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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

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

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

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

.

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

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

### EDSP: WEIGHT OF EVIDENCE ANALYSI S OF POTENTIAL
INTERACTION WITH THE ESTROG EN, ANDROGEN OR THYROID
PATHAWAYS

CHEMICAL: PHOSMET

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYPHOSMET 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..................................................14

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................18

IV. Committee’s Assessment of Weight of Evidence..........................................20

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................20

1. Tier 1 in vivo Assays.............................................................21

2. OSRI...............................................................................22

B. Estrogen Pathway...........................................................................24

C. Androgen Pathway.........................................................................24

D. Thyroid Pathway...........................................................................24

E. Conclusions.................................................................................25

V. EDSP Tier 2 Testing Recommendations....................................................25

VI. References.......................................................................................27

LIST OF TABLES: Table 1. Tier 1 Assay for Phosmet.....................................................................8
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Phosmet ....................................11
Table 3. Androgenic/Anti-Andr ogenic Pathway for Phosmet ....................................15
Table 4. Thyroid Pathway for Phosmet ............................................................19

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................30

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

APPENDIX 3. References Not Utilized in the Phosmet WoE Analysis...............................53PHOSMET 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 eneration
F2 Second filial g eneration
Fcd Fecundit yPHOSMET FINAL

iv Abbreviation Terminology

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 Concentration
MTD Maximum Tolerated Dose
N Ne g ative
NE Not examined/evaluate dPHOSMET FINAL

v Abbreviation Terminology

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)

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)PHOSMET FINAL

vi Abbreviation Terminology

VC Vehicle Control VO Vaginal Opening

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

Page 1 of 55 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 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

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

On April 9, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) anal ysis of the potential interaction of phosmet with the E, A or T hormone pathways. The T1 WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in

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

Phosmet is a member of the orga nophosphate class of insecticides whose primary mode of toxic
action is cholinesterase inhibi tion leading to neurotoxicity ( e.g ., cholinergic signs).

For the estrogen pathway, while the Tier 1 estrogen receptor (ER) binding assay was considered inadequate (due to solubility issues), the Tier 1 ER transactivation assay (ERTA), as well as an additional ERTA study which evaluated bot h agonism and antagonism was negative. Furthermore, the Tier 1 uterotrophic assay was negative. While the Tier 1 in vitro aromatase assay was positive, the steroidogenesis assay was negative. There were no estrogen-related
effects observed in the Part 158 mammalian and wildlife studies, except for an increased incidence of dilation and myometrial atrophy of the uterus observed at 12 and 24 months, respectively, in the mouse carcinogenicity study. In the female pubertal assay, although all test doses were considered to be overtly toxic, no estrogen-related effects were observed in this study. Similarly, the mid and high treatment groups in the fish short-term reproduction assay (FSTRA) were considered to be overtly toxic, and the information obtained from the low concentration was limited (essentially due to the loss of one of four replicat es). In the FSTRA at this low concentration, effects on female gonada l histopathology [decreased yolk synthesis, and occasional disruptions of follicular vitellogenin (V TG) concentration] were observed. It is notedPHOSMET FINAL

Page 2 of 55 that there were no other effects in the low c oncentration observed incl uding alterations in gonadal somatic index (GSI) and plasma VTG. Ther efore, the available da ta do not suggest that phosmet has the potential to act as an estrogen agonist based on the in vitro and uterotrophic study. Additionally, while the in vitro aromatase assay was positive which could indicate a potential decrease in es tradiol production, the in vitro steroidogenesis was negative and the results of both the female pubertal and FSTRA, do not support an anti-est rogenic effect.

For phosmet, while the Tier 1 AR binding a ssay was positive, the Tier 1 Hershberger was negative and no androgen-related eff ects were observed in the Tier1 male pubertal assay in the absence of overt toxicity. Additionally, the Tier 1 steroidogenesis assay wa s negative. In the FSTRA, based on the data available, there were no androgen-related effects observed in the absence of overt toxicity. The only androgen-rela ted effect observed in the mammalian Part 158
studies was an increase in the in cidence of testicular atrophy in the mouse carcinogenicity study. Overall there is no convincing evidence for pot ential interaction with the androgen pathway.

In the Tier 1 male pubertal assay, T4 levels were increased and changes in thyroid histopathology (decreased colloid ar ea and increases in follicular cell height) were observed at both doses, although the effects at the high dose occurred in the pr esence of overt toxicity. Similarly, thyroid effects (increas ed T4 levels and histopathology) were observed in the Tier 1 female pubertal assay; however, both test doses were considered to be overtly toxic in this assay.
In the Part 158 mammalian studi es, histopathological changes in the thyroid were observed at 10.9 mg/kg/day in the chronic rat study. In the amphibian metamorphosis assay (AMA), while developmental alterations were observed ( ↓ NF stage and HLL), there were no effects on thyroid histopathology. Therefore, the observed developmenta l delays are likely not thyroid-mediated.

Overall, for phosmet there is no convincing evidence for a potential interac tion with the estrogen or androgen pathway in mammals or wildlife. The available data indicate a potential interaction with the thyroid pathway in mammals, but not amphibians.

In the EDSP Tier 1 male and female pubertal and OSRI, there was evidence for potential interaction with the thyr oid pathway in studies conducted with adult animals, but no such data exists for potential thyroid t oxicity in the young animals.

