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 

# &

Health Effects Divison

# ::~:E£'~:::e 

# f] g:ams d f 'v--

Environmental

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

THR O UG H: Je ss Rowland

## ~ S

## ~~~ 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 EVIDENCE ANALYSI S OF POTENTIAL
INTERACTION WITH THE ESTROG EN, ANDROGEN OR THYROID
PATHWAYS

CHEMICAL: CYPERMETHRIN

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS


### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY


### U.S E NVIRONMENTAL 

### P ROTECTION 

### A GENCYCYPERMETHRIN 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...........................21

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

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

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

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

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

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

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

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

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

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

LIST OF TABLES: Table 1. Tier 1 Assays for Cypermethrin..............................................................8
Table 2. Estrogenic/Anti-Estroge nic Pathway for Cypermethrin................................11
Table 3. Androgenic/Anti-Androgeni c Pathway for Cypermethrin.............................17
Table 4. Thyroid Pathway for Cypermethrin.......................................................22

APPENDIX 1. EDSP Tier 1 Screening Assays.........................................................33
APPENDIX 2. Other Scientifically Relevant Information (OSRI)................................42

APPENDIX 3. References Not Utilized in the Cypermethrin WoE Analysis....................61CYPERMETHRIN 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 enerationCYPERMETHRIN 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 ConcentrationCYPERMETHRIN 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)CYPERMETHRIN 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-EvidenceCYPERMETHRIN FINAL

Page 1 of 64 Executive Summary

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether cypermethrin interacts with E, A, or T hormone pathways, the number and type of effects induced, th e magnitude and pattern of re sponses observed across studies,
taxa and sexes were c onsidered. Additionally, the conditi ons under which effects occur were considered, in particular, whet her or not endocrine-related resp onses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On June 25, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs and the Offi ce of Science Coordination and Policy conducted a weight-of-evidence analysis of the potential interac tion of cypermethrin with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE eval uation in this report are presented by pathway (E, A and then T) be ginning with the results of the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited OSRI for mammalian and wildlife studies ( 40 CFR Part 158 and literature).

Cypermethrin is a member of the synthetic pyrethr oid class of insecticide having similar toxicity profiles that elicit neurotoxic be haviors in laboratory animals. This assessment includes studies conducted on isomeric mixtures (i.e., cypermethrin and β -cypermethrin, a resolved mix of the biologically-active isomers).

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

vitro studies yielded conflicting resu lts; while some studies showed weak effects, other studies showed no effect on the estrogen pathway in ER binding or activation assays. The in vivo Uterotrophic and Female pubertal assays were negative, and no estrogen- related effects were
seen in the mammalian Part 158 studies. In th e fish short-term reproduction assay (FSTRA), there was a decrease in fecundity and some indica tion of mature oocyte atresia in females at the highest concentration tested (1.4 μg/L). These effects in female fish are further discussed under the androgen pathway section below.

There was convincing evidence for potential interaction with the androgen pathway. The effects
observed in the in vitro assays, along with eviden ce of androgen antagonist effects in some of the

in vivo mammalian assays, shows potential anti-androge nic effects in both mammals and fish.CYPERMETHRIN FINAL

Page 2 of 64 Positive responses were seen in the androge n binding and the androgen receptor (AR) binding and AR transactivation assays. Although no effe cts were observed in the Tier 1 mammalian assays, there were effects obs erved in mammalian literature studies of cypermethrin and β

cypermethrin, including d ecreased sperm production at ≥ 15 mg/kg/day (Liu et al . 2010) and damage to the seminiferous tubules and spermatids at ≥ 6.25 mg/kg/day (Hu et al ., 2013; Li et al

. 2013).

In the FSTRA, significant increases in male GSI were observed (22-27%) at the mid and high concentration along with a marginally significant d ecrease (24%) in male tubercle score at the high concentration. While there appears to be limited complementary effects within a sex, collectively the effects observed in the FSTRA across both males and females appear to be consistent with reported effects for other chem icals (n=3) reported to be potential androgen antagonists (US EPA 2007) including decreased fecundit y, increased female oocyte atresia, and decreased tubercle score. Effects on male or female gona dal staging index (GSI) were sometimes reported but the directi on of effect (increased/decreased ) was not consistent. It is
noted that increases in female vitellogenin (VTG) were also reported for two of the three chemicals, and this response was not observed in the FSTRA for cypermethrin.

There was no convincing evidence for potential interaction with the thyroid pathway. No thyroid-related responses were observed in mamm alian Tier 1 assays, Pa rt 158 studies, or other OSRI. Additionally, based on the lack of effect s on developmental endpoint s or alterations in thyroid histopathology, the amphi bian metamorphosis assay ( AMA) did not indi cate a potential for a thyroid interaction in amphibians for cypermethrin.

For cypermethrin, the current poin t of departure (POD) of 6.0 mg /kg/day used for human health
risk assessment is based on clini cal signs of neurotoxicity and mo rtality observed in male dogs and decreased body weight and body weight ga in observed in female dog at 18.1 mg/kg/day (LOAEL) in the chronic toxicity study in the dog s. This POD is used to derive the chronic Reference Dose (RfD) used for chronic dietary exposure risk assessments and for calculating the Margins of Exposure (MOEs) for non- dietary exposure risk assessments.

In general, if the POD is lower or equal to the dose(s) that caused endocri ne-related effects, there would be a concern that the POD may not be protective of potential estrogen, androgen or thyroid related effects. In the case of cyperm ethrin, the POD (6.0 mg/kg/day) is numerically identical to the doses (6.25- 7.25 mg/kg/day) that caused damage to the seminiferous tubules and spermatids in studies reported as OSRI (Hu et al., 2013 and Li et al . 2013). Additionally, decreased sperm production was seen at a dose (12 mg/kg/day) that is only approximately twofold higher than the POD. Therefore, the POD used in risk assessments does not address the concern for the adverse male reproductive effectsCYPERMETHRIN FINAL

Page 3 of 64 Although, adverse effects are seen, the ED SP Tier 2 study (post 1998 two-generation reproduction or the extended one-generation re production study) which evaluates male and female mammalian reproduction and fertility is not recommended since: 1) no estrogen-related
effects were seen in the Tier 1 female pubertal assay; 2) no estrogen-related effects were reported in the OSRI; and 3) the concern is only for the androgen-related eff ects in the adult male reproductive system.

Consequently, for mammals, a special study focuse d on evaluating the potential adverse effect of cypermethrin in the male reproductive system is recommended. The experimental design (i.e, protocol) for this study should be devel oped in consultation with the agency.

For non-mammalian species, in the problem fo rmulation for the registration review of cypermethrin, the chronic no observed adverse effect concentration (NOAEC) for fish was reported at 0.14 μg/L based on a 30-day exposur e fathead minnow early life-stage study. Reproductive effects were observed in the FSTRA at measured concentrations of ≥ 0.12 μg/L. A fish life-cycle study for cypermethrin with fath ead minnow is available with a reported NOAEC of 0.06 μg/L (MRID 40641701). However in this st udy, excessive analytical variation (high:low ratios of 3.0 to 7.8) was observed at all levels during the study, and the range of concentrations was so great that overlapping of concentrations occurred for all levels. Therefore, there is uncertainty with regards to the reported NOAEC in that study. Given the potential for antiandrogen effects observed in the Tier 1 weight of evidence analysis, the EDSP Tier 2 Medaka Extended One Generation Reproduction Toxicity (M EOGRT) study is recommended. With that recommendation, it is noted that given the physio -chemical properties of cypermethrin and the results in the fathead minnow life-cycle st udy, there may be difficulties in maintaining cypermethrin concentrations in aqueous systems over the duration of a long-te rm test such as the Tier 2 fish assay and careful attention and maintena nce of the test system will likely be required.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for cypermethrin. In both spec ies, no effects on reproductive parameters were observed; an additional study with alpha cypermethrin did report potential reproductive effects but there are uncertainties in this study. Th e type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are consid ered sufficient for evaluating potential reproductive effects to birds from cypermethrin exposure. Relative to the EDSP, additional testing is not recommended.

Given the lack of observed poten tial thyroid-related effects in the AMA, no additional testing with amphibians is recommended.CYPERMETHRIN FINAL

Page 4 of 64 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether cypermethrin interacts with E, A or T hormone pathways, the number and type of effects induced, the magnitude and pattern of respons es observed across studies, taxa and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whethe r or not endocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On June 25, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WOE) an alysis of the potenti al interaction of cypermethrin with the E, A or T hormone pa thways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Cypermethrin is a chiral compound with eigh t stereoisomers, each with an abundance of approximately 11-14%. Alpha( α )-cypermethrin consists of the ( R )-alcohol (1 S )- cis -acid and the ( S )-alcohol (1 R )- cis -acid; beta( β )-cypermethrin consists of the ( R )-alcohol (1 S )- cis and trans

acids and the ( S )-alcohol (1 R )- cis and trans -acids (alpha + theta forms); theta( θ )-cypermethrin consists of the ( R )-alcohol (1 S )- trans -acid and the ( S )-alcohol (1 R )- trans -acid; and zeta( ζ )- cypermethrin is an S-enantiomer-enriched formulation of the four ( S )-alcohol isomers of cypermethrin (24% each) with 1% each of the less active ( R )-alcohol isomers. Cypermethrin has a vapor pressure of 1.7 (or 3.1) × 10

− 7 mm Hg at 20°C; cypermethrin w ill exist as a particulate if released to the atmosphere and is expect ed to be removed from the atmosphere by photodegradation and deposition. Cypermethr in is poorly soluble in water (4- 8/7!qqc ), and has an octanol/water partition coefficient (log Kow) of 6.6 with an estimated bioconcentration factor range from 420-468. Cypermethrin is expected to have low mobility in soil, with no volatilization, but will rapidly degrade in soil under aerobic conditions.

The available information considered in determ ining the potential intera ction of cypermethrin with the E, A and/or T pathways include submitted EDSP Tier 1 assays and/or otherCYPERMETHRIN FINAL

Page 5 of 64 scientifically relevant information (OSRI) such as general toxicity st udies and other published
articles. These data are summarized in Sections III.A through III.C. An analysis of the data submitted to the Agency, using the WoE appro ach outlined by the Agency (USEPA, 2011), is presented in Section IV. The ED SP Tier 2 studies recommendations are presented in Section V.

II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix

1. B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Order recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). The Agency’s review of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team on cypermethrin (USEPA, 2010). Since then, the Agency has also conducted a more recent sear ch of available scientif ic literature for any
additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies c onsidered but not util ized 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.CYPERMETHRIN FINAL

Page 6 of 64 As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may 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 concordance or consistency (complementarity) of the responses in the individual study is evaluated. For the
Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a 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 expression of responses at higher levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide 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 cons iderations of whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemicCYPERMETHRIN FINAL

Page 7 of 64 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 July 2013 that evaluated scie ntific issues associ ated with the WoE evaluation of the EDSP Tier 1 screening proce ss. 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 effects in endocrine se nsitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

For these WoE analyses, overt toxicity was 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., changes 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.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/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 availabl e toxicity data into account. .CYPERMETHRIN FINAL

Page 8 of 64 The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

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

A. EDSP Tier 1 Screening Assays

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

Table 1. Tier 1 Screening Assay for Cypermethrin

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied by OSRI (Saito et al., 2000; Chen et al., 2002)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID 48670201)

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

Xu et al., 2008)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID 48670203)

Aromatase Assay (human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID 48670202)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID 48615701)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied by OSRI (Zhang et al. , 2008)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID 48683002)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID 48683003)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID 48683001)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied (MRID 49013801)CYPERMETHRIN FINAL

Page 9 of 64 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for cypermethrin to interact with the estrogen pathway is summarized in Table 2. The various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects. This
table also includes HPG-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 Agency determined that the requirement fo r an ER binding study was satisfied based on the
results of Saito et al. (2000) and Chen et al. (2002). The ER competitive binding assay by Chen

et al. (2002) was positive however the assay by Saito et al. (2000) and another OSRI assay by
Kim et al. (2004) were negative

. A yeast two-hybrid assay by Saito et al. (2000) was negative (using Nishikawa procedure) for ER interaction. In another published study, Tyler et al. (2000)
did not demonstrate hER activation in a recombinan t yeast expression system assay , but antiestrogenic activity was observed.

Cypermethrin was classified as negative in the Tier 1 ERTA. A mammalian ERTA luciferase assay by Saito et al. (2000) was also negative, however Kjeldsen et al. (2013) showed weak induction by cypermethrin in an ERTA a ssay with a maximum response of 119% (at 10

− 6 M); cypermethrin did not exert an ER-a ntagonistic effect in this assay

.
In pS2 (an ER responsive gene) gene expression assays, Chen et al. (2002) and Jin et al. (2010) observed an approximately 2-fold increase in pS2 expression at 10

− 6 M. Chen et al. (2002) and
Jin et al. (2010) showed that cypermethrin yielded a weak agonistic response in an E-Screen (MCF-7 cell proliferation) assay . However Kim et al. (2004) reported negative responses to cypermethrin in an E-Screen (MCF-7 BUS cell proliferation).

Cypermethrin was positive for estr adiol induction in the Tier 1 st eroidogenesis assay. The Tier 1 aromatase assay and an OSRI aromatase assay by Kjeldsen et al. (2013) were negative for
aromatase inhibition.

Cypermethrin was negative in the Tier in vivo uterotrophic assay and no estrogen-related effects
were seen in the fema le pubertal rat assay.

In the FSTRA, there were no treatment-related effects on plasma VTG concentration, secondary sex characteristics, or behavior in females. Fecundity was decreased by 42% and 84% in the medium- and high-treatments, (0.12 and 1.4 μg a.i. /L) respectively, but there were no treatmentrelated effects on fertilization success. An incr eased incidence of mature oocyte atresia was present at the low (0.013 μg a. i./L), medium, and high test c oncentration, respectively, (13%,CYPERMETHRIN FINAL

Page 10 of 64 7%, and 50%), compared to control females which had no reported incidents. While this histopathological finding was a ssociated with granulomatous inflammation and the presence of microsporidia in half of the cas es across all treatment levels, th e increased incidence of atresia was considered potentially treatment-related in the high dose. There were no differences observed in gonadal staging.

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

In Part 158 avian reproduction t oxicity studies (one bobwhite quail and one mallard duck) for cypermethrin, no treatment-rela ted effects were reported. In another Part 158 bobwhite quail reproduction toxicity study testing alpha cyperm ethrin, dietary administration resulted in significant decreases in the numbe r of hatchlings/eggs set (%) at the low dose of 48 mg/kg-diet, the adult male body weight gain at the mid- and high treatment groups (149 and 452 ng/kg-diet), and 14-day old offspring survivor s at the high treatment. However, in this study pen mate aggression was observed across all treatment levels and control gr oups, which likely resulted in the observed parental mortalities (one in cont rol, 2 in low treatment group, and 3 in high treatment group), and therefore, as there may have been poor husbandry in this study it may potentially have impacted the re sults of this reproduc tive study. In a Part 158 freshwater fish early life-stage toxicity test with fathead minnow, larval survival was significantly reduced in the highest test concentratio n (0.33 μg a.i./L). A Part 158 fish life-cycle toxicity test with fathead minnow is available, for which reduction in fecu ndity at 0.32 μg/L were observed, along with effects on parental survival and growth at th e same concentrations. However, excessive analytical variation (h igh:low ratios of 3.0 to 7.8) was obser ved at all levels during the study. The range of concentrations was so great that overlapping of concentra tions occurred for all levels. Therefore, there is uncertainty in the reported NOAEC, and this study is not included in
the table below.CYPERMETHRINFINALPage 11 of 64Table 2. Estrogenic/Anti-Estrogenic Pathway for CypermethrinLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Cypermethrin1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity OBserved 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER BindingRequirement satisfied by OSRI (Saitoet al.,2000, MRID 48176003 and Chenet al.,2002, MRID 48146201; see below)ERTA(MRID 48670201)NAromatase(MRID 48670202)NSteroidogenesis(MRID 48670203)P4Uterotrophic(MRID 48615701)NNEP5NFemale Pubertal Rat(MRID 48683002)NNNN N NNNFSTRA(MRID 48683001)NENP(H)6NFrt: N;Fcd:↓42, 84%(M, H)NNOSRIER BindingSaitoet al.(2000)MRID 48176003NNER BindingChenet al.(2002)MRID 48146201PPCYPERMETHRINFINALPage 12 of 64Table 2. Estrogenic/Anti-Estrogenic Pathway for CypermethrinLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Cypermethrin1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity OBserved 2 Overt Toxicity Observed 3Cell Proliferation/ERBinding(Kimet al., 2004)NNCell Proliferation/pS2Expression (Jinet al.,2010)PER Binding(Tyleret al., 2000)PERTA(Kjeldsenet al.,2013)PAromatase(Kjeldsenet al.,2013)NSubchronic toxicity(Dog; MRID44527002)NENENNEX(H)X(ML,MH, H)Developmentaltoxicity (Rat; MRID00056804)NENX(M, H)NDevelopmentaltoxicity (Rabbit;MRID 43776302)NENX(M, H)NDevelopmentalNeurotoxicity (Rat;MRID 46670402)NENNX(H)NCYPERMETHRINFINALPage 13 of 64Table 2. Estrogenic/Anti-Estrogenic Pathway for CypermethrinLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Cypermethrin1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity OBserved 2 Overt Toxicity Observed 3Three-generationreproduction (Rat;MRID 00112912)NR NR NNRNX(H)NThree-generationreproduction (Rat;MRID 00090040)NR NR NNRNNNChronic toxicity/carcinogenicity (Rat;MRID 00112910)NENNNX(H)NCarcinogenicity(Mouse; MRID00112911)NENENNENNChronic toxicity(Dog; MRID00112909)NENNNX(H)NAvian reproduction(Duck; MRID00090073)NN NAvian reproduction(Quail; MRID00090074)NN N1.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. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay.Changes in weight are absolute unless otherwise indicated.2.2. The systemic toxicity inthe Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated byan X in this column. For details see Section IV. A3.3. 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. ACYPERMETHRINFINALPage 14 of 644. Estradiol induced at 10 μM in Runs 3 and 4, and estradiol concentrations in all three runs at 10 μM were 111-117% of the solvent control.5. Treatment-related clinical signs of toxicity included one or two animals with dried red material around nose, hypersensitivity to handling, vocalizationupon handling, and salivation and/or wet clear material around mouth at 80 mg/kg/day (HTD).6. Increased incidence of mature oocyte atresiain highest test concentration (1.4 μg a.i./L)P Positive findingsN Negative (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedCYPERMETHRIN FINAL

Page 15 of 64 2. Effects on Androgen Pathway

The potential for cypermethrin to interact with the androgen pathway is summarized in Table 3. The various targets of the androgen pathway across the relevant 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. Effects that occurred in the presence of overt toxicity are di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

The Agency determined that the requirement for an AR binding study was satisfied by the results of AR transactivation (ARTA) studies by Zhang et al. (2008) and Xu et al. (2008). Zhang et al. (2008) examined the effects of cypermethrin and β -cypermethrin at a single concentration (1

×10

− 5 M) in an ARTA assay. The results indicate d that cypermethrin was anti-androgenic with an approximate 45% decrease in activity relative to the control; β -cypermethrin was negative in this assay. Xu et al. (2008) tested cypermethrin at 10

− 7 , 10

− 6 , and 10

− 5 M in an ARTA assay. Cypermethrin was reported to be an anti-androgen with an IC 50 of 6.80±2.3×10

− 5 M, with inhibition at the highest concentration (10

− 5 M). No androgen agonism was detected by Xu et al. (2008) ; Zhang et al. (2008) did not test androgen agonism

.

