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 ESTROGEN , ANDROGEN, OR THYROID

PATHWAYS

CHEMICAL: CYFLUTHRIN

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYCYFLUTHRIN FINAL

ii TABLE OF CONTENTS

Abbreviations...................................................................................................iii
Executive Summary..........................................................................................1

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

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

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

B. Other Scientifically Rele vant Information (OSRI)......................................5

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

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

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

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

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

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

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

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

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

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

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

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

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

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

LIST OF TABLES: Table 1. Tier 1 Assays for Cyfluthrin..................................................................8
Table 2. Estrogenic/Anti-Estroge nic Pathway for Cyfluthrin ....................................11
Table 3. Androgenic/Anti-Androge nic Pathway for Cyfluthrin .................................17
Table 4. Thyroid Pathway for Cyfluthrin ...........................................................21

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................34
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................44
APPENDIX 3. References Not Utilized in the Cyfluthrin WoE Analysis............................68CYFLUTHRIN 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 enerationCYFLUTHRIN 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 ConcentrationCYFLUTHRIN 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)CYFLUTHRIN 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-EvidenceCYFLUTHRIN FINAL

Page 1 of 68 Executive Summary

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 data, other scientifically releva nt information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether cyfluthrin interacts with E, A or T hormone path ways, the number and type of effects induced, the magnitude and pattern of responses observed acr oss studies, taxa and
sexes were considered. Additi onally, the conditions under which effects occu r were considered, in particular, whether or not e ndocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On July 23, 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Offi ce of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) analysis of the poten tial interaction of cyfluthrin with the E, A or T hormone pathways. The T1WoERC c onclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in

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

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

For the estrogen pathway, there was no evid ence of interaction either in the Tier 1 in vitro [estrogen receptor (ER) binding, ER transactivation assay (ERTA), and aromatase assay] or in
the Tier 1 in vivo [uterotrophic, female pubertal and fish short-term reproduction assay (FSTRA)] in the absence of overt toxicity. There were no estrogen- related effects were seen in the Part 158 studies [(effects on offspring surviv al were observed in the quail reproduction study, however, this endpoint is limited in its ability to inform potential interactions with the estrogen (and androgen) pathway)]. In the Tier 1 in vitro steroidogenesis assay, while a significant increase in estradiol production was observed at a mid-concentration, there was a lack of estrogen-related effects observed in the other in vitro and in vivo assays (in the absence of overt toxicity) .CYFLUTHRIN FINAL

Page 2 of 68 There was no evidence of interaction of cyflut hrin with the androgen pathway in the Tier 1 in

vitro assays [androgen receptor (AR) binding or steriodogenesis]. While potential antiandrogenic activity was re ported in three published in vitro AR transactivation assay (ARTA) studies, limited evidence of potential anti-androgeni c effects was observed in vivo in the absence of overt toxicity. While no androgen-related effect s were observed in Tier 1 assays (Hershberger, male pubertal and FSTRA) in the absence of overt toxicity, positive results were reported in a non-guideline Hershberger study by Zhang et a l, 2008. In that study, wh ich evaluated antiandrogenic activity on ly, cyfluthrin and β -cyfluthrin caused significant decreases in multiple secondary sex tissues at the 18 and 54 mg /kg/day doses. The dose levels (6, 18 and 54 mg/kg/day) in this study were comparable to the dose levels ( 5, 10 and 20 mg/kg/day) tested in the Tier 1 Hershberger assay. While Zhang et al. reported not observing clinical signs in their study, increased salivation, piloer ection and/or lack of coordina tion, which are indicative of neurotoxicity, were seen at doses greater than 20 mg/kg/day in the Ti er 1 and mammalian Part 158 studies. Therefore, the androgen-re lated effects observed in the Zhang et al. study may have occurred in the presence of overt toxicity. Since the effects may have potentially occurred in the presence of overt toxicity in addition to the other mammalia n studies indicating no androgenrelated effects up to overtly toxic doses, the conf idence in the results of this study are reduced. Androgen-related effects were not observed in the avian or fish Part 158 studies. Based on the available data, there is no convincing evidence of potential interacti on with the androgenpathway for cyfluthrin.

There was no convincing evidence of potential in teraction of cyfluthrin with the thyroid pathway. Thyroid parameters were not altered in Tier 1 mammalian in vivo studies. Additionally, there were no thyroid-related effects were seen in the Part 158 mammalian studies. In the amphibian metamorphosis assay (AMA), althou gh effects on developmen tal stage and growth parameters were observed in the mid- and hi gh-treatment groups compared to the negative control, similar effects (increas ed growth and development) were noted in the solvent control group. Therefore, it appears that the increased development and gr owth in the treatments and the absence of effects on thyroid histopathology may have been due to the presence of the solvent (may have resulted in increases in food resources) as oppose to a potential thyroid-related effect. Based on the results in the AMA, there appear s to be no convincing evidence of a potential thyroid-related intera ction at the concentr ations tested (up to 0.066 μg a.i./L). In the AMA, the achieved high-concentration group (0.066 μg a.i./L) did not exhibit any signs of toxicity. The achieved high concentration from the definitiv e AMA study is in the range of the no effect concentration reported in the rang efinder; however, due to issues in the range finding study, there is uncertainty in the actual e xposure concentration where toxicity was observed. Given the lack
of observed treatm ent-related effects in the definitive st udy along with having a clear understanding of the concentrati on where clinical signs occurred in the rangefinder, there is uncertainty whether the maximum concentration ach ieved in the AMA was adequate to test for an effect. However, as there is a lack of re dundancy across the available data with no evidenceCYFLUTHRIN FINAL

Page 3 of 68 for interaction in the AMA at the concentrations tested, overall there is a lack of convincing evidence of a potential interact with the thyroid pathway for cyfluthrin.

Based on weight of evidence considerations, mammalian and wildlife EDSP Tier 2 studies are not recommended for cyfluthrin since there was no convincing evidence of potential interaction with the estrogen, androgen or thyroid pathways.CYFLUTHRIN FINAL

Page 4 of 68 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 data, other scientifically releva nt information (OSRI), including general toxicity data and open lite rature studies of sufficient qu ality, were considered in this weight of evidence (WoE) assessment.

In determining whether cyfluthrin interacts with E, A or T hormone path ways, the number and type of effects induced, the magnitude and pattern of responses observed acr oss studies, taxa, and
sexes were considered. Additi onally, the conditions under which effects occu r were considered, in particular, whether or not e ndocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On July 23, 2014, the Tire 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Offi ce of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) analysis of the poten tial interaction of cyfluthrin with the E, A or T hormone pathways. The T1WoERC c onclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in

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

Cyfluthrin and β -cyfluthrin (an enriched isomer of cyfluthrin) are non-systemic pyrethroid
insecticides and both are mixtures of the same four diastereomer s. Isomers I and II are in the cis configuration, and Isomers III and IV are in the trans configuration. Cyfluthr in is comprised of approximately equal parts of the four isomers: Isomer I is approximately 25%, Isomer II is approximately 19%, Isomer III is approximately 34% and Isomer IV is approximately 23%. β -Cyfluthrin is enriched in Isomer II (approxima tely 35%) and Isomer IV (approximately 62%), and contains only minor amounts of Isomer I a nd Isomer III (less than 3% of total). This assessment includes studies conducted on isomeric mixtures (i.e., cyfluthrin and β -cyfluthrin, a resolved mix of the biologically-active isomers).

Cyfluthrin is poorly soluble in water (2.32 ppb), and has an octanol/water partition coefficient (log Kow) of 5.93. ! Data suggest that the primary routes of dissipation are aqueous and soil photolysis (4.5 and 5.6 days, respectively) and hydrolysis in alkali ne media (2.1 days). Cyfluthrin and beta -cyfluthrin are moderately persistent in the environment and immobile as cyfluthrin and beta -cyfluthrin bind strongly to soils ( aerobic soil metabolism 73.5-94.8 days, aerobic aquatic metabolism 32.9-42 days). The biocon centration factor for cyfluthrin in wholeCYFLUTHRIN FINAL

Page 5 of 68 rainbow trout was 854, whole fish. Residues were depurated at modera tely rapid rates in untreated water (apparent depura tion half-life of approximately ⊩ 3 days).

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

II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

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

1.

B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data 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 in itial review of the OSRI is provided in the Report of the Endocrine Disruptor Review Team (EDRT) on cyfluthrin (USEPA, 2010). Since then, the Agency has also conducted a more recent s earch of available scientific 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 presented 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 steps 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 theCYFLUTHRIN FINAL

Page 6 of 68 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.

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 consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a speci fic endpoint for discerning 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 interact 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. TheCYFLUTHRIN FINAL

Page 7 of 68 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 organizati on. Finally, under what conditions did the responses occur, including co nsideration of whether the responses were observed in the presence of overt or systemic toxicity. The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish 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 that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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

IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/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 addres sing the three components discussed 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 theirCYFLUTHRIN FINAL

Page 8 of 68 potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting data; 4) overall strength of eviden ce supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing th e need for additional data is based on a caseby-case analysis which took all availa ble toxicity data into account.

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

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

A. EDSP Tier 1 Screening Assays

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

.

Table 1. Tier 1 Screening Assays for Cyfluthrin

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID 48615603)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300;

OECD 455 Requirement Satisfied by OSRI (Kojima et al . ,2004); however, a Tier 1 assay was submitted (MRID No. 48615604) AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID 48615601)
Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550;

OECD 456 Requirement Satisfied (MRID 48615607)
Aromatase Assay (human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID 48615602)

Uterotrophic Assay (Rat) OCSPP 890.1600;

OECD 440 Requirement Satisfied (MRID 48615608)

Hershberger Assay (Rat) OCSPP 890.1400;
OECD 441 Requirement satisfied by OSRI (Zhang et al. ,2008); however a Tier 1 assay was submitted (MRID 48615605)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID 48615606)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied by OSRI (Du et al ., 2010; Kojima et al ., 2004; Zhang et al ., 2008); however a Tier 1 assay was submitted (MRID 48615606)CYFLUTHRIN FINAL

Page 9 of 68 Tier 1 Assays Test Guideline Test Order Status

Fish Short-term Reproduction Assay OCSPP 890.1350;

OECD 229 Requirement Satisfied (MRID 48670101)
Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100;

OECD 231 Requirement Satisfied (MRID 48682901)

B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for cyfluthrin to interact w ith the estrogen pathway is summarized in Table 2. The various targets of the estrogen pathway across the relevant Tier 1 a ssays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic or HPG axis effects. This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reporte d in the table and not considered further in the WOE assessment.

Cyfluthrin was negative (or not interactive) in the Tier 1 ER Binding, ERTA and aromatase assays. In the Tier 1 steroi dogenesis assay, cyfluthrin increas ed estradiol production (1.33 to 1.88 fold-change) across all three runs at concentrations of 10

− 10 to 10

− 5 M; however, the
increases in estradiol were not concentration dependent and were highl y variable, attaining significance only at a mi d-concentration of 10

− 7 M (p ≤ 0.05; 1.81 fold-change).

In a published study, Kojima et al. , (2004) evaluated cyfluthrin for its potential to activate two human estrogen receptor subtypes (hER α and hER β ) in luciferase-based transactivation assays in CHO-K1 cells. Cyfluthrin was evaluated for poten tial ER agonism and antagonism in this study. Cyfluthrin induced ER α (but not ER β ) activity with a 20% relative effective concentration (REC 20 ) of 5.9 × 10 -6 M, and relative luciferase activity (RLA) of 45% at 10 -5 M (100% activity defined as the activity achieved with 10 -10 M estradiol). Cyfluthrin was negative for both ER α and ER β antagonism in this test system. Ho wever, in another published study, Du et al. (2010) showed that cyfluthrin was negative for ER agonism and positive for ER antagonism using an ER-mediated luciferase reporter gene assay in CV -1 cells. In that study, cyfluthrin was found to have anti-estrogenic activit y, with a 20% relative inhi bitory concentration (RIC 20 ) of 1.36 x 10 -6 M. Additionally, cyfluthrin was negative for ER activity in a gene reporter assay in human ovarian carcinoma cells (BG1) (Kojima et al ., 2005).

In the Tier 1 in vivo mammalian assays, cyfluthrin was negative for estrogenic activity in the uterotrophic assay. In the Tier 1 female pubertal assay, at the high dose (20 mg/kg/day), the age at VO was increased by 2.7 days while body weight at VO was increased by 10%, compared to the control. Clinical signs of toxicity (increased salivation) we re noted in 5/15 rats at the highCYFLUTHRIN FINAL

Page 10 of 68 dose. No other estrogen-related effects were observed in the assay. There were no estrogenrelated effects observed in th e Part 158 mammalian studies.

In the FSTRA, effects were noted at the highest test concentration (0.421 μg/L) in female fish including changes in ovarian hi stopathology (a narrowing in the ra nge of ovarian gonadal stage) and a 92% decrease in fecundity. However, surv ival in the highest con centration group was only 50% in males and 18.8% in females, and the survivi ng fish showed clinical signs of toxicity and loss of typical reproductive behavior and color patte rn. No effects were noted in the two lower test concentration groups.

In a Part 158 one-generation re production study with mallard ducks effects at the mid and high dose (1000 and 4000 mg/kg-diet) in cluded a reduction in the numb er of eggs laid, embryo viability, hatchlings, and 14-day surv ivors. A high percentage of eggs cracked were observed at the low dose (250 mg/kg-diet), but this effects wa s not observed at the higher treatments. The study author reported a significant reduction in male and female body weight during the first four weeks of the study at the hi gh concentration. However, due to low control reproductive performance, including low embryo viability, hatc hling number and 14-day survivorship, there is uncertainty associated with th e results of this study. In a nother Part 158 one-generation reproductive study conducted on mallard ducks, there were no statistically-significant effects on reproductive parameters up to 250 mg/kg-diet. Th e reviewer noted that pathological results showed an increase in the incident of slight egg yolk peritonitis in the treatment-groups compared to the control ( ≥ 10 mg/kg-diet). In this study, there was also observed low control reproductive performance. Therefore, given these un certainties in control performance, these two mallard reproduction studies were not reported intable below. Finally, in a mallard onegeneration reproduction study with beta-cyfluthri n, no effects on reproductive parameters were observed up to the highest dose tested, 250 mg/kg-diet. In a Part 158 one-generation reproduction study with bobwhite qua il, two-week old chick survivor s were significantly reduced compared to the control at the high test conc entration (4000 mg/kg-diet ). For males, but not females, body weight and body weight gains were also significantly reduced at the high test concentration. Significant reductions in eggs hell thickness were detected at all test concentrations (250, 1000, and 4000 mg/kg-diet). However, in a follow-up study examining eggshell strength and thickness in bobwhite quail, there were no effects on eggshell thickness up to 4000 mg/kg-diet.

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

≥ 0.018 μg/L for rainbow trout, and reduced juve nile survival of sheepshead minnow at
concentrations of ≥ 0.084 μg/L. In a Part 158 fish life cycl e study with fathead minnow, there was a significant decrease in hatchab ility of F1 eggs at 0.25 μg/L as well as significant decreases in post-hatch survival of the parent and F1 ge nerations at the same test concentration.CYFLUTHRINFINALPage 11 of 68Table 2. Estrogenic/Anti-Estrogenic Pathway for CyfluthrinLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Cyfluthrin1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48615603)NERTA(MRID 48615604)NAromatase(MRID 48615602)NSteroidogenesis(MRID 48615607)NUterotrophic(MRID 48615608)NN NFemale Pubertal Rat(MRID 48615606)NNNN N NNX(H)4FSTRA(MRID 48670101)NENNNNNX5(H)OSRIER Transactivation(Kojima et al., 2004)PER Transactivation(Duet al., 2010)PER Transactivation(Kojima et al., 2004)NCYFLUTHRINFINALPage 12 of 68Table 2. Estrogenic/Anti-Estrogenic Pathway for CyfluthrinLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Cyfluthrin1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Subchronic toxicity(Rat; MRID41244108)NENENNENNSubchronic inhalation(Rat; MRIDs00157793, 40082901,40239301)NENNNENNDevelopmentaltoxicity (Rat gavage;MRID 44116501)NENNX(H)Developmentaltoxicity (Rabbit;MRID 42675401)NRNNNDevelopmentaltoxicity (Ratinhalation; MRID40780401)NRNNNDevelopmentaltoxicity (Ratinhalation; MRID43393401)NRNNNDevelopmentalNeurotoxicity (Rat;MRID 46054101)NENNX(H)NTwo-generationreproduction (Rat;MRIDs 44371401,44371402)NE N NNENNNCYFLUTHRINFINALPage 13 of 68Table 2. Estrogenic/Anti-Estrogenic Pathway for CyfluthrinLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Cyfluthrin1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Chronic toxicity/carcinogenicity (Rat;MRID 00137303)NENNNENNChronic toxicity/carcinogenicity (Rat;MRID 44459301)NENNNENNCarcinogenicity(Mouse; MRIDs44589701, 45228101)NENNNENNChronic toxicity(Dog; MRID44435401)NENNNNX(M, H)Avian reproduction(Duck; MRID48350615)NN NAvian reproduction(Quail; MRIDs00145330, 00152829)P6NN(H)6Fish life-cycletoxicity (Fatheadminnow, MRID41450401)NX(H)1.Key to responses: Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=Hightreatment. Arrows (↓or↑) indicate the direction of the response.A shaded cell indicates that is parameter is not routinely evaluated or is notapplicable in this assay. Changes in weight are absolute unless otherwise indicated. NR: Not Reported; NE: Not Examined; BW=Body weight; CS=Clinical signs.2.The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by anX in this column. For details see Section IV. ACYFLUTHRINFINALPage 14 of 683.The overt toxicity in the Tier 1 assays are presented inthis column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4.Clinical signs of toxicity (increased salivation)were noted in 5/15 rats at the high dose.5. The high dose of 0.421 μg a.i./L resulted in 50% mortality in males and 18.8% in females.6. Two-week old chick survivors were significantly reduced compared to the control at the high treatment concentration (4000 mg/kg-diet); however, forthe males, but not females, body weight and body weight gainswere significantly reduced at the high treatment concentration.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedCYFLUTHRIN FINAL

Page 15 of 68 2. Effects on Androgen Pathway

The potential for cyfluthrin to interact w ith 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.