For phosmet, the current Point of Departure (POD) of 3.56 mg/kg/day used for human health risk assessment is based on red blood cell (RBC) c holinesterase inhibition, which is the most sensitive endpoint for this class of chemicals, observed at 7.0 mg /kg/day in rats. This POD was used for deriving the chronic Reference Dose (RfD ) used for assessing chronic dietary risk for the general population. A second P OD of 0.16 mg/k/day used for deriving the chronic RfD for the young (the most sensitive population) is ba sed on RBC cholinesterase inhibition in the offspring. This POD was also used for calcula ting the Margins of Exposure (MOEs) used for non-dietary risk assessments.PHOSMET FINAL

Page 3 of 55 In the case of phosmet, although no data exists for potential thyroid to xicity in the young, a comparative thyroid assay (CTA) is not recomme nded since: 1) the POD used for assessing risk for the young is approximately 63-fold lower than the low doses (10 mg/kg/day) that caused thyroid-related effects in the Tier 1 in vivo pubertal assays; 2) the P OD for the adults and the young is based on the most sensitive endpoint (cho linesterase inhibition); 3) the CTA would be tested at higher doses and would not be expected to yield a POD lower than the level currently used for risk assessment; and 4) the chronic RfD and MOEs are protective of the thyroid related findings in the Tier 1 in vivo assays. Therefore, mammalian EDSP Tier 2 testing is not recommended for phosmet since additional testing is not expected to impact EPA’s current regulatory point of departur es and endpoints for human h ealth risk assessments.

As there was no evidence of potential interaction with the estrogen or androgen pathway in fish, or thyroid pathway in amphibians, additional testing is not recommended for these taxa.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for phosmet. In bobwhite quail, effects on reproduc tive parameters were observed at concentrations where mortality a nd adult body weight decreases were observed; no reproductive effects were observed in the mallard duck study. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from phosmet exposure. Relative to the EDSP, additional testing is not recommended.

.PHOSMET FINAL

Page 4 of 55 I. Introduction

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

In determining whether a pestic ide chemical interacts with E, A or T hormone pathways, the number and type of effects i nduced, the magnitude and pattern of responses observed across studies, taxa and sexes were considered. Additionally, the conditions under which effects occur were considered, in part icular, whether or not endocrine-rela ted responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On April 9, 2014, the EDSP Tire 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence analysis of the potential interac tion of phosmet 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).

Phosmet is a synthetic organophosphate pesticide, and its mode of action is based on altered neurotransmission due to cholinesterase (ChE) inhibition. Phosmet has a low vapor pressure (3.7–4.9 × 10 -7 mm Hg at 25°C), and therefore, volatili zation is not expected to significantly contribute to the dissipation of phosmet in the environment. The compound has a water solubility of 20–25 mg/L at (20–25°C) and an octanol /water partition coefficient (log K ow ) of 2.78 to 2.95. Phosmet is degraded by hydrolysis and atmos pheric photolysis. Phosmet has a whole body
bioconcentration factor value in fish ranging from 10 to 44.

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

Page 5 of 55 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 for phosmet 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 Orde r 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 re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review T eam on phosmet (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any
additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies 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-mediated processes ( i.e., E, , or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

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

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). Th e EDSP Tier 1 data include of elven assaysPHOSMET FINAL

Page 6 of 55 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 incl ude 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 in teract with E, A, and T consisting of different levels of biological organization from a molecular initiating ev ent, such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment and fecundity at the whole organism level. The extent of expression of responses at to high er levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biological organization? Finally, under what conditions did the responses occur including co nsideration of whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-mediated effect from a non-endocrine toxi c response.PHOSMET FINAL

Page 7 of 55 This uncertainty in distinguish ing whether the responses were endocrine-mediated was discussed at the FIFRA SAP meeting in that evaluated scien tific 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 ., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

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

Page 8 of 55 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 phosmet are shown below
in Table 1

.

Table 1. Tier 1 Screening Assays for Phosmet.

Tier 1 Assays Test Guideline Test Order Status
ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement not satisfied 1 (MRID 48618704)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement satisfied (MRID 48618705)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement satisfied (MRID 48618703)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement satisfied (MRID 48618701)

Aromatase Assay (Human recombinant microsomes) OCSPP 890.1200 Requirement satisfied (MRID 48618702)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement satisfied (MRID 48618707)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement satisfied (MRID 48618706)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement not satisfied 2 (MRID 48673003)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement satisfied (MRID 48673003)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement satisfied 3 (MRID 48673002)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement satisfied (MRID 48673001)

1 This assay is not satisfied due to issues with test chem ical solubility. However a repeat of this assay is not required since negative results were seen in the Tier 1 ERTA and OSRI which evalua ted potential interaction of phosmet with the ER.

2 This assay is not satisfied due to overt toxicity seen at both doses tested. However, a repeat of the assay is not required since it would have no impact on the risk assessment.

3 Although the mortality in the high treatment level, clinical signs of toxicity in the intermediate treatment level, and noteworthy rate of oocyte atresia in control females make the results difficult to interpret, when considered in context of other available Tier 1 EDSP data for phosmet, this study satisfies the EDSP Tier 1 Test Order requirement for a Fish Short-Term Reproduction Assay (OCSPP Guideline 890.1350).PHOSMET FINAL

Page 9 of 55 B. Effects on Hypothalamic-Pit uitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for phosmet to interact with 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.

