hAR assa y, very high concentrations of permethrin (~10

− 3 M) induced slight increases in activity above baseline values, thus indicating very weak agonist activity. Permethrin also exhibi ted weak anti-androgenic activity with a potency approximately 500-fold less than flutamide. The following activi ties were presented for permethrin: Estrogenic

LOEC = 3.1 ± 0.4 × 10

− 4 M; estrogenic EC 50 = 2 × 10

− 3 M; anti-androgenic LOIC = 3.0 ± 0.57 ×

10

− 5 M; anti-androgenic IC 50 = 7.3 ± 1.4 × 10

− 4 M.

All three of the permethrin metabolites had endocrine activity. 3-Ph enoxybenzyl alcohol had both estrogenic and anti-androgenic activity and was approximately 10

− 5 -fold less potent than E 2 , but only 4-fold less potent than flutamide. 3-Phenoxybenzoic acid and permethrin cyclopropane acid both had anti-estrogenic activity and were approximate ly 100-fold and 1,000-fold less potent than 4-OH-tamoxifen, respectively. In the anti-estrogen screen, an increasing concentration of 3-PBOH produced progressive enhancement of the E 2 -stimulated absorbance, showing an additive estrogenic response. At concentrations greater than 8 × 10

− 3 M, 3-PBOH was toxic to the yeast and caused lysis of the cells.

Uterotrophic Assay (Rat)

In a multi-component study, Kim et al. (MRID 48176002, 2005) examined the estrogenic effects of permethrin (96.6% purity) using Northern blot an alysis of uterine Calbindin-D 9k gene expression (CaBP-9k) and an uter otrophic assay using immature ( 18-day-old) female SD rats.
CaBP-9k is an intracellular calciu m binding protein that is estrogen- responsive in the rat uterus.
Uterine tissues obtained dur ing the uterotrophic assa y were used for the Northern blot analysis. Permethrin (5, 10, 50, 100, 200, or 800 mg/kg) alone, plus E 2 (3 μg/kg), or plus ICI 182,780 (3 μg/kg) was administered to imma ture female rats by s.c. injec tion. For the uterotrophic assay, animals were sacrificed 24 h after the 3-day trea tment, and the uterus a nd vagina were removed and weighed. For the CaBP-9k assay, total RNA from uterine tissues was extracted for Northern blot analysis of CaBP-9k mRNA. A descri ption of the probe us ed was not provided.

High dose (800 mg/kg) permethrin increased (p <0.05) relative (to body) uterine weights (data presented graphically). Permethrin also enhanced E 2 -induced uterine weight increases (data presented graphically); however, this was maxima l at 200 mg/kg (p<0.05). It was stated that the permethrin-induced increases in re lative uterine weights were inhibited by co-treatment with ICI 182,780; however, the inhibitory eff ect was statistically different only at 50 mg/kg (p<0.01), and control weights were not provide d. Relative vaginal weight was unaffected by treatment with permethrin. Northern blot analysis showed an up-regulation in CaBP-9k mRNA following permethrin exposure that was maximal at the lowe st dose (5 mg/kg). Perm ethrin also enhanced
the E 2 -induced increase in CaBP-9k mRNA leve ls in a non-dose-dependent fashion.PERMETHRIN FINAL

Page 42 of 67 Uterotrophic (Rat)

In a multi-component study, Kunimatsu et al. (MRID 48176008, 2002) performed a uterotrophic assay with permet hrin (97.4% purity) using 5-week-old ovariectomized female Sprague-Dawley [Crj:CD(SD)IGS] ra ts. Groups of six females were dosed with permethrin in corn oil via oral gavage at dose levels of 0, 37.5, 75, or 150 mg/kg/day for 3 days. Two additional groups received ethynyl estradiol (30 μg /kg/day) or methoxychlor (125 mg/kg/day) as reference chemicals. One day after the final dos e, rats were euthanized and uterus, liver, and kidneys were dissected and weighed. For the uterus, both wet (with luminal contents) and blotted (without luminal conten ts) weights were determined.

Tremor was observed in 3-6/6 ra ts in the high-dose (150 mg/kg/ day) group. No increases in uterus weight (wet or blotte d) were observed. Reference controls ethynyl estradiol and methoxychlor both showed the expected effects. The results of the study provide no evidence that permethrin exhibits any pot ential to cause adverse (anti-) estrogenic effects at dose levels below those causing excessi ve systemic toxicity.

Receptor Transactivation Assays

In a published study by Du et al . 2010 ( MRID 48196301 ), permethrin and its metabolite 3-PBA were tested for ER, AR, and thyroid hormone receptor (TR) binding activity. Agonist and antagonist interactions with the ER and TR we re examined using transfected African green monkey kidney CV-1 cells and AR activation was determined using transfected MDA-kb2 cells. The agonist assays were conducted using permethrin (94.0% purity) at concentrations ranging
from 10

− 9 M to 10

− 5 M; to test antagonist activity, the ER , AR, and TR tests were conducted at the same concentrations of pe rmethrin in the presence of E 2 , DHT, and T 3 , respectively. The

REC 20 (relative effective con centration, agonist) and RIC 20 (relative inhibito ry concentration, antagonist) were calculated.

Permethrin exhibited weak estrogen agonistic effects at concentrations from 10

− 8 to 10

− 5 M (REC 20 = 8.10×10

− 7 M) and also exhibited w eak antagonist effects at 10

− 6 and 10

− 5 M, in the presence of 1×10

− 9 M E 2 (RIC 20 >10

− 5 M). The metabolite 3-PBA showed antagonist effects at

10

− 6 and 10

− 5 M, in the presence of 1×10

− 9 M E 2 (RIC 20 = 8.84×10

− 8 M). No AR agonist activity was found for permethrin or 3-PBA; however, permethrin and 3-PBA showed AR antagonist activity with a calculated RIC 20 of 1.88 x 10

− 6 M for permethrin and >10

− 5 M for 3-PBA. Similarly, permethrin and 3-PBA did not exhibit TR agonist activity but did show TR antagonist activity with a calculated RIC 20 of 1.50×10

− 9 M for permethrin and 4.76×10

− 6 M for 3-PBA.

ER Transactivation and Binding Assays

In a published study by Chen et al. ( MRID 48160705 ), the effect of permethrin (purity ≥ 90%) on cell proliferation in human breast cancer estrog en-sensitive (MCF-7) cells in the absence ofPERMETHRIN FINAL

Page 43 of 67 estrogens (negative control) and in the presence of 17 β -estradiol (E 2 , positive control) was investigated (E-Screen assay). A binding assay using rat uterine cytosol ER was also studied and estrogen-responsive pS2 mRNA expression in MCF-7 cells treated with permethrin was measured by RT-PCR.

For the cell proliferation assay, MCF-7 cells were plated and permethrin was tested in log
increments at concentrations from 10

− 11 to 10

− 6 M. The bioassay was terminated on Day 6 and proliferation was determined by absorbance meas urements after addition of thiazolyl blue solution. The proliferative effect (PE) is meas ured as the ratio between the highest cell yield
obtained with the test chemical and the horm one free control and is expressed as -fold proliferation. Estrogenic activity was assessed by relative proliferative potency (RPP) which is
the ratio of maximum PE for the chemical to PE for 17 β -estradiol (E 2 ).

For the ER competitive-binding assay, 10

− 9 M [ 3 H]-estradiol was incubated with increasing concentrations of permethrin, uterine cytosol preparation, and buffer. The bound ligand was separated from the free ligand and radioactivity was measured. Da ta for permethrin and the E 2 standard curve were plotted as percent [ 3 H]-E 2 bound vs. molar concentration, and the IC 50 was determined. Permethrin was tested in log increments at concentrations from 10

− 12 to 10

− 3 M.

For the pS2 mRNA assay, MCF-7 cells in medi um were treated with permethrin or E 2 at various
concentrations. All cells were harvested at the same time, total RNA was extracted, and mRNA for pS2 and β -actin were amplified by RT-PCR.

In the E-Screen assay, an inverse U-shaped dose response curve was obtained that was significant at 10

− 10 to 10

− 7 M, with a maximum proliferation response at 10

− 8 M. The PE value for permethrin was 2.69. In comparison with the PE of E 2 (3.46), permethrin showed a partial agonistic response. In the ER competitive-bindi ng assay, permethrin inhibited the binding of [ 3 H]-E 2 at concentrations greater than 10

− 7 M, at a maximum of <67%. The pS2 mRNA expression increased 1.28-fold after 10

− 7 M permethrin treatment.

ER Transactivation and Binding Assays

In a published study by Kim et al. ( MRID 48176007 ), the estrogenic activity of permethrin was evaluated using the E-screen a ssay (with the MCF-7 cell line) and a competitive ER binding assay. Cells were incubated for six days in the presence of permethrin (98% purity; concentrations not reported) prior to being a ssayed for cell proliferation. The competitive ER binding assay was conducted using uterine cytoso ls isolated from Sprague-Dawley rats. Aliquots of rat uterus cytosol were incubated w ith selected concentrations of permethrin and [ 3 H]-estradiol.