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

In a published study, Kojima et al. , (2004) evaluated cyfluthrin for its potential to in teract with a human androgen receptor (hAR) in vitro in a transactivation assay us ing CHO-K1 cells. In this study, cyfluthrin tested positive for weak anti-androgenic activity, with a RIC 20 of 8.4 × 10 -6 M
and a RLA of 74% at 10 -5 M (100% activity defined as the activity ach ieved with 10 -10 M DHT). Cyfluthrin did not demonstrate androgenic transcriptional activit y of more than 20% of that induced by 10 -9 M DHT. In another published study, Du et al. , (2010) evaluated cyfluthrin for in

vitro androgen receptor binding acti vity (both as agonist and an tagonist) in a receptor-mediated luciferase reporter gene assay using a MDA-kb2 cell line. Cyflut hrin did not exhibit agonistic activity, but antagonistic activity was observed for cyfluthrin, with a RIC 20 of 8.84 × 10 -8 M. Finally, in another published in vitro study, Zhang et al. , (2008) examined the potential antiandrogenic effects of cyfluthrin and β -cyfluthrin with reporter gene assays using a MDA-kb2 breast cancer cell line. In this study, cyfluthrin inhibited (p<0.05) DHT-i nduced transcriptional
activation at concentrations from 10

− 7 to 10

− 5 M.

The Tier 1 Hershberger assay was negative, and no androgen-related effect s were observed in the Tier 1 male pubertal assay. These studies were conducted at dose levels up to 20 mg/kg/day; a dose level which showed signs of systemic toxic ity (piloerection and/or in creased salivation).

In the FSTRA, effects were noted only at th e highest test concen tration (0.421 μg/L) and included changes in GSI, testicul ar histopathology and secondary sex characteristics. However, overt toxicity was also noted at the highest test concentration ( i.e ., reduced survival and clinical signs of toxicity). There were no androgen-re lated effects in the mammalian or fish Part 158 toxicity studies. In the Part 158 avian reproduction studies, the only treatment-related effect was a decrease on offspring survival in quail study.

In a published, non-guideline Hershberger study, Zhang et al. (2008) also examined the potential anti-androgenic effects of cyfluthrin or β -cyfluthrin at doses up to 54 and 36 mg/kg/day, respectively. No mortality or trea tment-related clinical signs were noted in any of the groups in the Hershberger assay. There were also no sign ificant differences betw een initial or final body weights or weight gains of the cyfluthrin-treated groups compar ed to the control groups, andCYFLUTHRIN FINAL

Page 16 of 68 serum testosterone concentrations were not affected. However, treatment with either cyfluthrin

or β -cyfluthrin (in combination with TP) resulted in dose-dependent decreases at both the midand high-dose in the weights of the seminal vesi cle, ventral prostate, dorsolateral prostate, LABC and Cowper’s glands.CYFLUTHRINFINALPage 17 of 68Table 3. Androgenic/Anti-Androgenic Pathway for CyfluthrinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Cyfluthrin1Study Type/Literature Citation AR Binding AR Activation Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48615601)NSteroidogenesis(MRID 48615607)NHershberger(MRID 48615605)NENXNMale Pubertal Rat(MRID 48615606)NNNNNNENNNX(H)4FSTRA(MRID 48670101)NENNNNNX(H)5OSRIAR Binding &TranscriptionalActivation (Kojimaetal.,2004)PAR Binding &TranscriptionalActivation (Duet al.,2010)PAR TranscriptionalActivation (Zhangetal., 2008)PHershberger(Zhanget al., 2008)P(M,H)N NCYFLUTHRINFINALPage 18 of 68Table 3. Androgenic/Anti-Androgenic Pathway for CyfluthrinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Cyfluthrin1Study Type/Literature Citation AR Binding AR Activation Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Subchronic toxicity(Rat; MRID41244108)NNNENNENX(H)NSubchronic inhalationtoxicity (MRIDs00157793, 40082901,40239301)NNNENENENEN NDevelopmentalNeurotoxicity (Rat;MRID 46054101)NX(H)NTwo-generationreproduction (Rat;MRIDs 44371401,44371402)NNNENNENE NN NChronic toxicity/carcinogenicity (Rat;MRID 00137303)N N NENENE NN NChronic toxicity/carcinogenicity (Rat;MRID, 44459301)N N NENNE NN NCarcinogenicity(Mouse; MRIDs44589701, 45228101)N N NE N NE NEN NChronic toxicity(Dog; MRID44435401)NENNENNENNX(M, H)CYFLUTHRINFINALPage 19 of 68Table 3. Androgenic/Anti-Androgenic Pathway for CyfluthrinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Cyfluthrin1Study Type/Literature Citation AR Binding AR Activation Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Avian reproduction(Duck; MRID48350615)NN NAvian reproduction(Quail; MRIDs00145330, 00152829)P6NN(H)6Fish life-cycletoxicity (Fatheadminnow, MRID41450401)NENNEX(H)1.Key to responses: Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=Hightreatment. Arrows (↓or↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicablein this assay. 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).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 1 assays are presented inthis column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. A variety of clinical signs (increased salivation, piloerection, lack of grooming, uncoordinated movements, and/or tremors)were noted in 6/15 high-doseanimals on at least one occasion.5. The high dose of 0.421 μg a.i./L resulted in 50% mortality in males and 18.8% in females.6. Male, but not female, body weight and body weight gains weresignificantly reduced at the high treatment concentration. Significant decreases insurvival of 14-d old chick offspring observed at this high treatment.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedCYFLUTHRIN FINAL

Page 20 of 68 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

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

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

No effects on the thyroid pathway (thyroid horm one concentrations, weig ht, or histopathology)
were observed in the in vivo male and female pubertal assays. No thyroid-related effects were
observed in the Part 158 mammalian studies.

In the AMA, a co-solvent (DMF) was used in th e study and there were no significant differences between the negative and solvent control for any endpoint [(for Day 21 NF stage the significance was marginally close (p=0.058)], and as such, the reported results in Tabl e 4 reflect comparisons to the negative control. The Day 21 median NF developmental stage wa s significantly increased (p<0.05) by 1 stage in the mid and high treatm ent groups (0.032 and 0.066 μg/L) compared to the negative control (NF stage 58 vs. 57 in negative control), but not when compared to the solvent control (NF stage 58). Cyfluthrin did not significantly affect 7- or 21-Day normalized hind limb length (HLL), and no effects on thyroi d gland histopathology we re observed at any
treatment concentration. In re gard to the growth parameters , Day-21 snout-vent length (SVL) was significantly increased by 12.6% in the 0.032 μg a.i./L group and decreased by 10.8% in the 0.066 μg a.i./L group, compared to the negative c ontrol. Increases of 47.4 and 50.7% were also observed in wet body weight on Day 21 for th e 0.032 and 0.066 μg a.i./L groups, respectively, and wet body weight was increased (p<0.05) for Day 7 at the high concentration ( ↑ 31%).CYFLUTHRIN FINAL

Page 21 of 68 Table 4. Thyroid Pathway for Cyfluthrin.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Cyfluthrin 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 48615606) N N N N N N X (H) 4

Male Pubertal Rat (MRID 48615606 ) N N N N N N X (H) 4

AMA (Frog; MRID 48682901) N Day 21, +1 NF stage (M, H) 5 Day 7 BW: ↑ 31% (H); Day 21 BW:

↑ 47, 51% (M, H) 5 ; Day 21 SVL:

↑ 13, ↓ 11%

(M ,H) 5 N

OSRI Subchronic oral toxicity (Rat; MRID 41244108) NR N NR X (H) N
Subchronic oral toxicity (Dog; MRID 41267801) N N NR X (H) N

Subchronic inhalation (Rat; MRIDs 00157793, 40082901, 40239301) N N NR X (M, H) N

Developmental toxicity (Rat gavage; MRID 44116501) X (M, H) X (H)

Developmental toxicity (Rabbit; MRID 42675401) X (M, H) N

Developmental toxicity (Rat inhalation; MRID 43393401) X (ML, MH, H) N

Developmental Neurotoxicity (Rat; MRID 46054101) X (H) N

Two-generation reproduction (Rat; MRID 44371401 and 44371402) NE NE NR X (M, H) N

Chronic toxicity/ carcinogenicity (Rat; MRID 00137303) NR N NR X (H) NCYFLUTHRIN FINAL

Page 22 of 68 Table 4. Thyroid Pathway for Cyfluthrin.

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

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

Chronic toxicity/ oncogenicity (Rat; MRID 44459301) NR N NR X (M, H) X (H)

Carcinogenicity (Mouse; MRIDs 44589701, 45228101) NR N NR X (H) N

Chronic toxicity (Dog; MRID 44435401) N N N X (M, H) N

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

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 4. Clinical signs were noted in 6/15 males and 5/ 15 females at the high-dose on at least one occasion. 5. Treatment related effects noted at the mid and high concentrations were similar to the effects noted in the solvent control. P Positive finding N Negative findings NE Not examined NR Not reportedCYFLUTHRIN FINAL

Page 23 of 68 IV. Committee’s Assessment of Weight of Evidence

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

The WoE evaluation includes consid erations of biological plausibility of the findings from the different lines of evidence by examining the 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 sequelae of systemic effects. E ndocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses are mediated through an endocrine pathway, therefore nonendocrine toxic responses (incl uding but not limited to mortalit y or body weight changes) are
also considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence evaluation of the results of the Tier 1 assays stated that “In summary, the Panel agreed that little, if any, 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).

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).CYFLUTHRIN FINAL

Page 24 of 68 1. Tier 1 in vivo Assays

In the Hershberger assay, sporadic clinical si gns (increased salivation or piloerection) were observed in two high-dose (20 mg /kg/day) animals and one mid-dos e (10 mg/kg/day) animal. In the female pubertal rat study, increased salivation was observed in 5/15 animals in the high-dose group (20 mg/kg/day). In the male pubertal rat study, increased salivation was noted in one middose (10 mg/kg/day) animal on one day, and a vari ety of clinical signs (increased salivation, piloerection, lack of grooming, uncoordinated mo vements and/or tremors) were noted in 6/15 high-dose animals on at least one occasion. Sim ilar clinical signs (p iloerection, reduced or limited activity, uncoordinated movements and incr eased salivation) were also observed in one high-dose animal (20 mg/kg/day) in the uterotrophic assay.

In the FSTRA study, overt toxicity was observed at the highest tested cy fluthrin concentration (0.421 μg/L) based on mortality in both the males ( 50%) and females (18.8%); clinical signs of toxicity were also evident in the survivors at this concentration ( i.e., swimming at the surface, erratic behavior, loss of equilibr ium, labored respiration and decrea sed food intake). No signs of toxicity were noted in fish at test concentrations ≤ 0.149 μg/L. No overt or systemic toxicity was noted in the AMA study at test c oncentrations up to 0.066 μg/L.

2. OSRI

Systemic and/or overt toxicity of cyfluthrin and β -cyfluthrin in the OSRI in vivo studies consisted primarily of effects on body weight a nd body weight gains and ne urological effects.

Treatment-related mortality was not observed in th e oral subchronic toxicity studies on rats or dogs, but clinical signs of neurotoxicity were observed at the hi gh doses in both studies. In rats, lack of coordination and poor ge neral condition were noted at th e high dose (39-42 mg/kg/day), and body weight gains were reduced in both sexes at the high dose. In the dog study, neurological signs (awkward, staggering gait an d occasional buckling of the hind limbs) were noted in 4/8 dogs from both sexes at the hi gh dose (14-15 mg/kg/day). In the subchronic inhalation study on rats, clinical signs (unthriftiness/ unkempt fur and lethargy) were observed in all mid-dose females (0.16 mg/kg/day) during th e second half of the study, and similar signs were noted in all high-dose (0.91 mg/kg/day) males and females within 2 weeks of study initiation. Mid and high-dose males also had reductions in body weight and body weight gains compared to the control.

In the rat oral developmental study, overt and systemic toxicity we re noted in the high-dose dams (40 mg/kg/day); reductions were observed in maternal survival ( ↓ 10%), body weight gain ( ↓ 56%) and food consumption ( ↓ 20-49%). In addition, clinical signs of toxi city at the high dose included hypoactivity, locomotor in coordination, and salivation. Systemic toxicity was alsoCYFLUTHRIN FINAL

Page 25 of 68 noted at the mid-dose (10 mg/kg/ day) and consisted of reductions in food consumption and body weight gains during dosing. In the rabbit oral developmental study, there were no maternal
deaths or clinical si gns of toxicity during the study. However, food consumption and body weight gains were reduced in the mid- and high-dose groups (60 a nd 180 mg/kg/day) during dosing. In the developmental neurotoxicity stu dy, no overt or systemic toxicity was noted in dams orally dosed with β -cyfluthrin at up to 17.8 mg/kg/day.

In two rat inhalation developmental studies, there was no mortality, but clini cal signs of toxicity (reduced motility, dyspnea, p iloerection and ungroomed coat s) were noted at doses ≥ 0.0047 mg/L ( ≥ 1.28 mg/kg/day). However, food consumption and body weight gains were reduced in the mid- and high-dose groups (60 and 180 mg/kg/da y) during dosing. In another rat inhalation developmental study, maternal body weights and food efficiency were reduced in all dose levels (0.125-3.48 mg/kg/day) duri ng the dosing period. In addition, clinical signs (labored and irregular breathing, hypo activity, high-stepping gait, saliv ation, and reduced stool) were observed at the high-dose (3.23 mg/kg/day), but the effects were attributed to bradypnea in the dams since clinical signs were not observed in dams dosed at 3.48 mg/kg/day and supplemented
with O 2

.

Two 2-generation reproductive toxi city studies on rats are availa ble for cypermethrin In one study, no treatment related mortality was noted, but systemic toxicity was observed in adults in the mid- and high-dose groups ([M/F] 9/10 a nd 29/33 mg/kg/day). At the mid-dose, body weights were decreased in males and reduced fo od consumption was reduced in females during lactation. At the high-dose, there were reducti ons in body weight of both males and females as well as decreases in food consumption (relative to control) and clinical signs (splaying of hind limbs) in females during lactation. In the supp lemental reproductive study, no overt or systemic toxicity was noted in adult animals at doses up to 3.8/4.2 mg/kg/day.

In the Hershberger study from Zhang et al . (2008), no signs of overt or systemic toxicity were noted in castrated male rats following 7 days of dosing with cyfluthrin at up to 54 mg/kg/day or
with β -cyfluthrin at up to 36 mg/kg/day.

In a chronic toxicity study in rats, there were no treatment-related effects on survival or clinical signs at dietary levels of cyfluthrin up to 450 ppm, equivalent 22.8 a nd 28.3 mg/kg/day for males and females, respectively. At the mid-dos e (11.6/14.4 mg/kg/day), mean body weights were reduced by ≤ 7% in both sexes, and body we ight gains were decreased by ≤ 24%. At the highdose, mean body weights were decreased by ≤ 13% and ≤ 14% for males and females, respectively, and mean body we ight gains were decreased by ≤ 23% and ≤ 24%, respectively.CYFLUTHRIN FINAL

Page 26 of 68 In a mouse carcinogenicity study, there were no treatment-related effect on mortality, food efficiency, hematology, or organ weights for bot h sexes at all doses. At the high dose (233/310 mg/kg/day [M/F]), reductions in body weight were observed in both sexes ( ↓ 7-20%) throughout the study, and overall body weight ga in was decreased in the males ( ↓ 25%) and females ( ↓ 54%). Mean absolute food consumption was also decreased in the females ( ↓ 6-25%) throughout the study. In addition, increased incidence of rough co at (both sexes) and hunched back (females only) were noted in the hi gh-dose group of both sexes.

In the chronic toxicity study in dogs, no overt or systemic toxicity was not ed at the low and midlow doses (1.4/1.5 and 2.4/3.6 mg/kg/day [M/F]) . At the mid-high dose (10.6/10.7 mg/kg/day [M/F]), clinical signs of neurotoxicity were noted in both sexes, including gait and posture abnormalities. At the high dose [(15.5/18.0 mg/kg/da y (M/F)], the incidence and severity of posture and gait abnormalities in creased. Decreases (NS) in male body weights and body weight gains were also noted at the high dose.

No overt or systemic toxicity was observed in the mallard duck re productive study up to 250 mg/kg-diet for cyfluthrin (MRID 00158782). In another mallard duck re productive study testing up to 4000 mg/kg-diet, there was no treatment-relate d effect on adult mortality or clinical signs in any treatment group, and no significant diffe rence in food consumption (MRID 00145331). Significant reductions in body weight gains occurred at the high concentration from Weeks 0-4, but no significant differences in body weight or body weight gain were no ted from Week 8 until study termination. The mallard duck reproduction study with beta-cyfluthrin reported no overt or systemic toxicity up to 250 mg/kg-diet (MRID 48350615).

In a bobwhite quail reproductive study, there was no treatment-re lated mortality at dietary concentrations up to 4000 mg/kg-diet (MRID 00145330). There was no significant difference in food consumption, and no treatment-related eff ects were noted on body weight or body weight gain for the females. However, body weight a nd body weight gains for males were significantly reduced at the high concentration (4000 mg/kg-diet) in this study.