Phosmet was negative in the Tier 1 steroidogenesis assay and positive in t he T ier 1 aromatase assay in which aromatase activity was decreased to 34% at 10

− 4 M. Although there was no
evidence of an interactio n of phosmet with the ER in the Ti er 1 ER binding assay, issues with test chemical solubility were not adequately addressed which confounde d the interpretation of the assay and lessened confidence in the results. However, phosme t was negative in the Tier 1 ERTA and negative for ER agonism and antagonism i n a published literature study by Kojima et
al. (2004) which evaluated the potential to interact with two human estrogen receptor subtypes (hER α and hER β ) in transactivation assays in Chines e hamster ovary cells (CHO-K1 cells).

Phosmet was negative in the Tier 1 in vivo uterotrophic assay. In th e Tier 1 female pubertal

assay, persistent tremors were seen at both doses (10 and 20 mg/kg/day) tested, and body weight losses (9-10%) were seen at the high dose (20 mg/kg/day) indicating overt toxicity. Although
both doses were considered to be overtly toxic, it is noted that there were no treatment-related effects on vaginal opening, cycle status at necrop sy, mean age at first estrus, mean cycle length, percent cycling, percent regularly cycling or histopathological cha nges in the ovaries or uterus.

In the FSTRA, the mid-and high-treatment groups were considered to be overtly toxic as survival was 9% in the highest concentration tested and data from this treatment levelwere not statistically analyzed. In the mid-concentration, in addition to a 25% decrease in survival, there were generalized overt signs of t oxicity in female fish, includi ng lethargy in all surviving fish, and loss of equilibrium in females. At the low concentration, survival was reduced by 13%, whereas 3 out of 4 females in one replicate di ed, thereby reducing the ability to evaluate potential effects in this treatment. At the mid-concentration, there was a significant 81% decrease in fecundity, with a 7% decrease in fertilization. Additionally at this concentration, gonado-somatic index (GSI) was decreased ( ↓ 34%). Gonadal histopat hology was affected as evidenced by decreased yolk synthesis in th e low- and mid-concentration females, and occasional disruptions of follicular VTG con centration. There were no effects on plasma vitellogenin (VTG) concentrations at the low or mid-treatment.

In the Part 158 mammalian toxicity studies, in the 2-generation reproduc tion study in the rat, absolute and relative ovarian weight s were decreased in the high-dose P 0 females at doses that caused significant decreases ( ≥ 10%) in body weight. The viabili ty index was decreased at thePHOSMET FINAL

Page 10 of 55 high dose in the rat reproduction st udy; however, maternal overt toxicity and substantial ChE inhibition was also present at this dose. An increased incidence of dilation and myometrial atrophy of the uterus were observed at 12 and 24 months, respec tively, in the mouse carcinogenicity study at doses that caused si gnificant decreases in brain ChE activity.

In the Part 158 mallard duck reproduction study, th ere were no significant effects determined for any reproductive parameters assessed. In the bobwhite quail study, signif icant reductions were determined for eggs laid but these effects we re observed at the hi ghest treatment group (150 mg/kg-diet) where increased mort ality and decreased body weight relative to the control group were also observed. In the Part 158 fish early life stage study with ra inbow trout, effects on growth were observed at 6.1 μg a.i/L.PHOSMETFINALPage 11 of 55Table 2. Estrogenic/Anti-Estrogenic Pathway for PhosmetLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Phosmet1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histo p atholo gy 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 48618704)IERTA(MRID 48618705)NAromatase(MRID 48618702)PSteroidogenesis(MRID 48618701)NUterotrophic(MRID 48618707)NN NFemale Pubertal Rat(MRID 48673003)--4--4--4--4--4--4--4--4X(L,H)CS;BW(L,H)4FSTRA(MRID 48673002)NENP5(L)NN NX6(M,H)OSRIER transactivationassay;(Kojima,et al., 2004)NDevelopmental toxicity(Rat; MRID 41962902)NNX(H)NDevelopmental toxicity(Rat; MRID 00063192)NRNX(M, MH,H)X(MH, H)PHOSMETFINALPage 12 of 55Table 2. Estrogenic/Anti-Estrogenic Pathway for PhosmetLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Phosmet1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histo p atholo gy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt) / Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Developmental toxicity(Rabbit;MRID 41962901)NNX(H)X(H)Developmental toxicity(Monkey;MRID 00043397)NRNNNTwo-generationreproduction(Rat; MRID 41520001)NR N NNX(L,M,H)7X(H)Chronic toxicity(Rat; MRID 41916401)NENNNEX(L,ML,MH,H)X(H)Carcinogenicity(Mouse;MRID 00141659)P(H)NENNEX(M, H)NChronic toxicity(Dog;MRID 00076436)NRNRNRNRX(H)NAvian reproduction(Duck;MRID 00105999)NN NAvian reproduction(Quail;MRID 00125786)NNX(M, H)1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay.Changes in weight are absolute unless otherwise indicated. CS=Clinical signs; BW=Body weightPHOSMETFINALPage 13 of 552. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by anX in this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Both doses in the assay were considered to be overtly toxic, therefore no findings are reported in the table for these parameters..5. Gonadal histopathology was affected as shown by decreased yolk synthesis in the low-concentration females, and occasional disruptions of follicularVTG concentration.6. At the mid and treatment groups were considered to be overtly toxic as survival was 9% in the highest concentration tested and data from this treatmentwere not statistically analyzed. In the mid-concentration, inaddition to a 25% decrease in survival, there were generalized overt signs of toxicity infemale fish, including lethargy in all surviving fish, and loss of equilibrium in females7. Increased incidence of dilation and myometrial atrophy of the uterus were observed at 12 and 24 months, respectively.I InadequateP Positive findingsN Negative findings (in vitro) / No effect (in vivo)NE Not examinedNR Not reportedPHOSMET FINAL

Page 14 of 55 2. Effects on Androgen Pathway

The potential for phosmet to interact with the androgen pathway is summarized in Table 3. The various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for androgenic, anti-androgenic or HPG axis effects. This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reporte d in the table and not considered further in the WOE assessment.