In other OSRI studies, Du et al. (2010) demonstrated similar results to the earlier studies with cypermethrin displaying anti-androgenic activity with an IC 20 of 1.64×10

− 6 M, and no androgen agonism. However, negative responses for bot h androgenicity and an ti-androgenicity were determined by Kjeldsen et al. (2013) in ARTA assays. In addition, cypermethrin did not displace

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

.

Cypermethrin was classified as negative in the Tier 1 steroidogenesis assay. The Agency determined that the requirement for Hershberger assay was satisfied by Zhang et al. (2008). In that study, cypermethrin demonstrated an androg en-antagonist effect; an drogen agonism was not investigated. Treatment with cypermethrin at 50 mg/kg/day (only dose tested) resulted in decreased absolute weights for seminal vesicles , ventral prostate and dorsolateral prostate; however, there were no decrease s in LABC muscle, Cowper’s gl ands, or glans penis weights

. Treatment with β -cypermethrin at 50 mg/kg/ day did not result in any e ffects on the accessory sex organ (ASO) weights. However, there were no androgen-related effects observed in the Tier 1 male pubertal assay.

In the FSTRA, increases in gonado-somatic index (GSI) of 22 and 27% in the medium- and high-treatment groups (0.12, and 1.4 μg a.i./L), respectively, were observed compared to the negative control. Additionally, there was a marg inally significant (p=0.05 5) decrease of 24% in median tubercle score for males at the high treatment concentration. No other effects [ e.g. , body weight, plasma vitellogenin (VTG) concentra tion, gonadal stages, a nd histopathology] were
observed.CYPERMETHRIN FINAL

Page 16 of 64 No androgenic or anti-androgeni c effects on reproductive parameters due to cypermethrin treatment were reported in existing mammalian Pa rt 158 studies. However, several published mechanistic studies from the same laboratory (Hu et al. 2013, Li et al. 2013 and Liu et al. 2010) in orally dosed adult SD rats reported adverse eff ects of cypermethrin or β -cypermethrin on the reproductive system of male rats dosed for 15 days. At doses up to 50 mg/kg/day, Hu et al. (2013) observed decreased prosta te weights at the higher doses and decreased sperm production at the high dose; as well as damage to se miniferous tubules and spermatids, decreased
testosterone concentration and increased FSH and LH concentrat ions at the high dose. In a similar study by Li et al. (2013) at doses up to 60 mg/kg/da y, no effects on reproductive organ weights were noted, but decr eased sperm production, gross an d microscopic changes in seminiferous tubules and spermatids, decreased testosterone concentrat ions, and increased FSH concentration were noted. In addition, in a study of β -cypermethrin at doses up to 30 mg/kg/day (Liu et al. , 2010), no organ weight effects were seen, but sperm counts and sperm production
were decreased.

In another study, Wang et al. (2009) addressed effects of β -cypermethrin exposure in mice at 0, 1, 10 or 20 mg/kg/day for 35 days. Testes, epidid ymis, and seminal vesicles organ weights were dose-dependently decreased at 10 and 20 mg/kg, and prostrate orga n weights were decreased at 20 mg/kg only. Sperm counts, viability, motility, and testosterone concentrations were dosedependently decreased . However these effects were observed in the presence of decreased body weight gain of 52 and 81% at 10 and 20 mg/kg/da y doses (body weight data were not reported)

. Histological and ultrastructural changes due to β -cypermethrin exposure at the tw o highest doses were similar to changes observed in the Hu et al. 2013, Li et al. 2013 and Liu et al. 2010 studies, with damage to seminiferous tubules and sperm developmen t. There were no reported androgen-related endpoints affected in the ecotoxicology OSRI.CYPERMETHRINFINALPage 17 of 64Table 3. Androgenic/Anti-Androgenic Pathway for CypermethrinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Cypermethrin1Study Type/Literature Citation AR Binding AR Activation Steroidogenesis Testosterone Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity OBserved 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR BindingRequirement satisfied by OSRI (Zhanget al.,2008; MRID 48196302 and Xuet al.,2008; MRID 48176006 see below)Steroidogenesis(MRID 48670203)NHershbergerRequirement satisfied by OSRI (Zhanget al.,2008; MRID 48196302 see below)Male Pubertal Rat(MRID 48683003)NNNNNNNNNNFSTRA(MRID 48683001)NE↑22,27%(M, H)NTubercle:↓24%(H)NNNOSRIAR Binding(Zhanget al., 2002)MRID 48196302NAR Binding(Tyleret al., 2000)PARTAZhanget al.(2008)PARTAXuet al.(2008)MRID 48176006PCYPERMETHRINFINALPage 18 of 64Table 3. Androgenic/Anti-Androgenic Pathway for CypermethrinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Cypermethrin1Study Type/Literature Citation AR Binding AR Activation Steroidogenesis Testosterone Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity OBserved 2 Overt Toxicity Observed 3ARTA(Duet al., 2010)PARTA(Kjeldsenet al.,2013)NHershbergerZhanget al.(2008)MRID 48196302N↓SV,VP, DPN NSubchronic toxicity(Dog; MRID44527002)NNNNNENEX(H)X (ML,MH, H)DevelopmentalNeurotoxicity (Rat;MRID 46670402)NX(H)NThree-generationreproduction (Rat;MRID 00112912)NRNNRNNRNR NX(H)NThree-generationreproduction (Rat;MRID 00090040)NRNNRNNRNR NN NChronic toxicity/Carcinogenicity (Rat;MRID 00112910)N N NRNN NRX(H)NCarcinogenicity(Mouse; MRID00112911)N N NE N NE NEN NCYPERMETHRINFINALPage 19 of 64Table 3. Androgenic/Anti-Androgenic Pathway for CypermethrinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Cypermethrin1Study Type/Literature Citation AR Binding AR Activation Steroidogenesis Testosterone Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity OBserved 2 Overt Toxicity Observed 3Chronic toxicity(Dog; MRID00112909)NNNENNNEX(H)NAvian reproduction(Duck; MRID00090073)NN NAvian reproduction(Quail; MRID00090074)NN NMechanistic Study:Male ReproductiveSystem (Rat; Huetal., 2013)NNP(L, ML)NNRNX(ML,MH,H)X (MH,H)Mechanistic Study:Male ReproductiveSystem (Rat; Lietal., 2013)↓(MH,H)NP(L,ML,MH,H)NNRNN NMechanistic Study:Male ReproductiveSystem (Rat; Liuetal., 2010)NENP(L, H)NNRNN NRCYPERMETHRINFINALPage 20 of 64Table 3. Androgenic/Anti-Androgenic Pathway for CypermethrinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Cypermethrin1Study Type/Literature Citation AR Binding AR Activation Steroidogenesis Testosterone Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity OBserved 2 Overt Toxicity Observed 3Mechanistic Study:Male ReproductiveSystem (Mouse;Wanget al., 2009)8↓35,70%(M,H)↓21,39%(M, H)P(M, H)↓6,10%(M, H)NRSV:↓28,58%(M, H);PR:↓11%(H)X(M, H)NRAvian reproduction(Duck; MRID00090073)NN NAvian reproduction(Quail; MRID00090074)NN N1. Key to responses: Low treatment (L), Medium-Low treatment (ML), Medium treatment (M), Medium high treatment (MH), High treatment (H).Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in thisassay. Changes in weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventralprostate (VP), Dorsal prostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG),glans penis (GP),body weight gain (BWG).2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by anX in this column. 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. Fordetails see Section IV. AP Positive findingsN Negative (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedCYPERMETHRIN FINAL

Page 21 of 64 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 redundancy an d have the potential to detect changes in the HPT axis, i.e ., the pubertal female and male rat assays, and the AMA (frog).

The potential for cypermethrin to interact with thyroid regulation are summarized in Table 4. The various targets of the thyroi d pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

No effects on the thyroid pathway (thyroid weight, histopathology, or ho rmone concentrations)
were observed in the in vivo male and female pubertal assays.

In the AMA, cypermethrin did not delay or accelerate median Nieuwkoop-Faber (NF) developmental stage at Day 7 or 21 relative to the negative control. Alth ough, more than half of all individuals developed beyond NF stage 60 by Day 21 ( i.e. , NF stage 61-62), with 53%, 59%, 54%, and 52% of surviving frogs exhibiting de velopment beyond NF60 (termed “late-stage”) in the control, low-, medium-, and high-con centration groups (0.0019, 0.109, and 0.0867 μg a.i./L, respectively) it did not appear to be treat ment-related. No effects on thyroid gland histopathology were observed at any treatment con centration. There also was no effect on 7- or 21-day hind-limb length (HLL). In terms of growth endpoints, there was a decrease in snout-tovent length (SVL; ↓ 4%) and body weight ( ↓ ; 11%) at the high concentration on Day 7 only. Spinal deformities were observed in all trea tment groups including the negative control group (45% in the negative control a nd low treatment group and 37% in the medium and high treatment groups); these were determined to not be treatment related.

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

Page 22 of 64 Table 4. Thyroid Pathway for Cypermethrin

Lines of Evidence Indicating Potential Intera ction with the Thyroid Pathway for Cypermethrin 1

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmental stage(± or asynchronous,HLL)Growth (BW, BL,SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assay

Female Pubertal Rat (MRID 48683002) N N N N N N N

Male Pubertal Rat (MRID 48683003 ) N N N N N N N

AMA (Frog; MRID 49013801) N N Day 7:
BW: ↓ 11% (H); SVL: ↓ 4% (H) N

OSRI

Subchronic toxicity (Dog; MRID 44527002) N N NE X (H) X (ML, MH, H) Developmental toxicity (Rat; MRID 00056804) X (M, H) N
Developmental toxicity (Rabbit; MRID 43776302) X (M, H) N

Developmental Neurotoxicity (Rat; MRID 46670402) X (H) N

Three-generation reproduction (Rat; MRID 00090040) NR NR NR X (H) X (H)

Three-generation reproduction (Rat; MRID 00112912) NR NR NR N N

Chronic toxicity/ carcinogenicity (Rat; MRID 00112910) NE N N X (H) N

Carcinogenicity (Mouse; MRID 00112911) NE N NE N N

Chronic toxicity (Dog; MRID 00112909) N N N X (H) N
1. Key to responses: L=Low treatment, ML=Med ium-Low treatment, M=Medium treatment, MH=Medium high treatment, H=High treatment. Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated. 2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 3. The overt toxicity in the Tier 1 assays are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. ACYPERMETHRIN FINAL

Page 23 of 64 P Positive findings

N Negative findings NE Not examined NR Not reported

IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of cypermethrin to interact with endocrine related processes ( i.e., E, A, or T hormonal pathways) as well as 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 co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substance may contribute to the WoE 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 a sequelae of system ic effects. Endocrine 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 Tire 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).CYPERMETHRIN FINAL

Page 24 of 64 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).

1. Tier 1 in vivo Assays

Systemic or overt toxicity in the Tier 1 in vivo studies was minimal. In the female and male pubertal rat studies, one fema le in the 30 mg/kg/day group (HTD) was found dead on PND 25, and one male in the 15 mg/kg/day group was found dead approximately one hour after dosing on
PND 45. Neither death was considered related to treatment. All othe r rats survived until
scheduled sacrifice. There were no treatment-rela ted clinical signs of toxicity at any dose. Findings noted in the treated groups, including re d and/or clear material around the nose and/or mouth, occurred infrequently in single animals a nd/or in a manner that was not considered dose related. In the Uterotrophic assay, all animals survived until scheduled termination. Treatmentrelated clinical signs of toxicity included one or two animal s with dried red material around nose, hypersensitivity to handling, vocalization upon handli ng, and salivation and/or wet clear material around mouth at 80 mg/kg/day (HTD). No treatmen t-related mortalities or clinical signs were present in the FSTRA study. No trea tment-related overt toxicity or systemic signs of toxicity were observed in the AMA study at test termin ation. In the AMA, spinal deformities were observed in all treatment groups including the negative control group, and more than half of all individuals developed be yond NF stage 60 by Day 21 ( i.e. , NF stage 61-62), but these findings
were determined to not be treatment-related.

2. OSRI

Systemic or overt toxicity in the OSRI in vivo studies was also minimal. No mortality or treatment-related clinical signs were noted in any of the groups in a Hershberger assay (Zhang et
al. , 2008). No deaths occurred during a subchronic oral toxicity study in dogs. In the high-dose group, two males and one female exhibited slight tremor activity beginning on Days 49 (female), 60, or 71 through study termination. In addition, one male and one female exhibited moderate tremor activity either on Day 76/77 (male) or during the Week 8, 9, and 12 detailed physical examinations (female).

In a prenatal development study in rats, dams in the high-dose group displayed neurological signs such as splayed limbs, spasms, hypersensiti vity to noise, and convuls ions. In a prenatal development study in rabbits, four deaths occurred dur ing the study, but were not, or could notCYPERMETHRIN FINAL

Page 25 of 64 conclusively be, attributed to treatment. Exacerbation of some clinical signs such as anorexia, abdominal/genital staining, decrease d feces, and red or pink material in the pan occurred in the high-dose group and in a few does in the medi um-dose group, but these effects were not definitely attributed to treatment. In a deve lopmental neurotoxicity study in rats, one control female was found dead late in gestation; necropsy revealed a ruptured uterus and late resorptions; all remaining animals survived to scheduled sacrif ice. No treatment-related clinical signs of toxicity were observed during the daily examinations.

In a three-generation reproduction study in rats, one high-dose male (1000 pm) was sacrificed in extremis, and this death was considered to be treatment related. There were obvious clinical signs of neurotoxicity ( i.e. , ataxia, increased sensitivity to sound, and high-stepping gait) in the high-dose (1000 ppm) group that resu lted in a dose reduction to 750 ppm after twelve weeks. No
adverse health or behavioral e ffects were noted in a previous three-generation reproduction study in rats with a high dose of 500 ppm.

There were no treatment-related differences in surv ival in a chronic oral toxicity/carcinogenicity study in rats. Transient clin ical (behavioral) signs ( i.e. , frequent face washing, increased sensitivity to sound, hind limb incoordination) were noted during the first six days of dosing in six (6/52) high-dose males. Similarly, in th e carcinogenicity study in mice, there were no
treatment-related differences in survival and no c linical signs were present. In a chronic oral toxicity/carcinogenicity study in dogs, no mortality occurred during the study. Treated males and females displayed increases in vomiting dur ing Week 1, and high-dose animals displayed signs of nervous system stimulation in the form of body tremors, gait abnormalities, incoordination, disorientation, and hyper-s ensitivity to noise during Week 1.

There were no treatment-related mortalities or cl inical signs of toxicity reported in the avian reproduction studies with mallard ducks and bobwhite quail for cypermethrin. Although no reproductive effects were observed, numerous mort alities were observed, and prior to the onset of death birds were observed being “bullied”. In addition, alpha cypermethrin reproductive effects were observed in the pr esence of pen mate aggression, and the presence of aggressive behavior, was a sign that the animals were st ressed, which could have potentially biased the results of the reproductive study.