In the fish life-cycle study, no treatment-rela ted effects were noted on percent hatch in the parental or F 1 generations at test concentrations up to 0.14 μg/L (MRID 41450401). Standard length and wet weight as measures of growth were also not affected at any treatment level in either the parental or F 1 generations. However, there was a si gnificant decrease in hatchability of F1 eggs at the highest test concentration, 0.25 μg/L, and significant decreases in post-hatch survival of the parent and F1 generations at th e same test concentration. Part 158 fish early lifecycle studies reported significant effect s on growth at concentrations of ≥ 0.018 μg/L for rainbow trout (MRID 00155898), and reduced j uvenile survival of sheepshead minnow at concentrations
of ≥ 0.084 μg/L (MRID 00158781).CYFLUTHRIN FINAL

Page 27 of 68 B. Estrogen Pathway

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

in vitro (ER binding, ERTA and aromat ase) or in the Tier 1 in vivo (uterotrophic, female pubertal and FSTRA) assays in the absence of overt toxic ity. There were no estrogen-related effects were seen in the Part 158 studies [(effects on o ffspring survival were observed in the quail reproduction study, however, this endpoi nt is limited in its ability to inform potential interactions with the estrogen (and androgen) pathway)]. In the Tier 1 in vitro steroidogenesis assay, while a significant increase in estr adiol production was observed at a mid-concentration, there was a lack of estrogen-related effects observed in the other in vitro and in vivo assays (in the absence of overt toxicity) .

C. Androgen Pathway

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

vitro assays (AR binding or ster iodogenesis). While potential anti-androgenic activity was reported in three published in vitro ARTA studies, limited evidence of potential anti-androgenic
effects was observed in vivo in the absence of overt toxicit y. While no androgen-related effects were observed in Tier 1 assays (Hershberger, male pubertal and FSTRA) in the absence of overt toxicity, positive results were reported in a non-guideline Hershberger study by Zhang et a l, 2008. In that study, which evaluated anti-a ndrogenic activity only, cyfluthrin and β -cyfluthrin caused significant decreases in multiple secondary sex tissues at the 18 and 54 mg/kg/day doses. The dose levels (6, 18 and 54 mg/kg/day) in this study were comparable to the dose levels (5, 10 and 20 mg/kg/day) tested in the Tier 1 Hershberger assay. While Zhang et al. reported not observing clinical signs in their study, increased salivation, piloerection and/or lack of coordination, which are indicative of neurotox icity, were seen at doses greater than 20 mg/kg/day in the Tier 1 and mammalian Part 158 studies. Therefore, the androgen-related effects observed in the Zhang et al. study may have occurred in the pres ence of overt toxicity. Since the effects may have potentially occu rred in the presence of overt t oxicity, in addition to the other mammalian studies indicating no androgen-related effects up to overtly toxic doses, the confidence in the results of this study are reduced . No androgen-related effects were observed in
the avian and fish Part 158 studi es. Based on the available data, there is no convincing evidence of potential interaction with the androgen-pathway for cyfluthrin.

D. Thyroid Pathway

There was no convincing evidence of potential in teraction of cyfluthrin with the thyroid pathway. Thyroid parameters were not altered in Tier 1 mammalian in vivo studies. Additionally, there were no thyroid-related effects were seen in the Part 158 mammalian studies. In the AMA, although effects on developmental stage and growth param eters were observed in the mid- andCYFLUTHRIN FINAL

Page 28 of 68 high-treatment groups compared to the negative control, similar effects (increased growth and development) were noted in the solvent control group. Therefore, it appears that the increased development and growth in the treatments and the absence of effects on thyroid histopathology may have been due to the presence of the solvent (may have resulted in increases in food resources) as oppose to a potential thyroid-related effect. Based on the results in the AMA, there appears to be no convincing ev idence of a potential thyroi d-related interaction at the concentrations tested (up to 0.066 μg a.i./L). In the AMA, the achieved high-concentration group (0.066 μg a.i./L) did not exhibit any signs of toxicity. The achieved high concentration from the definitive AMA study is in the range of the no effect concentration reported in the rangefinder; however, due to issues in dosing in the range-finding study, there is uncertainty in the actual exposure concentration where the toxi city was observed. Given the lack of observed treatment-related effects in the definitive study along with having a clea r understanding of the concentration where clinical signs occurred in the rangefinder, th ere is uncertainty whether the
maximum concentration achieved in the AMA was ade quate to test for an effect. However, as there is a lack of redundancy across the available data with no evidence for interaction in the AMA at the concentrations tested, overall there is a lack of convincing evidence of a potential interact with the thyroid pathway for cyfluthrin.

E. Conclusions

Cyfluthrin demonstrates no convincing evidence of potential interaction with the estrogen, androgen or thyroid pathways.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations, mammalian and wildlife EDSP Tier 2 studies are not recommended for cyfluthrin since there was no convincing evidence of potential interaction with the estrogen, androgen or thyroid pathways.CYFLUTHRIN FINAL

Page 29 of 68 VI. References

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Banman, C.S.; Matlock, D.; and Lam, C.V.,(2012) . Short-term Reproducti on Assay with Fathead Minnow ( Pimephales promelas ) Exposed to Cyfluthrin Technical Under Flow-Through Conditions. Unpublished study prepared by Bayer Cr opScience, Stilwell, KS and Experimental Pathology Laboratories, Inc., Sterling, VA. Laboratory Repo rt Number: EBBDY005, March 30, 2012. (MRID 48670101)

Banman, C.S.; Alexander, T.M.; Lam, C.V.; an d Wolf, J.C., (2012). Amphibian Metamorphosis Assay with South African Clawed-Frog ( Xenopus laevis ) Exposed to Cyfluthrin Under FlowThrough Conditions. Unpublished study prepared by Bayer CropScience, Stilwell, KS and Experimental Pathology Laboratories, Inc., Sterling, VA. Laboratory Report Number: EBBDY004. July 19, 2012. (MRID 48682901).

Beavers, J. (1986) Baythroid Technical: A One-generation Reproduction Study with the Mallard ( Anas platyrhynchos ): Final Report: Project No.: 149-117. Unpublished Mobay Report No. 91888 pre- pared by Wildlife International Ltd. 118 p. (MRID 00158782)

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Blanck, O. (2011a) Cyfluthrin: Evaluation in the in vitro (HeLa-9903) estrogen receptor transcriptional activation assay. Unpublished st udy prepared by Bayer SAS, Sophia Antipolis Cedex, France. Laboratory Report No.: SA 11156, October 17, 2011. (MRID 48615604).

Blanck, M. (2011b). Cyfluthrin Evaluation in the Hershberger Bioassay. Unpublished study prepared by Bayer SAS, France. Laborato ry Report Number: SA 10150, May 23, 2011. (MRID 48615605).

Blanck, M. (2011c) Cyfluthrin: assessment of pubertal development and thyroid function in juvenile/peripubertal male and female rats. Unpublished study prepared by Bayer S.A.S., Bayer CropScience, Sophia Antipolis Cedex, France. Laboratory Study Number SA 10206, June 27, 2011. (MRID 48615606).CYFLUTHRIN FINAL

Page 30 of 68 Carlisle, J. (1984a) Effect of Cy fluthrin (Baythroid Technical) on Bobwhite Quail Reproduction: Study Number 83-675-08. Unpublished study prepar ed by Mobay Chemical Corp. 94 p. (MRID 00145330)

Carlisle, J. (1984b) Effect of Cyfluthrin (Technical Baythroid) on Mallard Duck Reproduction: Study Number 83-675-06. Unpublished study prepar ed by Mobay Chemical Corp. 85 p. (MRID 00145331)

Carlisle, J. (1985c) Effects of Cyfluthrin (Baythroid Technica l) on Bobwhite Quail Eggshells: Study No. 85-675-04: Mobay Report No. 90275. Unpublished study prepared by Mobay Chemical Corp. 52 p. (MRID 00152829)

Du, G., Shen, O., Sun, H., Fei, J., Lu, C., Song, L., Xia, Y., Wang, S., and Wang, X. (2010) Assessing Hormone Receptor Activities of Pyreth roid Insecticides and Their Metabolites in Reporter Gene Assays. Toxicological Sciences 116(1):58-66. (MRID 48196301).

Eigenberg, D.; Elcock, L. ( 1996) A Two-Generation Reproduction Study in Rats Using Technical Grade Cyfluthrin Administered Vi a the Diet: Lab Project Number: 93-672-UZ: 107408: 93-672UZ1. Unpublished study prepar ed by Bayer Corp. 973 p. (MRID 44371401)

Eigenberg, D. (1997) A Supplementary Two-Gene ration Dietary Reproduction Study in Rats Using Technical Grade Cyfluthrin: Lab Pr oject Number: 94-672-CK: 107474: 94-672-CKl. Unpublished study prepared by Bayer Corp. 771 p. (MRID 44371402)

Holzum, B.; Schmidt, U.; Pauluhn, J.; et al. (1994) FCR 1275: Explanatory Report on Results and Mechanistic Studies for Embryotoxic Effects in Rats after Inhalation: Lab Project Number: 06369: 23219: 22581. Unpublished study prepared by Bayer Ag. (MRID 43393401)

Jones, R.D. and Hastings, T.F. (1997) Technical Grade Cyfluthrin (FCR 1272): A Chronic Toxicity Feeding Study in the Beagle D og. Laboratory Study No.: 94-276-ZR. Unpublished study prepared by Bayer Corp. (MRID 44435401)

Jones, R and Hastings, T. (2000) Technical Gr ade Cyfluthrin (FCR 1272): A Chronic Toxicity Feeding Study in the Beagle Dog: Lab Pr oject Number: 94-276-ZR: 8365: 108007. Unpublished study prepared by Bayer Corporation. 14 p. Supplemental to MRID 44435401. (MRID 45189201)

Kojima, H., Katsura, E., Takeuchi, S., Niiyama, K., and Kobayashi, K. (2004). Screening for estrogen and androgen receptor act ivities in 200 pesticides by in vitro reporter gene assays using Chinese Hamster Ovary cells. Environ. Health Perspect. 112: 524-531. (MRID 48033008)CYFLUTHRIN FINAL

Page 31 of 68 Kojima, M., K Fukunaga, M. Sasaki, M. Nakamura, M. Tsuji, T. Nishiyama. (2005) Evaluation of estrogenic activities of pesticides us ing an in vitro reporter gene assay. Int. J. Environ. Health

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Pauluhn, J. (1983) Study of the Subchronic Inha lation Toxicity in Accordance with OECD Guideline No. 413: Baythroid: Supplemental Submission: Second Addendum to Bayer No. 12436.Unpublished study prepared by Bayer AG. 24 p. (MRID 40239301)

Pauluhn, J. (1984) Study for Subchronic Inhalation Toxicity to the Rat for 13 Weeks: FCR 1272 (Cyfluthrin): Rept. No. 12436. Unpublished Mobay study no. 86443 prepared by Bayer AG Institute of Toxicology and Hanover Me dical University. 170 p. (MRID 00157793)

Pauluhn, J. (1987) Study of the Subchronic Inha lation Toxicity in Accordance with OECD Guideline No. 413: FCR 1272: Cyfluthrin: Addendum to Bayer Report No. 12436, Dated Feb. 1, 1984: Study No. T9015085. Unpublished Mobay Re port No. 86443 prepared by Bayer AG. 157 p. (MRID 40082901)

Pauluhn, J. (1988) Addendum to MRID 40780401: FCR 1272: Cyfluthrin: Study for Embryotoxic Effects on Rats after Inhalation: Report N o. 97403; 16391-A. Unpublished study prepared by Bayer Ag. 12 p. (MRID 40968501)

Pauluhn, J. (1989) FCR: 4545 (C.N. Beta-cyfluth rin, Proposed) Subacute Inhalation Toxicity Study in the Rat: Lab Project No.: 146:100571; Report No.: 18146. Unpublished study prepared by Bayer Ag. 949p. (MRID 41783001)

Renhof, M.; Pauluhn, J. (1988a) FCR 1272: Comm on Name Cyfluthrin: Study for Embryotoxic Effects on Rats after Inhalation: Repo rt No. 16391: Report No. 97403. Unpublished study prepared by Bayer Ag. 169 p. (MRID 40780401)

Renhof, M.; Pauluhn, J. (1988b) FCR 1272 (Common Name Cyfluthrin): Study for Embryotoxic Effects on Rats after Inhalation (Supp. to MRID 40780401): Lab Proj ect Number: 97403: 16391. Unpublished study prepared by Bayer Ag, Wuppertal. 5 p. (MRID 42332001)

Rhodes, J.E.; McAllister, WA.; Leak, T.; Stue rman, L. (1990) Full Life-Cycle Toxicity of 14 CCyfluthrin (Baythroid") to the Fathead Minnow (Pimephales promelas) under flow-through conditions. Report N0.1 00097; 33821. Unpublishe d study prepared by ABC Labs. 905 p. (MRID 41450401)CYFLUTHRIN FINAL

Page 32 of 68 Rouquie, D. (2011a). Evaluation of Cyfluthr in in the Androgen Receptor Binding Assay. Unpublished study prepared by Bayer SAS, Soph ia Antipolis Cedex, France. Laboratory Study No.: SA 11190, November 18, 2011. (MRID 48615601).

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Sheets, L.P. (2003) A Developmental Neurotoxi city Screening Study with Technical Grade PCyfluthrin in Wistar Rats. Bayer CropSc ience LP, 17745. Laboratory Report No.: 200620; Unpublished study prepared by Bayer Corp (MRID 46054101)

Sheets, L.P and Dobbs, M. (2013) Weight of eviden ce to evaluate the potential for cyfluthrin to affect estrogen, androgen or t hyroid systems. Bayer CropScience, Research Triangle Park, NC. Report No. US0280. MRID 48682902

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Suberg, H.; Loeser, E.; Blass; et al. (1983b) FCR 1272 (Common Name : Cyfluthrin) Chronic Toxicological Study on Mice (Feeding St udy over 23 Months): Report No. 12035; 86107. (Unpublished study received Feb 13, 1984 under 3125-351; prepared by Bayer AG, W Ger., submitted by Mobay Chemical Corp., Kans as City, MO; COL: 072366-A). (MRID 00137304)

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Tinwell, H. (2011a) Evaluation of cyfluthrin in the H295R steroidogenesis assay. Unpublished study prepared by Bayer S.A.S., Bayer CropS cience, Sophia Antipolis Cedex, France. Laboratory Study No. SA 11067, October 18, 2011. (MRID 48615607)

Tinwell, H. (2011b). Evaluati on of Cyfluthrin in the Aromatase Assay. Unpublished study prepared by Bayer S.A.S. (Bayer CropScie nce [355 rue Dostoïevski, BP 153, 06903 Sophia Antipolis Cedex, France]). Laboratory Study Number: SA 11061. 21 October 2011. (MRID 48615602).CYFLUTHRIN FINAL

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von Keutz, E. (1987) FCR 4545: Subchronic Oral Toxicity to Dogs (13-Week Feeding Study): Project ID 98348. Bayer Report No.: 16180. Unpublis hed study prepared by Bayer Ag. 259 p. (MRID 41267801)

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Page 34 of 68 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100)

The 21-day assay (MRID 48682901) of cyfluthrin (94.04% purity) on amphibian metamorphosis of the South African clawed-frog ( Xenopus laevis ) was conducted under fl ow-through conditionsin an amphibian metamorphosis assay (AMA). Amphibian larva at developmental NieuwkoopFaber Stage (NF) 51 (80/control and treatment gr oup; 20/replicate) were exposed to a negative control, a solvent control (dimethylformamide, DMF; 0.1 mL/L), and cyfluthrin at nominal concentrations of 0.000024, 0.000060, and 0.00015 mg a. i./L. Mean-measured concentrations were 0.000013, 0.000032, and 0.000066 mg a.i./L, respectivel y. The test system was maintained within a temperature range of 20.86 to 22.25 o C and a pH of 7.5 to 7.9.

There were no significant differe nces between the negative and solvent control for any endpoint; however, for Day 21 median NF stage, the diffe rence of one NF stage (negative control: NF stage 57; solvent control: NF stage 58) was marginally significant (p=0.058). Unless otherwise indicated, all effects are reported based on comparison to the negative control.

By Day 21, two mortalities each were observed in the negative control, mid, and high treatment groups. No mortalities were observed in the solven t control or low treatment group. Bent tail was observed in 13-23 tadpoles in negative and solvent controls and each treatment group.

Day 7 snout-vent length (SVL) was marginally significantly increased (p=0.056) by 7.7% in the high treatment group compared to the negative control. Day 21 SVL was significantly increased (p<0.05) by 12.6% in the mid treatment group an d significantly decrease d (p<0.05) by 10.8% in the high treatment group compared to the negative control. Significant in creases (p<0.05) of 47.4 and 50.7% were also observed in Day 21 body we ight for the mid and high treatment groups, respectively, and Day 7 body wei ght was significantly increase d by 31% in the high treatment group (p<0.05) compared to the negative control.

Acceleration of median NF developmental stage and asynchronous development were not observed at Day 7. Day 21 median NF stage was si gnificantly increased (p<0.05) by one stage in the mid and high treatment groups (NF stage 58) compared to the negative control but was comparable to the solvent contro l. Cyfluthrin did not significan tly affect (p>0.05) Day 7 or 21 normalized (for snout-vent length) hind limb length (HLL). There were no treatment-related effects on thyroid gland hi stopathology observed at any treatment level.

The coefficient of variation (CV) for mean-measur ed concentrations ranged from 40 to 60% (due
to an error in the initial st ock solution), exceeding the guideli ne performance criterion of ≤ 20%. This deficiency introduces uncertainty into the interpretation of results; however, appropriateCYFLUTHRIN FINAL

Page 35 of 68 analytical measurements were provided to doc ument the exposure, and the study is otherwise scientifically sound.