In the E-screen assay, the positive control, E 2 , induced cell proliferat ion in a dose dependent
manner. Permethrin did not induce MCF-7B BUS ce ll proliferation at any of the concentrationsPERMETHRIN FINAL

Page 44 of 67 tested. To evaluate potential anti-estrogenic activity, cells were treated for six days at a
concentration of 10

− 6 M permethrin in the presence of either 10

− 10 or 10

− 11 M E 2 . 10

− 6 M permethrin significantly inhibited E 2 -induced cell proliferation. In the competitive ER binding assay, permethrin did not compete with [ 3 H]-estradiol at any of the concentrations tested

.

ER Transactivation Assays

In a published study by M. Jin et al

. ( 2010 ), permethrin (99% purity) and its metabolites PBCOH (98% purity), PBCHO ( ≥ 97% purity), and 3-PBA (98% purity) were tested for estrogenic activities in the MCF-7 cell line. A cell proliferation assay was conducted using MCF-7 cells incubated with medium containing 10

− 9 M E 2 (positive control), the test chemicals at 0.001-10 μM, or ethanol (0.1%, v/ v; negative control). After 5 days, cell proliferation was determined by measuring the absorbance with a micr o plate reader. The proliferative effect (PE), the ratio of the maximal cell yiel d of the test compound to the cell yield of the negative control, and the relative proliferative effect (RPE), the ratio of the PE for the test chemical to the PE of

E 2 , were calculated for assessment of the estrogenicity of the chemicals.

Estrogenic activity was also evaluated using a gene expression assay. MCF-7 cells were incubated for two days with the test chemicals at 0.001-10 μM, E 2 at 10

− 9 M (positive control), or ethanol at 0.1% (v/v; negative control). Total RNA was ex tracted and reverse transcribed for quantitative PCR analyses for the genes pS2 (an estrogen-responsive protein in breast tumors)
and ER α . Based on the results of preliminary experi ments, quantitative PCR measurements were
conducted with the test chemicals at 10

− 6 M.

Permethrin induced proliferati on (p<0.05) of MCF-7 cells at 10

− 8 to 10

− 6 M compared to the solvent control; RPE values at 10

− 7 M were determined to be 56.3% and 55.4%, compared to

10

− 9 M E 2 (100%). The three metabolites induced cell proliferation to various degrees with RPE values for PBCOH, PBCHO, and 3-PBA dete rmined as 62.5%, 15.2%, and 28.6%, respectively.
Permethrin and the metabolites PBCOH and 3-PB A induced proliferation of MCF-7 cells in a concentration-related manner; it was noted that the nonlinear response seemed to follow an inverted U-shaped dose-response pattern with the most significant effects observed at a
concentration of 10

− 7 M.

All test chemicals were found to up-regulate pS2 transcription. Permethr in and the metabolite PBCOH induced pS2 expression 2.59- and 3.45-fold, re spectively, compared to the control. The
effects of the metabolites PBCHO and 3-PBA on the pS2 expression were much lower (1.37- and 1.85-fold, respectively) than the other test chemicals. Permethrin and PBCOH at 10

− 6 M
decreased the ER α mRNA expression to levels similar to E 2 , which was found to down-regulate

ER α by 50% compared to the control. Metabolites PBCHO and 3-PBA down-regulated the ER α expression levels at lower levels (3.7% and 12.0 %, respectively). The relative inductive efficiency of the test chemicals on the up-regula tion of pS2 expression co mpared to that of E 2 and the relative inhibitory efficiency of the test chemicals on the down-regulation of ER αPERMETHRIN FINAL

Page 45 of 67 regulation compared to that of E 2 were calculated. In the pS 2 expression assay, PBCOH showed a fully estrogenic response (82%), and permet hrin and 3-PBA showed a partially estrogenic response (29-53%); PBCHO showed only a weakly estrogenic response (13%). Similarly, in the

ER α expression assay, PBCOH a showed fully es trogenic response (89%), and permethrin showed a partially estrogenic response (69%); 3-PBA showed onl y a weakly estrogenic response (24%), and the PBCHO response was <10% (negative response).

Estrogenic Potential

In a published study by Garey and Wolff (MRID 48176005, 1998) , permethrin (95.7% purity) was evaluated for estrogen and progestero ne agonist/antagonist activities using two in vitro systems based on human cells (Ishikawa Varian t-I human endometrial cancer cell line for estrogen effects and T47D human breast cancer cell line for proge sterone effects). Both cell lines produce alkaline phosphatase (ALP) as an indicator of hor monal activity. Test compounds
and standards were dissolved in ethanol and diluted in medium, added to cells in plates, and incubated. After a 48-h exposure period, cells were rinsed and a p- nitrophenyl phosphate solution was added to each well. The resulting formation of p -nitrophenol was detected using a plate reader.

In Ishikawa Var-I cells, permethrin did not dem onstrate estrogen agonist or antagonist activities; in T47D cells, permethrin did not exhibit pr ogesterone agonist or antagonist ac tivities.

Estrogenic Potential

In a published study by Go et al. (MRID 48176004) , permethrin (95.7% purity) was evaluated for estrogenic potential in the MCF-7 estrogen-se nsitive cell line, as measured by expression of pS2 and cell proliferation. For the pS2 expression assay, MCF-7 cells were incubated for 3 days and then exposed to permethrin for 48 hours. Total RNA was extracted and pS2 RNA was detected by Northern blot and autoradiography with quantificati on by densitometry. Estradiol (E 2 ) was used as the positive control. Results were expressed as the ratio of permethrin to the
vehicle control. In the MCF-7 cell proliferation assay, cells we re exposed to permethrin at concentrations of 0.001-100 μM. Cell proliferati on was determined on Day 6 by counting with a
Coulter counter.

Permethrin did not induce pS2 above the negative contro l or inhibit E 2 induction of pS2 in initial experiments at 5 μM, but at higher concentratio ns (100 μM), it weakly induced pS2 expression.
In combination with 0.1 nM E 2 , permethrin had little effect on pS2 expression levels stimulated by E 2 . At concentrations below 10 μM, permet hrin did not induce MC F-7 cell growth, but
induced modest increases in proliferation at 100 μM.PERMETHRIN FINAL

Page 46 of 67 ER Transcriptional Activation Assay

In a published study by Lemaire et al. (MRID 48074114) , permethrin was evaluated for ER α and ER β activation or inhibition in stable reporte r cell lines. Stable transfection of the ER α and

ER β constructs together with an estrogen reporter lu ciferase vector into HeLa cells resulted in

two E 2 -sensitive cell lines. The cells were incubated with permethrin ( ≥ 95% purity) for 16 h. At the end of incubation, a commerci al luciferase assay system wa s added and luciferase activity
was measured with a luminometer. For the ER α and ER β competitive-binding assay, ER α and

ER β cells were treated with 0.1 nM [ 3 H]-E 2 in the presence or absence of increasing
concentrations of unlabeled E 2 (as control) or permethrin. To tal cell lysate was counted by liquid
scintillation.

With the transactivation activity of E 2 set at 100%, the estrogenic activity percenta ges of 10 μM permethrin were 13.0 ± 0.4% for ER α and 14.2 ± 2.4% for ER β ; these were not statistically
different from the control values of 9.3 ± 1.3% and 10.8 ± 1.6%, respectively. Permethrin showed no anti-estrogenic activity in either ER α or ER β

.

AR Binding Assay

In a published study by Bauer et al

. ( MRID 48176001, 2002 ), permethrin (97.5% purity) was evaluated for its AR binding capability using an immuno-immobilized a ndrogen receptor assay (IRA) with recombinant human androgen receptor (rhAR). The receptor preparation was diluted
and incubated with 3 H-DHT in the presence or absence of increasing concentrations of permethrin or DHT. After washing unbound radioact ivity, wells were separated and transferred
into scintillation vial s and radioactivity was counted. Sp ecific binding was ca lculated as the difference of total binding and non-specific binding in the presence of a 200-fold surplus of unlabelled DHT. The relative binding affinity of permethrin was compared to DHT using the inhibition constants (K i ) of each compound to calculate the re lative binding affinity, where RBA = (K i (DHT) × 100) ÷ K i (permethrin).

The K i (permethrin) could not be determined becau se there was no detectable displacement of DHT up to the highest concentration tested (unr eported). Therefore the permethrin RBA was reported as <0.01%.

Androgen Receptor Transactivation Assays

In a published study by Xu et al

. ( MRID 48176006 ), the potential a ndrogenic and antiandrogenic activities of permethrin (>98% purity) were evaluated using a transiently-transfected human AR reporter gene assay in an African monkey kidney cell line (CV-1). After a 24 h incubation period, 0.1% ethanol (veh icle control) or 1 nM DHT ( positive control), and various concentrations of permethrin (10

− 7 to 10

− 5 M) were added for meas urement of agonistic or antagonistic activity. The cells were harvested 24 h after dosing, lysed, and the lysate analyzedPERMETHRIN FINAL

Page 47 of 67 for chloramphenicol transferase (CAT) and β -galactosidase ( β -Gal) activities using commercial kits. The amount of CAT for each lysate was normalized to the β -Gal activity.