Phosmet was negative in the Tier 1 steroidoge nesis assay. In the Tier 1 AR binding assay, phosmet showed >50% binding, with an IC 50 value of 10 -5 M. A published literature study that screened phosmet for its potential to interact with human AR in a transact ivation assay in CHO-K1
cells. In this, phosmet was reportedly negative fo r AR transactivation up to the highest concentration tested, 10 -5 M ( Kojima et al. 2004 )

In the Hershberger assay, there we re no increases in AST in the agonist phase of the assay. In the antagonist phase, decreases in all five AST wei ghts were observed at the high dose (25 mg/kg/day) which was determined to be overtly toxic ( 12% decrease in body we ight and tremors).

In the Tier 1 male pubertal rat assay, there were no effects on age or weight at preputial separation (PPS), hormone concentrations, or gross and micr oscopic pathology. Slight decreases in ventral prostate weights (NS; ↓ 9 and 15%) were observed in the lo w (10 mg/kg/day) and high-dose (20 mg/kg/day) males, respectively. tr emors were seen at both doses ( 10 and 20 mg/kg/day) tested. At the 10 mg/kg/day, mild tremors were seen during one day only in 6/16 animals. At 20 mg/kg/day, mild to moderate tremors were seen on multiple da ys in 11/16 animals. The high dose in this assay was determined to be overtly toxic based on the frequency and severity of the tremors.

In the FSTRA, as mentioned in th e estrogen pathway sec tion, the mid- and high treatments were overtly toxic, and a limited number of replicates in the low treatment available for evaluation. In this study, there was a 7% decrease in fertiliza tion at mid dose, and histological examination indicated an increase in the pr oportion of spermatogonia in the te stes at the high concentration. There were no other androgen-relate d effects observed in the study.

In the mammalian Part 158 studies, testicular atrophy was observed in hi gh-dose (14 mg/kg/day) males at termination (24 months) in the m ouse carcinogenicity study. In the 2- generation reproduction study, absolute testis wei ghts were decreased in high-dose F 0 and F 1 parents, with no treatment-related histopathologi cal lesions seen in the test es in either parental group. In both studies, significant reductions in brain and/or RBC ChE activity were reported at the doses that
showed effects on the testes. In a OSRI, phosmet had no effect on testis weight, sperm count or sperm morphology in mice dosed with phosme t (50 mg/kg/day) for 5 days (Quinto et al. 1989). There were no androgen-related e ffects identified for the available Part 158 wildlife studies.PHOSMETFINALPage 15 of 55Table 3. Androgenic/Anti-Androgenic Pathway for PhosmetLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Phosmet1Study Type / LiteratureCitation AR Binding Steroidogenesis Testosterone Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility (Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1AR Binding(MRID 48618703)PSteroidogenesis(MRID 48618701)NHershberger(MRID 48618706NENX(L, H)CS; BW(H)4Male Pubertal Rat(MRID 48673003)NNNNNNNNNX(L, H)X(H)5FSTRA (MRID 48673002)NNNNNX6(M,H)PHOSMETFINALPage 16 of 55Table 3. Androgenic/Anti-Androgenic Pathway for PhosmetLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Phosmet1Study Type / LiteratureCitation AR Binding Steroidogenesis Testosterone Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility (Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRIMale Reproductive toxicity(Mouse, Quintoet al., 1989)N NNTwo-generationreproduction(Rat; MRID 41520001)N NNX(L, M,H)7X(H)Chronic toxicity(Rat; MRID 41916401)N N NE N NE NX(L,ML,MH,H))NCarcinogenicity(Mouse;MRID 00141659)NP7NNNENEX(M, H);NChronic toxicity(Dog; MRID 00076436)NRNRNRNRNRNRX(H)NAvian reproduction(Duck; MRID 00105999)NN NAvian reproduction(Quail; MRID 00125786)NNX(M, H)1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changesin weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventralprostate (VP), Dorsalprostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), Glans penis (GP). CS=Clinical signs;BW=Body weight2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by anX in this column. For details see Section IV. A3. The overt toxicity in the Tier 1 assays are presented in this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. APHOSMETFINALPage 17 of 554. At the high dose, mean body weight was decreased (p≤0.05) by 12% on Day 11 (with a NS trend to decrease noted on Days 4 and 7), and body weightgains were decreased by 60-69%, compared to the control. Clinical signs of toxicity (tremors and uncoordinated movements) werealso noted at the highdose.5. Clinical signs (tremors) were seen at both doses tested (10 and 20 mg/kg/day)6. At the mid and treatment groups were considered to be overtly toxic as survival was 9% in the highest concentration tested and data from this treatmentwere not statistically analyzed. In the mid-concentration, inaddition to a 25% decrease in survival, there were generalized overt signs of toxicity infemale fish, including lethargy in all surviving fish, and loss of equilibrium in females7. Testicular atrophy at 24 months at the high dose (18 mg/kg/day)P Positive findingsN Negative findings (in vitro) / No effect (in vivo)NE Not examinedNR Not reportedPHOSMET FINAL

Page 18 of 55 C. Effects on Hypothalamic-Pitui tary-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 phosmet to interact w ith thyroid regulation is summarized in Table 4. The various targets of the th yroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. This table also includes HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the ta ble and not considered further in the WOE assessment.