Although significant increases in NF developmen tal stage as well as growth parameters, especially wet weight, were observed at the mi d and high treatment levels compared to the negative control, treatment-related alterations in thyroid histopat hology were not reported. Though not significant, increases in Day 21 devel opmental stage and growth were also observed in the solvent control compared to the negative c ontrol, and these increases in the solvent control were comparable to the increases observed in the treatment groups. Therefore, it appears that the increased development and growth in the treatme nts may have been due to increases in food resources as a result of the presence of the solvent with the solvent concentration being consistent between the solvent control and treatments groups.

Androgen Receptor (AR) Bi nding Assay (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48615601), ventral prostate cytosol from male Sprague Dawley rats was used as the source of AR to conduct a saturation binding experiment and a competitive binding experime nt. The saturation binding experiment was
conducted to demonstrate that the AR in the ra t prostate cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radio-labeled reference androgen (R1881) prior to routinely conducting AR compe titive binding experiments. The competitive binding assay was conducted to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentratio ns of cyfluthrin (loga rithmic increase from 10 -10 to
10 -4 M). DMSO was used as the vehicl e at a final assay concentration of ≤ 3.3%. The assay included dexamethasone as a weak positive contro l, and R1881 as the ligand reference standard.

In the saturation binding experiment , the mean maximum binding capacity (B max ) was 14 fmol/100 μg protein and the m ean dissociation constant (K d ) was 1.01 nM. The K d obtained from Saturation Assay #2 was slightly low (0.59 nM), but the K d s from the other two runs were within the recommended range (0.685 to 1.57 nM ) and the Scatchard plots for the three saturation assays were linear. The specific bi nding reached a plateau, and non-specific binding (NSB) was less than 20% of total binding (4.6- 12.6%). The saturation binding experiment demonstrated that the androgen receptor wa s present in a reasona ble concentration and functioning with appropriate a ffinity for the R1881 ligand.

In the competitive binding experiment, cyflut hrin was not fully soluble at the highest concentration (10 -3 M), therefore the highest concentration used in data interpretation was 10 -4 M cyfluthrin. Cyfluthrin is classified as non-binding as specific [ 3 H]-R1881 binding was >75% at all concentrations of cyfluthrin tested in all three runs. An IC 50 and relative binding affinity (RBA) could not be calcul ated for cyfluthrin.CYFLUTHRIN FINAL

Page 36 of 68 The mean log IC 50 s for R1881 and the weak positive contro l (dexamethasone) were -9.1 and -4.4 M, respectively, and the mean RBA for the w eak positive control was 0.0021%. Confidence in these numbers is high due to the small variation. The solvent control resp onses indicated no drift in the study assay. All performance criteria were met, with the ex ception that the ligand depletion and NSB were not reported, but the IC 50 s for R1881 and the weak positive control, dexamethasone, were within the expected ranges demonstrating that the ligand was available at an appropriate level.

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

Aromatase Assay (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48615602), cyfluthrin (95.8% a.i., Batch # AE F057122-01-05) was incubated with human recombin ant aromatase and tritiated androstenedione (1- β [ 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]-ASDN) in dimethyl sulfoxide (DMSO) at increasing logarithmic concentrations from 10 –10 M to 10

− 3 M for 15 minutes to assess the potential of cyfluthrin to inhibit aromatase activity.

Aromatase activity was determined by measuring the amount of tritiated water produced at the
end of a 15-minute incubation for each concentration of chemical. Tritia ted water was quantified using liquid scintillation counting (LSC). Th ree runs were conducted and each run included a full activity control, a bac kground activity control, a pos itive control series (10 –10 M to 10 –5 M) using a known inhibitor (4-hydroxyandrostenedione ; 4-OH ASDN), and the test chemical series (10 –10 M to 10 –3 M) with three repetiti ons per concentration.

Aromatase activity in the full act ivity controls ranged from 0.143 to 0.670 nmol/mg-protein/min for the three test runs, with a mean and sta ndard deviation of 0.353±0.183 nmol/mg-protein/min. Activity in the background controls ranged from 1.8 to 5.2 and averaged 3.4% of the full control activity. The % change for the background controls at the beginning and end of each run fell between the guideline recommendations of

− 5% and +6% of the overall average full activity. However, for the full activity data, a slight assay dr ift was observed in the first and third runs, as the % changes at the beginning and end of each run were between 88.2 and 111.8% for the first
run and between 87.4 and 112.6% for the second in stead of the recommended guideline range of between 90 and 110%. Overall, the response of the full activity controls and background controls was acceptable for each run.

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.341±0.182 nmol/mg-protein/min at the lowest tested concentration (10

− 10 M) and 0.004±0.001CYFLUTHRIN FINAL

Page 37 of 68 nmol/mg-protein/min at the highest tested concentration (10

− 5 M). For cyfluthrin, aromatase activity averaged 0.313±0.161 nmol/m g-protein/min at the lowest tested concentration (10

− 10 M) and 0.224±0.096 nmol/mg-protein/min at th e highest tested concentration (10

− 3 M). Analyses of the cyfluthrin data indicated they did not fit the 4-parameter regression model used for data interpretation. The mean aromatase activity of 10 –3 M cyfluthrin was determined to be 66% of control activity and the be st fit mean inhibition response curv e was a straight line (essentially no inhibition was observed at other doses). For 4-OH ASDN, the estimated Log IC 50 averaged

− 7.09 M, and the slope was

− 0.98. Confidence in these values is very high due to the small variations (<6% CV).

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

Estrogen Receptor (ER) Bi nding Assay (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48615603), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct saturation and competitive binding experiments. A saturation binding experiment wa s conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radio-labeled reference estr ogen prior to conducting ER competitive binding experiments. The competitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of increasing concentrations of cyfluthrin (logarithmic increase from 10 –10 to 10 –3 M). DMSO was used as the vehicle at a final concentration of 2%. The assay included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative co ntrol, and the natural ligand 17 β -estradiol.

For the saturation binding experiment, the approp riate protein concentration for the saturation binding assay was determined to be 0.5 mg/mL as it bound 25-35% of the [ 3 H]-17 β -estradiol.
The K d for [ 3 H]-17 β -estradiol was 0.070 nM, and the estimated B max was 0.034 nM for the prepared rat uterine cytosol. The K d for each run was within the expected range of 0.03 to 1.5

nM.

For the competitive binding experiment, the estimated mean IC 50 was not calculated for cyfluthrin as it did not achi eve 50% binding. The mean IC 50 for the positive control was 9 × 10 –6 M, with a mean relative binding affinity ( RBA) of 0.019% compared to the natural ligand; however, the Hill Slope fell outsi de the recommended range, at

− 2.0 to

− 1.7. Cyfluthrin was tested over a concentration range that fully defi ned the top of the curve. The maximum percent binding 108.8-117.4% was within approximately 25 percentage points of the lowest
concentration of the estradiol standard 83.5-116. 2%. The mean specific ER binding was >75% at cyfluthrin concentrations up to 10 –4 M in all three independent runs.CYFLUTHRIN FINAL

Page 38 of 68 Based on the results from the three runs, cyfluthrin is classified as Not Interactive in the Estrogen Receptor Binding Assay.

ER α Transcriptional Activatio n (ERTA) Assay (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48615604), hER α -HeLa-9903
cells cultured in vitro were exposed to cyfluthrin (95.8% a.i., Batch # FHER904218) at logarithmically increasing concentrations from 10

− 11 to 10

− 5 M in DMSO (0.1%) for 24 hours. The experiments were performed using 96-well plates and each cyfluthr in concentration was tested in triplicate (3 wells/plate). Cells were exposed to the test ag ent for 23±1 hr to induce reporter (luciferase) gene produc ts. Luciferase expression in response to activation of the estrogen receptor by cyfluthrin was measured upon addition of a luci ferase substrate and detection with a luminometer with acceptable sensitivity.

Cyfluthrin was tested up to the limit of cytotoxicity, 10

− 5 M. The laboratory proficiency assays using the required reference compounds adequately demonstrated that the assay was functioning properly. The only deviation was a weak positive re sponse in one run in response to the negative control, corticosterone. Perfor mance criteria were met for 17 β -estradiol and corticosterone, and the deviations from expected pe rformance criteria values for 17 α -estradiol and 17 α

methyltestosterone, indicate an increased sensitivity to weak agonists. The fold-induction was slightly lower than the required four -fold induction in the first run.

Cyfluthrin was only able to reach a maximum of 3.2-6.5% PC when tested up to the highest concentration possible based on cy totoxicity. Because the RPC Max < PC 10 in both assay runs, cyfluthrin was considered negative for estrogen r eceptor transcriptional ac tivation in this test
system.

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350)

The 21-day fish short-term reproduction assa y (MRID 48670101) of cyfluthrin (94.1% purity) with fathead minnows ( Pimephales promelas ) was conducted under flow-through conditions. Adult fish (16 spawning groups; 2 males and 4 fe males per group; ca. 4.5 months of age) were exposed to cyfluthrin at nominal concentr ations of 0 (negativ e control), 0.000108, 0.000325, and 0.000976 mg a.i./L, which resulted in mean-m easured concentrations of <0.0000066 (<LOQ, negative control), 0.000063, 0.000149, and 0.000421 mg a. i./L, respectively (59%, 46%, and 43% of nominal, respective ly). The test system was maintained at 24.5 to 25.6 o C and a pH of 7.6

to 8.1.CYFLUTHRIN FINAL

Page 39 of 68 Overall survival was 96% in th e negative control and 92% in th e low and mid treatment groups; high mortality was observed in the high treatment group, with overall survival of 29% (7 out of 24 fish surviving; 50% survival in males and 19 % survival in females). Effects on secondary sex characteristics and clinical signs ( i.e. , behavioral and other sublethal effects) were observed in all surviving fish at the high treatment level; effects included swimming at the surface, erratic behavior, loss of equilibrium, labored respiratio n, decreased food intake, and loss of typical reproductive behavior and color pa ttern. Body weight and length in males and females were not significantly different (p>0.05; Dunne tt) at any treatment level.

In the negative control, spawning frequency and fecundity were, respectively, at least every 4 days and 22.5 eggs/female/day/replicate; ferti lization success in the negative control was 98%. Fecundity was significantly reduced (p<0.05; Jonc kheere-Terpstra) by 92% at the high treatment level compared to the negative control. Fertilization success in the treatment groups was not significantly different (p>0.05; D unnett) from the negative contro l. Only two of the four exposure replicates in the high treatment group produced eggs; fertilization success for these replicates was 99%.

No significant difference (p >0.05; Mann Whitney U) in gonado-somatic index (GSI) was observed in females; however, male GSI wa s significantly reduced (p<0.05; JonckheereTerpstra) by 46% in the high treatment group. N uptial tubercle score was not significantly different (p>0.05; Dunnett) in males at any trea tment level; no nuptial tube rcles were present in females. Plasma vitellogenin (VTG) concentrat ions were not significan tly different (p>0.05; Mann Whitney U) in males or females. Testosterone and 17 β -estradiol concentrations were not determined. Treatment-related effects on gonada l histopathology were observed in male and female fish at the high treatment level. Hist opathological eff ects included a narrowing in the range of testicular stage scores in the high treatment group in males (Stage 2.5 only), with an absence of stages that indicate currently active or recent spawning, and a similar narrowing in the range of ovarian stage scores in females (Stages 3 to 3.5).

Hershberger Assay (OCSPP 890.1400)

In a Hershberger Assay (MRID 48615605) screeni ng for androgenic activity, cyfluthrin (95.8% purity, batch # FHER904218) in corn oil was admini stered daily via oral gavage to 58-day old, castrated male Sprague Dawley rats (6/dose) at dose levels of 0 (vehicle), 10 or 20 mg/kg/day. An androgenic positive control gr oup consisted of 6 castrated ra ts exposed to 0.4 mg/kg/day of testosterone propionate (TP) by s ubcutaneous (s.c.) injection.

To screen for potential anti-a ndrogenic activity, cyfluthrin (9 5.8% purity, batch # FHER904218) in corn oil was administered daily via oral gava ge to 58-day old, castrated male Sprague Dawley rats (6/dose) at dose levels of 0 (vehicle), 5, 10 or 20 mg/kg/day in conj unction with a daily doseCYFLUTHRIN FINAL

Page 40 of 68 of reference androgen TP at 0.4 mg/kg/day by s. c. injection. The anti-androgenic positive control group consisted of 6 castrated rats exposed to 0.4 mg/kg/day TP and 3 mg/kg/day flutamide (FT). TP alone was used as the anti-androgenic negative control.

For both components of the assay, body weights were determined da ily. The animals were dosed for 10 consecutive days and terminated a pproximately 24 hours after the final dose administration. At necropsy, the five androgen- dependent tissues were collected and weighed.

All animals survived until scheduled termination. Clinical signs of toxicity (piloerection or increased salivation) were observed in one anim al treated with cyfluthrin at the highest concentration (20 mg/kg/day) during the androg en agonist assay, and increased salivation was observed in one animal treated with cyfluthrin (+TP) at 20 and 10 mg/kg/day in the anti-agonist assay. No clinical signs of toxicity were obser ved at 10 mg/kg/day cyflut hrin or at 5 mg/kg/day cyfluthrin with TP.

In the androgen agonist assay, no adverse trea tment-related effects on body weights or overall body weight gains were observed in either cyfluthrin treated group co mpared to controls. In the 0.4 mg/kg/day TP group, overall bod y weight gain was increased (p<0.05) by 34% compared to the controls. Rats in the positive control (TP) group had accessory sex organ weight increases (p<0.01) as follows: 1002% in seminal vesicles; 867% in ventral prostate ; 199% in levator anibulbocavernosus (LABC) muscle complex; 474% in Cowper’s glands; and 108% in glans penis. The maximum guideline-specified %CV was slightly exceeded for the glans penis %CV in the control group (22.5% vs. 22%), but all other %CV values were less than the maximum permissible values.

In the anti-androgen assay, body weights and body we ights gains for all cyfluthrin (+TP)-treated groups and the positive control (FT +TP) group were comparable to the control (TP only) throughout the duration of the study. Rats in the positive control (FT) group had accessory sex organ weight decreases (p<0.01) as follows: 77% in seminal vesicles; 70% in ventral prostate; 48% in LABC; 63% in Cowper’s glands; and 29% in glans penis. All %CV values were less than the maximum permissible values.

The dosing for this study (high- dose of 20 mg/kg/day) was cons idered adequate based on the clinical signs of toxicity observed.

Statistically significant organ wei ght changes were not seen in tw o or more of the five androgen sensitive tissues. Cyfluthrin was negative fo r androgenicity and anti -androgenicity in the Hershberger assay.CYFLUTHRIN FINAL

Page 41 of 68 Female Pubertal Assay (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48615606), 15 Sprague Dawley [Crl:CD (SD)] rats/dose group were treated daily via oral gavage (dose volume 5 mL/kg) with cyfluthrin (95.8% a.i., Batch# FHER904218) in corn oil at doses of 0 (corn oil), 10, or 20 mg/kg/day from post-natal day (PND) 22 to 42. Animals were examined fo r vaginal opening (VO) daily beginning on PND 22, and weight at day of attainment was reco rded. Following sacrifice on PND 42, total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) le vels were determined using an enzymelinked immunosorbent assa y (ELISA). Weights were record ed for liver, kidneys, urogenital organs, pituitary, thyroid, and adrenal glands and microscopic examinations of the ovaries, uterus, thyroid, and kidneys were performed.

There were no effects of treatment on mortalit y, final body weights, body weight gains, mean age at first vaginal estrus, mean cycle length, perc ent cycling, percen t regularly cycling, clinical chemistry parameters, or microscopic pathology.

At 20 mg/kg/day, increased salivation was observed in 5 of the 15 rats. Additionally at this dose, the age at VO was delayed (p<0.01; 35.73 days treat ed vs. 33.00 days controls), the body weight at VO was increased (p<0.05) by 10%, and the mean age at first vaginal estrus was delayed (not significant; 36.36 days vs. 34.00 days) as a result of the delay in complete vaginal opening.

The doses tested were adequate ba sed on clinical signs of toxicity (increased salivation) at the high dose and the results of a dose range-finding study.

Male Pubertal Assay (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48615606), 15 Sprague Dawley [Crl:CD (SD)] rats/dose group were treated daily via oral gavage (dose volume 5 mL/kg) with cyfluthrin (95.8% a.i.; Batch # FHER904218) in corn oil at doses of 0 (corn oil), 10, or 20 mg/kg/day from post-natal day (PND) 23 to 53. Animals were examined for pr eputial separation (PPS) daily beginning on PND 30 and weight at day of attainment was record ed. Following sacrifice on PND 53, total serum testosterone levels were determined using enzyme immunoassay (EIA), and thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels were determined using enzyme-linked immunosorbent assay (ELISA). Weights were recorded for th e liver, kidneys, urogenital organs, pituitary, thyroid, and adrenal glands, and microscopic examinations of th e testes, epididymides, thyroid, and kidneys were performed.

No treatment-related effects were observed on mortality, final body weights, body weight gains, age or weight at attainment of PPS, organ weights, hormone (serum T 4 , TSH, or testosterone) levels, or histopathology para meters at any dose.CYFLUTHRIN FINAL

Page 42 of 68 In the 10 mg/kg/day dose group, increased salivation was observed in 1 of the 15 rats on Day 8. In the 20 mg/kg/day dose group, increased saliva tion, piloerection, lack of grooming, wasted appearance, uncoordinated movement s, and/or tremors were obser ved in 6 of 15 animals on at least one occasion. At 20 mg/kg/day, urea le vels were decreased (p<0.01) by 28%. This decrease was not considered a biologically advers e effect due to only 2 of 13 urea values in this group were lower than the lowest urea value in the control group. Serum TSH levels were decreased by 37% compared to controls, although the decrease was not st atistically significant.