An early life-stage study in fathead minnow was submitted, and after 30-day exposure to cypermethrin, survival was signifi cantly reduced compared to controls at 0.33 μg a.i./L. The resulting NOAEC value for survival was 0.14 μg a.i./L; and no treatment-related effects were observed for hatchability or growth.CYPERMETHRIN FINAL

Page 26 of 64 B. Estrogen Pathway

For cypermethrin the in vitro studies yielded conflicting resu lts; while some studies showed weak effects, other studies showed no effect on th e estrogen pathway in ER binding or activation assays. The in vivo Uterotrophic and Female pubertal assa ys were negative, and no estrogen
related effects were seen in the mammalian Part 158 studies. In the FSTRA, there was a decrease in fecundity and some indication of ma ture oocyte atresia in females at the highest concentration tested (1.4 μg/L). These effects in female fish are further discussed under the androgen pathway section below.

C. Androgen Pathway

For the androgen pathway, positive responses were seen for cypermethrin in AR Binding and ARTA studies. Although no effects were observed in the Tier 1 mammalian assays, there were effects observed in mammalian literature studies of cypermethrin and β -cypermethrin, including decreased sperm production at ≥ 15 mg/kg/day (Liu et al. 2010) and damage to the seminiferous tubules and spermatids at ≥ 6.25 mg/kg/day (Hu et al., 2013; Li et al. 2013).

In the FSTRA, significant increases in male GSI was observed (22-27%) at the mid and high concentration along with a marginally significant d ecrease (24%) in male tubercle score at the high concentration. While there appears to be limited complementary effects within a sex, collectively the effects observed in the FSTRA across both males and females appear to be consistent with reported effects for other chem icals (n=3) reported to be potential androgen antagonists (US EPA 2007) including decreased fecundit y, increased female oocyte atresia, and
decreased tubercle score. E ffects on male or female GSI were sometimes reported but the direction of effect (increased/dec reased) was not consistent. It is noted that increases in female VTG were also reported for two of the three chemicals, and this response was not observed in the FSTRA for cypermethrin.

However, on a weight of evidence basi s, given the effects observed in the in vitro assays along with evidence of potential androgen antagonist effects in some of the in vivo mammalian assays, there appears to be evidence for potential anti-andr ogenic effects in both mamma ls and fish.

D. Thyroid Pathway

No thyroid-related responses were observed in mammalian Tier 1 assays , Part 158 studies, or other OSRI. Additionally, based on the lack of effects on developmental endpoints or alterations in thyroid histopathology, the AMA did not supp ort a potential for a thyroid interaction in amphibians for cypermethrin.CYPERMETHRIN FINAL

Page 27 of 64 E. Conclusions

Cypermethrin demonstrates no convincing evidence of potential interaction with the estrogen or thyroid pathways. However there appears to be a potential for interaction with the androgen (anti-androgenic) pathway in mammals and fish.

V. EDSP Tier 2 Testing Recommendations

For cypermethrin, the current poin t of departure (POD) of 6.0 mg /kg/day used for human health
risk assessment is based on clini cal signs of neurotoxicity and mo rtality observed in male dogs and decreased body weight and body weight ga in observed in female dog at 18.1 mg/kg/day (LOAEL) in the chronic toxicity study in the dog s. This POD is used to derive the chronic Reference Dose (RfD) used for chronic dietary ex posure risk assessments and for calculating the Margins of Exposure (MOEs) for non- dietary exposure risk assessments.

In general, if the POD is lower or equal to the dose(s) that caused endocri ne-related effects, there would be a concern that the POD may not be protective of potential estrogen, androgen or thyroid related effects. In the case of cyperm ethrin, the POD (6.0 mg/kg/day) is numerically identical to the doses (6.25- 7.25 mg/kg/day) that caused damage to the seminiferous tubules and spermatids in studies reported as OSRI (Hu et al., 2013 and Li et al . 2013). Additionally, decreased sperm production was seen at a dose (12 mg/kg/day) that is only approximately twofold higher than the POD. Therefore, the POD used in risk assessments does not address the concern for the adverse male reproductive effects Therefore, the POD used in risk assessments does not address the concern for the adverse male reproductive effects

Although, adverse effects are seen, the ED SP Tier 2 study (post 1998 two-generation reproduction or the extended one-generation re production study) which evaluates male and female mammalian reproduction and fertility is not recommended since: 1) no estrogen-related
effects were seen in the Tier 1 female pubertal assay; 2) no estrogen-related effects were reported in the OSRI; and 3) the concern is only for the androgen-related eff ects in the adult male reproductive system..

For non-mammalian species, in the problem fo rmulation for the registration review of cypermethrin, the chronic no observed adverse effect concentration (NOAEC) for fish was reported at 0.14 μg/L based on a 30-day exposur e fathead minnow early life-stage study. Reproductive effects were observed in the FSTRA at measured concentrations of ≥ 0.12 μg/L. A fish life-cycle study for cypermethrin with fath ead minnow is available with a reported NOAEC of 0.06 μg/L (MRID 40641701). However in this st udy, excessive analytical variation (high:low ratios of 3.0 to 7.8) was observed at all levels during the study, and the range of concentrations was so great that overlapping of concentrations occurred for all levels. Therefore, there is uncertainty with regards to the reported NOAEC in that study. Given the potential for antiCYPERMETHRIN FINAL

Page 28 of 64 androgen effects observed in the Tier 1 wei ght of evidence analysis, the EDSP Tier 2 Medaka Extended One Generation Reproduction Toxicity (M EOGRT) study is recommended. With that recommendation, it is noted that given the physio -chemical properties of cypermethrin and the results in the fathead minnow life-cycle st udy, there may be difficulties in maintaining cypermethrin concentrations in aqueous systems over the duration of a long-te rm test such as the Tier 2 fish assay and careful attention and maintena nce of the test system will likely be required.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for cypermethrin. In both spec ies, no effects on reproductive parameters were observed; an additional study with alpha cypermethrin did report potential reproductive effects but there are uncertainties in this study. Th e type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are consid ered sufficient for evaluating potential reproductive effects to birds from cypermethrin exposure. Relative to the EDSP, additional testing is not recommended.

Given the lack of observed poten tial thyroid-related effects in the AMA, no additional testing with amphibians is recommended.CYPERMETHRIN FINAL

Page 29 of 64 VI. References

Bauer, E.R.S., Bitsch, N., Brunn, H., Sauerwein, H., and Meyer, H.H.D. (2002) Development of an immune-immobilized androgen receptor a ssay (IRA) and its application for the characterization of the receptor binding affinity of different pesticides. Chemosphere 46:1107-

1115.

Chen, H., Xiao, J., Hu, G., Zhou, J., Xiao, H., and Wang, X. (2002) Estrogenicity of organophosphorus and pyret hroid pesticides. Journal of Toxicology and Environmental Health

, Part A 65:1419-1435.

Daly, I.W. (1994) A Subchronic (3-Month) Oral Toxicity Study of FMC 30980 Technical in the Dog via Dietary Administration. Unpublishe d study performed by Pharmaco LSR Inc., Toxicology Services North America, Mettle rs Road, East Millstone, NJ. Study No. 92-3114. FMC No. A92-3706. March 31, 1994. (MRID 44527002)

Du, G., Shen, O., Sun, H., Fei, J., Lu, C., S ong, L., Xia, Y., Wang, S ., and Wang, X. (2010) Assessing hormone receptor activities of pyrethroid insecticides and their metabolites in reporter

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

Freeman, C. (1994) Cypermethrin Technical Or al Teratology Study in Rabbits. Unpublished study performed by FMC Corporation Toxico logy Laboratory, Study No.: A93-3822, October 28, 1994. (MRID 43776302)

Hend, R.W., Hendy, R., and Fleming, D.J. (1979) T oxicity studies on the insecticide WL 43467: A three generation reproduction study in rats . Unpublished study performed by Shell Toxicology Laboratory, Study No.: TLGR.0188. 78, February, 1979. (MRID 00090040)

Herberth, M.T. (2011) Cypermethrin-Uterotrophi c Assay in Ovariectomized Rats. Unpublished study performed by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Report No.: WIL-639150, November 10, 2011. (MRID 48615701)

Herberth, M.T. (2012) Cypermethrin – Pubertal Development and Thyroi d Function in Intact Juvenile/Peripubertal Female Rats. U npublished study performed by WIL Research Laboratories, LLC, Ashland, OH. Laborator y Project ID Number : WIL-639152, April 9, 2012. (MRID 48683002)

Herberth, M.T. (2012) Cypermethrin – Pubertal Development and Thyroi d Function in Intact Juvenile/Peripubertal Male Rats. Unpublished study performed by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Project ID Number: WIL-639151, April 9, 2012. (MRID 48683003)CYPERMETHRIN FINAL

Page 30 of 64 Hu, J.X., Li, Y.F., Li, J., Pan, C., He, Z., D ong, H.Y., and Xu, L.C. ( 2013) Toxic effects of cypermethrin on the male reproductive syst em: with emphasis on the androgen receptor. J Appl
Toxicol . 33(7):576-585.

Jin, M., Li, L., Xu, C., Wen, Y., and Zhao, M. (2010) Estrogenic activit ies of two synthetic pyrethroids and their metabolites. J. Environ. Sci. (China) 22(2):290-296.

Kalinowski, A.E., Banham, P.B., Chart, I.S., Co ok, S.K., Gore, C.W., and Moreland, S.F. (1982) Cypermethrin: One Year Oral Dosing Study in Dogs, Unpublished study performed by ICI, Report: CTUP/703, FMC study number A82-763. (MRIDs 00112909, 42068503, and 92027037)

Kim, I.Y., Shin, J.H., Kim, H.S., Lee, S.J., Kang, I.H., Kim, T.S., Moon, H.J., Choi, K.S., Moon, A., and Han, S.Y. (2004) Assessing estrogenic activity of pyrethroids insec ticides using in vitro combination assays. Journal of Reproduction and Development 50(2): 245-255.

Kjeldsen, L. S., Ghisari, M., a nd Bonefeld-Jørgensen, E.C. (2013) Currently used pesticides and
their mixtures affect the function of sex hor mone receptors and aromatase enzyme activity. Toxicol Appl Pharmacol . 272(2): 453-464.

Lee, M.R. (2012) Cypermethrin: Amphibian Me tamorphosis Assay with African Clawed Frog ( Xenopus laevis) . Unpublished study performed by Smith ers Viscient, Inc., Wareham, MA. Laboratory report number 1781-6765. Study spons ored by Syngenta Crop Protection, LLC, Greensboro, NC. Study completed November 28, 2012. (MRID 49013801)

Lindsay, S., Banham, P.B., Chart, I.S., Chal mers, D.T., Godley, M.J., Taylor, K. (1982 ) Lifetime Feeding Study in Mice, Unpublished study performed by ICI, Report: CTUP/687, FMC study number A82-762. (MRIDs 00112911 and 92027038)

Li, Y.F., Pan, C., Hu, J.X., Li, J., and Xu, L. C. (2013) Effects of cypermethrin on male reproductive system in adult rats. Biomed Environ Sci. 26(3):201-208.

Liu, L., Hu, J.X., Wang, H., Chen, B.J., He, Z., a nd Xu, L.C. (2010) Effects of beta-cypermethrin on male rat reproductive system. Environ Toxicol Pharmacol . 30(3):251-256.

Milburn, G.M., Forbes, D., Banham, P.B., Chart, I. S., Godley, M.J., Gore, C.W., Pratt, I., Scales, M.D.C., Stonard, M.D., and Woollen, B.H. (1982 ) Cypermethrin: 2 Year Feeding Study in Rats. CTL report number: CTUP/669. FMC study number A82-761. (MRID 00112910)

Milburn, G.M., Banham, P.B., Birtly, R.O.N ., Godley, M.J., and Moreland, S.F. (1982) Cypermethrin: Three-Generation Reproduction Study in the Rat. Unpublished study performed by ICI Central Toxicology Laboratory, Study No.: RR0143, July 9, 1982. (MRIDs 00112912, 42068504, 92027040)CYPERMETHRIN FINAL

Page 31 of 64 Nemec, M.D. (2005) A dietary developmental neur otoxicity study of zeta-c ypermethrin technical in rats. Unpublished study performed by WIL Re search Laboratories, LLC, Ashland, Ohio. Study Number WIL-105018; August 24, 2005. (MRID 46670402)

Roberts, N.L., Fairley, C., Chanter, D., McAllis ter, A., and Almond, R. (1981) The effect of dietary inclusion of cypermethrin on reproduction in the bobwhite quail. ICI 342/81341 (FMC no. Cy 10.1.2/1) . Unpublished study performed by The Huntingdon Research Centre, Cambridgeshire, UK ; submitted by ICI Americas Inc., Wilmington, Delaware. (MRID 00090074)

Roberts, N.L., Fairley, C . ( 1981) The effect of dietary inclus ion of cypermethrin on reproduction
in the Mallard duck. Unpublished study performed by Huntingdon Research Centre, Cambridgeshire, UK. Report no.: ICI 34118164 (FMC no

. Cy 10.1.2/2). (MRID 00090073)

Saito, K., Tomigahara, Y., Ohe, N., Isobe, N., Nakatsuka, I., and Kaneko, H. (2000) Lack of significant estrogenic or antiestroge nic activity of pyrethroid insectic ides in three in vitro assays based on classic estrogen recep tor alpha-related mechanisms. Toxicol Sci. 57(1):54-60.

Stevens, J (2013). Cypermethrin - Weight of Evidence Evaluation of Tier 1 Endocrine Disruptor Screening Program Results Assessment. Report Number: CCWOE-001 FMC Corporation, Philadelphia, PA 19103. S yngenta Crop Protection, LLC, Greensboro, NC 27419-8300 and United Phosphorus, Inc.,King of Prussia, PA 19046. MRID No. 49068201

Tapp, J.F. et al. 1998. Cypermethrin: determinati on of chronic toxicity to fathead minnow (Pimephales promelas) full lifecycle. ICI PL C, Wilmington DE, Br ixham Laboratory, Deveon England. Laboratory Report EL/B/3173. MRID 40641701.

Tesh, J.M., Tesh, S.A., and Davies, W. (1978) WL 43467: Effect upon the progress and outcome of pregnancy in the rat. Unpublished study perf ormed by Life Science Research, Study No.: 78/SHL2/364, October 4, 1978. (MRID 00056804, 92027039 or 92027061)

Tyler, C.R., Beresford, N., Van der Woning, M., Sumpter, J.P., and Thorpe, K. (2000) Metabolism and environmental degradation of pyre throid insecticides pr oduce compounds with
endocrine activities. Environ. Toxicol. and Chem. 19(4):801-809.

USEPA (2010) Cypermethrin - Report of the En docrine Disruptor Review Team; Test Order# EDSP-109702-75, 76, and 78. December 14, 2010. Offi ce of Chemical Safety and Pollution
Prevention. U.S. Environmental Pr otection Agency, Washington, D.C.

USEPA. (2011) Endocrine Disruptor Screeni ng Program – Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.CYPERMETHRIN FINAL

Page 32 of 64 USEPA (2013) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting held July 30 - August 1, 2013 on Weight-of-Ev idence: Evaluating Resu lts of EDSP Tier 1 Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

Wagner, H. (2012) Cypermethrin - H295R St eroidogenesis Assay. Unpublished study performed by CeeTox, Inc., Kalamazoo, MI. Laborat ory Study No.: 9047V-100353STER, January 17, 2012. (MRID 48670203)

Wang, X.Z., Liu, S.S., Sun, Y., Wu, J.Y., Zhou, Y. L., and Zhang, J.H. (2009) Beta-cypermethrin impairs reproductive function in male mice by inducing oxidative stress. Theriogenology 72(5):599-611.

Wilga, P. (2012) Cypermethrin: Human Reco mbinant Aromatase Assay. Final Report. Unpublished study performed by CeeTox, Inc., Ka lamazoo, MI. Laboratory Report No.: 9047V100353AROM, January 17, 2012. (MRID 48670202)

Willoughby, J.A. (2012) Cypermethrin: Estrogen Receptor Transcriptiona l Activation (Human Cell Line (HeLa-9903)) Final Report. U npublished study performed by CeeTox, Inc, Kalamazoo, MI. Laboratory Report No.: 9047V-100353ERTA, January 16, 2012. (MRID 48670201)

Xu, L.C., Liu, L., Ren, X.M., Zhang, M.R., Cong, N., Xu, A.Q., and Shao, J.H. (2008) Evaluation of androgen receptor transcriptional activities of some pesticides in vitro. Toxicology 14; 243(1-2):59-65.

York, D.O. (2012) Cypermethrin – Fish Shor t-term Reproduction Assay with the Fathead Minnow ( Pimephales promelas ). Performed by Smithers Viscient, Wareham, Massachusetts, Laboratory Project No. 1781.6766. Submitted by S yngenta Ltd, Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom; FMC Corporation, Philadelphia, Pennsylvania; Unit ed Phosphorus, Inc., King of Prussia, Pennsylvania. Completion date April 18, 2012. (MRID 48683001)

Zhang, J., Zhu, W., Zheng, Y., Yang, J., and Z hu, X. (2008) The antiandrogenic activity of pyrethroid pesticides cyfluthrin and β -cyfluthrin. Reproductive Toxicology 25:491-496.CYPERMETHRIN FINAL

Page 33 of 64 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA): (OCSPP 890.1100)

The 21-day assay (MRID 49013801) of cypermethrin (95.2% purity) on amphibian metamorphosis of African Clawed Frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Fabe r (NF) stage 51 (80 la rvae per group) were exposed to a negative control and test ch emical nominal concentrations of 0.0000030, 0.000030, and 0.00030 mg a.i./L. The time-weighted aver age (TWA) concentrations were <0.0000010 (<LOQ, control), 0.0000019, 0.0000109, and 0.0000867 mg a.i/L; the %CV exceeded 20% for all treated levels. The test system was main tained at 22 to 23°C and a pH of 7.2 to 8.2.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. Two mortalities o ccurred at the low treatment level and one mortality occurred at
the mid treatment level. No other mortalities were observed. Clinical signs including spinal deformities were observed in 27 tadpoles at the ne gative control and low treatment levels and in 22 tadpoles at the mid and high treatment levels.