In the Tier 1 female pubertal assay, T 4 concentrations were increased by 24 and 38%, respectively, in the low- and high-dose groups, but no treatment-r elated effects were noted for thyroid weight or TSH concentration. However, bot h doses in this assay were considered to be overtly toxic based on persistent tremors seen at both doses (10 and 20 mg/kg/day) and body weight losses (9-10%) seen at the high dose (20 mg/kg/day). In the male pubertal assay, T 4 levels were increased by 48 and 43%, respectively, in the low ( 10 mg/kg/day)- and high-dose (20 mg/kg/day) groups, with no trea tment-related effects noted for thyroid weight or TSH concentration. The high dose in th is assay was determined to be overtly toxic based on the frequency and severity of the tr emors observed. Marginal decreas es in thyroid colloid area and increases in follicular cell height were observe d in all animals in the pubertal rat studies. No effects on pituitary weight were not ed in either pubertal study.

In the AMA, there were no histological effect s on the thyroid. Phosmet decreased normalized hind-limb length (HLL) at the intermediate ( ↓ 12 and 15%) and high concentrations ( ↓ 10 and 29%) on Days 7 and 21, respectively, with a 6.7% decrease in snout-vent length on Day 7 in the high-dose group. Median NF stage was unaffected on Day 7, although a delay was observed on Day 21 in the intermediate and hi gh treatment groups (Stage 58 vs . 59); no asynchronous development was observed.

In the mammalian Part 158 studies, no changes in thyroid weight or hist ology were seen except for mineralization of the thyroid in medium -high dose females (5/50 vs. 0/50) at study termination (24 months) in the mouse carcinogenicity study.PHOSMET FINAL

Page 19 of 55 Table 4. Thyroid Pathway for Phosmet

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

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

EDSP Tier 1 Assays

Female Pubertal Rat (MRID 48673003) -- 4

-- 4

-- 4

-- 4

-- 4 X (L, H) CS; BW (L, H) 5
Male Pubertal Rat (MRID 48673003) N P 6 ↑ 48% (L) N N X (L, H) CS (H)

AMA (Frog; MRID 48673001) N HLL: ↓ 12, 9.6% Day 7, ↓ 15,

27% Day 21 (M, H) SVL: ↓ 6.7% (Day 7, H) N

OSRI Chronic toxicity (Rat; MRID 41916401) N P (MH) NE NE NE X (L,ML, MH,H) X (H)

Chronic oncogenicity (Mouse; MRID 00141659) N N NE NE N X (M, H) N

Chronic toxicity (Dog; MRID 00076436) NR NR NR NR NR X (H) N

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

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 4. Both doses in the assay were considered to be overtly toxic, therefore no findings are reported in the table for these parameters. 5. Persistent clinical signs (tremors) observed at both doses tested (10 and 20 mg/kg/day) and body weight loss (9- 10%) at 20 mg/kg/day. 6. Marginal decreases in thyroid colloid area and increases in follicular cell height were observed in all animals N Negative findings P Positive findings NE Not examined NR Not reportedPHOSMET FINAL

Page 20 of 55 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 phosmet to interact w ith endocrine mediated processes ( i.e., E, Aor 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 analysis of those lines 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 evalua tion in helping elucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively a sequelae of system ic effects. Endocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses 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. In addition to the endocrine -related effects described above
for the in vivo Tier 1 assays and OSRI, other eff ects observed which may be considered overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. However, in other instances, mo re than one parameter was needed to determine overt toxicity. Effects which were considered to be signs of system ic toxicity were generally less severe forms of toxicity (e.g., changes in organ w eights or blood chemistry).PHOSMET FINAL

Page 21 of 55PHOSMET FINAL

Page 22 of 55 1. Tier 1 in vivo Assays

ChE activity was not measured in any of the mammalian in vivo Tier 1 assays. In the Hershberger assay, signs of clinical toxicity in cluded tremors and/or uncoordinated movement up to four hours post-dose were seen in most of the high- dose (25 mg/kg/day) animals; no clinical signs were observed at doses ≤ 15 mg/kg/day. At the high dose, body weight gain was decreased (p<0.05) by 69% in the androgenic po rtion of the Hershberger assay. In the anti-androgenic portion of the assay, body weight and body weight gain were decreased (p<0.05) by 12% and 60%, respectively, at Study Day 11. Organ weights, other than for the acce ssory sex organs, were not evaluated in the androgenic and anti-androgenic parts of the assay.

In the uterotrophic assay, no clin ical signs of toxicity were observed at either dose (5 or 20 mg/kg/day). After the 5-day exposure period, final body weight s and body weight gains at both doses were comparable to control.

In the female pubertal assay, tremors were seen in 10/16 rats at the 10 mg/kg/day dose level (during 10 days) and all animals at the 20 mg/kg/da y dose (during 18 days). The severity of the tremors ranged from “not characterized to moderate.” Decreased body weights (9 to 10%) were observed at the high dose during PND 26 to 36. Additionally, intermittent body weight gains in this group were decreased (p<0.05) by up to 30%, but overall body weight gain was decreased (NS) by only 4%.

In the male pubertal assay, tremors were observed in one 10 mg/kg/day rat (time of occurrence not provided) and in 11 of 16 rats at 20 mg/kg/day. Body weight differences intreated animals versus control were <10%, and overall body weight gain was decr eased (p<0.05) only 6 to 7%.