The dosing levels utilized in th e current study are considered appr opriate based on clinical signs of toxicity (increased salivation and/or tremors) at the high dose and the results of a dose rangefinding study.

Steroidogenesis Assay (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48615607), H295R cells cultured in vitro in 24-well plates were incubated with cyfluthrin (>95.8% purity, Batch # AE F057122-01-05) at logarithmic
concentrations from 10

− 10 to 10

− 4 M for 48 hours in triplicate for th ree independent experiments. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.1%.

Testosterone and estradiol levels were measured using specific EIA kits. A Quality Control (QC) plate was run concurrently with each i ndependent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels. The positive controls included a known inhibitor (prochloraz) and a known inducer (forskolin) of estradiol a nd testosterone production.

Guideline acceptability recommendations and requirements were generally met, including lack of cytotoxicity, adequate producti on of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction an d inhibition with positive contro ls. The exceptions included that testosterone was only induced by 1.8-fold rather than the Guideline requirement of ≥ 2-fold, and the between plate %CV for the SC in the test and QC plates was 33-34% for estradiol, which is above the Guideline recommendation ( ≤ 30%).

Treatment with cyfluthrin at 10

− 9 to 10

− 4 M had no significant effect on testosterone production, although there was a marginal reduction (0.70- fold) in testosterone observed at 10

− 4 M. The only statistically significant change in testosterone produc tion was a 0.65-fold change (p ≤ 0.05) at the lowest concentration tested (10

− 10 M); however, this reduc tion was not concentration dependent. Cyfluthrin generally increased es tradiol production (1.33- to 1.88-fold) across all
three runs at concentrations of 10

− 10 to 10

− 5 M. However, the increases in estradiol were not concentration dependent, and were highly variable with the increases at taining significance onlyCYFLUTHRIN FINAL

Page 43 of 68 at 10

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

− 4 M), but the change was not significant.

Based on hormone responses in three independent runs, cyfluthrin treatment did not result in statistically significant and repr oducible alterations in testoste rone or estradiol production.

Uterotrophic Assay (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48615608) conducted to screen for potential estrogenic activity, cyfluthrin (95.8%% purity, batch #FHER904218) in corn oil was administered daily via oral gavage to groups of 6 immature female, Sprague-D awley rats at dose levels of 0 (vehicle), 5, 10, or 20 mg/kg/day on post-natal days (PND) 19-21. A positive control group was treated with

17 α -ethynyl estradiol (EE) in corn oil by gavage at a dose level of 1 μg/kg/day. All animals were terminated and necropsied on PND 22 appr oximately 24 hours after the final dose to
determine wet and blo tted uterine weights.

All animals survived until scheduled termination. Clinical signs of toxicity were observed in one animal treated with cyfluthrin at the highest concen tration (20 mg/kg/day); no treatment related effects were observed at the lower dosing levels . No precocious vagina l opening was observed in treated females. Body weights and overall body weight gains (Days 1- 4) in the cyfluthrin treated groups were comparable to the contro ls throughout the study. Uterine weights in the cyfluthrin treated groups were also comparable to the controls. Absolute wet and blotted uterus weights for the positive EE group were increase d (p<0.01) by 195% and 201%, respectively, as expected.

The high dose tested in this st udy (20 mg/kg/day) is adequate ba sed on the results of the rangefinding study which showed mortal ity, severe clinical signs and reduced body weight gain at 40 mg/kg/day.

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

Page 44 of 68 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Receptor Transcriptional Activation

The purpose of this study was to screen and ev aluate 200 pesticides, c overing nine chemical classes, for their potential to interact with two human estrogen receptor subtypes (hER α and
hER β ) and one human androgen receptor (hAR) in tr ansactivation assays with Chinese hamster ovary cells (CHO-K1 cells) ( Kojima et.al

. , 2004 ). Cyfluthrin was dissolved in dimethylsulfoxide to a final con centration of 10 mM and diluted to the desired concentrations in Dulbecco’s modified Eagle medium plus Ham’ s F-12 nutrient mixture. The final solvent
concentration in the culture me dium did not exceed 0.1% and did not affect cell yields. The human estrogen receptor (hER α ) and androgen receptor (hAR) expression vectors (pcDNAER α and pZeoSV2AR, respectively) were used in the study. Estrogen or androgen responsive elements containing reporter plasmid (pGL3-tkER E and pIND-ARE, respectively) were used for the binding experiments. The pRL-SV40 containing the Renilla luciferase gene was used as an internal control for transfection efficiency.

Cyfluthrin tested positive for both estrogeni c and anti-androgenic activities with the in vitro screening assays, indicating pleiotro pic effects on hER and hAR via ER α and AR. Cyfluthrin was found to induce estroge nic activity in the ER α transactivation assay, with a REC 20 of 5.9 x

10 -6 M, and relative luciferase activity (RLA) of 45% at 10 -5 M (100% activity defined as the activity achieved with 10 -10 M E 2 ). Cyfluthrin also had weak an ti-androgenic activity in the AR transcriptional assay, with a RIC 20 of 8.4 x 10 -6 M and RLA of 74% at 10 -5 M (100% activity defined as the activity achieved with 10 -10 M DHT). Cyfluthrin did not demonstrate androgenic transcriptional activity of more than 20% of that induced by 10 -9 M DHT (cyfluthrin concentration tested was not reported).

In a published study by Kojima et al . (2005) , the response of an in vitro ER–related reporter gene assay was investigated af ter exposure to thirty two pest icides either alone or in combination. The source of the test compounds was not reported; however, cyfluthrin had a reported purity of >96%. The test compounds were prepared in DMSO at 10

− 2 M.

The E-CALUX Assay (Xenobiotic Detection Systems, Inc., NC, USA) uses a human ovarian carcinoma cell line (BG1) transfected with a luciferase reporter gene under the control of a mouse mammary tumor virus (MMTV) promoter . Estrogen-responsive concatemers (repetitive DNA sequences), inserted directly above the pr omoter, confer estrogen responsiveness to the viral promoter and reporter gene. The transfected cells are made st able by an antibiotic resistance gene resulting in the cell lin e used in the assay (BG1Luc4E 2 ). Compounds that interact with estrogen-responsive DNA are identif ied by the luciferase gene re sponse and quantified in terms of Relative Light Units (RLU).CYFLUTHRIN FINAL

Page 45 of 68 The BG1Luc4E 2 cells were grown in medium for f our days, then the assay medium was replaced, and cells were transferred (2 × 10 5 cells/mL) into 96-well pl ates and incubated at 37  ̊ C
with 5% CO 2 for 24 hours. The medium was removed, and the test compound or estrogen standard (E2) was added. After incubation at 37  ̊ C with 5% CO 2 for another 24-hour period, the medium was removed and the cells were lyse d. Luciferase activity was measured with a luminometer in order to compare treatment RLU to the E2 standard. For comparative purposes,
the EC 10 (Effective Concentration at which 10% of the maximum response was observed) was
used. The data were not sufficien t to allow calculation of an EC 50 . Although the article does not specifically identify the E 2 standard, the reviewers assumed that E2 referred to 17 β -estradiol. Cyfluthrin did not induce an estroge nic response in this assay system.

In a non-guideline study by Du et al. , 2010, nine pyrethroid insecticid es, including cyfluthrin, were evaluated for estrogen, androgen, and thyroid hormone receptor activity (both as agonist and antagonist) using receptor-mediated lucifera se reporter gene assays. Two cell lines (CV-1 and MDA-kb2) were employed; both were stably transfected to express either the estrogen
receptor α (ER α ), androgen receptor (AR), or thyroid hormone receptor β (TR β ) genes.

Cyfluthrin did not exhibit agoni stic activity for estrogen, andr ogen, or thyroid hormone. The following antagonistic activitie s were presented for cyfluthrin: anti-estrogenic RIC 20 = 1.36 x

10 -6 M; anti-androgenic RIC 20 = 8.84 x 10 -8 M; and thyroid hormone RIC 20 = 5.39 x 10 -7 M.

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).

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%. Cyfluthrin and β -cyfluthrin were each tested at concentrations ranging from 10

− 8 to 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 sa line, and 25 μL of lysis buffer was added per well. After incubation for 25 minutes, the cells we re lysed, 100 μL of lu ciferase reaction agent was added, and luciferase activity was determined. The results of the assay were expressed as the percent of androgenic activity for each compound relative to the DHT control.

In the in vitro reporter gene assay, cyfluthrin inhib ited (p<0.05) DHT-induced transcriptional
activation at concentrations from 10

− 7 to 10

− 5 M. The positive control performed as expected within the same dose range.CYFLUTHRIN FINAL

Page 46 of 68 Prenatal Development in Rodents (Rat; OSCPP 870.3700)

In a developmental toxicity study ( MRID 44116501

, 1996 ), β -cyfluthrin (96.5-97.3% purity) was administered by gavage to 30 female Wistar rats/dose in aqueous 1% Cremophor solution at dose levels of 0, 3, 10, or 40 mg/kg/day from gest ation days (GD) 6 through 15, inclusive. Dams
were sacrificed on GD 20.

Maternal toxicity was noted at 10 mg/kg/day by reduction in mean food consumption (10-15%) with recovery of appetite following treatment. Mean body weights and mean body weight gains were not significantly reduced, however, mean adjusted terminal weights were significantly reduced (14%) at 10 mg/kg/day. Clin ical signs of toxicity were ab sent. Maternal toxicity at 40 mg/kg/day included reduced survival (10%), reduced mean body weight gains (56%, Days 6-16 and 16%, Days 0-20), reduced weight gain (32%, p ≤ 0.01) corrected for gravid uterine weights, and reduced mean food consumption during treatment (20-49%). Clinical signs of toxicity at the high dose included hypoactivit y, locomotor incoordina tion, and salivation.

No treatment-related changes were seen in pregnancy rate, number of corpora lutea, implantation sites, the extent of resorptions /i mplantations, number of live fetuse s/litter, fetal sex ratio, or soft tissue abnormalities at any dose.

The maternal LOAEL was 10 mg/kg/day, based on reduced body weight gain and reduced food consumption with post-treatment recovery. The maternal NOAEL was 3 mg/kg/day. The developmental LOAEL was 40 mg/kg/day, based on reduced fetal body weights and increased
skeletal variations. The deve lopmental NOAEL was 10 mg/kg/day.

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

In a developmental study ( MRID 42675401, 1992 ), cyfluthrin (96% purity) in corn oil was administered via gavage to pr egnant female Chinchilla (CH bb: CH, Hybrids, SPF Quality) rabbits (25/dose) during GD 6-18 of gestation at dose levels of 0, 20, 60, or 180 mg/kg/day.

There were no maternal deaths or clinical signs of toxicity during the study, and no treatmentrelated gross pathology was observed. All groups lo st weight during the dosing period. The midand high dose groups lost a significantly (p ≤ 0.01) greater amount of we ight than the control group: a loss of 40 grams in the control group versus losses of 189 and 233 grams in the midand high-dose groups, respectivel y. Increases in body weight (N S) were observed in the two highest dose groups during the post-dosing peri od. At 180 mg/kg/day, food consumption was decreased during days 6-15 ( ↓ 41-48%, p ≤ 0.01) and Days 15-19 ( ↓ 27%). At 60 mg/kg/day, food consumption was decreased during days 6-19 ( ↓ 24-29%). Food consumption in the mid- and high-dose groups also increased dur ing the post-dosing period.CYFLUTHRIN FINAL

Page 47 of 68 There were no treatment-related effects on the numb er of live litters, fetal weights, implantations, external, visceral or skeletal malformations and variations. Post-implant ation loss (expressed as a percentage of the number of implantati on sites) was increased at 60 mg/kg/day (p ≤ 0.05) and 180 mg/kg/day (p ≤ 0.01). “Embryonic resorptions” (early resorptions, when expressed as a percentage of the number of implantation sites: 3.6, 6.3, 11.5 and 15.1for the 0, 20, 60 and 180 mg/kg/day groups, respectiv ely, were increased (p ≤ 0.01) at 60 and 180 mg/kg/day. The total number of fetuses (also expressed as a percen tage of the number of implantation sites) was significantly decreased at 60 and 180 mg/kg/day: (89.1, 89.1, 80.3 and 71.5 for the 0, 20, 60 and 180 mg/kg/day groups, respectively). Upon examinati on of the data, it appear s that the increases in resorptions over the control group are not likely to be to xicologically significant because the number of live pups/litter are co mparable across the dose groups and although the control group has slightly more, there is no indication of a dose-response across a dose range of 1 order of magnitude. In addition, the individual animal data indicate that the does with a higher number of implantation sites generally have a higher number of resorptions. This is probably because the does cannot support a higher number of fetuses and thus, some are resorbed. The highest dose group had five does with 15-19 implantation sites. They each resorbed 4-9 embryos/fetuses. The control group had two does with 15 implanta tion sites. These 2 does resorbed 2 and 5 embryos/fetuses, respectively. The mid-dose group had one doe with 18 implantation sites. This doe resorbed 7 embryos/fetuses.

The maternal LOAEL was 60 mg/kg/day, based on decreased body weight gain and food consumption during the dosing period; th e maternal NOAEL was 20 mg/kg/day. The developmental NOAEL was 180 mg/kg/day, and a developmental LOAEL was not observed.

Inhalation Developmental Toxici ty in Rodents (Rat; OCSPP 870.3600)

Two developmental toxicity studies via inhalation ( MRIDs 40780401, 40968501 and 42332001,

1988 ) were conducted. In the first study, 4 groups of 30 female Bor:WISW (SPF Cpb) rats were inseminated by being housed overnight with males. The presence of sperm in the vaginal smears following mating established GD 0. The dams were dynamically exposed head-only to cyfluthrin dissolved in a 1:1 mi xture of Lutrol and ethanol at analytical concentrations of 0, 0.0011, 0.0047 or 0.0237 mg/L/day for 6 hours/day on GD 6 through 15. A second study was conducted in order to establish a NOAEL for offspr ing toxicity. In that study, the dams were exposed to analytical concentrations of 0, 0.00009, 0.00025, 0.00059 or 0.0042 mg/L of the test
material. The MMAD was 1.23 - 1.45 μ m in the first study and 1.29 - 1.53 μ m in the second study. An oxygen enriched atmosphere (30%) wa s provided for the 0.0042 mg/L group to see if the embryotoxic effects seen in th e first study at this concentrati on could be lessened. The rats were observed several times on the exposure da ys except during the exposures (because of restraint for head-only exposur e). They were weighed on GDs 0, 6, 9, 12 and 20. The dams were sacrificed on day 20 and their pups rem oved by caesarian section. Their ovaries and uteriCYFLUTHRIN FINAL

Page 48 of 68 were examined for implantations, live young, embryoni c and fetal deaths, fetal sex and weights,
and external fetal abnormalities.

Combining the results of the two studies, ma ternal effects were observed at 0.0047 mg/L and above, and clinical signs included reduced mo tility, dyspnea, piloerection, ungroomed coats and eye irritation. The symptoms were mainly obs erved after the end of exposure in the 0.0047 mg/L concentration group and were largely gone by the next exposure day. At 0.0237 mg/L concentration, the symptoms were observed at gr eater intensity over the entire exposure period.

No treatment-related changes were seen in pregna ncy rate, implantation sites, the mean number of resorptions /implantations, number of live fe tuses/litter, fetal sex ratio, or soft tissue
abnormalities at doses of ≤ 0.0047 mg/L. However, e ffects were observed on the pups at ≥ 0.0011 mg/L. At 0.0011 mg/L and above, a dose-related incr ease in the incidence of runts and skeletal
anomalies in the sternum were observed. At 0.0047 mg/L and above, biologically significant decreases in pup weights were observed. At 0.0237 mg/L, increases in post-implantation loss, late embryonic deaths and in sk eletal anomalies in the extremities were observed as well as microphthalmia.

The maternal NOAEL was 0.0011 mg/L and the maternal LOAEL was 0.0047 mg/L (reduced motility, dyspnea, piloerection, ungroomed coat s and eye irritation). The developmental NOAEL was 0.00059 mg/L and the developmental LOAEL was 0.0011 mg/L (increases in the
incidence of runts and skeletal anomalies in the sternum).

An ad hoc committee met on 4/22/93 to discuss the developmental toxicity data base for cyfluthrin. At that time, the Committee reco mmended that due to deficiencies that were mentioned in the review of the study, this study should be re-examined if it is to be used as a regulatory endpoint. Although the study had been graded Acceptable and NOAELs and
LOAELs had been established for maternal and developmental toxicity, comments had also been made that developmental anomalies in the study had not been adequately reported. The dams in this study had reflex apnea, al though it was poorly characterized.

Inhalation Developmental Toxici ty in Rodents (Rat; OCSPP 870.3700)

Cyfluthrin (96.2% purity) ( MRID 43393401

, 1994 ) was administered by inhalation to 25 pregnant female Wistar rats/group at the followi ng analytically determined concentrations: air control, vehicle control (polyethylene glycol-400: 50% ethanol), 0.00046, 0.00255, 0.0119 or 0.0128 mg/L plus 39% oxygen for GD 6 through 15 in a dynamic nose-only inhalation chamber. The rats were exposed to the test material 6 hours/day, 7 days/week. The MMAD ± geometric
standard deviation was 1.1 ± 1.5 μ m for all the groups. Additional satellite rats (5/group) wereCYFLUTHRIN FINAL

Page 49 of 68 exposed similarly from GD 6 through day 13 to de termine the effect of cyfluthrin exposure on body temperature, ventilation rate and plasma cyfluthrin levels.