Cypermethrin caused no significant increase or decrease (p>0.05) in 7-day hind-limb length (HLL) at any treatment level. There was no significant accelerati on or delay (p>0.05) of median NF developmental stage observed at Day 7 or 21 at any treatment level relative to the negative control. Further, no asynchronous development wa s observed. More than ha lf of all individuals developed beyond NF stage 60 by Day 21 (i.e., NF stage 61-62), with 53%, 59%, 54%, and 52% of living frogs exhibiting late stage (NF>60) development in the control, low, mid, and high groups, respectively. There was no i ndication of a dose response pattern for occurrence of latestage development and both the reviewer and stud y author conducted sepa rate analysis of only late-stage individuals. The revi ewer’s analysis indicated a si gnificant dose-dependent reduction (p<0.05; Jonckheere-Terpstra) in both Day 7 snout-vent length (SVL) (4%) and wet weight (12%) of tadpoles exposed to the high treatmen t group, relative to the control. No other growth or developmental effects were not ed in this study, including for th e additional analysis of latestage frogs on Day 21. No effects on thyroid gland histopathology were observed at any
treatment level.

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

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

Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP19) assay (MRID 48670202) , cypermethrin (95.2% a.i. Lot # PL10-0492) was incubated with human recombinant aromatase and tritiated androstenedione (1-CYPERMETHRIN FINAL

Page 34 of 64 β [ 3 H(N)]-androst-4- ene-3,17-dione; [ 3 H]ASDN) in dimethyl sulfoxide (DMSO) at log
concentrations of 10 -10 to 10 -4 M for 15 minutes to assess the potenti al of cypermethrin to inhibit aromatase activity.

Aromatase activity was determined by measuring the amount of tritiated water produced at the
end of a 15-minute incubation for each concentration of chemical. Tritia ted water was quantified using liquid scintillation counting (LSC). Three independent runs of the aromatase assay were
conducted and each test concentrat ion of cypermethrin was tested in triplicate. Each run included a full activity c ontrol, a background activity control, a positive control series (10 -10 to
10 -5 M) using a known inhibito r (4-hydroxyandrostenedione; 4- OH ASDN). The highest concentrations of cypermethrin tested in Run 1 (10 -3 M) and in Run 2 (10 -3.5 M) showed marked precipitation during the 15-minute incubation period and these data were not evaluated. The highest concentration for all three runs used for data interpretation was 10 -4 M because it did not result in marked precipitation.

Aromatase activity in the full activity controls averaged 0.222 ± 0.049 nmol/mg-protein/min. The response of each full activity control within a run was between 90 to 110% of the average full activity. Activity in the background cont rols ranged from 0.45 to 0.78% and averaged 0.64% of the full activity control.

For the positive control substance (4-OH ASDN), aromatase activity results were within the recommended ranges for the performance criteria. The estimated log IC 50 for 4-OH ASDN averaged -7.17 M and the slope was -0.94.

For cypermethrin, aromatase activity averaged 0.170 ± 0.018 nmol/mg-protein/min at the lowest
tested concentration 10

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

− 4 M, used for data interpretation. The average dose-res ponse curve indicated that the aromatase activity at all concentrations of cypermethrin te sted was essentially equivalent to the activity observed in the full activity control (>75% activ ity= non-inhibitor).

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

Page 35 of 64 ER α Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48670201), hER α -HeLa-9903
cells cultured in vitro were exposed to cypermethrin (95.2% a.i., Batch PL10-0492) at logarithmically increasing concentrations from 10

− 10.3 to 10

− 3.3 M in DMSO (0.1%) for approximately 24 hours in three independent runs . A total of 8 runs were conducted, but only the data from three runs were reported. The experiments were performed using 96-well plates and each cypermethrin concentration was tested in replicates of 6 wells/pla te in each run. Cells were exposed to the test agent for 24±2 hours at 37±1°C to induce report er (luciferase) gene products. Luciferase expression in response to activation of the estrogen receptor by cypermethrin was measured using a proprietary luciferase assay.

Cypermethrin was tested up to the limit of solubility, 10 -4 M. The RPC Max was 16.3% at 10 -4 M
and the PC 10 was 10 -4.6 M in Run 1, indicating a positive response to the assay. However, in
Runs 2 and 3 the RPC Max for cypermethrin was ≤ 0.9% at concentrations up to 10 -3.3 M, indicating that cypermethrin was negative for estrogenic activity under the conditions of the assay. Review of the performance criteria parameters for Run 1 (Table 3) shows that Run 1 was unusually sensitive, with all parameters fo r all reference chemicals at lowe r levels than expected according to the guidance.

In all runs, cypermethrin response was similar to that of the negative control (corticosterone),
and in no run did it reach levels similar to the weak estrogen 17 α -methyltestosterone. For Run 2, Log PC 50 values could not be determined for 17 α -methyltestosterone as it did not reach 50% activation at concentrations up to 10 -5 M. This indicates a decreased sensitivity of cells. However in that same run, responses were clos e to zero at all cypermethrin concentrations.

Although the conditions of this a ssay were not optimal, in general cypermethrin responses were similar to those of the negative control corticos terone and not comparable to the responses of

17 α -methyltestosterone, which was able to r each maximums of 124.3, 16.5, and 52.8% PC in Runs 1, 2, and 3, respectively. Cypermethrin wa s only able to reach maximums of 16.3, 0.9, and 0.6% PC in Runs 1, 2, and 3, respectively, when tested up to the highest concentration possible based on cytotoxicity. Because the RPC Max < PC 10 in both assay runs, cypermethrin was considered negative for estrogen receptor tran scriptional activation in this test system.

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

The 21-day short-term reproduction assay (MRID 48683001) of cypermethrin with fathead minnows ( Pimephales promelas ) was conducted under flow-through conditions. Adult fish (16 spawning groups, 2 males and 4 females in each group; 5 months old) were exposed to cypermethrin (95.2% purity) at nominal c oncentrations of 0 (control), 0.000030, 0.00030, andCYPERMETHRIN FINAL

Page 36 of 64 0.003 mg a.i./L; mean-measured con centrations were <LOQ (<0.000001), 0.000013, 0.00012, and 0.0014 mg a.i./L. The test system was maintained at 25 to 26°C and a pH of 7.0 to 7.7.

There were no significant differences in either male or female survival at any treatment levels compared to the negative control. Percent survival was 100% for all groups of males and was 100, 94, 94, and 88% for the negative control and lo w, mid, and high treatment concentrations, respectively, for females. During the in-life e xposure, there were no notable observations that occurred with regards to behavi or, coloration/banding, changes in ovipositor appearance, or size of the dorsal nape pad, and no clinical signs of toxicity were observed. There were no treatmentrelated effects on male and fe male body weight and length.

Spawning occurred at least every 4 days in thee of the four control replicates and mean fecundity in control averaged 14 eggs/female/day/rep licate (range: 13-18 eggs/female/day among the replicates); fertilization success in the control group averaged 96%. Fecundity was decreased (p<0.05) by 42% and 84% in the mid- and high-tr eatment females, respectively. There were no treatment-related effects (p> 0.05) on fertilization success.

Male gonadal somatic index (GSI) was increased (p<0.05) by 22 and 27% at the mid and high treatment concentrations, respectively, compared to the negative control. There were no
treatment-related effects on female GSI (p>0.05) or male and female plasma vitellogenin (VTG). Additionally, there were no significa nt effects on median male t ubercle score, and no tubercles were observed on any females.

Although not analyzed statis tically, in male fish, there were only background or sporadic gonadal histopathological findings noted th at were not considered related to treatment. In female fish,
there was an increased inciden ce of mature oocyte atresia at the high treatment concentration, which was associated with granulomatous infl ammation and the presence of microsporidia in
half the observed cases across all treatment leve ls. There were no signifi cant findings observed in the gonadal stage for either males or female s, and no notable observations in secondary sex characteristics for any treatment group compared to the controls.

The performance criteria for fecundity was not me t. There were one replicate in the control group (Replicate C) where there was a period of 4 days in which no spawning occurred and fecundity was 13 eggs/female/day. Therefore, this re plicate did not meet the criteria of either at least 15 eggs/female/day or spawning at least ev ery 4 days. However, it is noted that the overall control mean fecundity value (14) was just sli ghtly below 15 eggs/female/day, and the other two replicates did achieve 15 or gr eater eggs/female/day and the rema ining replicate spawned at least every 4 days (11 eggs/female/day). Also, the %C V values for the mean recoveries at the two highest treatment concentrations were 45.6 and 51.2%, which did not meet the validity criteria of <20% over the 21-day test. There was no patter n of decline during th e study period and no undissolved test substance was obser ved in the dilution system, as su ch, the recoveries indicate the test material was generally poorly recovered in solution under the test conditions. Given theCYPERMETHRIN FINAL

Page 37 of 64 physicochemical properties of cypermethrin, these results may be considered consistent with expectations for the chemical and previous expe rience with the test item. The remaining validity criteria for OCSPP 890.1350 were met. Therefore, wh ile these deviations ar e noted, they did not sufficiently impact the interpretation of the assay results.

Hershberger Assay; (OCSPP 890.1400)

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

Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48683002), 15 Sprague Dawley rats/dose group were treated daily via oral gavage with cypermethrin (95.2% a.i.; cis/trans ratio: 49.8/50.2; Batch #PL10- 0492) in corn oil at doses of 0 (corn oil), 15 or 30 mg /kg/day from post-natal day (PND) 22 to 42/43. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and weight at day of attainment was recorded. Following sacrifice on PND 42/43, total thyroxine (T 4 ) levels were determined using an electr ochemi-luminescent immunoassay and thyroid stimulating hormone (TSH) levels were determin ed using a radioimmunoassay. Adrenal, liver, thyroid, pituitary, and ur ogenital organ weights were recorde d, and microscopic examinations of the uterus, ovaries, thyroid, and kidney were performed.

One animal in the 30 mg/kg/day group was found dead on PND 25. No internal findings were noted for this animal during gross examination at necropsy. All other rats survived until
scheduled sacrifice. As there wa s no other evidence of toxicity in this study, this single death
was considered incidental. There were no treatmen t-related clinical signs of toxicity at any dose level; findings noted in the treated groups occurr ed infrequently in single animals and/or in a
manner that was not considered dose related. Mean body weights, daily body weight gains, cumulative mean body weight gain, and food consum ption in the treated groups were similar to the control group throughout the study.

No treatment-related effects were observed on m ean age or weight at vaginal opening (VO), and mean age at first estrus. The cycle status at necr opsy (diestrus, proestrus, estrus, or not cycling) appeared similar across all groups. There were no treatment-related cha nges in serum hormone or clinical chemistry levels. In addition, there were no effects of treatment observed in gross or microscopic pathology.

There was no evidence of direct test substance-related effects on pubertal development or thyroid function in the juvenile/peripube rtal female rat at cypermet hrin dose levels of 15 and 30 mg/kg/day.CYPERMETHRIN FINAL

Page 38 of 64 No evidence of toxicity was observed in th e study up to the highest dose tested; however, treatment-related clinical signs were seen in the preliminar y range-finding study at 30 and 65 mg/kg/day. In the preliminary study, tremors and clear material around the mouth were seen at 30 mg/kg/day, 4 hours after administration. At 65 mg/kg/day (after 4 hours), tremors, splayed limbs, and clear material around the mouth, ventra l neck and thoracic area, and forelimbs were seen. It is possible that th e effects observed in the prelim inary study were missed in the definitive study since clinical signs were meas ured 1 hour after chemical administration. Clinical signs were measured 4 hours after ad ministration in the preliminary study, which is closer to the time-to-peak effect for cypermethrin. Therefore, dosing levels tested in this assay are considered appropriate.

Male Pubertal A ssay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48683003), 15 Sp rague Dawley rats/dose group were treated daily via oral gavage with cypermethrin (95.2 % a.i.; cis/trans ratio: 49.8/50.2; Batch #PL10- 0492) in corn oil at doses of 0 (corn oil), 15, or 30 mg /kg/day from post-natal day (PND) 23 to 53/54. Animals were examined for preputial separation (PPS) daily beginning on PND 30 and weight at day of attainment was recorded. Following sacrifice on PND 53 or 54, total serum

testosterone a nd thyroxine (T 4 ) levels were determined usi ng an electrochemiluminescent immunoassay, and thyroid stimulating hormone (TSH) levels were determined using a radioimmunoassay. Adrenal, li ver, thyroid, pituitary, and ur ogenital organ weights were recorded, and microscopic examinations of th e testis, epididymis, thyroid, and kidney were performed.

One male in the 15 mg/kg/day group was found dead approximately one hour following dosing on PND 45, which was likely due to dosing error. All other rats survived until scheduled
sacrifice. There were no treatm ent-related clinical si gns of toxicity at any dose level. Findings noted in the treated groups, in cluding red and/or clear materi al around the nose and/or mouth, occurred infrequently in single animals and/or in a manner that was not considered dose related. Mean body weights, daily body weight gains, cumulative mean body weight gain, food consumption, mean age of attainment of comp lete PPS, and mean body weight at the age of attainment in the 15 and 30 mg/kg/day groups we re similar to the control group throughout the study.

No treatment-related gross observa tions were noted at necropsy and no significant effects of treatment were observed on the weights of a ny of the organs and tissues collected.

There were no test substance-related changes in serum T 4 , TSH and testosterone values at any dose level. Urea nitrogen ( ↓ 17%) and bile acid ( ↓ 52%) were decreased (p<0.05) in the 30 mg/kg/day group compared to controls. Howeve r, decreases in bile acids have not beenCYPERMETHRIN FINAL

Page 39 of 64 associated with toxicological effects. The slig ht decrease in blood urea nitrogen was within the historical control reference range and considered an incidental finding. There were no other serum chemistry changes in the cypermethrin -dosed groups compared to the control group.

There were no test substance-rela ted histologic changes in the t hyroid, testes, epididymides, and kidneys. The amount of colloid and follicular height of thyroi d epithelial cells were similar
between control and test substance-treated animals.

No evidence of toxicity was observed in th e study up to the highest dose tested; however, treatment-related clinical signs were seen in the preliminar y range-finding study at 30 and 65 mg/kg/day. In the preliminary study, tremors and clear material around the mouth were seen at 30 mg/kg/day, 4 hours after administration. At 65 mg/kg/day (after 4 hours), tremors, splayed limbs, and clear material around the mouth, ventra l neck and thoracic area, and forelimbs were seen. It is possible that th e effects observed in the prelim inary study were missed in the definitive study since clinical signs were meas ured 1 hour after chemical administration. Clinical signs were measured 4 hours after ad ministration in the preliminary study, which is closer to the time to peak effect for cyperm ethrin. The dose levels used in the study are considered adequate based on the results of the range-findi ng study.

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48670203), H295R cells cultured in vitro in 24-well plates were incubated with cypermethrin (95.2% purity, Lot # PL10-0492) at concentrations of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 μM for 48 hours in triplicat e for four independent experiments. Dimethyl sulfoxide (DMSO) was used as the vehi cle, at a final concentration of 0.05%. Because precipitation was observed at 100 μM, this group wa s not evaluated statistically. Run 1 was not analyzed because one of the forskolin concentrat ions on the quality control plate was incorrect (3.3 μ M was used instead of 1 μ M).

Testosterone and estradiol levels were measured using HPLC-MS/MS. A Quality Control (QC) plate was run concurrently with ea ch independent run of a test chem ical plate to demonstrate that the assay responded properly to positive contro l agents at two concentration levels; positive
controls included the known inhi bitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production.

Guideline recommendations and requirements we re met, including adequate production of testosterone and estradiol, accep table reproducibility (low %CV) , and appropriate induction and inhibition with positive controls. Cytotoxicity greater th an 20% was not observed.CYPERMETHRIN FINAL

Page 40 of 64 Statistically significant increases in estradiol were noted at 10 μM in Runs 3 and 4, and estradiol levels in all three runs at 10 μM were 111-117% of the solvent cont rol (SC). All other values of
testosterone and estradiol in the tr eated cells were similar to the SC.

Based on the hormone responses in each of the th ree independent runs, cypermethrin treatment resulted in statistically si gnificant and reproducible increas es in estradiol production, but cypermethrin treatment did not result in statisti cally significant and reproducible alterations in testosterone production.