There were no indications of cy totoxicity at the dose levels tested; there was no significant
difference in the amounts of β -Gal between the treated groups and vehicle control groups. Permethrin did not exhibit androgenic activity at the tested concentrations. Permethrin did
attenuate an increase in the CA T amount induced by DHT; the inhibitory effect was detected at

1 × 10

− 5 M. The IC 50 value was (5.86 ± 2.20) × 10

− 5 M.

Androgen Receptor Transactivation Assays

As part of a multi-component published study by Zhang et al. (MRID 48196302, 2008) , the potential anti-androgenic effects of permethrin (purity not repo rted) were examined using an androgen-responsive cell line (MDA-kb2). Various concentrations of permethrin with and without DHT (1 nM) were tested.

Permethrin was found to significantly inhibit DHT-induced transcrip tional activation at a
concentration of 10

− 5 M (data presented graphically).

Hershberger Assay (Rat)

In a multi-component study, Kim et al. (MRID 48176002) conducted a Hershberger assay with permethrin (96.6% purity) using 5-week-old male SD rats. Male SD rats were castrated at 5 weeks of age. Permethrin was administered by oral gavage at 10, 50 or 100 mg/kg/day for 10 days (n=6–8). As a reference androgen, 0.4 mg/k g TP was injected subcutaneously. To examine anti-androgenic activity in a second group of rats, TP-treated rats (n=6–8) were administered permethrin at 10, 50 or 100 mg/kg/day for 10 days. Flutamide (25 mg/kg/day) and p , p  ́-DDE (100 mg/kg/day) were used as reference anti-a ndrogens. Animals were evaluated daily for mortality, injury and general appearance, and in dividual body weights were recorded every three days. Twenty-four hours after th e final dose the five androgen-de pendent tissues were excised and weighed.

No statistically si gnificant changes in body weights were not ed in any of the treatment groups. The positive control, TP, induced statistically si gnificant increases in the absolute and relative weights of all of the androgen-dependent sex accessory tissues. Permethrin did not show an agonist effect on androgen-dependent tissue we ights at any dose tested. Flutamide and p , p  ́- DDE reduced sex accessory tissue weights in TP-tre ated castrated males verses the TP-treated group. Administration of permethrin to TP-treated castrated rats led to statistically significant reductions (antagonism) in the five sex accessory tissue weights compared to TP at all doses tested, though not in a dose-dependent manne r, nor to the extent of flutamide or p , p  ́-DDE.PERMETHRIN FINAL

Page 48 of 67 Hershberger Assay (Rat)

In a multi-component study, Kunimatsu et al. (MRID 48176008) conducted a 5-day Hershberger assay with permethrin (97.4% purity ) using 9-week-old castrated male SpragueDawley [Crj:CD(SD)IGS] rats. Groups of six male s were dosed with permethrin in corn oil via oral gavage at dose levels of 0, 25, 50, or 75 mg/kg/day for 5 days with or without 0.25 mg/kg/day TP administered by s.c. injection as a reference andr ogen (eight groups). For the 4 groups treated with TP, an addi tional group received 100 mg/kg/day p , p  ́-DDE (gavage) as a reference control for assessing w eak anti-androgenic activity. For the groups receiving no TP, an additional group received 100 mg/kg/ day methyltestosterone via ga vage as a reference control for androgenicity. One day after the final administ ration of permethrin, rats were euthanized, the sex accessory glands/tissues (ventral prostate, dorsolateral prostate, seminal vesicles with coagulating glands and LABC), liver a nd kidneys were dissected and weighed.

Tremor was noted in 2-6/6 rats at 75 mg/kg/day. No toxicologi cally significant changes were observed in body weight, food consumption, or organ weights. No androgenic or antiandrogenic effects resulted from treatment with permethrin. Reference controls p , p  ́-DDE and methyltestosterone produced the expected effect s. The results of the study provide no evidence that permethrin exhibits any poten tial to cause adverse (anti-) a ndrogenic effects at dose levels below those causing excessi ve systemic toxicity.

Hershberger Assay (Rat)

As part of a multi-component published study by Zhang et al. (MRID 48196302, 2008) , a Hershberger assay was conducted with permethrin (purity not reported) using 3-week-old male Sprague-Dawley rats. After 7 days acclimation, th e rats were castrated and allowed to recover for 2 weeks. Following recovery, rats were we ighed and randomly assigned to experimental groups (n=6). The groups consisted of controls (0.5 mg/kg/day TP s.c.), TP + flutamide (50 mg/kg/day p.o.), and TP + permethrin (50 mg/kg/ day orally). The trea tment period was 7 days for all groups. Following the last dose, rats were euthanized by exsanguination. The seminal vesicles, ventral prostate, dorsolateral prostate , LABC muscle, Cowper’s glands, glans penis, liver, paired adrenal glands, spleen and paired kidneys were dissected and weighed. Serum testosterone was determined using commercially available FIA kits.

No mortality or treatment-related clinical signs were not ed in any of the groups. It was reported that there were no significant differences betwee n initial or final body weights, or weight gains of the groups compared to the control group. Perm ethrin caused significant decreases in seminal vesicle and ventral prostate weig ht (data presented graphically).PERMETHRIN FINAL

Page 49 of 67 Three-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a two-generation repr oduction toxicity study ( MRID 00120271 ), permethrin (94.0-98.8% purity) was administered to groups of 12 male and 24 female Wistar rats in the diet at concentrations of 0, 500, 1000, or 2500 ppm (0, 25, 50, and 125 mg/kg/day, respectively, using a standard conversion factor of 0.05). Two litters were produced by each generation. Parental animals in each generation received test or cont rol diet for 12 weeks post-weaning and were then paired for mating to produce the A litters. Afte r various rest periods, th e parental animals were mated again to produce the B litters. Test diet s were administered during mating, gestation and lactation for three successive genera tions throughout the study. The F 2 parents were mated for a third time, using the same breeding pairs as for the B litters, producing the C litters for a developmental toxicity evaluation. Ten males of the F 1 generation were maintained on experimental diets until they were 54-55 weeks old and were submitted for microscopic
examination of selected neurological tissues.

There were no treatment-related parental deaths. A few parental animals were euthanized for
humane reasons. There were no treatment-rela ted effects on body weights, body weight gains, food consumption, or food efficiency.

Treatment-related clinical si gns in high-dose parental anim als were limited to whole body tremors, occurring in all parental generations (exception: tremors were not observed in the P males) during the first few days of the prema ting period. In the 2500 ppm groups, the incidence
rates for the tremors were 20/24 (P females), 11/12 and 24/24 (F 1 males and females, respectively), and 12/12 and 24/24 (F 2 males and females, respectively). Tremors were also observed in pregnant and lacta ting females exposed to 2500 ppm permethrin. The tremors were intermittent and transient. Neuropathy was not obs erved in a special microscopic examination of selected neurological tissues from F 1 males continued on test for one year.

Gross examination at necropsy did not reveal any dose- or treatment-re lated findings, nor did microscopic examination of grossly abnormal tissu es from all parents surviving to scheduled termination and of reproduc tive tissues from animals suspected of infertility.

The LOAEL for systemic toxicity is 2500 ppm (125 mg/kg/day) based on tremors observed in the P females, and the F 1 and F 2 males and females. The systemic toxicity NOAEL is 1000 ppm (50 mg/kg/day).

There were no treatment-related effects in th e reproductive performances (male or female fertility indices, gestation inde x, gestation length, live birth in dex, viability index, lactation index, parturition, or fetal sex ratio) of the F 1 , F 2 and F 3 generations. In the F 3c litters, there were no developmental effects associated with th e administration of permethrin over three generations. The percentages of male fe tuses of the 1000 and 2500 ppm groups (39.0 and 44.7%, respectively) were lower than the control value (53.2%), but the effect was not associatedPERMETHRIN FINAL

Page 50 of 67 with increased resorptions and wa s not dose-related. Also, no consis tent effect on sex ratios was
observed in other litters or generations of the study.

Microscopic examination of F 3b weanlings revealed dose-relate d increases in centrilobular hypertrophy of the liver. The incidences of s light and moderate centrilobular hypertrophy were dose-related, ranging from 0 to 80% for the males and from 10 to 100% for the females. The hypertrophy of the liver was an ad aptive and reversible effect a nd was not considered adverse.

The reproductive toxicity and offspring toxicity NOAEL is ≥ 2500 ppm (125 mg/kg/day) and the reproductive toxicity and offspring t oxicity LOAEL is not identified.

Developmental Toxicity (Rat, OCSPP 870.3700)

In a developmental toxicity study ( MRID 40943603 ), 24 presumed pregnant Wistar rats per group were administered 0, 15, 50, or 150 mg/kg/day of permethrin (93.9% purity; 38 cis:62 trans isomers) in corn oil by oral gavage on ge station days (GD) 7-16, in clusive. On GD 22, all surviving dams were sacrificed and all fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations/variations.