Maternal body weight was statistically significantly decreased at ≥ 0.00046 mg/L for the interval GD 6 to 15 ( ≥ 26% from the vehicle control) and GD 0 to 20 ( ≥ 16% from the vehicle control). The relative efficiency of food utilization appeared to be decreased at ≥ 0.00046 mg/L ( ≥ 9% of the vehicle control). Increases in untidy and ruffl ed fur and retarded breathing were observed at 0.0119 and 0.0128 (with O 2 ) mg/L. Labored and irregular breathing, hypoactivity, high-stepping gait, salivation and reduced stool were observed only at 0.0119 mg/L. The satellite animals showed a decreased (but relatively flat dose-re sponse) breathing rate/minute in the treated animals versus the air and vehicle control gr oups. Minimum volume (ml/m in, ml/min/kg and as % of vehicle control) were decreased in a dose- related manner (bradypnea). Rectal temperatures were slightly decreased in the tr eated animals. Placental weights ( ≥ 7% from the vehicle control) and fetal weights ( ≥ 11% from the vehicle cont rol) were decreased at ≥ 0.00255 mg/L. Developmental toxicity was also expressed in the form of retarded skeletal ossification in the phalanx, metacarpals, cervical verteb rae, sacral and caudal arches at ≥ 0.00255 mg/L. Doserelated total fetal malformations such as microphthalmia a nd skeletal dysplasia were evident at 0.0119 mg/L (8.8% in fetuses/43% in letters vs. vehicle controls with 1.0% in fetuses/14% in litters).

The evidence suggests that the cyfluthr in exposure at 0.0119 and 0.0128 mg/L caused the developmental toxicity partly through the bradyp nea in dams. While the bradypnea in dams at 0.00255 mg/L may have caused the reduced fetal a nd placental weight and retarded ossification, the data presented were insuffici ent to draw this conclusion.

The maternal LOAEL is 0.00046 mg/L based on decreased body weight gain and reduced relative food efficiency. The NOAEL is not determined. The developmental NOAEL is 0.00046 mg/L and the developmental LOAEL is 0.00255 mg /L based on reduced fetal and placental weights and reduced ossification in th e phalanx, metacarpals and vertebrae.

Developmental Neurotoxicity (Rat; OCSPP 870.6300)

In a developmental neurotoxicity study ( MRID 46054101

, 2003 ), β -Cyfluthrin (95.1-97.6% a.i.) was administered to 30 parent fema le Wistar rats/dose in the diet at nominal concentrations of 0, 30, 125, or 200 ppm from GD 0 through postnatal day 21. The average daily intake of β

Cyfluthrin was 0, 2.4, 11.0, and 17.8 mg/kg/da y during gestation and 0, 5.9, 25.4, and 40.9 mg/kg/day during lactation for the 0, 30, 125, and 200 ppm groups, respectively. Pup physical development was assessed by body weight, and se xual maturation of females was assessed by age at VO. Maturation of males was a ssessed by age at completion of PPS.CYFLUTHRIN FINAL

Page 50 of 68 In dams, no treatment-related effects were seen on mortality, clinical signs, body weight, body weight gain, or reproductive performances (matin g, fertility, gestation, li ve birth, viability or lactation indices). The maternal NOAEL wa s 17.8 mg/kg/day; a maternal LOAEL was not
established.

In offspring, treatment had no adverse effects on survival, clinical signs, birth weight, developmental landmarks, FOB parameters, motor or locomotor activity, auditory startle reflex, learning and memory, brain mo rphology or neuropathology. Body weight and body weight gain were comparable across all dos e groups at birth and on PND 4. However, pup body weight was decreased by 8-11 % in the high-dose group ( 17.8 mg/kg/day) on subs equent days through weaning. Weight gain of high-dose pups wa s decreased 8.5%-13% from PND 4-21. After weaning, pups received only untreat ed diet, and the only compound-re lated effects were noted in high-dose males and females and were associated with the decreased body we ight that developed during lactation. High-dose male s weighed 10% less than controls during the first week postweaning and 7% less during the last week of the study. High-dose females weighed 7.5% less than controls during the first week post-weaning and had weight comparable to controls by the last two weeks of the study. At terminati on, absolute brain weight was decreased 5.8% (p ≤ 0.05) in high-dose females compared to controls. Ac quisition of VO and PPS in F1 offspring were unaffected by treatment even in the presence of up to 10% decrease in body weight and body weight gain.

The offspring LOAEL was 17.8 mg/kg/day base d on decreased body weight and body weight gain and decreased brain weights in female s at termination. The offspring NOAEL was 11.0 mg/kg/day.

Multi-Generation Reproduction in Rodents (Rat; OCSPP 870.3800)

In a 2-generation reproduction study ( MRID 44371401, 1996 ) cyfluthrin (95.4% purity) was administered in the diet to 30 Sprague Dawley rats/sex/dose at dose levels of 0, 50, 125, or 400 ppm (equivalent to doses of 0, 3/4, 9/10, or 29/33 (M/F) mg/kg/day during premating and gestation and 0, 7, 19, or 59 mg/kg/ day during the first two weeks of lactation). Exposure to P animals (30/sex/dose) began at 7 weeks of age and lasted for 10 weeks prior to mating to produce F1 pups. Upon weaning, F1 pups (30/sex/dos e) selected to become parents of the F2 generation were fed cyfluthrin in test diets at the same concen tration their dam received. F1 animals were given test diets for 11 weeks prio r to mating. All animals were mated on a 1:1
ratio.

Parental toxicity of cyfluthrin was evident at 125 and 400 ppm. At 125 ppm, marginal treatmentrelated reductions in mean body weight ( ↓ 6-8%) were observed during the premating period and terminal body weights were similarly reduced ( ↓ 6%) in F1 males. During lactation there was a treatment related decrease in food consumption ( ↓ 9-11%, p<0.05 or 0.01) for F1 females at thisCYFLUTHRIN FINAL

Page 51 of 68 dose. At 400 ppm, treatment-related splaying of hind limbs was observed in P and F1 females during lactation (15/30 P females, 9/30 F1 fema les vs. 0/30 in both P and F1 controls). Treatment-related reductions in body weights ( ↓ 7-14%) and food consumption ( ↓ 11-22%) compared to lactating controls were also not ed in lactating females of both generations. Marginal treatment-related decr eases in mean body weights ( ↓ 7-8%) were observed in F1 males during the premating period. The re duction of terminal body weights ( ↓ 6-8%) in F1 males and
females was also considered tr eatment-related. Food consumpti on for all dams (treated and controls) was markedly increased during lactation when compared to the premating and gestation periods. Although a decrease in food consump tion was observed in the mid- and high-dose groups during lactation, this was in comparison to the control group. Food consumption in the treated groups was still increas ed in comparison to food consumption in these same groups during the premating and gestation periods. As a result, cyfluthrin intake by dams was approximately doubled during lactation compared to premating and gestation periods.

Therefore, since most of the tr eatment-related effects of cyfluthr in in females (decreases in food consumption and body weights and splayed hind limbs) were observed during this period, they were likely associated with the increase in dose during lactation. In contrast, during the premating period most of the obser ved parental treatment related changes consisted of marginal reductions in body weights. There were no treatm ent related mortalities or treatment-related effects on reproductive parameters or function.

No treatment-related effects were observed in the reproductive performance (male or female fertility indices, gestation inde x, gestation length, live birth in dex, viability index, lactation index, parturition, or fetal sex ratio) at any dose. No changes were seen in absolute or relative testicular weights, but ab solute ovarian weights were significantly decreased ( ↓ 25%) in the F0 animals. However, this isolated finding was not attributed to treatment due to lack of a dose response, no corresponding decrease in relative weight, no corrobor ative ovarian lesions, and the change was not seen across generations in this study nor in the follow-up study. No treatmentrelated histopathological changes we re seen in the testes, epididym ides, seminal vesicle, prostate, coagulating gland, ovaries, uterus, vagina, cervix or the pituitary glands at any dose in either generation.

The LOAEL for parental toxicity was 125 ppm (9/10 mg/kg/day, M/F) based on reductions in body weight and food consumption. The parent al NOAEL was 50 ppm (3/4 mg/kg/day, M/F).

Offspring toxicity of cyfluthr in was manifested at 125 and 40 0 ppm. At 125 ppm, treatment
related coarse tremors were observed in 16% of the F1 pups and 73% of the F2 pups during lactation days 5-17. Treatment-related decrea ses (decreased 6-14%) in mean body weight were observed between 4-21 days in F1 and F2 pups. At 400 ppm, treatment-related coarse tremors were observed in F1 and F2 pups between days 5 and 17 of lactation. These tremors wereCYFLUTHRIN FINAL

Page 52 of 68 observed in 54% of the F1 pups and 36% of the F2 pups. A treatm ent-related reduction (decreased 6-26%) in mean litter weights was observed in F1 and F2 pups between days 0 and 21 of lactation. The offspring LOAEL was 125 ppm (19 mg/kg/day) base d on coarse tremors during lactation and decreases in mean litter weights. The offspring NOAEL was 50 ppm (7 mg/kg/day).

Multi-Generation Reproduction in Rodents (Rat; OCSPP 870.3800)

In a supplemental 2-generation reproduction study ( MRID 44371402

, 1997 ) cyfluthrin (95.5% purity) was administered to 30 Sprague Dawley ra ts/sex/dose at dose leve ls of 0, 25, or 50 ppm in the diet (equivalent to dos es of 0, 1.9 or 3.8 mg/kg/day in males; 0, 2.1, or 4.2 mg/kg/day in females during premating; and 0, 2.0 or 3.9 mg/kg/ day during gestation in females). Exposure to P animals (30/sex/dose) began at 7 weeks of ag e and lasted for 10 weeks prior to mating to produce F 1 pups. Upon weaning, F 1 pups (30/sex/dose) selected to become parents of the F 2 generation were fed cyfluthrin in test diets at the same concen tration their dam received. The F 1 animals were given test diets for 11 weeks prio r to mating. All animals were mated on a 1:1
ratio.

The objectives of the present study were to de termine the reproducibility of the effect on pup weight and to verify and/or establish a reproductive NOAEL for cyfluthrin at 50 ppm. There was no evidence of parental toxic ity of cyfluthrin at 25 or 50 ppm. There were no treatmentrelated clinical findings or incr eases in mortality. There were no significant differences in body weights between the controls, treated P males, P females, F 1 males, or F 1 females during the premating interval. There were no treatment-rela ted changes in food consumption in males or females. During gestation and lactation there were no treatment-related effects on body weights, body weight gains, or food consumption of dams. No treatment-related effects were observed in the reproductive performance (male or female fert ility indices, gestation index, gestation length, live birth index, viability index, la ctation index, parturition, or feta l sex ratio) at any dose tested.

No changes were seen in absolute or relative ov arian weights in either generation. The absolute and relative testicular weights were significantly (p<0.05) decreased ( ↓ 6-8%) in the F1 males, but this decrease was not considered to be biolog ically significant due to lack of a dose response, and no corresponding decrease in relative testes we ight. In addition, the change was not seen across generations either in this study or in the previous study (d iscussed above). No treatmentrelated histopathological changes we re seen in the testes, epididym ides, seminal vesicle, prostate, coagulating gland, ovaries, uterus, vagina, cervix or the pituitary glands at any dose in either generation.

The LOAEL for parental toxicity was not observed. The parental NOAEL is 50 ppm (3.8/4.2 mg/kg/day). The reduction in pup weights observe d at 50 ppm in the previous two-generationCYFLUTHRIN FINAL

Page 53 of 68 reproductive study (MRID 44371401) could not be re produced in this study. The LOAEL for offspring toxicity was not observed. The offspring NOAEL is 50 ppm (3.8/4.2 mg/kg/day).

Hershberger Assay Rat

In a study examining the potential anti-androgeni c effect of several pyr ethroid pesticides ( Zhang
et al. , 2008 ), cyfluthrin or β -cyfluthrin (purities not reported; cis/trans ratio is not stated) were
assessed in an in vivo Hershberger assay. Three-week old male SD rats were maintained under standard laboratory conditions and provided with standard laboratory diet and water ad libitum

. After an acclimation period of seven days, the rats were castrated on PND 28 and allowed to recover for two weeks prior to in itiation of dosing on PND 42. Af ter the recovery period, rats were weighed and randomly assigned to dose groups (n=6). The anti-androgenic Hershberger assay included ten treatment groups: a sham control (uncastrated); a vehicle control group (peanut oil only); a control group dosed with peanut oil (orally) and testosterone propionate (TP) by s.c. injection at 0.5 mg/kg/day ; a positive co ntrol group dosed orally with flutamide (50 mg/kg/day) plus TP; three groups dosed orally cyfluthrin at 16, 18 or 54 mg/kg/day plus TP; and three groups dosed orally with β -cyfluthrin at 4, 12 or 36 mg/kg/ day plus TP. All groups were dosed for seven days. After the final dose, rats were anaesthetized and sacrificed by exsanguination. Blood from each rat was collect ed for serum analysis of testosterone concentration. The seminal vesicles, ventral pr ostate, dorsolateral pr ostate, LABC, Cowper’s glands, glans penis, liver, paired adrenal glan ds, spleen, and paired kidneys of each rat were dissected and weighed. Serum testosterone c oncentrations 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 assay. There were no significant diffe rences between initial or final body weights, or weight gains of the cyflut hrin-treated groups compared to the control groups. Serum
testosterone concentrations were not shown for the cyfluthrin-treat ed rats, but it was reported that no treatment-related effects were observed. Comp ared with the control group (TP plus peanut oil), there was no treatment-related effect on wei ghts of liver, kidney and sp leen in the flutamide and cyfluthrin groups. Pairs of adrena l glands weights of the 36 mg/kg/day β -cyfluthrin plus TP group were significantly highe r than those of peanut oil plus TP control group

As expected, dosing castrated rats with TP incr eased the accessory sex organ weights to normal levels compared to hormone deficient peanut oil control group, and fl utamide, a known antiandrogen, significantly (p<0.05) inhibited the TP-i nduced growth of accessory sex organs. Both cyfluthrin and β -cyfluthrin demonstrated anti-androgenic activ ity. With the exception of the glans penis, treatment with either cyfluthrin or β -cyfluthrin (in combination with TP), resulted in dose-dependent decreases in the weights the se m inal vesicle, ventral prostate, dorsolateral prostate, LABC, and Cowper’s glands, when comp ared to the TP+peanut oil control, These weight decreases attained significant (p<0.05) at both the mid- and high-dose for each chemical.CYFLUTHRIN FINAL

Page 54 of 68 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 cyfluthrin and β -cyfluthrin (no androgenic por tion), and the castrated rats were dosed with TP at 0.5 mg/kg instead of 0.2-0.4 mg/kg/day. However, because the control group (castrated + TP) exhibited similar growth of accessory sex tissues to the sham-operated group (uncastrated), the 0.5 mg/kg dose of TP was acceptable.

Subchronic Oral Toxicity in Rodents (Rat; OCSPP 870.3050)

In an subchronic oral toxicity study ( MRID 41244108, 1986 ), Wistar (BOR:WJSW strain) rats (15/sex/group) were treated with beta-cyfluth rin (99.7% purity) in the diet at 0. 30, 125 or 500 ppm (0. 2.3/2.5. 9.5/10.9 or 38.9/42.4 mg /kg/day, males/females. resp ectively). In addition.
15/sex were treated with beta-c yfluthrin in the diet at 0 or 500 ppm (0 or 37.0/43.0 mg/kg/day, males/females. respectively) and then allowed to recover on control diet for an additional period of 4 weeks The dietary mixtures were prepared weekly and based on stability analyses were
considered to be stable.

Three deaths at 30 ppm and 1 death at 125 ppm we re observed and were be lieved to have been due to improper blood collection techniques. At 5 00 ppm, 2 deaths occurred, 1 each in the main study and in the recovery study. The cause of deat h at this dose level was not determined and may have been due to the test material. Lack of coordination and poor general condition were
also observed at this dose leve l during the first week of trea tment (not reported by day). Necrosis in the head and neck regions was seen in 6 males and l female. Two males also had a "sore". In males, the mean body weight gain was lower throughout the study (weekly range of 8- 80% of the control value). In females, the m ean body weight gain was lower during weeks l, 6 and 10 (range of 16-67% of the control value). In addition, water consumption was reduced by 19% when compared to the control groups. No treatment-related change s were seen in the absolute or relative weights of the liver, te stes or adrenal glands , and no treatment-related histopathological lesions were seen in the testes, uterus, ma mmary, thyroid, adrenal, or pituitary glands.

The NOAEL for the 3-month rat feeding study was 125 ppm (9.5/10.9 mg/kg/day for males/ females, respectively). The LOAEL was 500 ppm (37.0/43.0 mg/kg/day for males/females, respectively) based on lack of coordination, poo r general condition, decreased body weight gain and water consumption, necrosis and sores in the head and neck regions and mortality.CYFLUTHRIN FINAL

Page 55 of 68 Subchronic Oral Toxicity in Non-Rodents (Dog; OCSPP 870.3050)

In subchronic oral toxicity study ( MRID 41267801, 1987 ), beta-cyfluthrin (99% purity) was administered to beagle dogs (4/sex/dose) at dose levels of 0, 10, 60, or 360 ppm in the diet for 13 weeks; actual achieved doses were 0, 0.39/0.39, 2.36/2.5 or 13.9/15.4 mg/kg/day [M/F], respectively. The following observations/measurements were conducted: clinical signs, body weights, food consumption, ophthalmological exam inations, body temperature, femoral pulse, neurological examinations, hematology, clinical chemistry, urinalysis, organ weights, gross examinations and microscopic examinations.