Uterotrophic; (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48615701) conducted to screen for potential estrogenic activity, cypermethrin (technical, 95.2% a.i., Batch # PL10- 0492) in corn oil was administered daily via oral gavage (dosing volume 5 mL/kg bw) to gr oups of six ovariectomized female Sprague Dawley rats/dose at dose levels of 0 (vehicle), 25, or 80 mg/kg/day on post-natal days (PND) 66 to 68. The positive control group was treated with a daily dose of 17 α -ethynyl estradiol (EE) in corn oil at 0.3 mg/kg/day by oral gavage (dosing volume 5 mL/kg bw ). The selected dose levels were based on toxicity observed in the dose range-finding study (MRID 48615702), in which clinical signs and body weight gain effects were observe d at 80 and 100 mg/kg/day, and marginal effects at 65 mg/kg/day. Body weights and food consumption were determined daily. All animals were terminated and necropsied on PND 69 approximately 24 hours after the final
dose administration to determine wet and blotted uterine weights.

All animals survived until scheduled termination. Treatment-related clinic al signs of toxicity included one or two animals with dried red material around nose, hypersensitivity to handling, vocalization upon handling, salivation and/or wet clear material around mouth at 80 mg/kg/day. Body weights in the treatment groups were co mparable to vehicle controls throughout the duration of the study. At 80 mg/kg/day, the overall body weight gain was significantly decreased (p<0.05; ↓ 51%). The overall aver age food consumption (Day s 1-4) was decreased (p<0.05) by 15% at 80 mg/kg/day. Overall av erage food consumption in the 25 mg//kg/day group was comparable to the vehicle control. Uterine weights in the cypermethrin treatment groups were comparable to the vehicle controls.

In the positive control (EE) group, body weights were decreased (p<0.05) by 11% on Day 4 compared to the vehicle cont rol, leading to an overall body weight loss (p<0.05) of –14.9 g compared to a gain of 14.6 g in the vehicle controls. Overall average food consumption in the positive control (EE) group was decreased (p<0.0 5) by 46%. Absolute wet and blotted uterus weights for the positive control (EE) group were increased (p<0.05) by 370% and 171%, respectively, as expected.CYPERMETHRIN FINAL

Page 41 of 64 The clinical signs and 51% d ecrease in overall body weight gains in the high-dose group (together with consistent findings in th e range-finding study at doses of 65, 80 and 100 mg/kg/day) indicated that the te sted dose levels were adequate.

No statistically significant changes were seen in uterine weight in this assay. Cypermethrin is negative in the uterotrophic assay.CYPERMETHRIN FINAL

Page 42 of 64 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 ge neral toxicity studies considered relevant for determining the potential interaction of cypermet hrin with the endocri ne system are summarized below. Given the breadth of toxicity data available on cypermet hrin, the only Part 158 st udies discussed in this appendix are those that fulfill current guideline requirements and are directly relevant to assessing potential endocrine effects ( i.e. , multiple-generation rodent reproductive studies, development studies, chronic toxicity studies, etc.).

AR Binding

In a published study, Bauer et al. (2002) assessed cypermethrin (91.0% purity; cis/trans ratio is
not stated ) binding to recombinant human AR (hAR) with an immuno-immobilized AR binding

assay.

The cDNA of the hAR was inserted into th e transfer plasmid pAcSG-His NT-C. The recombinant Baculovirus was obtained by transfec tion of insect cells (Sf9) with linearized Baculo gold virus DNA and the recombinant plasmi d. After 40 hours, the cells were collected by centrifugation, combined with disruption buffe r, and disrupted by three freezing and thawing cycles. Supernatant was collected after a 100,000×g centrifugation for 15 minutes at 4°C.

The receptor preparation was diluted 1:60 in assay buffer and a 100 μL aliquot/well was
incubated for 16 hours at 4°C with 0.44 nM 3 H-DHT in the presence or absence of increasing concentrations of the pesticides or DHT. Th e receptor was fixed on the microtiter plate with 50 μL of the 1:500-diluted specific hAR antibody. Receptor binding was determined by LSC. Specific binding represents the difference of total binding and non-specifi c binding in presence of a 200-fold surplus of unlabelled DHT. Cypermethrin was tested at multiple concentrations up to the maximal soluble concentration (concentra tion range not reported). Cypermethrin did not displace 3 H-DHT; therefore, a K i was not determined and a relati ve binding affinity of <0.01 was assigned.

In another published study, Tyler et al. (2000) assessed cypermethrin (95.8% purity; cis/trans
ratio is not stated ) binding to hAR in a recombinant y east expression system (PGKhAR) assay. In the presence of an active ligand, such as th e positive control (DHT), the androgen receptor binds to an androgen-responsive element on a pl asmid, thereby initiating transcription of the
reporter gene lacZ, which encodes the enzyme β -galactosidase. This produces a change in color from yellow to red that is determined by measur ing the absorbance at 540 nm. A stock solution of cypermethrin was serially diluted in ethanol , and 10-μL portions of th e appropriate dilutions were added to sterile incubation plates and ev aporated to dryness. The androgenic positive control was DHT, the anti-androge nic positive control was flutamide, and the negative control was ethanol. For the anti-androgenic assay, DHT wa s added to the medium at a concentration of

2.5×10

− 9 M. Portions (200 μL) of th e assay medium that contained the recombinant yeast wereCYPERMETHRIN FINAL

Page 43 of 64 added to the plates; plates were sealed and shaken for two minutes prior to incubation at 28°C for 24 hours followed by room temperature incubation until optimal color development had occurred (details not reported). The plat es were read at 540 and 620 nm (turbidity correction for yeast growth).

Cypermethrin was assessed for androgenic and anti-androgenic activity across a concentration range of 10

− 6 to approximately 10

− 3 (possibly 10

− 2.8 ). Cypermethrin did not demonstrate any androgenic activity ; therefore, EC 50 and LOEC 50 estimates could not be determined. Antiandrogenic activity was observed at cypermet hrin concentrations approximately >10

− 3 M, yielding an LOIC value of 1×10

− 3 M (1000-fold less potent compared to flutamide).

AR Transcriptional Activation Assays

In a published study, Zhang et al. (2008) examined the potential an ti-androgenic effects of cyfluthrin and β -cyfluthrin with reporter gene assays in vitro . A comparison of anti-androgenic activities was also made to four structurally -related pyrethroids (permethrin, cypermethrin, β -cypermethrin, and bifenthrin). This study was eval uated with specific attention to cypermethrin
and β -cypermethrin (purities not reported; cis/trans ratio is not stated).

For the in vitro reporter gene assay, th e MDA-kb2 breast cancer cell line, which was stably transfected with an androgen-re sponsive reporter gene (pMMTV .neo.luc), was treated when the
cell reached 80% of confluence with the various pyrethroids or the anti-androgen hydroxyflutamide (OHF), both with and without dihydrotestosterone (DHT; 1 nM). Each test substance was prepared in ethanol with a final ethanol concentration in the medium of 0.1%. Cypermethrin and β -cypermethrin were each tested at a concentration of 1 × 10

− 5 M. Cells were
incubated at 37°C with CO 2 overnight for 24 hours, the dosing medium was removed, cells were washed once with phosphate-buffered saline, and 25 μL of lysis buffer was added per well. After incubation for 25 minutes, the cells were lysed, 100 μL of luciferase reaction agent was added, and luciferase activity was determined. Th e results of the assay were expressed as the percent of androgenic activ ity for each compound rela tive to the DHT control.

In the in vitro reporter gene assay, cypermethrin inhi bited (p<0.05) DHT-induced transcriptional
activation at a concentration of 1×10

− 5 M (the only concentration tested), but β -cypermethrin had no effect. Although the data were only presented graphically, treatment with cypermethrin resulted in approximately a 45% decrease in a ndrogenic activity compared to control (1 nM DHT). The positive control (OHF) resulted in an approximate 65% decrease in androgenic activity.

The authors concluded that cypermet hrin has anti-androgenic effects in vitro ; however, β -cypermethrin did not demonstr ate anti-androgenic activity.

In another published study, Xu et al. (2008) conducted transcriptional ac tivation assays to detect AR agonists and antagonists using an African monkey kidney cell line (C V-1 cells) transfectedCYPERMETHRIN FINAL

Page 44 of 64 with the reported plasmid pCAT3-Basic (contai ning mouse mammary tumor virus sequences for four androgen receptor elements) and a pSV-Gal re porter. Cells were transfected, and incubated for an additional 24-hour period prior to addition of DHT (1nM) and test chemicals. Cypermethrin (>98% purity; cis/trans ratio is not stated ) was tested at 10

− 7 , 10

− 6 , and 10

− 5 M (in ethanol, 0.1%) with and without DHT against a ve hicle control (medium with 0.1% ethanol). The highest concentration (10

− 5 M) was the limit of cytotoxicit y. Cypermethrin was reported to be an anti-androgen with an IC 50 of 6.80 ± 2.3×10

− 5 M, with a decrease (p<0.01) in androgenic activity compared to DHT at 10

− 5 M cypermethrin. No androgen agonism was detected.

In a published study, Du et al. (2010) examined the potential androgenic and anti-androgenic effects of pyrethroids (incl uding cypermethrin, purity 92%; cis/trans ratio is not stated). For the

in vitro reporter gene assay, the MDA-kb2 breast cance r cell line was stably transfected with the androgen-responsive reporter gene (pMMTV.neo.luc) as used by Zhang et al. (2008). The cells were plated, allowed to attach (4-6 hours) , followed by treatment with cypermethrin (10

− 9 to 10

− 5 M) with or without DHT, 5 α -DHT (10

− 12 to 10

− 7 M; positive control), or 0.1% DMSO (negative control). Cells were treated with the cypermethrin alone and with 1×10

− 6 M flutamide, a potent antagonist of AR, to distinguish between AR - and GR-related activity. Determination of luciferase activity was similar to that described in Zhang et al. (2008). No AR agonist activity was observed for cypermethrin; however, an anti-androgenic effect was observed with a
calculated relative IC 20 of 1.64×10

− 6 M.

In a published study, Kjeldsen et al. (2013) determined the androge nic and anti-androgenic properties of cypermethrin (and other pesticides) with in vitro tests including an ARTA assay that used the Chinese hamster ovary cell line (CHO-K1). Cypermethrin (>95% purity) was dissolved in DMSO, with final DMSO concentra tions of 0.1%. Cypermethrin was tested in various concentrations within the range of 1×10

− 10 to 1×10

− 5 M, without removal of the transfection reagent and cDNA. Cypermethrin was tested alone and with 25 pM of one of the AR agonists (R1881 or DHT, corresponding to appr oximately half of the maximal effect concentration). Cypermethrin di d not display cytotoxicity at th e highest tested concentration (1×10

− 5 M), and it did not demonstrate an agonistic or antagonistic AR effect up to the highest
cocentration.

Aromatase

In another published study, Kjeldsen et al. (2013) also assessed effects of cypermethrin on aromatase enzyme activity with the human c horiocarcinoma JEG-3 cell line. Cypermethrin (>95% purity) was dissolved in DMSO, with final DMSO concentrations of 0.1%. Cypermethrin was tested at various c oncentrations within the range of 1×10

− 9 to 1×10

− 4 M. Cypermethrin was found to be cytotoxic at concentrations ≥ 5×10

− 5 M, but did not inhibit aromatase activity at any tested concentration ≤ 5×10

− 5 M.CYPERMETHRIN FINAL

Page 45 of 64 ERTA/ ER Binding/Cell Proliferation

In a published study, Saito et al. (2000) investigated the activity of several pyrethroids (including cypermethrin, purity >93%; cis/trans ratio is not stated ) on the ER α pathway with three different assay systems: a mammalian ER TA assay with HeLa cells and a luciferase reporter, a yeast two-hybrid system (Nishikawa procedure), and hER competitive ligand-binding with fluorescence polarization (P an Vera). Positive contro ls were run for agonism (17 β -estradiol and 4-nonylphenol) and antagonism (4-hydroxytamoxifen). 17 β -estradiol displayed the expected responses in all three assays, with dose-dependent induction in the ERTA and the yeast twohybrid assay, and dose-dependent inhibition (d isplacement of fluorescence standard) in the competitive ligand-binding assay. Cypermethrin was tested at 10

− 7 , 10

− 6 , and 10

− 5 M in the competitive binding assay, at 1 and 10 μM in th e HeLa cell assay, and 10 μM in the yeast twohybrid assay; tested dose(s) were determined on the basis of solubility. The results were displayed graphically; negative results for al l pyrethroids (including cypermethrin) were observed in all assays.

In a published study, Chen et al. (2002) tested organophosphorus and pyr ethroid pesticides in an E-Screen (MCF-7 cell proliferation) assay and an ER Competitive-Bind ing assay. MCF-7 cells were further analyzed with the pS2 mRNA gene expression assay as assessed by qRT-PCR. MCF-7 cells were plated with medium at an initial concentration of 10 3 cells per well and
allowed to attach for 24 hours. The medium wa s removed and replaced by medium containing cypermethrin at a concentration range of 10

− 11 to 10

− 6 M. The bioassay was terminated on Day 6 (late exponential phase) by remova l of the medium and addition of a thiazolyl blue solution (5 mg/mL PBS buffer). Absorbance was determin ed, after 4-hour incubation, at 490 nm. Fold proliferation of each exposure group was determined as the ratio of group absorbance to control
absorbance. Uterine cytosol from female SD rats wa s prepared for isolation of ER. The Competitive Binding assay was conducted similarl y to the Guideline ER Binding assay with [ 3 H]-estradiol and 17 β -estradiol.

Cypermethrin (>90% purity; cis/trans ratio is not stated ) was tested in all three assays. The PE (proliferation effect) value for cypermethr in was 1.46. In comparison with the PE of 17 β

estradiol (3.46), cypermethrin showed a partial agonistic response. Cypermethrin inhibited the binding of [ 3 H]-17 β -estradiol to ER with an IC 50 of 0.562 mM. These results indicate an equivocal or very weak positive response with percent bound of approximately 50%. In the pS2 gene expression assay (10

− 10 to 10

− 6 M cypermethrin), cypermethrin was reported to be positive

at 10

− 6 M with an approximately 2-fold response relative to β -actin mRNA.

Kim et al. (2004) conducted a similar investigation on seven pyrethroid pest icides (including cypermethrin) in an E-Screen (MCF-7 BUS cell proliferation) assay and an ER CompetitiveBinding assay. The E-Screen and ER Competitive -Binding assays were conducted in a similar manner as described previously for Chen et al. (2002). In addition, anti-est rogenic activity in the E-Screen assay was examined by combining cypermethrin (10

− 6 M) and 17 β -estradiol (10

− 10 orCYPERMETHRIN FINAL

Page 46 of 64 10

− 11 M) in MCF-7 BUS for six days. Anti-estroge nic activity was assessed relative to the negative control, ICI 182,780 (10

− 9 M).

Cypermethrin (98% purity; cis/trans ratio is not stated ) was tested in both assays. Cypermethrin did not induce MCF-7 BUS cell proliferation at any concentration, and also did not demonstrate anti-estrogenic activity against cell proliferation. Cypermethr in was also negative in the Competitive Binding assay against [ 3 H]-estradiol

.
In a published study, another similar investigation was conducted by Jin et al. (2010) with cypermethrin and permethrin in an E-Screen (MCF -7 cell proliferation) a ssay. MCF-7 cells were further analyzed with the pS2 mRNA gene expression assay as a ssessed by qRT-PCR. MCF-7 cells were plated with medium at an initial concentration of 10 3 cells per well and allowed to
attach for 24 hours. The medium was rem oved and replaced by medium containing cypermethrin at a concentration range of 10

− 9 to 10

− 5 M, 17 β -estradiol at 10

− 9 mol/L (positive control), or ethanol at 0.1% (v/v; negative control). The bioassay was terminated on Day 6, and
absorbance was determined at 490 nm. Fo ld proliferation of each exposure group was determined as the ratio of group ab sorbance to control absorbance.

Cypermethrin (98% purity; cis/trans ratio is not stated ) was tested in both assays. Cypermethrin induced (p<0.05) proliferation of MCF-7 cells with a PE of 1.63, and a RPE of 56.3% at a cypermethrin concentration of 10

− 7 M. In comparison with the PE of 17 β -estradiol (2.12), cypermethrin showed a partial agonistic response. It was noted that the full dose response of cypermethrin was an inverted U-shaped dose-response curve. In the pS2 gene expression assay (10

− 9 to 10

− 5 M cypermethrin), cypermethrin was reported to be positive at 10

− 6 M with a 2.15-fold response relative to GAPDH mRNA.

In a published study, Tyler et al. (2000) assessed cypermethrin (95.8% purity; cis/trans ratio is
not stated ) binding to hER in a recombinant yeast expression system assay with Saccharomyces

cerevisiae yeast. In the presence of an active liga nd, such as the positive control (17 β -estradiol), the estrogen receptor binds to an estrogen-res ponsive element on a plasmid, thereby initiating transcription of the reporter gene lacZ, which encodes the enzyme β -galactosidase. This produces a change in color from yellow to red that is determined by measuring the absorbance at 540 nm. A stock solution of cyperm ethrin was serially diluted in ethanol, and 10-μL portions of the appropriate dilutions were adde d to sterile incubation plates a nd evaporated to dryness. The estrogenic positive control was 17 β -estradiol, the anti-estrogenic positive control was 4- hydroxytamoxifen, and the negative control was et hanol. For the anti-estrogenic assay, 17 β

estradiol was added to the medium at a concentration of 2.5 × 10

− 10 M. Portions (200 μL) of the assay medium that contained the recombinant yeast were added to the plates; plates were sealed and shaken for two minutes prior to inc ubation at 28°C for 24 hours followed by room temperature incubation until optimal color development had occurred (details not reported). The plates were read at 540 and 620 nm (tur bidity correction fo r yeast growth).CYPERMETHRIN FINAL

Page 47 of 64 Cypermethrin was assessed for estrogenic and anti-estrogenic activity across a concentration range of 10

− 6 to approximately 10

− 3 M (possibly 10

− 2.8 M). Cypermethrin did not demonstrate any estrogenic activity; therefore, EC 50 and LOEC 50 estimates could not be determined. Antiestrogenic activity was observed at cypermethrin concentrations approximately >10

− 4 M, yielding an LOIC value of 7±3×10

− 4 M (between 1000- and 10,000-fold less potent compared to 4-hydroxytamoxifen).