All dams survived to scheduled termination and no treatment-related abnormalities were noted at necropsy. There were no effects of treatment on clinical signs, body weight gains, or food consumption in the low- or mid-dose groups. In the high-dose group, clini cal signs of toxicity
seen between GD 8-19 included tremors in 21/24 rats and head flicki ng in 6/24 rats. Body weight gains and food consumption by the high-dose dams were less (p<0.05) than that of the controls throughout the dosing in terval. For GD 7-10, 10-13, a nd 13-16, body weight gains were decreased by 88%, 32%, and 18%, respectively.

The maternal LOAEL is 150 mg/kg/day based on c linical signs of toxi city and decreased body weight gain and food consumption. Th e maternal NOAEL is 50 mg/kg/day.

No dose- or treatment-related effects were obser ved on gravid uterine weights, fetal sex ratios, pre- or post-implantation losses, or numbers of corpora lutea/dam or live fetuses/dam. Mean fetal body weight of the high-dose group was 3.2% (p ≤ 0.05) less than that of the controls. However, mean litter weight of the high-dose group was 3% (NS) greater than that of the controls. Therefore, the reduced fetal body weights were considered a questionable toxic

response.

No treatment-related external or visceral fetal malformations/variations were noted. The fetal and
litter incidence rates of short length extra ribs were increas ed (p<0.05) in the high-dose group as compared with the controls. Short length extr a ribs were observed in 31% of the high-dose
fetuses vs. 11% of the control fetuses and in 87% of high-dose litters vs. 57% of control litters.PERMETHRIN FINAL

Page 51 of 67 The developmental LOAEL is 150 mg/kg/day base d on decrease in fetal body weights and an
increase in the incidence rate of short le ngth extra ribs. The developmental NOAEL is 50 mg/kg/day.

Developmental Toxicity (Rabbit, OCSPP 870.3700)

In a prenatal developm ental toxicity study ( MRID 40943602 ), presumed pregnant Dutch rabbits were administered 0, 600, 1200, or 1800 mg/kg/ day of permethrin (92.5% purity; 32.3 cis:60.2 trans isomers) in 0.5% aqueous Tween 80 by oral gavage on GD 6-18, inclusive. The number of does mated for each group was 19, 21, 20, and 23, respectively. On GD 29, all surviving does
were sacrificed and all fetuses were weighed and examined for external malformations/variations. Approxi mately one-half of the fetuses were processed for skeletal examination and the remaining one-half was fi xed and examined for visceral anomalies.

A total of 0, 5, 5, or 4 does died or were sacrificed moribund in the control, low-, mid-, or highdose groups, respectively. Due to the lack of a dose-response, the deaths could not be definitively attributed to test article administration. Clinical signs of toxicity included body tremors observed in 5 of the high-dose animals only. Little or no feces or urine was noted on at least one occasion for 2/19 (11%), 4/21 (19%), 6/20 (30%), and 8/23 (35%) animals in the control, low-, mid-, and high-dose groups, respectively.

Absolute body weights were similar between the treated and control grou ps throughout the study. However, after examining the replotted body weight data, there was a sharp drop in weight for the low, mid, and high dose groups after Day 6 a nd only a slight drop for the control that was noticeable after Day 12. Body weight gains by the low-, mid-, and high-dose groups were decreased by 79%, 50%, and 91%, respectivel y, during GD 0-18 with significance (p<0.05) attained for the low- and high-dose groups. Du ring the post-dosing interval, recovery of body weights was noted for the low- and mid-dos e groups, but not for the high-dose group.

The maternal LOAEL is estimated to be <600 mg/kg/day based on decreased body weight gain.
The maternal NOAEL is not identified.

The number of live fetuses and mean litter size was decreased for all dose groups compared to the control group (110(15), 80(13), 69(14), and 72 (13) for control, low-, mid-, and high-dose groups, respectively). However, no dose-response was evident or statistical significance noted.

Post-implantation loss was increased (p<0.05) in the mid- and high-dose groups to 155% and
248% of the control level. Corre spondingly, the number of early and late resorptions was higher in these groups as compared to the control group values. Mean fetal body weights in the highdose group were slightly ( ↓ 9%; NS) less than that of the contro ls and attributed to maternal body weight decreases. No dose-relate d or statistical differences we re observed between the treated and control groups for number of fetuses/li tter or mean gravid uterine weights.PERMETHRIN FINAL

Page 52 of 67 No treatment-related external or visceral fetal ma lformations/variations were noted. In the midand high-dose groups, reduced ossification of the fore- and hindlimbs was indicated by slightly (NS) greater ossification scores as compared with the controls. Mean scores for the control, low-, mid-, and high-dose groups were 1.92, 1.99, 2.00, and 2.25, respectively, for the forelimb and 1.65, 1.56, 1.89, and 1.90, respectively, for the hindlimb.

Therefore, the developmental LOAEL is 1200 mg /kg/day based on increased post-implantation loss, greater numbers of early a nd late resorptions and an equivocal decrease in ossification of the fore- and hindlimbs. The developmental NOAEL is 600 mg/kg/day.

Chronic Toxicity/Carcinoge nicity (Rat, OCSPP 870.4300)

In a combined chronic toxicity/carcinogenicity study ( MRID 00069701 ), permethrin was administered to 60 rats/sex/group in the feed at doses of 0, 500, 1000, or 2500 ppm (mean estimated intake 0/0, 19.4/19.1, 36.9/40.2, and 91.5/104 mg/kg/day in males/females, respectively). Twelve rats/sex/g roup were sacrificed at 52 week s and the surviving rats were sacrificed at 104 weeks exposure.

There was no treatment-related effect on mortality or tumor induction. During the first two weeks of the study, treatment-related tremors a nd hypersensitivity were observed in the highdose males and females. There were no e ffects on body weight, body weight gain, food consumption or food efficiency, ophthalmologi c endpoints, hematologi c endpoints, clinical chemistry, or urinalysis parameters.

Liver changes suggestive of adaptive hype rtrophy included in creased aminopyrine- N

demethylase activity in all male treatment groups, in the mid- and high-dose female at 52 weeks, and in the high-dose male and female groups at 104 weeks. This was coupled with modestly
increased absolute and relati ve liver weights in the high- dose males and high and low-dose females at 52 weeks and in all male treatment groups and mid-dose females at 104 weeks. Further evidence for adaptive cha nges included hypert rophy of centrilobular hepatocytes with increased cytoplasmic eosinophili a in the mid- and high-dose male and females at 104 weeks’ exposure and increased smooth endoplasmic reti culum proliferation in all treatment groups except low-dose males at 52 weeks and high-dose groups at 104 weeks. Liver changes also included fatty vacuoles confirme d by electron microscopy in th e mid- and high-dose males at both 52 and 104 weeks and in the high-dose female s at 104 weeks. It was concluded that the increased liver weight and hypert rophy observed in the liver were adaptive and reversible and
were not adverse.

Under the conditions of this study, the LOAEL is 2500 ppm (91.5/104 mg/kg/day in males/females, respectively) based on trem ors and hypersensitivity. The NOAEL is 1000 ppm (36.9/40.2 mg/kg/day in males/ females, respectively).PERMETHRIN FINAL

Page 53 of 67 At the doses tested, permethrin did not affect the incidence of tumor-bearing animals or the incidence of any specific tumor type in either sex. Permethrin was not carcinogenic to the rat. Dosing was considered adequate based on liver effects and on tremors and hypersensitivity in
male and female rats.

No treatment-related changes were seen in absolute or relative weights of the testes, ovaries, pituitary, or adrenal glands at any dose level. No treatment- related histopathol ogical lesions were seen in the testes, epididymides, semina l vesicle, prostate, ovaries, uterus, cervix, mammary, thyroid, adrenal or pituit ary glands at any dose level.

Chronic Toxicity (Dog, OCSPP 870.4100)

In a chronic toxicity study ( MRID 00129600 ), permethrin (92.5%, pur ity, cis/trans 32.3/60.2) was administered to beagle dogs (6 /sex/group) in corn oil by gelati n capsule at dose levels of 0, 5, 100, or 1000 mg/kg/day for one year. The high dose was lowered from 2000 mg/kg/day after 2 days due to overt toxic r eaction to the test material.

There were no mortalities. Neur ological clinical signs (tremors , uncoordinated gait, nervousness and convulsions, also excessive salivation and vomiting) were observed in the high-dose group. At the high-dose, decreased body weight gain (3 7% for males and 33% for females less than control, respectively), de creased food consumption (increased food left uneaten), increased liver weight (+30% and +36% for males and females, respectively) and alkaline phosphatase level (+377% and +220% for males and females, respectiv ely) were reported. At mid-dose, increased liver weight (+25% both sexes) and alkaline phosphatase levels (+134% for males and +99% for females) were observed.

The thyroid showed apparent increases in weig ht for the males (+27%, +28% and +17%) and thefemales (-5%, +27% and +33%) for the low, mid and high dose test groups. No treatmentrelated changes were seen in absolute or relati ve weights of the testes , ovaries, adrenal or pituitary glands at any dose level. No treatment-related histopathological lesions were seen in the testes, epididymides, prostate, ovaries, uter us, cervix, mammary, thyroid, or pituitary glands at any dose level. Microscopi c evaluation of the adrenals showed focal degeneration and
necrosis in the cortex with variable infla mmatory cell infiltration along with swelling and
vacuolization of the cells in th e inner cortex at high-dose males and females (5/5 males and 4/6 females) and at mid-dose males (1/5). The liver also showed hepatic ce llular swelling at midand high-dose males and females.