No treatment-related effects were observed at the low and mid-doses. At the high dose, the following clinical signs were noted : awkward, staggering gait and occasional buckling of the hind limbs (both sexes, 4/8 dogs) and an increase in incidence of vomiting (bot h sexes). In 3/8 dogs, the gait abnormalities began 6-8 hours after feeding during Week 1 (not reported in terms of days; in the fourth dog, the gait abnorma lities did not begin until after week 1). In addition, a suggestive decrease in body weight gain was observed in high-dose females (0.35 kg versus 1.0 kg in controls). No treatment-related changes were seen in absolute or relative weights of the testes, prostate, ovaries, thyroid or adre nal glands. No treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate ovaries, uterus , vagina, thyroid, adrenal or pituitary glands.

The NOAEL is 2.36/2.5 mg/kg/day for males/fe males, respectively. The LOAEL is 13.9/15.4 mg/kg/day for males/females, respectively, based on gait abnormalities, increased incidence of vomiting, and suggestive decr eased body weight gain.

4-Week Inhalation Toxicity in Rodents (Rat; Non-guideline)

In a subchronic inhalation toxicity study ( MRID 41783001, 1989 ), Bor:WISW (SPF-Cpb) rats (10/sex/concentration) were dynamically exposed by head/nose only inhalation to β -cyfluthrin (97.9% purity) in ethanol/polyethyl ene glycol 400 (1: 1) at concen trations of 0 (air control, 0 (vehicle control), 0.00026, 0.0027 or 0.023 mg/L for 6 hours/day, 5 days/week for 4 weeks (equivalent to 0, 0.07, 0.73 or 6.3 mg/kg/day).

At 0.0002 mg/L, no treatment-related effects were observed. Although the decrease in mean body weight gain at this dose level a ppears to be biologically significant ( ↓ 67-80%) compared to the vehicle control group, it was not considered to be toxicologically significant, as mean body weights were 96% of the vehicle control valu es. At 0.0027 mg/L, sta tistically significant decreases in body weight were observed in both sexes (p ≤ 0.01, ≤ 10%) as well as decreased urinary pH in males. In addition, there was a decr ease in mean respiratory rate in males. At the highest concentration tested (0.023 mg/L), additio nal observations include clinical signs in both sexes (10/10/sex: ungroomed fur and piloerecti on; increased activity with slightly reducedCYFLUTHRIN FINAL

Page 56 of 68 motility at all times); decrease in absolute and relative spleen a nd thymus weights in both sexes (p ≤ 0.01); increase in alka line phosphatase (p ≤ 0.01, 21% in females); d ecrease in cholesterol (p ≤ 0.01, females) and slight decreases in protein, potassium, calcium, phosphorus and lymphocytes in females (p ≤ 0.01 for all).

With regard to specific endocrine endpoints, n o treatment-related changes were seen in absolute or relative weights of the testes, ovaries, thyr oid or adrenal glands. In addition, no treatmentrelated histopathological lesions we re seen in the testes, epididym ides, seminal vesicle, prostate, ductus deferens, ovaries, uterus, mammary, thyroid, adrenal or pituitary glands.

The LOAEL is 0.0027 mg/L (0.73 mg/kg/day), based on decreases in body weight in both sexes and decreased urinary pH in males. The NOAEL is 0.00026 mg/L (0.07 mg/kg/day).

90-Day Inhalation Toxicity (Rat; OCSPP 870.3465)

In a subchronic inhalation toxicity study ( MRIDs 00157793, 40082901, 40239301; 1983-1987 ), Bor:WISW (SPF-Cpb) rats (10/se x/dose) were dynamically exposed by head-only inhalation to cyfluthrin (94.9% purity) in etha nol/polyethylene glycol 400 (1:1) at concentrations of 0 (air control), 0 (vehicle control), 0.00009, 0.00071, or 0.00451 mg/L for 6 hours/day, 5 consecutive days/week for 13 weeks (estimated to be e quivalent to 0, 0.02, 0.16 or 0.91 mg/kg/day).

All animals survived the 13-week study, and no treatment-related changes were observed in organ weight, gross pathology, and histopathology. At 0.00009 mg/L, total decreases in mean body weight (6%, p<0.05) and mean body weight gain (21%) were observed in males by study termination, when compared to the control group. Because there only appeared to be a slight effect on body weights in males and there were no ot her effects at this dose level, the apparent decrease in body weight gain is not considered to be toxicologically sign ificant. At 0.00071 mg/L, total decreases in mean body weight (8%, p<0.01) and mean body weight gain (21%) were observed in males by study termination when compar ed to the control gro up. Ten of 10 females were observed to display unthr iftiness/unkempt fur and lethargy following exposure on Days 42 to 86. These signs were not present on days where no exposure took place. In addition, increased urinary protein was noted in mid- and high-dose males at 6 and 12 weeks. At 0.00451 mg/L, 10/10 of both sexes were observed to disp lay the same clinical signs as the mid-dose group on Days 13-88 for males and 9-86 for female s. Agitation and “erect tails” were also observed in these animals, starti ng at Week 5. On days when no exposure took place, these signs were still present, although only slightly. Mean body weights in high-dose males were decreased by 14% at study termination and mean body weight gain (weeks 0-13) was decreased by 46% when compared to the control group. Decreas ed urinary pH was also noted; however, no corresponding lesions were found to ve rify any possible nephritic damage.CYFLUTHRIN FINAL

Page 57 of 68 With regard to specific endocri ne endpoints, no treatment-related ch anges were seen in absolute or relative weights of the testes, ovaries, thyr oid or adrenal glands. In addition, no treatmentrelated histopathological lesions were seen in the testes, ovaries, thyroid or adrenal glands.

The LOAEL is 0.00071 mg/L (0.16 mg/kg/day), ba sed on decreases in body weight and body weight gain in males and clinical sign s in females. The NOAEL is 0.00009 mg/L (0.02 mg/kg/day).

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

In a chronic/carcinogenicity study ( MRID 00137303

, 1983 ), cyfluthrin (49.7-51.0% purity in a premix concentrate in Wessalon S) was administered for 24 months in the diet to Wistar SPF rats (65/sex/dose) at dose levels of 0, 50, 150 or 450 ppm (equivalent to 0, 2.02, 6.19 or 19.20 mg/kg/day in males and 0, 2.71, 8.15 or 25.47 mg/kg/day in females).

There were no treatment-related deaths and no tr eatment-related changes noted in the clinical observations, food consumption, hematology, urinalys is, and the gross or microscopic data. No ophthalmologic examinations were performed. Mean body weights of the high-dose males and females were lower ( ↓ 5-10%) than the controls throughout the study. Mean body weights of the
mid-dose males were also lower than the c ontrols during the first year of the study ( ↓ 4-5%), with a body weight gain 6% less than controls), but the animals re covered thereafter.

No treatment-related changes were se en in absolute or relative we ights of the testes, ovaries or adrenal glands at any dose level, and no treatment-related histopathological lesions were seen in the testes, seminal vesicle, prostate, ovaries, ut erus, thyroid, adrenal or pituitary glands at any dose level. Significant (p<0.05) in creases were observed in the in cidences of inflammatory foci of the kidneys in the mid-dose females (7/50 treated versus 1/50 controls) and the high-dose females (7/49 treated) and in hyperplastic nodules of the adre nals in the high-dose animals (males: treated = 20/50, control = 10/48; females: treated = 18/49, control = 4/50). Medullary hyperplasia was also observed in high-dose males (14/50 treated vs 4/48 c ontrols). In addition, compared to concurrent controls, the following we re observed: (i) increased (75%; p<0.01) liver N-demethylase activity in the high-dose females after 7 days of dosing; and (ii) increased
fluoride levels in the bones of the mid- a nd high-dose males (11-21%; p<0.05) and the high-dose females (19%; p<0.01), as well as in the te eth of the high-dose males (20%; p<0.01).

The chronic LOAEL was 450 ppm (equivale nt to 19.20 mg/kg/day in males and 25.47 mg/kg/day in females) based on decreased body wei ghts (up to 10%) in both sexes. The chronic NOAEL was 150 ppm (equivalent to 6.19 mg/kg/day in males and 8.15 mg/kg/day in females). There was no evidence of carcinogenicity in the study.CYFLUTHRIN FINAL

Page 58 of 68 Chronic Oral Toxicity/Carcinogenici ty in Rodents (Rat; OCSPP 870.4300)

In another combined chronic toxicity/carcinogenicity study ( MRID 44459301

, 1997 ), cyfluthrin (94.7% purity), was administered to separate 1- and 2-yr sacrifi ce groups of Fischer 344 rats. The 1-year sacrifice grou p consisted of 40 animals (20 males and 20 females) in both the control and high dose groups and 20 animals (10 males and 10 females) in both the low and intermediate
dose levels for a total of 120 animals. The tw o-year sacrifice group c onsisted of 100 animals (50males and 50 females) in all 4 dose groups for a total of 400 animals. Nominal dietary concentrations of 0, 50, 225, 450 ppm corresponde d to mean daily cyfluthrin intakes of approximately 0, 2.6, 11.6, 22.8 mg/kg/day for males and 0, 3.3, 14.4, 28.3 mg/kg/day for
females.

At 225 ppm, mean body weights were reduced by ≤ 7% in both sexes and mean body weight gains were decreased by ≤ 12% and ≤ 10% for males and females, respectively, compared to the control group. At 450 ppm, mean body weights were decreased by ≤ 13% and ≤ 14% and mean body weight gains were decreased by ≤ 23% and ≤ 24% for males and females, respectively. An increased frequency of alopecia was also obser ved in 450-ppm males and females (M: 17, 16, 17, 30; F: 30, 35, 34, 43). There were treatment-re lated effects on mortality, food consumption, hematology, urinalysis, or gross pathology. In addition, there were no tr eatment-related changes in absolute or relative testes and ovary weights. No treatment-related histopathological lesions were seen in the testes, epididymides, seminal ve sicle, prostate, preputia l gland, ovaries, uterus, vagina, cervix, clitoral gland, thyroi d, adrenal or pituitary glands at any dose level. There was a statistically significant increase in the incidences of mammary gland adenocarcinomas in females at the high dose (4/50; 8%) compared to controls (1150; 2%). However, with the exception of one animal that died on Study Day 668 with a fibroa denoma, all the tumors were seen in animals
that were sacrificed at the end of the study.

The LOAEL was 225 ppm (11.6 and 14.4 mg/kg/day fo r male and female rats, respectively), based on decreases in body weight gain of 12% and 10% in male s and females, respectively. The NOAEL was 50 ppm (2.6 and 3.3 mg/kg/day [M/F]).

Carcinogenicity in Rodents (Mouse; OCSPP 870.4200)

In a carcinogenicity study ( MRID 00137304, 1983 ), cyfluthrin (49.7-51.0% purity as a premix concentrate in Wessalon S) was administered in the diet for 23 months to SPF mice (50/sex/dose) at dose levels of 0, 50,200 or 800 ppm (equivalent to 0, 11.6, 45.8 or 194.5 mg/kg/day in males and 0, 15.3, 63.0 or 259.9 mg/kg/day in females).

There were no treatment related changes noted in the clinical observa tions, food consumption, hematology, gross observations, organ weight s and microscopic pathology data. No ophthalmologic examinations or urinalyses were performed. The mortality data were equivocalCYFLUTHRIN FINAL

Page 59 of 68 with respect to treatment. The incidence of mo rtality in the mid- and high-dose females was somewhat higher than the c ontrols throughout the study. Ho wever, only the mid-dose at termination was significantly (p ≤ 0.05) different from controls. At study termination, mortality in the females was 52, 60, 74 and 68% for the c ontrol, low-, mid- and high-dose groups,respectively. Mortality in the males was 80, 78, 82, and 88% for the control, low, mid- and highdose groups, respectively at study termination. M ean body weights relative to controls were no more than 10% less for mid- and high-dose male s and 9% less for the mid- and high-dose females throughout the study. The mean body weight s of the treated group s, however, were not significantly different at termin ation, possibly due to the morta lity differences in the dosed groups compared to the concurrent controls.

In the clinical chemistry data, alkaline phosphatase activity was elevated in treated males at all dose levels when compared to controls at each time point except termination where there was evidence that the samples were hemolyzed, leadin g to spurious results. A dose-response was evident at months 6 (143-230%; p ≤ 0.05 or 0.01) and 12 (137-73%; p ≤ 0.05 or 0.01); however, it
was somewhat flat for the low- and mid-dose groups at Month 12. At Month 18, there was no dose-response. The low-dose group had the highest elevation in alkaline phosphatase ( 1 114%,

p ≤ 0.05) while the mid- and high-dose groups remained somewhat flat (161 and 68%, respectively (p<0.05 and 0.01: the odd significance values indicate some wide-ranging individual values in at least one dose group) . The histopathology data did not confirm the liver as a target organ. Under the conditions of this study, th ere was no evidence of carcinogenic potential.

With regards to specific endocrine endpoints, there were no treatmen t-related changes in absolute or relative weights of the testes or ovaries at any dose level, and no treatment-related histopathological lesions were seen in the testes, seminal vesicle, prostate, ovaries, uterus, mammary glands, thyroid, adrenal or pituitary glands at any dose level.

Carcinogenicity in Rodents (Mouse; OCSPP 870.4200)

In a mouse oncogenicity study ( MRID 44589701, 1998 ), cyfluthrin ( ≥ 93.9% purity) was administered in the diet to CD-1 mice ( 50/sex/group) for up to 80 weeks at 0, 200,750, or 1400/1600 ppm (equivalent to 0/0, 31.9/ 38.4, 114.8/140.6, and 232.7/309.7 mg/kg/day [M/F], respectively). A supplementa ry submission was made ( MRID 45228101, 2000 ) containing the neoplastic incidence summary. The high-doses we re chosen based on the results of a 6-week range-finding study, and the selected doses incl ude the estimated maximum tolerated doses for each sex for a lifetime study in the mouse. Mo rtality, food efficiency, hematology, and organ weights for both sexes at all doses were unaffected by treatment with cyfluthrin. There were no observations of toxicological c oncern in either sex at 200 ppm or in the females at 750 ppm.

At 750 ppm in males, a toxic effect on the ears was observed and considered to be treatmentrelated based on macroscopic and microscopic pathology findings. Ma croscopic findingsCYFLUTHRIN FINAL

Page 60 of 68 included crusty zone of the skin of the ear in the males (11150 treated vs 5/50 controls). Microscopic findings involving the skin of the ear in the males included: acanthosis (16/50 treated vs 9/50 controls); chronic active in flammation (11150 treated vs 4/50 controls); inflammation, all types (13/50 treat ed vs 6/50 controls); ulcer ( 12/50 treated vs 5/50 controls); and debris (13/50 treated vs 4/50 controls). Monthly body weight gain was reduced at the first month in the males ( ↓ 21 %), but not fo r the overall study.

At 1400/1600 ppm [M/F], a toxic effect on the ears was also observed and considered to be treatment-related based on clinical signs a nd macroscopic and micr oscopic findings. The following were increased in the females at clinic al examination: ear lesion redness (5/50 treated vs 0/50 controls) and ear lesion scab (22/50 treated vs 2/50 cont rols). Related gross necropsy findings included crusty zone of the skin of the ear in the males (12/50 tr eated vs 5/50 controls) and females (13/50 treated vs 1/50 controls). Re lated microscopic findings observed in the skin of the ear both sexes, including: acanthosis (16- 17/50 treated vs 2-9/50 controls); chronic active inflammation (8-10/50 treated vs 1-4/50 controls); inflammation, a ll types (9-16/50 treated vs 1- 6/50 controls); ulcer (5-1150 trea ted vs1-5/50 controls); and debr is (8-11150 treated vs 2-4/50 controls). In addition, at clinical examina tion, an increased incidence of rough coat was observed in the males (30/50 treated vs 9/50 controls) and females (20/50 treated vs 9/50 controls) and hunched back was observed in the females (5/50 treated vs 0/50 controls). Additional gross necropsy findings included rough coat in the males (22/50 treated vs 7/50 controls) and females (16/50 treated vs 8/50 controls) and wet/stained ventrum in the males (7/50 treated vs 0/50 controls). Re ductions in mean body weight we re observed in the males and females ( ↓ 7-20%) throughout the study. Monthly body we ight gain was reduced for the first month in the males and females ( ↓ 60-69%) as compared to contro ls. Overall body weight gain, as calculated by the reviewers, was decreased in the males ( ↓ 25%) and females ( ↓ 54%). Mean absolute food consumption was decreased in the females ( ↓ 6-25%) throughout the study.

With regards to specific endocrine endpoints, no treatment-related changes were seen in absolute or relative weights of the testes, ovaries, or adrenal glands at any dose level. In addition, no treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, uterus, vagina , cervix, clitoral gland, mammar y glands, thyroid, adrenal or pituitary glands at any dose level.

The LOAEL was 750 ppm for males (equivalent to 114.8 mg/kg/day) base d on macroscopic and microscopic ear skin lesions and reduced body weight gains, and 1600 ppm in the females (equivalent to 309.7 mg/kg/day) based on clinical signs, macros copic and microscopic pathology findings, and reduced body weights, body weight gains, and food consumption. The NOAEL for males was 200 ppm (equivalent to 31.9 mg/ kg/day). The NOAEL for females was 750 ppm (equivalent to 140.6 mg/kg/day). There was no evidence of carc inogenic potential under the conditions of this study.CYFLUTHRIN FINAL

Page 61 of 68 Chronic Oral Toxicity in No n-Rodents (Dog; OCSPP 870.4100)

In a chronic toxicity study ( MRIDs 44435401, 1997; and 45189201, 2000 ), cyfluthrin (94.8- 95.1% purity) in corn oil was administered to beagle dogs (4/sex/dose) in th e diet at dose levels of 0, 50, 100, 360, or 500 ppm for 12 months; actual achieved doses were of 0/0, 1.36/1.46, 2.43/3.61, 10.64/10.74, or 15.47/17.99 mg/kg/day [M/F], respectively. High-dose animals initially received 640 ppm, but the dose was re duced to 500 ppm at Week 8 due to excessive toxicity.