In another published study , Kjeldsen et al. (2013) determined the estroge nic and anti-estrogenic properties of various pesticides (including cypermethrin) with in vitro tests including an ERTA assay with the MVLN cell line. Cypermethrin (> 95% purity) was dissolved in DMSO, with final DMSO concentrations of 0.1%. Cypermethrin wa s assessed alone in various concentrations within the range of 1×10

− 10 to 1×10

− 4 M, and 17 β -estradiol co-exposure experiments were carried out with pesticide con centrations ranging from 1×10

− 10 to 1×10

− 5 M. In the ERTA assay, cytotoxicity was observed with cypermethrin ( ≥ 5×10 -5 M). Cypermethrin weakly induced the luciferase activity with a maximum response of 119% (at 10

− 6 M) compared to the solvent control (set to 100%). Co-exposure with 25 pM 17 β -estradiol (E 2 -EC 65; 65% of maximum response) did not enhance the 17 β -estradiol -induced response of cyperm ethrin; cypermethrin did not exert an ER-antagonistic effect in this assay.

Hershberger

In a study examining the potential anti-androgeni c effect of several pyr ethroid pesticides ( Zhang
et al. , 2008 ), cypermethrin or β -cypermethrin (puriti es not reported; cis/trans ratio is not stated)
were assessed in an in vivo Hershberger assay. The study eval uated the androgenic (cyfluthrin
and β -cyfluthrin only) and anti-androge nic activities of cyfluthrin, β -cyfluthrin, and four structurally-related pyrethroi ds (permethrin, cypermethrin, β -cypermethrin, and bifenthrin). All
test substances were admi nistered in peanut oil.

For the in vivo portion, three-week old male SD rats we re maintained under standard laboratory conditions and provided with stan dard laboratory diet and water ad libitum . After an acclimation period of seven days, the rats were castrated on PND 28 and allo wed to recover for two weeks prior to initiation of dosing on PND 42. After the recovery period, rats were weighed and randomly assigned to dose groups (n=6). The an drogenic portion of the Hershberger assay was conducted with a sham-operated control group, but th is assessment did not include cypermethrin

or β -cypermethrin. The anti-androgenic portion of the Hershberger assay included eight treatment groups: a vehicle control group (peanut oil only); a positive control group dosed orally with flutamide (50 mg/kg/day); and six groups e ach dosed orally with testosterone propionate (TP) and a pyrethroid. Cypermethrin and β -cypermethrin were each dosed orally at 50 mg/kg/day for seven days in addition to a dail y s.c. injection of TP at 0.5 mg/kg/day. After the final dose, rats were anaesthetized and sacrificed by exsanguinati on. Blood from each rat was collected for serum analysis of testosterone concentration. The seminal vesicles, ventral prostate, dorsolateral prostate, LABC, Cowper’s gla nds, glans penis, liver, paired adrenal glands,CYPERMETHRIN FINAL

Page 48 of 64 spleen, and paired kidneys of each rat were dissected and weighed. Serum testosterone concentrations were analyzed by time-resolved fluorescence with commercially available FIA
kits.

No mortality or treatment-related clinical si gns were noted in any of the groups in the Hershberger assays. The text states there were no significant differences between initial or final body weights, or weight gains of the pyrethroid-treated groups co mpared to the control groups, but the actual data for the cypermethrin-treated groups were not presented. Serum testosterone concentrations were not shown for the cypermet hrin-treated rats, but it was reported that no
treatment-related effects were observed. Treatment with cypermethrin at 50 mg/kg/day resulted in a decrease (p<0.05) in absolute weights of the seminal vesicles, ventral prostate, and dorsolateral prostate compared to the castrated control + TP; however, th ere were no significant decreases in the weights of the LABC muscle, Cowper’s glands, or glans penis. However, the exact percentage decreases, relative to control, could not be ascertained as the data were only reported graphically. Treatment with β -cypermethrin at 50 mg/kg/da y had no effect on any of the androgen-sensitive accessory sex tissues co mpared to the castrated control + TP. The weights of all accessory sex tissu es for the castrated control +TP were similar to the uncastrated males, indicating that the dosage of TP (0.5 mg/kg/day) was appr opriate, and the positive control (castrated + TP + flutamide) displayed a grea ter decrease in tissue weights compared to cypermethrin and β -cypermethrin. The authors concluded th at cypermethrin has anti-androgenic
effects in the in vivo assay; however, β -cypermethrin does not have anti-androgenic activity.

Although the study was conducted prio r to the establishment of the OPPTS 890 Test Guideline for the Hershberger (890.1400) assay, the in vivo portion of the study generally conforms to the
Guideline. Differences from the Guideline were th at the rats were castrat ed at PND 28 instead of PND 42, as recommended by the Guideline, and dos ing was conducted for seven days instead of the recommended ten days. In addition, the Hershberger assay investigated only the antiandrogenic activity of cypermethrin and β -cypermethrin (no androgenic portion), and the castrated rats were dosed with TP at 0.5 mg/ kg instead of 0.2-0.4 mg/kg/day. However, because the control group (castrated + TP) exhibited similar growth of acce ssory sex tissues to the shamoperated group (uncastrated), the 0.5 mg/kg dose of TP was acceptable. The study is considered acceptable for use in a WoE evaluation.

Subchronic Oral Toxicity in Non-Rodents (Dog; OCSPP 870.3150)

In a 13-week, subchronic toxicity study in dogs ( MRID 44527002, 1994 ), cypermethrin (95.7% purity; cis/trans ratio is not stated) was administer ed to Beagle dogs (4/sex/group) at concentrations of 0, 300, 600, 800, or 1100 ppm in the diet (achieved doses of 0/0, 10.4/12.2, 20.7/25.4, 24.6/34.3, and 37.0/45.2 mg/kg/day [M/F], resp ectively) for three months. Organs that were weighed included the liver, kidneys, testes/epididymid es, and thyroid/parathyroids. Gross and microscopic pathological examinations were conducted on these tissues as well as the pituitary, adrenals, ovaries, pros tate, m ammary gland, and uterus.CYPERMETHRIN FINAL

Page 49 of 64 No deaths occurred during the study. In th e high-dose group, two males and one female exhibited slight tremor activity beginni ng on Days 49 (female), 60, or 71 through study termination. In addition, one male and one female exhibited moderate tr emor activity either on Day 76/77 (male) or during the Week 8, 9, and 12 detailed physical examinations (female). Males and females displayed decreases in body weights throughout the study in the high-dose males and females ( ↓≤ 13 and 19%, respectively); d ecreases in medium-low ( ↓≤ 14%) and medium-high ( ↓≤ 11%) females were observed, but were generally <10%. Reduced body weight gains were generally noted throughout the study in both sexes, with negative or no body weight gains observed in high-dose males (Weeks 0-9) , medium-high females (Weeks 0-7), and highdose females (Weeks 0-13, except between Weeks 2 and 3).

No treatment-related differences in food consum ption, hematological, clinical chemistry, or urinalysis parameters, organ weight, or gro ss or microscopic pathol ogy were observed. No neoplastic tissue was observed. The LOAEL is 1100 ppm (equiva lent to 37.0/45.2 mg/kg/day [M/F]), based on tremor activity in both sexe s and decreased body weight and body weight gains
in the males and females. The NOAEL is 800 ppm (equivalent to 24.6/ 34.3 mg/kg/day [M/F]).

Prenatal Development in Rodents (Rat; OSCPP 870.3700)

In a developmental toxicity study in rats ( MRIDs 00056804, 92027039 and 92027061; 1978 ), cypermethrin (98.2% purity; cis/trans ratio is not stated) was admi nistered by gavage to mated CD rats (25/dose group) at doses of 0 (c orn oil), 17.5, 35, or 70 mg/kg/day on GDs 6-15,
inclusive. The rats were sacrif iced at GD 21 of gestation and s ubjected to gross necropsy. All fetuses were weighed, sexed and examined for gr oss external alterations. One-third of the
fetuses in each litter were examined for viscer al soft tissue alterati ons by a free-hand serial sectioning technique. The remaining two-thirds of the fetuses in each litter were examined for
skeletal alterations.

Dams in the high-dose group displayed neurologic al signs such as splayed limbs, spasms, hypersensitivity to noise, and convulsions. Decreased (p<0.05 and 0.001, respectively) body weight gains of 12 and 28% were observed in the medium- and hi gh-dose groups during GDs 6-15. Overall body weight gain was decrease d (p<0.05) in the high-dose dams by 8%. Reproductive parameters were not affected by tr eatment with cypermethrin; pregnancy rates ranged from 88-100%. No effects on either skeletal or visceral st ructures were reported. The maternal LOAEL is 35 mg/kg/day, based on body we ight decreases, and the maternal NOAEL is 17.5 mg/kg/day. The developmental LOAEL is not determined (>70 mg/kg/day), and the developmental NOAEL is ≥ 70 mg/kg/day.

Prenatal Development in No n-Rodents (Rabbit; OCSPP 870.3700)

In a developmental toxicity study in rabbits ( MRID 43776302, 1994 ), cypermethrin (94-96% purity , cis/trans ratio approximately 1:1) was administer ed to female New Zealand White rabbitsCYPERMETHRIN FINAL

Page 50 of 64 (20/dose group) by gavage at doses of 0 (c orn oil), 100, 450 or 700 mg /kg/day from GDs 7-19, inclusive. Cypermethrin was administered as a 50% w/v solution in corn oil at varying volumes. On GD 29, all surviving does were sacrificed, and all fetuses we re weighed and examined for external malformation/variations. Each fetus was examined viscerally by fresh dissection and
the sex determined. All carcasses were eviscera ted and processed for skeletal examination.

Four deaths occurred during the study, but were not, or could not conclusively be, attributed to treatment. Exacerbation of some clinical sign s such as anorexia, abdominal/genital staining, decreased feces, and red or pink material in the pan occurred in the high-dose group and in a few does in the medium-dose group. Due to the infre quency of occurrences, these effects were not definitely attributed to treatment. Body wei ght gain was decreased during the dosing period (GDs 7-19) at the medium (NS; ↓ 25%) and high (p<0.05; ↓ 30%) doses, with compensatory increases during GDs 19-29. There were no indi cations of reproductive or developmental toxicity; pregnancy rate range d from 85-100%. The maternal LOAEL is 450 mg/kg/day, based on body weight gain, and the maternal NOAEL is 100 mg/kg/day. The LOAEL for developmental toxicity is not determ ined (>700 mg/kg/day), and the NOAEL is ≥ 700 mg/kg/day.

Developmental Neurotoxicity in Rodents (Rat; OCSPP 870.6300)

In a developmental neurotoxicity study in rats ( MRID 46670402, 2005 ), zeta-cypermethrin (81.8% purity) was administered in the diet to mated female Crl:CD®(SD)IGS BR rats (25/group) at nominal concentrations of 0, 50, 125, or 300 ppm from GD 6 through LD 21. Mean-measured doses to the animals, adjust ed for purity, were 0, 2.9, 7.4, or 17.3 mg/kg/day, respectively, during gestation and 0, 7.1, 17.5, or 39.8 mg/kg/day, respectively, during lactation. Functional Observations (FO) were conducte d on 25 dams/group on GDs 10 and 15, and on all surviving dams that delivered offspring on LD s 10 and 21. On PND 4, litters were culled to eight offspring, with at least th ree males and three females per li tter. Offspring were allocated
for detailed clinical observat ions (FO) and assessment of motor activity, auditory startle response, and learning and memory, as well as for neuropathology at study termination (PND 72). On PND 21, the whole brain was co llected from 10 pups/sex/dose group, weighed, and subjected to micropathologic examinati on and morphometric an alysis. Pup physical development was evaluated by body weight. The ag e of sexual maturation (VO in females and PPS in males) was assessed.

One control female was found dead late in gesta tion; necropsy revealed a ruptured uterus and late resorptions; all remaining an imals survived to scheduled sacrifice. No treatment-related clinical signs of toxici ty were observed during th e daily examinations.

Absolute body weight of dams was similar be tween the treated and control groups throughout gestation. However, body weight gain in the high -dose group was slightly le ss (NS) than that of control for each interval, resulti ng in an overall gestational weightgain (GDs 0-20) in the highdose animals that was decreased (p<0.05; ↓ 10%). Food consumption by the high-dose groupCYPERMETHRIN FINAL

Page 51 of 64 was decreased (p ≤ 0.05 or 0.01) compared to control during GDs 6-9 ( ↓ 13%) and GDs 6-20 ( ↓ 4%). Throughout lactation, mean body weight of the high-dose group was decreased (p ≤ 0.05 or 0.01; ↓ 5-8%) compared to control; overall body wei ght gain (LDs 1-21) was also decreased (NS; ↓ 15%). Food consumption by the high-dose group was decreased (p ≤ 0.01; ↓ 11%) throughout lactation. The observed changes in body weight, body we ight gain, and food consumption were not considered to be adverse due to the small magnitude of the change. No
treatment-related effects were observed in re productive parameters, and gross necropsy was
unremarkable.

No treatment-related effect on the mean number of pups born, mean liv e litter size, percentage of males per litter, pup survival, or clinical si gns was observed. Pup body weights were similar
between the treated and contro l groups on PNDs 1-11. On PNDs 13-21, mean body weight was decreased (p ≤ 0.05 or 0.01) in the high-dose female offspring ( ↓ 8-10%) and was slightly decreased (NS) in the high-dose male offspring ( ↓ 6-7%). Mean body weight gain was decreased (p ≤ 0.05 or 0.01) in the high-dose females ( ↓ 11-14%) for all intervals during PNDs 7-17 and for PND 4-21 ( ↓ 13%). Mean weight gain was decreased (p ≤ 0.01) in the high-dose males ( ↓ 14%) during the PND 11-13 interval. Post-weaning body weight and body weight gain were similar
between the treated and control groups. The average age and b ody weight at attainment of sexual maturation (VO) was not affected by treatment.

No significant difference in total motor activ ity was found between the treated and control groups on any testing day. Aud itory startle response, learni ng and memory, and gross and qualitative microscopic findings were not affected by treatment.

A slight, non-dose-related increase (NS) in male brain weights was noted at the high dose on PND 21 ( ↑ 4%). An increase (p ≤ 0.05) in the vertical thickness of the cortex was observed in high dose PND 21 females ( ↑ 6%). This sole brain morphometric change was determined to occur in isolation, only in female pups on PND 21, and was not considered biolog ically significant.

The maternal NOAEL is 300 ppm (17.3 mg/kg/day, HDT); a LOAEL is not established. The offspring LOAEL is 300 ppm (17.3 mg/kg/day to dams), based on decreased body weight and body weight gain in females. The offspr ing NOAEL is 125 ppm (7.4 mg/kg/day to dams).CYPERMETHRIN FINAL

Page 52 of 64 Multi-Generation Reproduction in Rodents (Rat; OCSPP 870.3800)

In a three-generation reproduction study in rats ( MRIDs 00112912, 42068504, and 92027040;

1982 ), cypermethrin (90.6 to 93.1%, cis/trans ratio: 53.4/46.6%) was admi nistered to Wistarderived SPF strain rats (15 males and 30 fema les/dose group) at nominal doses of 0, 50, 150 or 1000/750 ppm (high dose was reduced to 750 ppm after twelve weeks because of severe neurological symptoms). These doses corres pond to estimated intakes of 0, 2.5, 7.5, or 50/37.5 mg/kg/day. Three successive generations were produced, each consisting of two separate breedings yielding six sets of litters.

One high-dose male (1000 pm) was sacrificed in ex tremis, and this death was considered to be treatment-related. There were obvious clinical signs of neurotoxicity ( i.e. , ataxia, increased sensitivity to sound, and high-stepping gait) in the high-dose (1000 ppm) group. Parental weight gain was decreased in the medium- and hi gh-dose groups throughout the study; selected generations include F 2 males ( ↓ 7 and 9%, respectively) and F 0 and F 2 females ( ↓ 5 and 10%, and ↓ 9 and 11%, respectively). Food consumption was also decreased (signifi cance not reported) in the medium- and high-dose groups. There were no treatment-related effects on the fertility, gestation, viability, or lactation indices [a non-dose-dependen t decrease (p<0.05) in fertility in
the F 1 a low-dose females was not considered a treatme nt effect]. There were also no treatmentrelated effects on litter parameters, with the exception of decreased mean litter weight gains during lactation ( i.e.

, ↓ 12 to 21% and ↓ 12 to 17% for F 1 b males and females, respectively, for
PNDs 10 to 28 and ↓ 8 to 11% and ↓ 9 to 13% for F 2 b males and females, respectively, for PNDs 21 to 28). There were no consistent, treatment-re lated histopathological ch anges in pups at birth or in parental rats.