In the FSTRA, survival was decreased in all tr eatment groups; 83, 75 and 9% in the low-, midand high-dose groups. However, 3 of the 4 females that died in the low treatment group were in the same replicate (replicate A). There were clin ical signs of overt toxi city in the mid and high treatments (e.g., lethargy, loss of equilibrium). Body weights in the intermediate-concentration group were decreased (NS) by 12 to 15%. In add ition, there was increased oocyte atresia in all treatment groups (69% in the control group and 36–83% in the treatment groups) due to microsporidia infections but fecundity in th e control and low treatment groups, 16 and 19 eggs/female/day/replicate, respectivel y, did not appear to be affected.

In the AMA, no overt or systemic signs of toxici ty were observed at test concentrations up to 1.0 mg/L. Bent tail (spinal curvat ure) was observed (30 to 49%), but this finding occurred in the control and the two lower phosmet concentratio ns compared to the high concentration.PHOSMET FINAL

Page 23 of 55 2. OSRI

In the Part 158 studies, the most sensitive endpoint across studies was cholinesterase inhibition.

In the subchronic neurotoxicity study in rats , ChE activity was determined in whole blood, plasma, RBC, and six separate regions of th e brain. In the low-dose group (1.5/1.6 mg/kg/day, m/f), mean RBC ChE activity was decrea sed (p<0.01) in males at Week 13 ( ↓ 19%) and females at Week 7 ( ↓ 42%). Decreases (p<0.05 or 0.01) were also observed at Week 13 in mean plasma ChE activity in females ( ↓ 29%), mean whole blood ChE activity in both sexes ( ↓ 16–19%), and ChE activity of the olfactory bulb ( ↓ 36%) and brain stem ( ↓ 21%) regions in females. In the middose group (2.7/3.1 mg/kg/day, m/f), decreased (p <0.05 or 0.01) mean ChE activities were observed in plasma ChE in the males at Week 3 ( ↓ 21%) and females at Weeks 3 and 13 ( ↓ 27-46%); RBC ChE in the males at Weeks 3, 7, and 13 ( ↓ 26–39%) and females at Week 7 ( ↓ 38%); whole blood ChE at Weeks 3, 7, and 13 in both sexes ( ↓ 24–36%); and whole brain ChE at Week 7 in the females ( ↓ 20%) and at Weeks 3 and 7 in the males ( ↓ 11–17%). In the high-dose group (9.4/11 mg/kg/day, m/f), d ecreased (p<0.05 or 0.01) mean ChE activities were observed in plasma ChE at Weeks 3, 7, and 13 in both sexes ( ↓ 23–71%); RBC ChE at Weeks 3, 7, and 13 in males ( ↓ 65–70%) and at Weeks 3 and 7 in females ( ↓ 66–89%); whole blood ChE in both sexes ( ↓ 59–74%) at Weeks 3, 7, and 13; whole brain ChE in both sexes ( ↓ 43–68%) at Weeks 3 and 7; and in all six brain regions in the females ( ↓ 36–67%) and in all brain regions (except for olfactory region) in the males ( ↓ 27–52%) at Week 13.

In the two-generation reproduction study, tremors we re exhibited in the hi gh-dose (23 mg/kg/day)

F 0 and F 1 animals. Serum ChE activity was decreased in mid-dose (6.1 mg/kg/day) F 0 females (p<0.01, ↓ 24%) and high-dose F 0 /F 1 males (p<0.01, ↓ 30/41%) and females (p<0.01, ↓ 65/63%). RBC ChE activity was decreased in low-dose (1.5 mg/kg/day) F 0 males (p<0.05, ↓ 8%), low-dose

F 0 /F 1 females (p<0.01, ↓ 11/16%), mid-dose F 0 /F 1 males (p<0.01, ↓ 37/48%) and females (p<0.01, ↓ 48/59%), and high-dose F 0 /F 1 males (p<0.01, ↓ 74/85%) and females (p<0.01, ↓ 81/80%).

In the chronic oral toxicity/carcinogenici ty study in rats, dose -related inhibition (p ≤ 0.01 or 0.05) of serum ChE activity occurred in males in the high-dose (23/ 27 mg/kg/day, m/f) group at all sample times and in the high-medium group ( 9.4/10.9 mg/kg/day, m/f) at 18 months, and in females in the high-medium- and high-dose groups at all sample times and in the low-medium group at 6 months. RBC ChE was inhibited (p ≤ 0.01 or 0.05) in males and females in the lowmedium (1.8/2.1 mg/kg/day, m/f), high-medium and high-dose groups at all sample times, except
at 12 months in the low-medium animals. RBC ChE also was inhibited in the low-dose males (1.1 mg/kg/day) only at the 6-month sa mple time. Brain ChE was inhibited (p ≤ 0.01) in males and females in the high-medium- and high-dose gr oups at all sample times, and in the 24-month low-dose samples in males (p<0.05).PHOSMET FINAL

Page 24 of 55 In the mouse carcinogenicity study, a dose-relate d trend in the incidence of convulsions was attributed to phosmet administration. Inhibiti on (significance not repor ted) of plasma ChE activity occurred in males in the low (1.0/1.2 mg/kg/day, m/f) and mid- (4/5 mg/kg/day, m/f) dose groups and mid-dose females ( ↓ 11 to 13%) at the interim sacr ifice, with lower inhibition ( ↓ 4 to 10%) observed at study term ination. Greater inhibition (s ignificance not reported) of plasma ChE activity was noted in high-dose (1 4/18 mg/kg/day, m/f) males and females ( ↓ 54 to 56%) at the interim sacr ifice, with a similar degree of inhibition ( ↓ 47 to 52%) observed at study
termination. RBC ChE activities were not determin ed at the interim sacrifice, but were not different from control at study termination. Brain ChE was inhibi ted (significance not reported) in males and females in all dose groups to a similar degree ( ↓ 22 to 34) at the interim sacrifice; at study termination, brain ChE activity in males was not different from control, and was decreased only in mid- and high-dose females ( ↓ 14 and 22%, respectively).