One high-dose female was sacrificed on Day 56 after exhibiting gait (i ncluding incoordination and increased stride width), postural, and beha vioral abnormalities, convulsions, tremors, and cranial and peripheral nerve deficits. The high- dose was reduced to 500 ppm for the remainder of the study. One control male died on Day 318 and one control female died on Day 210 from asymptomatic idiopathic epilepsy.

In the 360 ppm animals, clinical signs (males and females combined) included abnormal posture (1/8 treated vs 0/8 controls, st arting on day 183) and a higher inci dence of vomiting (8/8 treated vs 4/8 controls, starting on day 1). Gait abnormalities, includi ng hypermetria, abnormal stride width, and reluctance to walk, were found at the 6 month (2/4 males, 1/ 4 females) and 12 month (1/4 males, 2/4 females) neurological exam inations. Abnormal postural reactions, including “wheelbarrowing” with head position abnorma l or dorsally flexed, abnormal front foot placement during lateral hopping, hemistanding, and abnormal foot placement or weight support during backward stepping, were also found at the 6 month (4/4 males, 3/4 females) and 12 month (4/4 males, 3/4 females) examinations. No postural or gait abnormalities were observed in
controls.

At 500 ppm, clinical signs (males and female s combined) included gait abnormalities such as reluctance/inability to walk, stiff-leggedness, atax ia, and abnormal stride width (1-2/8 treated vs 0/8 controls); seizures (2/8 trea ted vs 0/8 controls); convulsions and tremors (2/8 treated vs 0/8 controls); and abnormal posture (1/8 treated vs 0/8 controls). Vomiting (8/8 treated vs 4/8 controls), diarrhea (4/8 treated vs 0/8 controls) and soft feces (2/8 treated vs 0/8 controls) were also observed. Gait abnormalities similar to those observed in the 360 ppm group were found at the 6 month (3/4 males; 1/3 females) and 12 mo nth (2/4 males; 1/3 females) examinations. Abnormal postural reactions sim ilar to those observed in the 360 ppm group, but also including
abnormal time to initiate front leg lateral hopping, were found at the 6 month (4/4 males; 3/3 females) and 12 month (4/4 males; 3/3 female s) examinations. Again, no postural or gait
abnormalities were observed in controls. Non-sta tistically significant decreases in body weights were observed in the males throughout the study (decreased 6-20%). Mean body weight gains, as calculated by the reviewers, were decreased at the end of treatment in the males ( ↓ 53%).CYFLUTHRIN FINAL

Page 62 of 68 Hepatic N

− demethylase activity was increased in males at termination, the onl y interval tested (increased 45%).

There were no differences of t oxicological concern observed in rectal body temperature, food consumption, electrocardiogra phy, blood pressure, hematological , clinical chemistry or urinalysis parameters, ophthalmoscopic, necropsy, or histopathological findings, absolute or relative organ weights, and plasma cholinesterase, hepatic O

− demethylase or cytochrome P450 activities. No neoplastic tissue was ob served in dogs from any test group.

With regard to specific endocri ne endpoints, no treatment-related ch anges were seen in absolute or relative weights of the testes, ovaries, thyroid, adrenal or pituitar y glands at any dose level. In addition, no treatment-related hist opathological lesions were seen in the testes, epididymides, prostate, ovaries, uterus, cervix, mammary glands, thyroid, adrenal or pituitary glands at any
dose level.

The LOAEL was 360 ppm (equivalent to 10.64/10.74 mg/kg/day [M/F]), base d on clinical signs, gait abnormalities, and abnorma l postural reactions in males and females. The NOAEL was 100 ppm (equivalent to 2.43/ 3.61 mg/kg/day [M/F]).

Avian Reproduction (Duck; OCSPP 850.2300)

In an avian reproductive study, ( MRID 00158782, 1986) 16-week old mallard ducks ( Anas platyrhynchos ) were administered cyfluthr in (94% purity) in the feed at nominal concentrations of 0 (acetone/corn oil, 0.06/0.02% v/w), 10, 50 or 250 ppm. Actual mean measured concentrations in the diets were 9.87, 42.7 and 262 ppm, respectively. The ducks were dosed for a total of 21 weeks, beginning 9 weeks prior to the start of egg production and then through 12 weeks of egg production. Each treatment group was comprised of 16 mating pairs, with one mating pair per pen. A progres sive photo period wa s employed in this study: 8 hours/day through Week 9 and 17 hours/day until the end of the study. Temperature was maintained at 74±7°F throughout the study and humidity was main tained at ca. 82%. Individual adult body weights were recorded at Week 0, 4, 6, 8 and st udy termination, and food consumption in each replicate was measured weekly throughout the study. Eggs were collected, assessed for cracks, and incubated; eggs were checked for viability on days 14 and 21. On day 24 the set eggs were transferred to a hatchling compartment. Indi vidual chick body weight s were recorded after hatching and on Day 14 after hatching; egg sh ell thickness was measured every two weeks during egg production. Adult birds and chicks were observed routin ely for mortality and clinical signs.

No mortality or clinical signs of toxicity were no ted at any dose level. At dietary concentrations up to 250 ppm, the study author reported ther e was no treatment-related effect on adult body weight, body weight gain, or food consumption. Th ere were also no signif icant treatment-relatedCYFLUTHRIN FINAL

Page 63 of 68 effects on any reproductive parameters. In th is study, there was also observed low control reproductive performance, as indicated by a low number of eggs lai d, embryo viability, and hatching rate compared to guideline criteria. The reviewer did note that the treated males lost more weight than control males from week 0 to 8 and gained less weight than control males by study termination. Additionally, the reviewer note d that pathological resu lts showed an increase in the incident of slight egg yolk peritonitis in the treatment- groups compared to the control ( ≥ 10 mg/kg-diet). However, these reported observa tions by the reviewer did not impact the overall NOAEC. The NOAEC for the study is 250 ppm, the hi ghest dose tested, and a LOAEC was not
observed.

In another duck reproduction study ( MRID 00145331, 1984 ), 16-week old mallard ducks ( Anas platyrhynchos ) were administered cyfluthrin (90 .5 % purity) in the feed at nominal concentrations of 0 (acetone/corn oil, 0.03/ 0.01% v/w), 250, 1000, or 4000 ppm for 24 weeks (10 weeks prior to the start of egg production and 14 weeks of egg production). Each treatment group was comprised of 12 mating pairs, with on e pair per pen. A progressive photo period was employed in this study: 7 hours/day through Week 9 and 17 hours/day until the end of the study. Temperature was maintained at 64-73°F througho ut the study and humidity was maintained at 30-78%. Individual adult body weights were reco rded at Week 0, 4, 8 and 24, and body weight changes were determined for each interim period. Food consumption in each replicate was measured weekly throughout the pr e-treatment and treatment periods of the adult phase. Eggs were collected beginning on Week 10, and egg ha ndling and hatching procedures, measurements and observations were as per guidelines. Eggs hell thickness and strength measurement were made on Weeks 1, 3, 5, 7 and 9 of laying. Indivi dual chick body weights were recorded after hatching and on Day 14 after hatching; egg sh ell thickness was measured every two weeks during egg production. Adult birds and chicks were observed routin ely for mortality and clinical signs.

There was no reported treatment-related effect on adult mortality or clinical sign in any dose group, and no significant difference in food c onsumption. The study author’s reported a significant reduction in male and female body weight during the firs t four weeks of the study at the high concentration.

With regards to reproductive effects, there was a dose-dependent reduction in the number of eggs laid/hen at the mid and high doses, which attained significance at the high dose. At both the mid and high doses, there were also significant reducti ons in the number of eggs laid, eggs set, fertilized eggs, 3-week viable embryos, hatchlings and 14-day su rvivors. A high percentage of eggs cracked were observed at the low dose (250 mg/kg-diet), but this effects was not observed at the higher treatments. However, due to low control reproductive perf ormance, including low embryo viability, hatchling number, and 14-day survi vorship, there is uncertainty associated with the results of this study.CYFLUTHRIN FINAL

Page 64 of 68 A strong trend of significant repr oductive impairment is evident at > 250 ppm. The impairment at 250 ppm is tentative but not resolvable by th is study because of poor control response and because of too many cracked eggs at 250 ppm.- A definitive NOEL is not established.

The one-generation reproductive toxici ty of beta cyfluthrin to 16 pairs per level of 22-week old mallard duck ( Anas platyrhynchos ) was assessed over ca . 21 weeks ( MRID 48350615, 1990 ). Beta cyfluthrin was administered to the birds in the diet at nominal concen trations of 0 (negative control), 10, 50, and 250 mg ai/kg diet. Mean -measured concentrations were <0.5 (LOQ, control), 10.5, 53.1, and 269 mg ai/kg di et. No mortalities or signs of toxicity were observed in the control or any of the treatment groups during the 21-week study. Lesions or abnormal behavior noted during the study were attributed to pen wear and tear, and in some instances, excessive interaction between pen mates. No tr eatment-related effects were observed for feed consumption, changes in body weight, or reproduc tive parameters. No statistically significant differences upon egg shell thickness were obser ved at any treatment level. The NOEL was 250 mg ai/kg diet.

Avian Reproduction (Quail; OCSPP 850.2300)

In an avian reproduction study ( MRID 00145330, 1984 ), 20-week old bobwhite quails ( Colinus virginianus ) were administered cyfluthr in (90.5 % purity) in the feed at nominal concentrations of 0 (acetone/corn oil, 0.03/0.01% v/w), 250, 1000, or 4000 ppm for 23 weeks (8 weeks prior to the start of egg production and 15 weeks of e gg production). Mean measured concentrations were 275, 1010 and 3825 ppm, respectively. Each treatment group was comprised of 24 mating pairs, with one pair per pen. A progressive pho to period was employed in this study: 7 hours/day through Week 8 and 17 hours/day until the end of th e study. Temperature was maintained at 64- 73°F throughout the study and humidity was ma intained at 30-78%. Individual adult body weights were recorded at Week 0, 4, 8 and study termination, and body weight changes were determined for each interim period. Food consump tion in each replicate was measured weekly throughout the pre-treatment and tr eatment periods of the adult phase. Eggs were collected beginning on Week 11, and egg handling and hatching procedures, measurements and observations were as per guideli nes. For eggshell thickness and strength measurements were made every 2 weeks beginning on Week 14. Indi vidual chick body weights were recorded after hatching and on Day 14 after hatching. Adult bi rds and chicks were observed routinely for mortality and clinical signs.CYFLUTHRIN FINAL

Page 65 of 68 One control female, and one male and one female in the high-dose died on test, but no clinical signs for toxicity were reported sign in any dose group. There was no significant difference in food consumption, and no treatment-related eff ects were noted on body weight or body weight gain for the females. For the males, body we ight and body weight gains were significantly reduced at the high dose (4000 ppm). No reproductive effects were noted in the low- and middose groups. The high-dose group had fewer eggs laid/hen, a lower hatching percentage, and fewer 2-week old chicks, compared to the cont rol; however, only 2-week old chick survivors were significantly different from the control. Si gnificant reductions in egg shell thickness were observed on Weeks 16, 18 and 20 at the low dose, on Weeks 16 and 18 at the mid-dose, and on Weeks 16, 18, 20 and 22 at high dose.

In a follow-up study examining eggshell strength and thickness ( MRID 00152829, 1985 ), bobwhite quail were administered cyfluthrin at nominal concentratio ns of 12.5, 49.9, 228, 926 or 4298 ppm in the diet. Adult birds were observed daily for clinical signs and mortality. Body weights were recorded at weeks 0, 5, and 14. Th e first egg was laid at the end of week 9; thereafter, eggs were collected daily through the first two days of week 13. Al1 eggs laid were tested for shell strength and th ickness. Thickness and strength were compared between groups for the study as a whole and for each day on wh ich eggs were laid. The NOAEC for eggshell thinness was determined to be >4000 pp m based on a 31⁄2 week egg laying period.

Fish Life-cycle Toxicity (Fathead Minnow; OCSPP 850.1500)

In a fish life-cycle study ( MRID 41450401, 1990 ), newly fertilized eggs of Fathead minnows (less than 24 hours post-fertilization) were obtained from U.S. Fish and Wildlife Service, Columbia, MO. The embryos resulting from thes e fertilized eggs formed the stocks for two generations of testing for survival, growth and reproduc tion. Mean measured water concentrations of cyfluthrin were 0.0 18, 0.033, 0.065, 0.14 and 0.25 μg/L representing 100 to
116% of nominal.

In the F 0 phase of the study, percent hatch ranged from a low of 75.7% at 0.033 μg/L to a high of 92.1% at 0.018 μg/L, with no statistical signifi cance found for any of the groups when compared
to the controls. For the F 1 generation, hatching ranged betwee n 72.9% at 0.25 μg/L to 94.3% at 0.14 μg/L. Within replicates success ranged betw een 46% at the highest concentration to 100% at 0.14 μg/L.

The F 1 generation hatching ranged between 72.9% fo r 0.25 μg/L to 94.3% in 0.14 μg/L. Within replicates success ranged between 46% in 0.25 μg/L and 100% in 0.14 μg/L. Frequency analysis gave a significant reduc tion in hatch in the F 1 generation at the highest test concentration.CYFLUTHRIN FINAL

Page 66 of 68 Parental survival showed signi ficant differences from the c ontrols only at the highest concentration of 0.25 ug/L (53%) for the Day-7 to 60 post-hatch group. The to 60 to 120 posthatch groups as far as Day-301 showed no signi ficant differences from the controls although mortality started to increase in the pe riod between Day 153 to termination at 301.

For the F 1 generation the controls were pooled because frequency analysis and the Fisher's Exact test showed no significant differe nces. Further, only at the hi ghest concentration (0.25 μg/L) significant differences in survival were found when compared to the controls after approximately 60 days of post-hatch exposure.

Standard length and wet weight as measures of growth were not a ffected at any treatment level in either the parental or F 1 generations. Five parameters we re used to indicate reproductive success: spawns/pair; number of eggs/pair; eggs/spawn; number of reproductive days and number of eggs/pair/ reproducti ve day. These parameters were tracked from study Day 159 to study day 307. The total level of spawns for the parental generation showed a significant decrease at 0.033 μg/L, when compared to co ntrols. At 0.14 μg/L. no overall statistical significant difference with the controls was obs erved. The control had the largest number of eggs/spawn.

There were no significant differenc es between the pooled controls a nd any of the test levels for the mean number of eggs/spawn in either gene ration, but the number of spawns and eggs per adult or pair of adults, as we ll as, the number of reproductive days, were lowest for the 0.033 and 0.25 μg/L. The solvent control and the 0.25 μg/l treatment level had the lowest eggs/pair/ reproductive day. The greatest numbe r of reproductive days (days from the first to the last spawn) was observed at 0.018 μg/L. None of th e test concentrations showed a significant decrease in this parameter. No significant differe nces were observed, at an y test level, with the controls for the mean number of eggs/pair/reproductive day parameter. For the parental generation, mean values for this parameter ra nged between a high value of 38.2 for the blank control and a low value of 13.6 at 0.25 μg/L. For mean percent hatch, survival, standard length and wet weight results in the F 1 , no significant reduction from the pooled controls was observed.

Fish Early-Life Stage Toxicity (OCSPP 850.1400)

In a sheepshead minnow ( Cyprinodon variegatus ) early life-cycle study ( MRID 00155898,

1986 ) using cyfluthrin (90.5%), fi sh were exposed to cyfluthrin concentrations of 50, 100, 200, 400, 800, and 1600 ng a.i./L for 39 days. Additionally, a negative and solvent control were used. Mean measured concentrations ranged from 12.2 to 527 ng a.i./L, and high variability within a treatment was observed. Endpoints analyzed included: embryo mortality and time to hatch, larval survival and growth (dry weight). Redu ced juvenile survival of sheepshead minnow was
observed at concentrations ≥ 0.084 μg/L. The no-effect concentration was 24.7 ng a.i./L.CYFLUTHRIN FINAL

Page 67 of 68 In a rainbow trout ( Oncorhynchus mykiss ) early life-cycle study ( MRID 00158781, 1985 ) using cyfluthrin (96%), fish (embryos /larvae) were exposed to cyflut hrin concentrations of 25, 50, 100, 200, and 400 ng a.i./L for 58 days. Additionally, a so lvent control, but not negative control was used. Mean measured concentrations ranged from 10 to 160 ng a.i./L, and high variability within a treatment was observed. Significant effects on growth were reported at concentrations of

≥ 0.018 μg/L for rainbow trout. A number of fish showed behavioral sign s at concentrations of 50 ng/L (nominal concentrations) and higher. Based on mean measured concentrations, the NOAEC was 10 ng a.i./L.CYFLUTHRIN FINAL

Page 68 of 68 APPENDIX 3: References Not Utilized in the Cyfluthrin 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 Cyfluthrin but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the com pound of interest was not us ed in the study; 3) the test material was not adequately described; 4) a formulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only da ta on invertebrates.

Deanovic, L.A., et al . (2013). Comparing the Effectiveness of Chronic Water Column Tests with
the Crustaceans Hyalella azteca (Order: Amphipoda) and Ceriodaphnia dubia (Order: Cladocera) in Detecting Toxicity of Current-Use Insecticides. Environmental Toxicology and Chemistry . 32(3):707-712.

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