The observations of increased liver weight, al kaline phosphatase levels , and hepatic cellular swelling were considered adaptive and reversible and were not adve rse. Therefore, the systemic toxicity LOAEL is 1000 mg/kg/day based on c linical neurotoxic signs and decreased body weight gain and food consumpti on. The NOAEL is 100 mg/kg/day.PERMETHRIN FINAL

Page 54 of 67 28-Day Oral (Feeding) Toxicity (Rat)

In a 28-day oral toxic ity range-finding study ( MRID 00120267 ), permethrin (90.5% purity) was administered to Wistar rats (8/sex/dose) in the diet at dose levels of 0, 200, 500, 1000, 2500, 5000, or 10,000 ppm (equivalent to 0/0, 20/ 20, 51/51, 106/76, 267/207, and 319/428 mg/kg/day in males/females, respectively). The mg/kg equivalent of the 10,000 ppm dose could not be calculated, since all rats receiving that dose died within three days.

At 5000 ppm, one female and four males died by Day 18. No other deaths were observed. Surviving rats receiving 2500 ppm and above be came hypersensitive during the first week of treatment and remained so throughout the study. Body weight of the 5000 ppm males was 16-
22% below that of controls thr oughout the treatment period, and overa ll body weight gain of that group was decreased by 31%. Li ver weight was increased 28- 33% in the 2500 and 5000 ppm females. The relative (to body) liver weight was increased in the 2500 and 5000 ppm males, and in all treated females except those receiving 500 ppm. The liver changes were judged to be an adaptive response. Testis weight was decrea sed 10-13% in 2500- and 5000-ppm males, and relative adrenals weight was decr eased 29% in 5000-ppm females; neither of these organ weight changes showed a dose-response relationship. Th ere were no treatment-re lated changes in food consumption, hematology, clinical chemistry, or ur inalysis of any group of rats. No treatmentrelated changes were seen in absolute or relative weights of the ovaries or pituitary glands at any
dose level. No treatment-related histopathological lesions were se en in the testes, epididymides, seminal vesicle, prostate, ovaries, uterus, mammary, thyroid, adrenal or pituitary glands at any
dose level.

Under the conditions of this study, the LOAEL for pe rmethrin in rats is 2500 ppm (equivalent to 267/207 mg/kg/day for males/females, respective ly) based on hypersensitivity. The NOAEL is 1000 ppm (equivalent to 106/ 76 mg/kg/day for males/females, respectively).

Testicular Toxicity (Mouse)

In a published study by Y. Jin et al

. ( 2012 ), 5 male ICR mice/dose group were administered one of the four permethrin enantiomers, (+)-cis-, (

− )-cis-, (+)-trans-, and (

− )-trans-permethrin (separated from racemic permethri n, >95% purity), in DMSO orally at dose levels of 0 (DMSO), 25, 50, or 100 mg/kg/day from PND 21 to PND 42. Mice were s acrificed on the last day of treatment. Blood was collected, and serum te stosterone levels we re measured using a radioimmunoassay. The testes were weighed, and the left testis in each group was fixed, processed, sectioned, and stained with hematoxylin and eosin for histolog ical analysis. Total
RNA was isolated from the testes of mice and th en reverse transcribed for quantitative PCR to determine the expression of 3-hydroxy-3-methylgl utaryl-CoA synthase (H MG-CoA synthase), 3- hydroxy-3-methylglutaryl-CoA re ductase (HMC-CoA reductase ), low-density lipoprotein receptor (LDL-R), scavenger receptor class B ty pe 1 (SR-B1, high-density lipoprotein receptor), peripheral benzodiazepine receptor (PBR), ster oidogenic acute regulatory protein (StAR),PERMETHRIN FINAL

Page 55 of 67 cytochrome P450 cholesterol side-c hain cleavage enzyme (P450scc), 3 β -hydroxysteroid dehydrogenase (3 β -HSD), cytochrome P450 17 α -hydroxysteroid dehydrogenase (P450 17 α ), and

17 β -hydroxysteroid dehydrogenase (17 β -HSD).

No clinical signs of toxicity were observe d and there were no effects of treatment on body weight. At 100 mg/kg, both abso lute and relative weights of testes in the (+)-cis, (

− )-cis and (

− )-trans-permethrin treated gr oups were decreased significan tly (p<0.05) compared to the control; no significant differences were found in the (+)-trans-permethrin treated group. No histological changes in the test es were observed in mice exposed to 25 or 50 mg/kg of any of the permethrin enantiomers. However, the number of spermatogenic cells was decreased in the (+)-cis, (

− )-cis, and (

− )-trans-permethrin treated groups at 10 0 mg/kg. Large inte rstitial spaces
in the seminiferous tubules were also observed in the 100 mg/kg (+)-cis, (

− )-cis, and (

− )-transpermethrin treated groups. At 100 mg/kg (+)-cis, (

− )-cis, and (

− )-trans permethrin exposure,
serum testosterone levels were significantly reduced compared to the control. However, when the groups treated with the different enantio mer types were compared to each other, no significant differences in te stosterone concentration we re observed at any dose.

The effects of permethrin enantiomer exposure on the mRNA levels of the main genes related to cholesterol synthesis in the testes were evaluated, as cholesterol is the main precursor for steroid
hormones such as testosterone. The mRNA levels of HMG-CoA synthase decreased significantly in the test es of the mice after treatment w ith 25, 50, and 100 mg/kg (+)-cis, (

− )-cis, (+)-trans, and (

− )-trans enantiomers compared to the c ontrol; however, no si gnificant differences were observed between the 4 enantiomers. For HMG-CoA reductase, significant differences in
mRNA levels were not observed between the 25 mg/kg enantiomer-treated mice and control or between the different enantiomer groups. At 50 and 100 mg/kg, enantiomer-specific responses
were observed. The mRNA levels of HMG-CoA reductase were only significantly decreased compared to the control in the 50 and 100 mg/kg (+)-cis enantiomer treated groups ( ↓ 40% and 48%, respectively). Significant differences were also found between the (+)-cis and (

− )-cis or (

− )-trans groups at 50 mg/kg dosage. The mRNA levels of testicular SR-B1 in all permethrin enantiomer treated groups were sli ghtly inhibited, but did not attain statistical significance. No significant differences in LDL- R mRNA levels were observed in any permethrin enantiomertreated group compared to the control

PBR and St AR play key regulator y roles in cholesterol transpor t from the outer to the inner
mitochondrial membrane. PBR mRNA levels were decreased by permethrin treatment in dosedependent and enantiomer-specific manners. At 25 and 50 mg/kg exposure le vels, the testicular mRNA level of PBR was decreased significantly only in the (+)-cis en antiomer treatment group, while at 100 mg/kg it was signifi cantly decreased in all enantio mer groups compared with the control group ( ↓ 39%, 47%, 47%, and 33%, respectively). An enantiomer-specific effect on PBR expression was observed at both 25 and 50 mg/kg concentrations; a significant difference in PBR mRNA expression was found be tween the (+)-cis and (

− )-trans groups. The testicular StARPERMETHRIN FINAL

Page 56 of 67 mRNA levels were down-regulated significan tly in the 25, 50, and 100 mg/kg (+)-cis and (

− )-trans enantiomer-treated groups compared to control. Additionally, StAR mRNA levels were down-regulated in the (

− )-cis and (+)-trans enantiomer-tr eated groups at 50 mg/kg and the (

− )-cis enantiomer group at 100 mg/kg. Moreove r, significant differences were also found between the (+)-trans and (

− )-trans groups at 100 mg/kg.

Key genes in the testosterone synt hetic pathway, including P450scc, 3 β -SHD, P450 17 α and

17 β -HSD, in the testes of mice we re also examined. A significant decrease in P450scc mRNA levels appeared in the 25 mg/kg (+)-cis and (

− )-trans enantiomer groups compared to controls. At 50 or 100 mg/kg, the testicular P450scc mRNA le vels were significantly inhibited by all four enantiomers. Treatment with the (+)-cis enan tiomer induced a dose-dependent decrease in 3 β

HSD mRNA levels, with significance reached at 50 and 100 mg/kg (+)-cis enantiomer ( ↓ 30% and 46%, respectively), and treatment with the ( -)-cis enantiomer resulted in a significant decrease at 100 mg/kg. Th e mRNA levels of P450 17 α were not influenced significantly regardless of the permethrin enan tiomers or administration doses. Significant differences in the mRNA levels of testicular P450scc, 3 β -HSD and P450 17 α were not observed between the permethrin enantiomers for any dose level. However, the effects on 17 β -HSD appeared in a different manner. First, a signi ficant decrease was observed in the (+)-cis enantiomer in all groups (25, 50, and 100 mg/kg) compared to the c ontrol. Second, at 100 mg/kg, gene expression was decreased significantly for all the permethr in enantiomers. Third, there was a significant difference between the (+)-cis and (+)-trans gro ups and between the (-)-cis and (+)-trans groups at 25 and 50 mg/kg.