In the chronic dog study, brain and RBC ChE inhibition was observed in the high-dose(10 mg/kg/day) animals (significance and details were not reported); no additional treatmentrelated effects on ChE activity were observed at the mid (1.0 mg/kg/day) or low (0.5 mg/kg/day)
doses.

In the 21-day dermal toxicity study in rats, brain ChE activity was decreased (p ≤ 0.01, ↓ 36%) at study termination in high-dose (60 mg/kg/day) ma les, but plasma and RBC ChE activities were unaffected by treatment. In high-dose females, ChE activity was decreased in plasma (p ≤ 0.01, ↓ 38%) on Day 22 and decreased in RBC (p ≤ 0.05, ↓ 29%) on Day 8, and brain ChE activity was inhibited (p ≤ 0.01) in a dose-dependent manner ( ↓ 13, 20, and 61%) in the low (15 mg/kg/day)-, mid (22.5 mg/kg/day)-, and high- (60 mg/kg/day) dose groups, respectively.

In the non-Guideline developmental study (Staples et al ., 1976), animals in the high-medium and high-dose gavage groups (25 and 30 mg/kg/day) died. Additionall y, in the rabbit developmental toxicity study, deaths and signs of clinical toxicity occurred in the high-dose group (15 mg/kg/day).

Several wildlife Part 158 studies were also available. However, it should be noted that ChE was not measured in any of these studies. Observa tions were primarily base d on clinical signs of toxicity. Two avian reproduction studies were conducted including the ma llard and the bobwhite quail, both at 25, 60, and 150 ppm dose levels. Ther e were no reported signs of clinical or overt toxicity in either study.PHOSMET FINAL

Page 25 of 55 B. Estrogen Pathway

While the Tier 1 ER binding assay was considered in adequate (due to solubility issues), the Tier 1 ERTA assay as well as an additional ERTA study which evaluated both agonism and antagonism was negative. Furthermore, the Tier 1 uterotrophic assay was negative. While the
Tier 1 in vitro aromatase assay was positive, the steroidogenesis assay was negative. There were no estrogen-related effects observed in the Part 158 mammalian and wildlife studies, except for
an increased incidence of dilati on and myometrial atrophy of the uterus observed at 12 and 24 months, respectively, in the mouse carcinogenic ity study. In the female pubertal assay, although
all test doses were considered to be overtly toxic, no estrogen- related effects were observed in this study. Similarly, the mid and high treatment groups in the FSTRA were considered to be overtly toxic, and the information obtained fr om the low concentration was limited (due to essentially loss of one of four replicates). In the FSTRA at th is low concentration, effects on female gonadal histopathology (decreased yolk synthe sis, and occasional disruptions of follicular VTG concentration) were observed. It is note d that there were no ot her effects in the low concentration observed including alterations in GSI and plasma VT G. Therefore, the available data do not suggest that phosmet has the potential to act as an estrogen agonist based on the in

vitro and uterotrophic studies. Additionally, while the in vitro aromatase assay was positive which could indicate a potential de crease in estradiol production, the in vitro steroidogenesis was negative and the results of both the female pubertal and FSTRA, do not support an antiestrogenic effect.

C. Androgen Pathway

While the Tier 1 AR binding assay was positive, the Tier 1 Hershberger was negative and no androgen-related effects were observed in the Tier 1 male pubertal assay in the absence of overt toxicity. Additionally the Tier 1 steroidogenesi s assay was negative. In the FSTRA, based on the data available, there were no androgen-rela ted effects observed in the absence of overt toxicity. The only androgen-rela ted effect observed in the mammalian Part 158 studies was an
increase in the incidence of te sticular atrophy in the mouse car cinogenicity study. Overall there is no convincing evidence for potential interaction with th e androgen pathway.

D. Thyroid Pathway

In the Tier 1 male pubertal assay, T4 levels were increased and changes in thyroid histopathology (decreased colloid ar ea and increases in follicular cell height) were observed at both doses, although the effects at the high dose occurred in the pr esence of overt toxicity. Similarly, thyroid effects (increas ed T4 levels and histopathology) were observed in the Tier1 female pubertal assay; however, both test doses were considered to be overtly toxic in this assay.
In the Part 158 mammalian studi es, histopathological changes in the thyroid were observed at 10.9 mg/kg/day in the chronic rat study. In the AMA, while developmental alterations werePHOSMET FINAL

Page 26 of 55 observed ( ↓ NF stage and HLL), there were no effects on thyroid histopathology. Therefore, the observed developmental delays are likely not thyroid-mediated.

E. Conclusions

There is no convincing evidence for a potential interaction with the estrogen or androgen pathway in mammals or wildlife. The available data indicate a potential interaction with the thyroid pathway in mammals, but not amphibians.

V. EDSP Tier 2 Testing Recommendations

In the EDSP Tier 1 male and female pubertal and OSRI, there was evidence for potential interaction with the thyr oid pathway in studies conducted with adult animals, but no such data exists for potential thyroid t oxicity in the young animals.