The parental/offspring LOAEL is 750 ppm (50/37.5 mg/kg/day) based on consistent decreased body weight gain in both sexes in the parents and decreased mean litte r weight gain during lactation. The parental/offspring NOAEL is 15 0 ppm (7.5 mg/kg/day; decreases in mean body weight gain at the medium dose are not cons idered to be toxicologically significant).

In a previous three-generation reproduction study in rats ( MRID 00090040, 1979 ), cypermethrin (98% purity) was administered in the diet to Wistar SPF strain rats (30/sex /dose group) at nominal doses of 0, 10, 100 or 500 ppm (estimated intakes of 0, 0.5, 5, or 25 mg/kg/day). The first parental group (F 0 ) produced two litters (F 1 a and F 1 b), the F 1 b litter was culled to produce
the F 2 a and F 2 b litters, and the F 2 b litter was culled to produce the F 3 a and F 3 b litters.

No adverse effects were noted for the general heal th or behavior of rats in any treatment group. In the high-dose group, there was decr eased parental weight gain ( ↓ 3% for males and 7% for females) for the F 1 generation. There were no treatment-re lated effects on the fertility, gestation, viability, or lactation indices. There were also no treatment-related effects on litter parameters, with the exception of decr eased (p<0.01) pup weight at PND 21 in the medium- ( ↓ 2-3%) and high-dose pups ( ↓ 4%). This parameter was only significan t for the combined weights from allCYPERMETHRIN FINAL

Page 53 of 64 litters; these differences were not apparent for individual generation weights. The small decrement at 100 ppm is not considered of suffici ent magnitude to be in cluded in the LOAEL. There were no consistent, treatme nt-related histopathological ch anges in pups at birth or in parental rats. The LOAEL is 500 ppm (25 mg/ kg/day) based on decreased weight gain. The NOAEL is 100 ppm (5 mg/kg/day).

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

In a chronic toxicity/carci nogenicity study in rats ( MRID 00112910, 1982 ), cypermethrin (88- 93% purity, cis/trans ratio: 55/45%) was administered in the diet to Wistar-derived SPF strain rats (52/sex/dose group) at nomin al doses of 0 (two vehicle control groups), 20, 150 or 1500 ppm (estimated intakes of 0, 0, 1, 7.5, or 75 mg/kg/day) for 2 years. The high-dose animals received diets containing nominal doses of 1000 ppm instead of 1500 ppm fo r the first six weeks of the study, but no explanation for this difference was provided. Satellite groups of 12/sex/dose group were sacrificed after one year of dosing. At study termination, su rviving rats in all groups were sacrificed for gross and histopat hological evaluation. Select organs weighed at Week 52 and at termination included the liver, kidneys, testes, ov aries, adrenals, pituit ary gland, and brain. Gross and microscopic pathological examinations were conducted on 42 ti ssues (complete list of organs was not included).

There were no treatment-related differences in survival. Transient clinical (behavioral) signs ( i.e. , frequent face washing, increased sensitivit y to sound, hind limb uncoordination) were noted during the first six days of dosing in six (6 /52) high-dose males. Body weight gain was decreased throughout the study in the high-dose animals ( i.e.

, ↓ 10% for males and 13% for
females at Week 13 and ↓ 12% for males and 18% for females at study termination). Lesser decreases in body weight gain (<10%) were oc casionally noted for the medium-dose groups. Relative liver weight was increa sed (significance not reported; ↑ 21%) in high-dose females at the interim sacrifice (Week 52). Absolute and relati ve kidney weights were decreased (significance not reported; ↓ 17%) in high-dose females at study termin ation. No treatment-related effects on gross and microscopic pathology were observed in examined organs, including the testes, epididymides, seminal vesicle, prostate, ovaries , uterus, mammary, thyroi d, pituitary or adrenal glands. Cypermethrin was not considered to be oncogenic in this st udy. A possible treatment
association with increased testicul ar interstitial tumors was not considered definite. Frequent
occurrences of adenomas and o ccasional carcinomas of the pitu itary were noted in all test animals (females>males), but no dose respons e was evident. The LOAEL is 1500 ppm (75 mg/kg/day) based on body weight gain decreases, and the NOA EL is 150 ppm (7.5 mg/kg/day).

Carcinogenicity in Rodents (Mouse; OCSPP 870.4200)

In a chronic toxicity/car cinogenicity study in mice ( MRIDs 00112911 and 92027038, 1982 ),

cyperm ethrin (91.5% and 94-94.2% purity, cis/trans ratio: 53-54/46-47%) was administered to Swiss-derived SPF mice (70/sex/dos e group) at doses of 0 (two vehicle control groups), 100,CYPERMETHRIN FINAL

Page 54 of 64 400, and 1600 ppm (estimated intakes of 0, 0, 14, 57 or 229 mg/kg/day) for 97 weeks for males
and 101 weeks for females. Interim sacrifices were conducted after one year of dosing with 9-10/sex/dose group. At study termin ation, all surviving mice in al l groups were sacrificed for gross and histopathological evaluation. Select organs weighed at Week 52 and at termination included the liver, kidneys, testes, and brain. Gr oss and microscopic pathological examinations were conducted on 45 tissues (complete list of organs was not included).

There were no treatment-related differences in su rvival, and clinical sign s were not present. Decreased body weight gains were observed in the high-dose animal s, with significant decreases (significance not reported) noted only during the first year; no tr eatment-related differences were
noted in the low- and medium-dose animals. Li ver weights were affected at the interim and
terminal sacrifices in both sexes. At the interi m sacrifice, absolute liver weights were increased (p<0.05) by 19 and 20% in the medium- and hi gh-dose males (with 28 and 16% increases in relative liver weight in the high dose males and females, respectivel y). Liver weight effects were
not observed at the terminal sacr ifice, but increased liver weight s were noted in one of the two vehicle control groups for both males and females; this liver weight variability apparently
masked increased absolute and relative liver weights in the high-dose males at the terminal sacrifice. Absolute and relative testis weight was decreased in high-dose males (significance not reported; ↓ 18%) at the interim sacrifice only. No treatment-related effects on histopathology were noted in the testes, epididymides, semina l vesicle, prostate, ova ries, uterus, cervix, mammary glands, thyroid, pituitary or adrenal glands at any dose level.

The LOAEL is 400 ppm (57 mg/kg/day) based on liver weight decreases, and the NOAEL is 100 ppm (14 mg/kg/day). This st udy was determined to be positi ve for the induction of benign alveologenic neoplasms, with a treatment-related increase (p<0.05) in high-dose females (14 vs. 5 control).

Chronic Oral Toxicity/Carcinogenici ty in Non-Rodents (Dog; OCSPP 870.4100)

In a chronic toxicity/car cinogenicity study in dogs ( MRIDs 00112909, 42068503, and 92027037; 1982 ), cypermethrin (90.6% purity, cis/trans ratio: 53.9/46.1%) dissolved in corn oil was administered to Beagle dogs (6/sex/dose gro up) in gelatin capsules at doses of 0, 1, 5, or 15 mg/kg/day (adjusted for purity) for 52 weeks. Organ weights determined included adrenals, kidneys, liver, ovaries, testes, pituitary, and thyroid; histopathologica l examinations were conducted on 37 tissues (complete li st of organs was not included).

No mortality occurred during th e study. Treated males and females displayed increases in vomiting during Week 1, and high-dose animals disp layed signs of nervous system stimulation in the form of body tremors, gait abnormalitie s, uncoordination, disorientation, and hypersensitivity to noise during Week 1. Males and females in the medium-dose group had increased incidences of passage of liquid stools throughout the study period (approximately 5- and 10-fold, respectively), and the incidence of this condition was increased approximately 30-fold at theCYPERMETHRIN FINAL

Page 55 of 64 high-dose. Decreased body wei ght/body weight gain and food consumption was noted in the high-dose animals (extent and significance not re ported). Non-significan t increases in liver weight were noted (only high-dos e females were reported), and a potential increase (NS) in adrenal weight in high-dose males ( ↑ 19 or 11%). There were no treatment-related changes in the absolute or relative weights of the testes, ovaries , thyroid or pituitary glan d. There were also no treatment-related effects on gross or micr oscopic pathology, incl uding in the testes, epididymides, prostate, ovaries, uterus, mammary glands, thyroid, pituitary or adrenal glands at any dose level. There was no evidence for cypermethrin being oncogenic in the dog.

The LOAEL is 15 mg/kg/day based on neurological symptoms (body tremors, gait abnormalities, uncoordination, disorientation, and hypersensitivity to noise) and decreased body weight, and the NOAEL is 5 mg/kg/day.

Avian Reproduction (OCSPP 850.2300)

In an avian reproduction study ( MRID 00090073, 1981 ), Mallard ducks ( Anas platyrhynchos ) were administered cypermethrin (purity and cis/trans ratio not reported) in the feed. No treatment-related effects on repr oduction or any other endpoint were seen up to 50 ppm. In the study report, although no reproduc tive effects were observed, nu merous mortalities were observed, and prior to the ons et of death birds were observed being “bullied”.

In an avian reproduction study ( MRID 00090074, 1981 ), young Bobwhite quail approaching their first breeding season ( Colinus virginianus ) were administered c ypermethrin (87.8% purity,

cis/trans ratio: 53/47%) in the feed at nominal concen trations of 0, 5, or 50 ppm (adjustment for purity was not reported) for 31 weeks (nineteen weeks prior to the start of egg production and during twelve weeks of egg production).

There were increased (NS) proportions of e ggs cracked or broken in the control ( ↑ 19.2%) and high-dose ( ↑ 17.6%) groups compared to the low-dose group (10.9%), and all were above the Guideline upper limit of 2%; no differences in mean egg weight or egg shell thickness were
noted. There were no treatment-related differences in the numbers of viable embryos/eggs set, eggs laid/hen, and normal hatchlings. Finally, m ean body weights at time of hatching and at Day 14, showed no treatment-related difference, as well as no differences in survival of chicks to 14 days.

The reproductive NOAEC for bobwhite quail exposed to cypermethrin in the diet for 31 weeks was 50 ppm (50 mg/kg diet). In the study report, although no reproductive effects were observed, numerous mortalities were observed, an d prior to the onset of death birds were observed being “bullied”.

In an avian reproduction study ( MRID 47944018, 2001 ), bobwhite quail (20 pairs per level of

ca. 12-month old) were administered alpha cypermet hrin in diet at nominal concentrations of (negative control), 50, 150, and 450 mg ai.kg diet . Mean-measured concentrations were <1.125CYPERMETHRIN FINAL

Page 56 of 64 (LOQ, control), 8, 149, and 452 mg ai/kg diet, respectively. Six adult mortalities occurred during the study one from the control group, two from th e 48 mg ai/kg diet group, and three from the 452 mg ai/kg diet group. Most findi ngs were reported to be rela ted to the long-term housing of birds in cages. Several chicks at the 452 mg ai/kg diet level were found dead without prior clinical signs. Post mortem examination show ed no evidence of treatment-related abnormalities. Significant reductions (p<0.05) were detected in adult male body we ight gain at the two highest treatment levels. At the 452 mg a i/kg diet level, treatment-rela ted effects included 14-day old survivors of number hatched. There was a significant difference observed at the 48 mg ai/kg dose for # hatchlings/egg set (%). Parents were c onfined to very small cages (0.31 x 0.39 x 0.29 m) for a protracted period of time before the study even began (10 week acclimation period). These conditions may have been responsible for the bi rd aggression observed across all treatment and control groups, which likely result ed in the reported mortalities. Fighting between birds is likely a sign that the animals were stressed, which may have biased the result s of the reproductive study. This can be seen in the reproductive succe ss of the control group, where one pair of birds did not lay any eggs and anothe r pair of birds laid 48 eggs but had zero viable embryos. Due to poor husbandry of test birds, this study can be used for purposes of characterization only.

Fish Early Life-Stage Toxicity; (OPPTS 850.1400)

In a freshwater fish early life-stage toxicity test ( MRID 00089039, 1981 ), Fathead minnow ( Pimephales promelas ) embryos and larvae were exposed to cypermethrin (91.5% purity) at nominal concentrations of water (negative) control, solvent control, 0.031, 0.062, 0.12, 0.25, and 0.50 μg/L (mean-measured concentrations of negative control, solv ent control, 0.048, 0.045, 0.063, 0.14, and 0.33 μg a.i./L in a flow-through system for 30 days. Observations were made on embryo hatching (%), and larval survival and gr owth (length and weight). Each test chamber contained 60 embryos, 40 larvae were selected from the surviving embryos for each concentration. First day of hatch was not reported. Test were run at 25°C, rather than 22 or 27°C. Data were analyzed by analysis of variance (ANOVA), parameters examined were percentage of hatch and the surv ival, length, and weight of larvae after 30 days of exposure. If treatment effects were indicated, th e means of the test parameters were compared to those of the controls using Dunnett’s procedur e. No treatment-related effects were observed on hatchability, or larval growth. Larval surv ival was significantly reduced in the highest test concentration (mean-measured concentration 0.33 μg a.i./L), p<0.05 compared to the controls. The NOAEC for fathead minnow exposed to cypermethrin wa s 0.14 μg a.i./L, based on reduced survival.

Fish Life-Cycle Toxicity; (OCSPP 850.1500)

A life-cycle toxicity test with fathead minnow ( Pimephales promelas ) was conducted using cypermethrin (92.9-93.2% purity). Newly fertilized embryos (<48 hours) were exposed to cypermethrin at nominal concentrations of 0.06, 0.12, 0.25, 0.5, and 1.0 μg/L (mean measured concentrations of 0.051, 0.077, 0.15, 0.32, and 0.65 μg/L) ( MRID 40641701, 1998 ). F0 fish were monitored for survival and growth, and once fish attained sexua l maturation, reproductionCYPERMETHRIN FINAL

Page 57 of 64 was monitored, and F1 generation was exposed for 30 days. The study duration was 300 days. The reviewer's analysis detected significant (p<0.05 ) reductions in % survival of F0 fish 30 and 60 days post hatch at 0.32 μg/L, with the most sensitiv e endpoint being % survival of fish after 30 days post-hatch (based on the number of larvae released) with a LOAEC of 0.15 μg/L (NOEAC 0.077 μg/L). The reviewer's analysis additionally detected significant treatment-related effects on length and weight of F0 female fish 300 days posthatch at 0.32μ/L, and on reproduction (i.e., # eggs, # eggs/batch, # eggs/spawn) at the highest treatment level (0.32μ/L) evaluated for those endpoints. No effects on F1 hatch, survival or growth was observed. However, this study is supplemental, as excessive analytical variation (high:low ratios of 3.0 to 7.8) was observed at all levels during the study. The range of concentrations was so great that overlapping of concentrations occurred for all
levels.

Mechanistic Studies: Male Reproductive System (Rat)

In a published study, Hu et al. , (2013 ) investigated the re productive toxicity of cypermethrin in intact adult male rats. Adult male SD rats (8 0-90 days old) were ra ndomly separated, by weight, into 5 groups (12 rats/group) a nd orally dosed by gavage with cypermethrin (97.53% purity;

cis/trans ratio is not stated) in corn oil at 0, 6.25, 12.5, 25, or 50 mg/kg/day for 15 days. Rats were weighed and observed for signs of treatment-related effects da ily. At the conclusion of the treatment period, rats were sacrificed by d ecapitation, and the livers, kidneys, testes, epididymides, seminal vesicles, and prostates were excised and weighed.

One testis from each rat was frozen (

− 20°C) to be used for testicular sperm head counts and daily sperm production determinations. The frozen testis was thawed and decapsulated prior weighing and homogenization in ice-cold 0.9% sodium chloride solution containing 0.01% Triton X-100. The spermatozoa in the sample were counted with a hemocytometer. The other excised testes were used for histological evaluations. The te stis was fixed in Bouin’s fixative 10% neutral buffered formalin, embedded in paraffin, sectione d, and stained using the standard hematoxylineosin double staining procedure for microscopic examination. Additionally, fragments of the testis from fresh tissue were prepared for electron microscope examination. Immunohistochemical investigation of the AR were conducted, but wi ll not be discussed further.

Serum was collected (procedure no t described), and concentrations of testosterone, folliclestimulating hormone (FSH) and luteinizing horm one (LH) were measured with commercial radioimmunoassay kits.

Food consumption for all treatment groups was not different compared to control throughout the treatment period, and no mortalities, signs of toxicity, or abnorma lities in behavior or appearance were observed. Body weight gains (Treatment Days 11-15) were decreased (p<0.05) in rats dosed with cypermethrin at 12.5, 25, and 50 mg/k g/day. A downward trend (NS) in liver and kidney weights was observed in trea ted rats. Prostate absolute weights were decreased by 24 and 22% (p<0.01) in rats treated with cypermethrin at 25 and 50 mg/kg/day, respectively. No differences were noted for the testes, epididymid es, and seminal vesicles weights in any of theCYPERMETHRIN FINAL

Page 58 of 64 treatment groups. Daily sperm production was decreased (p<0.01) in rats treated with cypermethrin only at 50 mg/kg/day.