Testicular Toxicity (Mouse)

In a published study by Wang et al

. ( 2012 ), nine-week-old male Sv/129 mice were dosed by daily oral gavage with 10 μmol/kg diazinon (equ ivalent to 3 mg/kg), 90 μmol/kg cis-permethrin (99.5%; equivalent to 35 mg/ kg), or diazinon+cis-permethrin at 100 μmol/kg for 6 weeks; a control group was dosed with corn oil alone. Afte r the first treatment and just before the last treatment, mice were placed in individual cages to collect urine for 24 hours; levels of cispermethrin and metabolite 3-PBA were determined in urine by GC/MS. Mice were terminated
16 hours after the final treatment. Blood was coll ected and plasma cis-permethrin (GC/MS) and testosterone (enzyme immunoassay) levels were measured. Live r, epididymis, testis, seminal vesicle, and prostate were collect ed and weighed. The levels of cis-permethrin in testis were measured by GC/MS. The mRNA levels of St AR (steroidogenic acute regulatory protein), TSPO (translocator protein), and P450scc (cytochrome P450 side-cha in cleavage) genes in testes were measured by real-time quantitative PCR. Epididymal sperm suspension was used to

m icroscopically assess sperm count, sperm mo tility, and abnormalities in sperm morphology. Western blot analyses were conducted on testis homogenate with prim ary antibodies against StAR, TSPO, and P450scc. Histopathological evaluation of the right testes and caput epididymides was conducted after the tissues were fixed in modified Davidson’s solution,PERMETHRIN FINAL

Page 57 of 67 processed, and stained with periodic acid Schiff’s reagent then hematoxylin. Seminiferous tubules were classified into three stage groups (I-VI, VII-VIII, and IX-XII) and examined for changes of tubule atrophy, cytoplasm vac uolation, and germ cell degeneration.

No signs of systemic toxicity were observ ed during dosing, and there were no effects of treatment on body weights, body weight gains, or reproductive organ weights. Urinary excretion of 3-PBA was decreased (p<0.05) in mice in the mixture group compared to the cis-permethrin group ( ↓ 30-50%). Levels of cis-permethrin in plasma and testes were 1.5- and 1.8-fold higher, respectively, in mice in the mixture group compar ed to the cis-permethrin group. These results
indicate that metabolism of cis-permethrin is inhibited by diazi non. Plasma testosterone levels in the diazinon and cis-permethrin treatment groups were similar to controls; however, plasma
testosterone levels were decr eased (p<0.01) in the mixture group compared to controls ( ↓ 45%) and compared to the cis-permethrin group ( ↓ 32%). Testicular mRNA and protein expression of StAR were not significantly different in any of the treatment groups compar ed to the controls. TSPO mRNA levels and protein expressions were significantly d ecreased in the cis-permethrin ( ↓ 46% and 47%, respectively) and mixture ( ↓ 53% and 55%, respectively) groups compared to controls. There were no signifi cant differences between the cis- permethrin and mixture groups for TSPO mRNA and protein expressions. Fo r P450scc, mRNA levels were significantly decreased ( ↓ 70%) in the mixture group compared to c ontrols; P450scc mRNA levels in the cispermethrin groups were similar to controls. Protein expression of P450scc was significantly decreased in all treatment gr oups compared to controls ( ↓ 24% and 43% for the cis-permethrin and mixture groups, respectively). Two-way ANOVA did not show any significant interaction between exposure and testosterone levels or mR NA or protein expressi ons of StAR, TSPO, or

P450scc.

Histopathological examination of the testes sh owed no significant increase in abnormalities in the cis-permethrin groups compared to controls; however, the numb ers of degenerate germ cells in seminiferous tubules was signi ficantly increased in the mixtur e group compared to controls. The percentage of seminiferous tubules in st age group VII-VIII was significantly decreased in the cis-permethrin and mixture groups. In the caput epididymis, cytoplasmic vacuolation was observed more frequently in all treatment groups th an in the controls; median vacuolation scores were significantly higher in the treatment groups. Two-way ANOVA did not show any significant interaction between exposu re to both chemicals and testic ular or epididymal changes. Caudal epididymal sperm count was signifi cantly reduced in th e cis-permethrin ( ↓ 60%) and mixture ( ↓ 51%) groups compared to controls. Sperm motility was also significantly decreased in the cis-permethrin and mixture groups. The percentage of sp erm with normal morphology was significantly decreased in the mixture ( ↓ 17%) group compared to controls; the decrease in normal sperm morphology in the mixture group was al so significant when compared to the cispermethrin group. Two-way ANOVA did not show any significant interaction between exposure
to both chem icals and sperm count, percent motility, or percent abnormal morphology.PERMETHRIN FINAL

Page 58 of 67 Avian Reproduction Toxicity (Quail, OCSPP 850.2300)

In an avian reproduction toxicity study ( MRID 42322901, 1992 ), groups of 32 bobwhite quail ( Colinus virginianus ) were fed diets containing permethr in at 0, 25, 125, or 500 ppm. The birds were fed the diets beginning at 20 weeks of age and continuing through egg laying, final incubation, hatching, and a 14-day offspring rearing period; the total approximate duration of
treatment was 27 weeks. The bi rds were housed with one male and one female per pen, with 16 pens per dose group.

Adult quail showed no overt signs of toxicity and no treatment-rela ted clinical signs. There were
no treatment-related mortalities. There were three deaths in the 25 ppm group which were
considered to be incidental to treatment as no treatment-related findings were seen at necropsy.

There were no significant di fferences in body weights, feed consumption, reproductive parameters, egg shell thickness, or offspring body weight between the cont rol and any treatment

group.

Avian Reproduction Toxicity (Duck, OCSPP 850.2300)

In an avian reproduction toxicity study ( MRID 42322902 ), groups of 32 Mallard ducks ( Anas platyrhyncos ) were fed diets containing permethrin at 0, 25, 125, or 500 ppm. The birds were fed the diets beginning at 23 weeks of age a nd continuing through egg laying, final incubation, hatching, and a 14-day offspring r earing period; the total approximate duration of treatment was 24 weeks. The birds were housed with one male and one female per pen, with 16 pens per dose

group.

There were two mortalities in the control group and one each in the 25 and 125 ppm groups;
these mortalities which were considered to be incidental to treatment as no treatment-related findings were seen at necropsy. No overt signs of toxicity were observed in any group. One female duck in the 125 ppm group had a prolapsed cloaca during week 13; after the prolapse was reduced, the duck appeared normal for the remainder of the study.

There were no significant diffe rences in body weights, egg sh ell thickness, or offspring body weight between the control and any treatment group. At 500 ppm, feed consumption was slightly decreased (NS) throughout the study, with a significant difference from controls during weeks 6, 12, 14, and 19. There were no significant differences or apparent treatment-related effects in reproductive parameters at 25 and 125 ppm. However, while not significant, there appeared to be a slight reduction in the number of eggs laid by hens at 500 ppm during the last two weeks of egg production. During terminal necropsy, this reduction in egg production was
correlated with an increase in the number of hens in the 500 ppm group with a regressing ovary.PERMETHRIN FINAL

Page 59 of 67 Fish Full Life Cycle Toxicity (Fathead minnow, OCSPP 850.1500)

In a fish full life cycle toxicity study ( MRID 00102096, 1977 ), groups of 60 fathead minnows ( Pimephales promelas ) embryos (60 embryos/aquarium; 2 aqua ria/dose level) were exposed to permethrin (95.7% purity) in a flow-through system at nominal concentrations of 0 (control), 0, solvent control (7.2 mg/L DMSO), 0.063, 0.13, 0.25, 0.50, and 1.0 μg/L. During the initial 35 days of the chronic test, mean measured concen trations of permethrin were well below nominal
concentrations and this trend increased as fish increased in size. To reverse the trend, nominal permethrin concentrations were increased to 0.094, 0.19, 0.38, 0.75, and 1.5 μg/L. The measured concentration upon which survival and pe rcent hatch were compared to controls were 0-35 days: <0.023, <0.032, 0.092, 0.14, and 0.41 ppb; and 0-63 days: <0.042, 0.083, 0.17, 0.23, and 0.55 ppb. Percent hatch and survival was signi ficantly different at th e 0.41 ppb level. After 156 days exposure, surviving females were return ed to spawning chambers in the ratio of 3 males to 7 females. The levels tested (measured concentrations) were <0.073, 0.16, 0.22, 0.32, and 0.87 ppb. At the 0.87 ppb level, there were no female survivors. At ≤ 0.32 ppb, survival, weight, length, and eggs/female di d not differ significantly from c ontrols. The second generation eggs were then exposed to mean measur ed concentrations of <0.11, 0.17, 0.30, 0.41 and 0.91 ppb. The % hatch for these levels did not differ si gnificantly from controls but the survival of second generation fry at ≥ 0.41 ppb was significantly different than controls. To observe if there were residual effects of the chemical, fry were transferred from control groups to the 0.41 ppb test level and vice-versa; surviv al of 0.41 ppb fry transferred to c ontrol was significantly greater
than those transferred to the 0.41 ppb test level.

Mean measured concentrations of 0.91 and 0.41 ppb significantly reduced the percent survival of fry during 30 days exposure. Minnows which su rvived the initial expos ure period demonstrated normal ranges of measured parameters of surviv al, growth, reproduction, a nd egg hatchability. Based on the data, the researcher conclude d the maximum acceptable concentration of permethrin for fathead minnows is estimated to be between 0.30 and 0.41 ppb. Residue analysis
of fish showed bioaccumulation in female mi nnows as high as 4600x the water concentration. The majority of these residues were eliminat ed after 14 days in uncontaminated water.

Estrogenic Effects (Trout and Medaka)

In a published study by Nillos et al . (2010), permethrin stereoisomers were examined for estrogenic activity in vitro using transcriptional measurements of the egg-yolk precursor VTG in rainbow trout primary hepatocyte cultures, and in vivo using VTG protein measurements in male medaka. Additionally, the estrogenic activity of three permethrin metabolites [3-PBOH), 3-(4’- hydroxyphenoxy)benzyl alcohol (3,4 ’-PBOH), and 4-hydroxy permet hrin (4-OH permethrin)] was investigated in the trout in vitro study.

For the in vivo VTG expression assay with medaka, adu lt male medaka were held at static conditions for 8 days with individual pe rmethrin enantiomers, 1S-(+)-cis-, 1R-(

− )-cis-,PERMETHRIN FINAL

Page 60 of 67 1S-(+)-trans-, and 1R-(

− )-trans-permethrin (>99% purity), at 10 μg/L, 17- β estradiol (positive control; concentration not specif ied), or 1% acetone (solvent control). Based on the reported results, the experiment included “control” and “mixture” test groups which were not defined; the study reviewers assumed that the control group in cluded no chemical or solvent and that the mixture group was a mixture of all four permethrin enantiomers. On the last day of exposure,
fish were anesthetized and the livers were exci sed and pooled for each individual replicate. The VTG protein level in the liver was determined using a commercial ELISA kit; the VTG concentration was normalized to the total prot ein in the liver homoge nate. VTG production between the permethrin enantiomers was compared to determine enantio-selectivity.

For the in vitro VTG expression assay, trout hepatocyte s were isolated from juvenile trout (confirmed to be without endogenous expression of VTG) and the hepatocytes were seeded in 48-well plates. Cells were incubated (18 °C fo r 24 h) with racemic permethrin at 50 μM, the individual permethrin enantiomers at 50 μM each , permethrin metabolites at 5, 10, 25, or 50 μM each, 17- β estradiol (positive control) at 0.036 μM, or 1% acetone (solvent control); the experiment included a control group and treatmen ts were conducted in triplicate. The VTG mRNA expression in fish hepatocytes was measur ed using quantitative PCR after isolation of total mRNA from the cells using a commercial RNA extraction kit.

Microsomal biotransformation was investigated in livers pooled from five trout. The livers were homogenized and the microsomal fractions we re resuspended. A reaction mixture of microsomal protein, substrate (racemate and en antiomers), NADPH, and Tris-HCl buffer was incubated. The supernatant was isolated by cen trifugation for reverse-ph ase HPLC/UV analysis for metabolic products. Negative controls omitted the NADPH or included boiled microsomal protein. Cytochrome P450 inhibition tests were conducted by co-incubation with ketoconazole, which was added after addition of NADPH, and the mixture was incubated for 5 minutes prior to addition of the individual permethrin enantiomers.

After exposure of medaka in vivo for 8 days to the individual permethrin stereoisomers and permethrin mixture at 10 μg/L, hepatic VTG production was increased (p<0.05) compared to controls. Additionally, significant differences in the relative estrogenic potential of the enantiomers were reported: 1S -cis- and 1S-trans-permethrin had responses 2.5 and 1.3 times greater than their respective R enantiomers. The expression of VTG mRNA was significantly increased in trout primary hepatocytes exposed in vitro to the individual permethrin stereoisomers and permethrin mixture at 50 μM compared to controls. Additionally, a significant difference in the relative estrogenic potential of 1S-cis-p ermethrin and 1R-cispermethrin was reported: 1S-cis-permethrin had ~2-fold higher VTG mRNA expression levels than 1R-cis-permethrin. A similar (but not significant) trend was observed for the transpermethrin enantiomers.PERMETHRIN FINAL

Page 61 of 67 The estrogenic activity of the permethrin me tabolites (4-OH permet hrin, 3-PBOH, and 3,4’- PBOH) was also investigated in the in vitro trout hepatocyte experi ment. A concentrationrelated increase in the VTG mR NA expression level was observed in trout hepatocytes exposed to 4-OH permethrin and 3,4’-PBOH at 5, 10, 25, and 50 μM (the study au thors did not state whether increases were statistically significant). For 3-PBOH, a significa nt increase in the VTG mRNA expression was only reported at 10 μM ; the increase was not dose-dependent.

Stereoselective formation of three metabolites (4-OH permethr in, 3-PBOH, and 3,4’-PBOH) was observed following the incubation of permethrin in rainbow trout liver microsomes. Transpermethrin underwent ester cleav age (resulting in formation of 3-PBOH and 3,4’-PBOH) more extensively than cis-permethrin. However, the predominant metabolite produced was the hydroxylated derivative of intactpermethrin (4-OH permethrin). Furthermore, the cispermethrin diastereomers appeared more sus ceptible to hydroxylation, yielding >2 times the amount of the hydroxylated product than the trans-permethrin diastereomers.

1S-cis-Permethrin had the highest NADPH-depe ndent metabolic conversion of the four permethrin stereoisomers, with the hydroxylat ed metabolite (4-OH permethrin) comprising 99% of the metabolite profile. Formation of 4-OH pe rmethrin from 1S-cis-permethrin was ~25 times greater than the formation from 1R-cis-permeth rin, demonstrating a signi ficant stereoselectivity to 1S-cis-permethrin for the hydroxylation of permethrin. However, the NADPH-catalyzed cleavage of the cis-permethrin enantiomers was enantioselective for 1R-cis-permethrin.

Of all permethrin stereoisomers, 1S-trans-perme thrin was found to be the most susceptible to ester cleavage. Although the trans-permethrin st ereoisomers appeared more susceptible to ester cleavage than the cis-permethrin stereoisomers, hydroxylation in the fish liver microsomes was still higher than ester cleavage; 1S-trans-perme thrin was more extensively hydroxylated than 1Rtrans-permethrin.

Generally, ketoconazole signifi cantly inhibited metabolite formation and enantiomer conversion
when co-incubated with the indi vidual enantiomers. The highes t inhibition (98% reduction in conversion) was observed for 1S-cis-permethri n, likely due to diminished 4-OH permethrin
formation.

The study authors concluded that the results of the in vivo and in vitro studies indicated stereoselective estrogenic activity of permethrin resulting from biotransformation of the parent compound to more estrogenic metabolites. 1S-cis -Permethrin was observed to have significantly higher activity than the 1R-cis enantiomer in both studies. All permethrin enantiomers were oxidized to the 4-OH permethrin metabolite a nd underwent esterase cleavage to 3-PBOH and 3,4’-PBOH. Racemic 4-OH permethrin as well as 3-PBOH and 3,4’-PBOH possessed significant estrogenicity.PERMETHRIN FINAL

Page 62 of 67 Estrogen Activity in Fish

In a published in vivo study by Brander et al . (2012 ), evaluation of the concentration response to permethrin (99% purity; 50/50 mixture of isomers) with ju venile (65- to 70-day old) Menidia beryllina was examined in a 14-day static aqueous exposure bioassay. The biomarker selected was choriogenin (Chg), an estrogen-dependent egg coat protein, which was assessed with an indirect enzyme-linked immunos orbent assay (ELISA) relati ve to a positive control (ethinylestradiol). Fish were exposed to perm ethrin (0.1, 1, and 10 μg/L) or ethinylestradiol (1, 10, 50 ng/L) by adding stock solutions prepared in methanol (a control of 0.01% methanol was included). At study termination, fish were anes thetized (on ice), frozen, and homogenized in buffer, and the resultant supernatant afte r centrifugation was analyzed by ELISA. An in vitro chemical-activated luciferase gene expression (CALUX) bioassay was also conducted, using the human ovarian carcinoma (BG-1) cell line stably transfected with an estrogen-responsive luciferase reporter plasmid. The CALUX bioassa y was used to determine the concentrationdependent agonist (pesticide alone) and antagonist (p esticide in presence of estradiol) effects of permethrin. Plated cells were incubate d with carrier solvent (ethanol) and 17 β estradiol (E 2 ; 1 nM), permethrin, or permethrin plus 1 nM E 2 ; permethrin was tested at 0.1-1,000 μg/L.