Slight distortion of seminiferous tubules in rats treated at 6.2 5 and 12.5 mg/kg/day was associated with changes in the lumen of the seminiferous tubules and decreased spermatids. In rats treated at 25 and 50 mg/kg/ day, atrophy and distorted semini ferous tubules, reduction and deformation of germ cells, and disordered arra ngement of spermatoblasts were observed. The
number of seminiferous tubules was decreased (p<0.01) in males treated with cypermethrin at 6.25 and 12.5 mg/kg/day; it was observed that the development of spermatogonia was obstructed and the arrangement of spermatid s was disordered. The perimeter of the seminiferous tubule was decreased (p<0.05) and the number of t ubules exhibiting degeneration was increased (p<0.01) in all treatment gr oups compared to control.

The diameters of the seminiferous lumen al so were decreased (p<0.05) at different spermatogenesis stages in rats treated with cypermethrin at 6.25, 12.5, and 25 mg/kg/day, and the number of cell layers of the seminiferous tubul es was decreased in rats treated at 12.5, 25, and 50 mg/kg/day. Significant ultras tructural changes of the seminiferous epithelium were observed with cypermethrin treatment. In addition, the number of AR positive Sertoli cells, Leydig cells, and peritubular myoid cells were decreased (p<0.01) in rats tr eated at 12.5, 25, and 50 mg/kg/day.

Finally, serum testosterone concen trations were decreased by 51% (p <0.05) in rats treated at 50 mg/kg, and serum FSH and LH concentrations were increased by 35 and 50%, respectively (p<0.05 and 0.01, respective ly) at 50 mg/kg/day.

In a similar published study from the same group, Li et al. (2013 ), also investigated the reproductive toxicity of cypermethrin in intact adult male rats. A dult male SD rats (9 weeks old) were randomly separated, by weight, into 5 groups (12 rats/group) and orally dosed by gavage with cypermethrin (98% purity; cis/trans ratio is not stated) in corn oil at 0, 7.5, 15, 30, or 60 mg/kg/day for 15 days. Rats were weighed an d observed for signs of treatment-related effects daily. At the conclusion of the treatment peri od, rats were sacrificed by decapitation, and the livers, kidneys, testes, epididymides, seminal ve sicles, and prostates we re excised and weighed.

The right testis from each rat was frozen (-80°C) to be used for testicular sperm head counts and daily sperm production determinations. The frozen testis was thawed and decapsulated prior weighing and homogenization in ice-cold 0.9% sodium chlori de solution containing 0.01% Triton X-100. The spermatozoa in the sample we re counted with a hemocytometer. The left testes were used for histological evaluations. Th e testis was fixed in mo dified Davidson’s fluid, embedded in paraffin, sectioned, and stained using the standard hematoxylin-eosin double staining procedure for microscopic examination.CYPERMETHRIN FINAL

Page 59 of 64 Serum was collected (procedure no t described), and concentrations of testosterone, FSH and LH were measured with commercial radioimmunoassay kits.

Food consumption for all treatment groups was not different compared to control throughout the treatment period, and no mortalities, signs of toxicity, or abnorma lities in behavior or appearance were observed. Body weights and absolute orga n weights for mice treated with cypermethrin were similar to control. Daily sperm production was significantly (p<0.0 5) decreased in rats treated with cypermethrin at 30 and 60 mg/kg/day.

Histopathological examination of the testes iden tified a slight distortion of the seminiferous tubules in rats dosed with cypermethrin at 7.5 and 15 mg/kg/day. In rats dosed with cypermethrin at 30 and 60 mg/kg/ day, atrophic and distorted semi niferous tubules, and deformed and disordered arrangement of germ cells were ob served in the testes. Additionally, the numbers of germ cells, Sertoli cells, and Leydig cells we re reduced, and the number of cell layers of the
seminiferous tubules was decr eased. High-magnification dark-f ield microscopy (100×) showed that the arrangement of germ cells was in disorder. Intraepithe lial vacuolization and multinucleated formation of spermatocyte were observed in the treatment groups. In addition, impairments of Sertoli cells and Leydig cells were seen in cypermethrin-treated rats;
vacuolization was found in Sert oli cells, and deformed nucleus was noted in Leydig cells. The perimeters of the seminiferous tubule were decr eased (p<0.05) in all cypermethrin-treated rats;
the diameters of the seminiferous lumen were de creased (p<0.01) in ra ts treated at 7.5, 15, and 30 mg/kg/day; and the number of cell layers of seminiferous tubules was decreased (p<0.01) in rats treated at 15, 30, and 60 mg/kg/day.

Serum testosterone concentrations were decreased (p<0.05) in rats treated with cypermethrin at 30 and 60 mg/kg/day, and serum FSH concentrations were increased (p<0.01) in rats treated at 60 mg/kg/day compared to control. An upward trend in serum LH concentrations was observed in cypermethrin-treated rats, but increases we re not significantly different from the control

group.

An earlier published study from the same group, Liu et al. (2010 ), investigated the reproductive toxicity of β -cypermethrin in intact adult male rats. Adult male SD rats were randomly assigned to three groups (10 rats/group), and orally dosed by gavage with β -cypermethrin (>95.0% purity) in corn oil at 0 (vehicle only), 15, or 30 mg/kg for 15 days.

Following 15 days of treatment, rats were sacrif iced by decapitation and th e testes, epididymides, and seminal vesicles were excised and weighed; relative weights were calculated. After weighing, one testis was frozen (

− 20 °C) for testicular sperm head counts and daily sperm production determinations; the other tes tis was fixed with 10% formalin for immunohistochemistry testing of th e AR (not discussed further). Frozen testes were thawed, decapsulated, and homogenized with ice-cold 0. 9% sodium chloride solution containing 0.01% Triton X-100. The number of sperm heads in th e sample was counted with a hemocytometer.CYPERMETHRIN FINAL

Page 60 of 64 There were no treatment-related effects on body or organ weights. Sperm head counts and daily sperm production were decreased (p<0.05) in a do se-dependent manner in rats treated with β -cypermethrin at both 15 and 30 mg/kg compared to control.

Finally in another published study from a different group ( Wang et al. , 2009 ), reproductive toxicity of β -cypermethrin was investigated in intact adult male mice. Adult male Kunbai mice were randomly assigned to six groups (10 mi ce/group), and orally dosed by gavage with β

cypermethrin (>99.0% purity) in corn oil at 0 (vehicle only); 1, 10, or 20 mg/kg of β

cypermethrin; 20 mg/kg of β -cypermethrin plus 20 mg/kg Vita min E; or 20 mg/kg Vitamin E for 35 days.

After 35 days of treatment, rats were sacrifi ced by decapitation and blood was collected for analysis of serum testosterone concentration with a commercial radioimmunoassay kit. The testes, epididymides, seminal vesicles, and pr ostate were excised a nd weighed; overall body weight gain was also recorde d. For histological examinati ons, samples of the testes and epididymides were fixed in Bouin’s solution, em bedded in paraffin, sectioned, and then stained with hematoxylin and eosin for examination by light microscopy. Some testes samples were sectioned and prepared for ultrastructure analysis.

The caudal epididymis was cleared of blood and ad ipose tissue, and opened to release sperm. The sperm were collected by centrifugation with saline-merthiolate-Triton, and the number of sperm was determined with a hemocytometer. Sperm viability was evaluated with eosin Y staining which determines whethe r the membrane is physically intact or if the membrane is damaged or broken; the percentage of viable sperm was calculated. Sperm malformation was assayed by the motility of the sperm and intact nature of the acrosome under a light microscope.

Vitamin E treatment alone had no effect on any pa rameter values; all responses were similar to control. Body weight gains a nd testes, epididymis, and seminal vesicles organ weights were dose-dependently decreased (p<0.05) in mice treated with β -cypermethrin at 10 and 20 mg/kg, and prostrate organ weights were decreased (p< 0.05) in mice treated at 20 mg/kg only. In mice
treated with β -cypermethrin and vitamin E, body weight gain and testosterone-sensitive organ weights were increased compared to wei ghts in animals dosed only with 20 mg/kg β -cypermethrin. Sperm count, viability, and mo tility were also dose-dependently decreased (p<0.05) in mice treated with β -cypermethrin at 10 and 20 mg/kg. At both 10 and 20 mg/kg, the rate of acrosome deformity was increased by approximately 183% and 296%, respectively. In
mice dosed with β -cypermethrin and vitamin E, these sperm parameters were improved compared to those dosed only with 20 mg/kg β -cypermethrin. Finall y, serum testosterone concentrations were dose-depende ntly decreased (p<0.05) after β -cypermethrin treatment at 10 and 20 mg/kg ( ↓ approximately 35 and 70%, respectively). Histological a nd ultrastructural changes due to β -cyperm ethrin exposure at the two highe st doses were similar to changes observed in the previously disc ussed studies, with effects on se miniferous tubules and sperm development.CYPERMETHRIN FINAL

Page 61 of 64 APPENDIX 3: References Not Utilized in the Cypermethrin 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 Cypermethrin but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the compound of interest wa s not used in the study; 3) the test material was not adequately describe d; 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) the study contained only data on invertebrates

Al-Hamdani, N.M. and Yajurvedi, H.N. (2010) Cypermethrin reversibly alters sperm count without altering fertility in mice. Ecotoxicol. Environ. Saf . 73(5):1092-1097.

Aït-Aïssa, S., Laskowski, S., Laville, N., Porche r, J. M., and Brion, F. (2010) Anti-androgenic activities of environmental pesticides in the MDA-kb2 reporter cell line. Toxicol. In Vitro. 24:

1979-1985.

Boone, M.D. (2008) Examining the single and in teractive effects of three insecticides on amphibian metamorphosis. Environmental Toxicology and Chemistry Vol. 27, No. 7, pp. 1561–

1568.CYPERMETHRIN FINAL

Page 62 of 64 Bossi, R., Vinggaard, A.M., Taxvig, C., Boberg, J., and Bonefeld-Jørgensen, E.C. (2013) Levels of pesticides and their metabolites in Wistar rat amniotic fluids and maternal urine upon gestational exposure. Int. J. Environ. Res. Public Health 10(6):2271-2281.

Carriquiriborde, P., Díaz, J., López, G.C., Ronc o, A.E., and Somoza, G.M. (2009) Effects of cypermethrin chronic exposure and water temper ature on survival, growth, sex differentiation, and gonadal developmental stages of Odontesthes bonariensis (Teleostei). Chemosphere 76(3):374-380.

Dahamna, S., Bencheikh, F., Harzallah, D., B oussahel, S., Belgeit, A., Merghem, M., and Bouriche, H. (2010) Cypermethrin toxic e ffects on spermatogenesis and male mouse reproductive organs. Commun. Appl. Biol. Sci . 75(2):209-216.

Garey, J. and Wolff, M.S. (1998) Estrogenic and Antiprogestagenic Activities of Pyrethroid
Insecticides. Biochemical and Biophysical Research Communications 251, 855–859.

Ghodageri, M.G. and Katti, P. (2013) In vitro induction/inhibition of germinal vesicle breakdown (GVBD) in frog ( Euphlyctis cyanophlyctis ) oocytes by endocrine active compounds. Drug and Chem. Toxicol. 36(2), 217-223.

Glaister, J.R., Gore, C.W., Marsat, G. J., Phillips, C., and Pratt, I. (1980) PP383: 90 Day Feeding Study in Rats. CTL report number: CTUP/ 327, ICI signed on 12-14-79. FMC study number

A81

- 563. (MRID 00056802)

Go, V., Garey, J., Wolff, M.S., and Pogo, B.G. T. (1999) Estrogenic Potential of Certain Pyrethroid Compounds in the MCF-7 Human Breast Carcinoma Cell Line. E nvir onme nt a l
Health P e rsp e ct iv es, V o lum e 107

, Number 3, M arch.

Hinfray, N., Porcher, J.M., and Brion, F. (2006) Inhibition of rainbow trout (Oncorhynchus mykiss) P450 aromatase activities in brain and ovarian microsomes by various environmental substances. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 144(3):252-62.

Hu, J.X., Li, Y.F., Pan, C., Zhang, J.P., Wang, H. M., Li, J., and Xu, L.C. (2012) Anti-androgen effects of cypermethrin on the amino- and ca rboxyl-terminal interact ion of the androgen
receptor. Toxicology 292(2-3):99-104.

Jin, Y., Wang, L., Ruan, M., Liu, J., Yang, Y., Zhou, C., Xu, B., and Fu, Z. (2011) Cypermethrin exposure during puberty induces ox idative stress and endocrine disruption in male mice. Chemosphere 84(1):124-130.

Kim, S.-S., Lee, R.-D., Lim, K.-J., Kwack, S.-J., Rhee, G.-S., Seok, J.-H., Lee, G.-S., An, B.-S., Jeung, E.-B., and Park, K.-L. (2005) Potential Estrogenic and Antiandrogenic Effects of Permethrin in Rats. Journal of Reproduction and Development Vol. 51, No. 2.CYPERMETHRIN FINAL

Page 63 of 64 Kunimatsu, T., Yamada, T., Ose, K., Sunami, O., Kamita, Y., Okuno, Y., Seki, T., and Nakatsuka, I. (2002) Lack of (Anti-) Androgeni c or Estrogenic Effects of Three Pyrethroids (Esfenvalerate, Fenvalerate, and Permethrin) in the Hershberger and Uterotrophic Assays. Regulatory Toxicology and Pharmacology 35, 227–237.

Laville, N., Balaguer, P., Brion, F., Hinfray, N., Casellas, C., Porche,r J.M., and Ait-Aissa, S. (2006) Modulation of aromatase activity and mRNA by various selected pesticides in the human
choriocarcinoma JEG-3 cell line. Toxicology 228(1):98-108.

Lemaire, G., Mnif, W., Mauvais, P., Balaguer, P., and Rahmani, R. (2006) Activation of α - and β -estrogen receptors by persistent pesticides in reporter cell lines. Life Sciences 79:1160- 1169.

Morinaga, H., Yanase, T., Nomura, M., Okabe, T ., Goto, K., Harada, N., and Nawata, H. (2004) A benzimidazole fungicide, benomyl, and its meta bolite, carbendazim, induce aromatase activity in a human ovarian granulos e-like tumor cell line (KGN). Endocrinology 145(4): 1860-9.

Nataraj, M.B. and Krishnamurthy, S.V. (2012) Effects of Combinations of Malathion and Cypermethrin on Survivability and Time of Meta morphosis of Tadpoles of Indian Cricket Frog ( Fejervarya limnocharis ). Journal of Environmental Science and Health 47:67-73.

Pan, C., Liu, Y.P., Li, Y.F., Hu, J.X., Zhang, J. P., Wang, H.M., Li, J., and Xu, L.C. (2012) Effects of cypermethrin on the ligand-independent interaction between androgen receptor and steroid receptor coactivator-1. Toxicology 299(2-3):160-164.

Pan, C., Wang, Q., Liu, Y.P., Xu, L.F., Li, Y.F., Hu, J.X., Jiang, M., Zhang, J.P., Zhang, M.R., Yu, H.M., Zhou, J.L., Zhou, X.L., and Xu, L.C. ( 2013) Anti-androgen effects of the pyrethroid pesticide cypermethrin on interactions of androgen receptor with corepressors. Toxicology 311(3):178-183.

Singh, A., Yadav, S., Srivastava, V., Kumar, R ., Singh, D., Sethumadhavan, R., and Parmar, D. (2013) Imprinting of cerebral and hepatic cytochrome P 450s in rat offsprings exposed prenatally to low doses of cypermethrin. Mol Neurobiol . 48(1):128-140.

Sumida, K., Ooe, N., Nagahori, H., Saito, K., Is obe, N., Kaneko, H., and Nakatsuka, I. (2001) An in vitro reporter gene assay method incorporatin g metabolic activation with human and rat S9 or liver microsomes. Biochem. Biophys. Res. Commun. 280(1):85-91.

Taxvig, C., Hadrup, N., Boberg, J., Axelstad, M., Bossi, R., Bonefeld-Jorgensen, E.C., Vinggaard, A.M. (2013). In vitro – in vivo correlations for endocrine activity of a mixture of currently used pesticides. Toxicol Appl Pharmacol . 272: 757-766.

Taylor, J.S., Thomson, B.M., Lang, C.N., Sin, F.Y., and Podivinsky, E. (2010) Estrogenic pyrethroid pesticides regulate expression of estr ogen receptor transcripts in mouse Sertoli cells differently from 17beta-estradiol. J. Toxicol. Environ. Health, Part A 73(16):1075-1089.CYPERMETHRIN FINAL

Page 64 of 64 Wang, H., Wang, Q., Zhao, X.F., Liu, P., Meng, X. H., Yu, T., Ji, Y.L., Zhang, H., Zhang, C., Zhang, Y., and Xu, D.X. (2010) Cypermethrin exposure during puberty di srupts testosterone synthesis via down regulating StAR in mouse testes. Arch. Toxicol . 84(1):53-61.

Wang, H., Wang, S.F., Ning, H., Ji, Y.L., Zhang, C., Zhang, Y., Yu, T., Ma, X.H., Zhao, X.F., Wang, Q., Liu, P., Meng, X.H., and Xu, D.X. (2 011) Maternal cypermethrin exposure during lactation impairs testicular development and spermatogenesis in male mouse offspring. Environ. Toxicol . 26(4):382-394.

Yuan, C., Wang, C., Gao, S.Q., Kong, T.T., Che n, L., Li, X.F., Song, L., and Wang, Y.B. (2010) Effects of permethrin, cypermethrin a nd 3-phenoxybenzoic acid on rat sperm motility in vitro evaluated with computer-assisted sperm analysis. Toxicol. In Vitro 24(2):382-386.