Mean survival in all in vivo treatments after 14 days was ≥ 80%, with the exception of 70% mean survival observed in the 10 μg/L permethrin group. Juvenile M

. beryllina exposed to all three concentrations of permethrin and ethinylestradiol had higher (p<0.05) rela tive concentrations of Chg expressed in whole-body homogenates. None of the permethrin treatments were different from one another when compared with ANOVA ( p>0

. 05); however, a trend toward an inverse correlation between response and increasing concentration with permethrin was apparent. Permethrin treatments were not significantly different from positive control concentrations of 1 and 10 ng/L ; h owever, all permethrin concentrations tre nded toward a higher relative expression of Chg compared to the 1 ng/L ethinylestradiol treatment and a lower relative expression than the 10 ng /L ethinylestradiol treatment. All study concentrations of permethrin induced the expression of Chg in juvenile M. beryllina relative to control, and 1 ng/L permethrin induction yielded greater Chg expression compared to 1 ng/L ethinylestradiol

.
For the CALUX assay, ER agonism was not obs erved for any of the permethrin test concentrations. For permethrin, some reduction in the ability of E 2 to induce ER-dependent reported gene activity was observed (~30-40% of maxi mal estradiol activity), but this inhibitory effect was not concentration-dependent (data pr esented graphically). It was concluded that permethrin was estrogenic in the in vivo test, but anti-estrogenic in cells in vitro , and noted that
the difference observed between in vivo and in vitro results may be attributable to the lack of appropriate metabolism in the CALUX cell line.PERMETHRIN FINAL

Page 63 of 67 Estrogen Effects (Embryo-Larval Zebrafish)

In a published study by Y. Jin et al . (2009), the estrogenic effects of permethrin enantiomers were evaluated by detecting the expression levels of estrogen-responsive genes in embryo-larval zebrafish. Permethrin (99.2%) was dissolved in acetone and diluted to 100, 250, 500, and 1000 ng/L in rearing water containing 0.1 % acetone (v/v). The four pe rmethrin enantiomers, (+)-cis-, (

− )-cis-, (+)-trans-, and (

− )-trans-permethrin, were also dilu ted to 250 or 1000 ng/L in water containing 0.1% acetone. Embryo- larval zebrafish were exposed to the above testing solutions under static renewal conditions for 7 days (0-7 days post-fertilization). Control embryos and larvae were exposed in water containing 0.1% acetone (v/v) only. The exposure solutions were renewed daily. No difference in survival rate was found during the study. Separate groups of embryo-larval zebrafish were expos ed for 7 days to estradiol (E 2 ) at concentrations of 25, 50, 100, 250, 500, 1000 ng/L in water containing 0.1% acetone. Samples were homogenized and
total RNA was isolated and reverse transcribe d for real-time quantitative PCR analyses to determine the expression of vtg1, vtg2, esr α , cyp19a, and cyp19b genes.

The expression levels of vtg1, vtg2, esr α , cyp19a, and cyp19b mRNA in the zebrafish larvae were induced effectively when exposed to E 2 for 7 days. The lowest concentration of E 2 to significantly induce transcripts of these estroge nic-responsive genes was 50 ng/L, and the induction in mRNA levels of vtg1, esr α , cyp19a, and cyp19b were 5.0- , 2.6-, 2.3-, and 5.4-fold respectively, at this concentration compared with those in the control; vt g2 was the only one of these transcripts which was significantly i nduced at 25 ng/L E2. In addition, the E 2 -induced increase in the mRNA level of the five genes was concentration-dependent. The sensitivity of these genes (in terms of the con centration needed to elicit a significant response) was the same, with the exception of vtg2 which was slightly more sensitive than the others. Thus, the expression of vtg1, vtg2, esr α , cyp19a, and cyp19b genes were adopted as biomarkers, and the 50 ng/L E 2 group was selected as the positive co ntrol in subsequent experiments.

Gene-specific responses to exposure to racemic permethrin were observed at concentrations

≥ 250 ng/L. The mRNA levels of three genes, vtg1, esr α , and cyp19b were higher (p<0.05) than those of the control group at exposure concentrations of 250, 500, and 1000 ng/L. In addition, permethrin induced the expression of the esr α in a concentration-dependent manner. Significant induction was observed for cyp19a only in the 50 0 and 1000 ng/L permethrin treatment groups. No significant difference was observed between the control and treatment groups for vtg2 expression, even at 1000 ng/L. Maximum induction in the tran script levels of vtg1, esr α , cyp19a, and cyp19b occurred in th e highest exposure group, being about 3.8-, 5.0-, 4.8- and 3.0- fold higher than the control group, re spectively. In the case of esr α and cyp19a, the induction folds in the highest permethrin treatment group were higher than that of the positive control group of 50 ng/L E2. From these results, embryo-larval expressions of vtg1, esr α , cyp19a, and cyp19b genes were adopted as biomarkers, and the 250 and 1000 ng/L racemic permethrin groups were selected as the positive contro l in the enantiomer exposure experiments.PERMETHRIN FINAL

Page 64 of 67 Generally, at 250 ng/L each permethrin enantio mer exhibited a simila r effect on the mRNA levels of the four selected genes as the r acemate did. The only significant difference between enantiomers was found in cyp19b mRNA i nduction between the (+)-trans and (

− )-trans enantiomer treatment groups. The mRNA levels of cyp19b in larval zebrafish receiving (

− )-trans
treatment were about 1.6-fold hi gher than those in the group treated with (+)-tran s enantiomer. When the exposure concentration of permethrin enantiomers was increased to 1000 ng/L, the enantioselectivity in the induction of embryo- larval estrogenic-res ponsive gene expression
became more obvious. The maximum induction in the mRNA levels of all four genes was observed in the group treated with (

− )-trans enantiomer, being much higher even when compared with that of the racemate group. Moreover, a sign ificant difference in the mRNA levels of vtg1, cyp19a, and cyp19b existed between the (+)-trans and (

− )-trans enantiomer treatments. The mRNA levels of vtg1, cyp19a, and cyp19b of (

− )-trans treatment were about 3.2-, 1.8- and 1.5- fold higher than that in the group treated with (+ )-trans enantiomer, respectively. There was also a significant difference in mRNA levels of the cyp19b gene between th e two cis-enantiomer treatment groups; the mRNA level of cyp19b in (+)-cis treatment group was about 1.5-fold higher than that in th e group treated with (

− )-cis-enantiomer. No significant difference was observed in expression of vtg1 or cyp19a be tween the two cis-enantiomers. For esr α , no significant difference was found between (+)-trans and (

− )-trans or (+)-cis and (

− )-cisenantiomers, although the inductive tendency wa s similar to vtg1, cyp19a, and cyp19b. The study authors concluded that the results of these experiments st rongly indicate the occurrence of significant enantioselectivity in estrogenic ac tivity of permethrin en antiomers exposed to embryo-larval zebrafishPERMETHRIN FINAL

Page 65 of 67 APPENDIX 3: References Not Utilized in the Permethrin WoE Analysis

In 2009, after public review and comment, a fina l list of 67 chemicals and schedule for issuing
Test Orders for the EDSP Tier 1 screening battery was made av ailable in a Federal Register Notice issued October 21, 2009 (74 FR 54422). The ag ency’s review of the initial data submitted as “other scientifically releva nt information (OSRI) was provided in the Report of the Endocrine Disruptor Review Team (USEPA, 2010).

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies previously submitted to the agency for registra tion/reregistration, published literature articles and/or Tier 1 assays. The agency also c onducted a more recent search (2009 to 2014) of
available scientific literature fo r any additional relevant informa tion for their weight of evidence (WoE) evaluations. These articles were evalua ted in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/s cience/efed/policy_guidance/team _authors/endangered_species _reregistration_workgroup/PDF_ro t/esa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature To xicity Studies to Suppor t Human Health Risk Assessment ( http://www.epa.gov/pesticides /science/lit-studies.pdf).

The following published and unpublished references were considered for use in the WoE analysis for Permethrin but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the com pound of interest was not us ed in the study; 3) the test material was not adequately described; 4) a formulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only da ta on invertebrates.

Aït-Aïssa, S, Laskowski, S, Laville, N, et al . (2010) Anti-androgenic act ivities of environmental pesticides in the MDA-kb2 reporter cell line. Toxicol. In Vitro 24 : 1979-1985.

Boone, M (2008) Examining the single and interactiv e effects of three insecticides on amphibian metamorphosis. Environ. Toxicol. Chem

. 27 (7) : 1561-1568.

DeLorenzo, M, Serrano, L, Chung, K, et al . (2006) Effects of the inse cticide permethrin on three life stages of the grass shrimp, Palaemontes pugio

. Ecotoxicol. Environ. Saf. 64 : 122-127.

DeMicco, A, Cooper, K, Richardson, J, et al . (2010) Developmental ne urotoxicity of pyrethroid insecticides in zebrafish embryos. Toxicol. Sci. 113 (1) : 177-186.PERMETHRIN FINAL

Page 66 of 67 Hinfray, N, Porcher, J, and Brion, F (2006) Inhibition of rainbow trout ( Oncorhynchus mykiss )
P450 aromatase activities in brain and ovarian mi crosomes by various environmental substances.
MRID 48203005. Published: Comp. Biochem. Physiol. Part C. 144: 252-262.

Issam, C, Zohra, H, Monia, Z, et al . (2011) Effects of dermal s ub-chronic exposure of pubescent male rats to permethrin (PRMT) on the histological structures of genital tract, testosterone and lipoperoxidation. Exp. Toxicol. Pathol. 63 (4) : 393-400.

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