For phosmet, the current Point of Departure (POD) of 3.56 mg/kg/day used for human health risk assessment is based on red blood cell (RBC) c holinesterase inhibition, which is the most sensitive endpoint for this class of chemicals, observed at 7.0 mg /kg/day in rats. This POD was used for deriving the chronic Reference Dose (RfD ) used for assessing chronic dietary risk for the general population. A second P OD of 0.16 mg/k/day used for deriving the chronic RfD for the young (the most sensitive population) is ba sed on RBC cholinesterase inhibition in the offspring. This POD was also used for calcula ting the Margins of Exposure (MOEs) used for non-dietary risk assessments.

In the case of phosmet, although no data exists for potential thyroid to xicity in the young, a comparative thyroid assay (CTA) is not reco mmended since: 1) the POD for the young is approximately 63-fold lower than the low doses (10 mg/kg/day) that caused thyroid-related
effects in the Tier 1 in vivo pubertal assays; 2) the POD for the adults and the young is based on the most sensitive endpoint (cholinesterase inhibi tion); 3) the CTA would be tested at higher doses and would not be expected to yield a POD lower than the level currently used for risk assessment; and 4) the chronic RfD and MOEs ar e protective of the thyr oid related findings in
the Tier 1 in vivo assays. Therefore, mammalian EDSP Tier 2 testing is not recommended for phosmet since additional testing is not expected to impact EPA’s current regulatory point of departures and endpoints for huma n health risk assessments.

As there was no evidence of potential interaction with the estrogen or androgen pathway in fish, or thyroid pathway in amphibians, additional testing is not recommended for these taxa.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for phosmet. In bobwhite quail, effects on reproduc tive parameters were observed at concentrations where mortality a nd adult body weight decreases were observed; no reproductive effects were observed in the mallard duck study. The type of data obtained fromPHOSMET FINAL

Page 27 of 55 Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from phosmet exposure. Relative to the EDSP, additional testing is not recommended.PHOSMET FINAL

Page 28 of 55 VI. References

Cappon, G.D. (1999). A Dietary S ubchronic (90-Day) Neurotoxicity Study of Phosmet in Rats. Unpublished study performed by WIL Research Laboratorie s, Inc. Study No. WIL-331002. (MRID 44811801).

Chang, J.C.F., Morrissey, R.L., and Wyand, S. ( 1991). 2-Year Chronic Toxicity/Oncogenicity Study with R-1504 in Rats. Unpublished study performed by Ciba-Geigy Corp. Laboratory Report No. T-13241. (MRID 41916401).

Cohle, P

. (1988). Early Life Stage Toxicity of 14 C-Phosmet to Rainbow Trout (Salmo gairdneri) in a Flow-Through System. Unpublished study performed by Analytical Bio-Chemistry Laboratories, Inc. Laborator y Report No. 35777. (MRID 40938701).

Courtney, K.D. (1968). Teratological investigation of Captan, Imidan and Thalidomide in
Macaca mulatta . Final report. Unpublished study performed by Bi onetics Research Laboratories, Inc. (MRID 00043397).

Fletcher, D., Jenkins, D., and Leonard, M. (1982a) . Report to Stauffer Chemical Co.: Toxicity and Reproduction Study with Imidan Technical in Bob-white Quail. Unpublished study performed by Bio-Life Assoc., Ltd Laboratory Report No. BLAL 81 QR2 (T-10817). (MRIDs 00129519, 00125786).

Fletcher, D., Jenkins, D., and Leonard, M. (1982 b). Report to Stauffer Chemical Company: Toxicity and Reproduction Study with Im idan Technical in Mallard Ducks. Unpublished study performed by Bio-Life Associates, LTD Laborator y Report No. BLAL 81DR2 (T-10816). (MRID 00105999).

Hilaski, R.J. (1999). A 21-Day Dermal Toxicity Study of Imidan in Rats. Unpublished study performed by MPI Research. Laboratory Re port No.: 815-002. (MRID 44795801).

Hodge, M.C.E. (1991). Phosmet: Te ratogenicity Study in the Rat. Unpublished study performed by ICI Central Toxicology Laboratory Report No .: CTL/P/3360: RR0544

. (MRID 41962902).

Katz, A.C., Sprague, G.L., Frank, D.W., Turnier, J.C., Zwicker, G.M., and Freundenthal, R.I. (1984). Two-year Dietary Oncogenicity Study in Mice with lmidan Technical: Final Report:

T-10719. Unpublished study performed by Stauffer Chemical Compa ny Laboratory Report No.: T-10719. (MRIDs 00141659, 0016114, and 40595501).

Kojima, H., Katsura, E., Takeuchi, S., et al . (2004). Screening for Estrogen and Androgen Receptor Activities in 200 Pesticides by In Vitro Reporter Gene Assays Using Chinese Hamster Ovary Cells. Environ. Health Perspect. 112(5):524–531. (MRID 48033008).PHOSMET FINAL

Page 29 of 55 Lee, M.R. (2012). Phosmet—Amphibian Meta morphosis Assay with African Clawed Frog (Xenopus laevis) Following OPPTS Test Gu ideline 890.1100 and OECD Test Guideline No. 231. Unpublished study performed by Smithers Viscient, Wareham, Massachusetts 02571. Laboratory report number 12791.6142. Study comple ted February 27, 2012. (MRID 48673001).

Meyer, L.S. and Walberg, J.A. (1990). A Tw o-Generation Reproduction Study in Rats with

R-1504. Unpublished study performed by Ciba-Geigy Corporation Laboratory Report No. T-13260. (MRID 41520001).

Moxon, M.E. (1991). Teratology Study in the Rabbit. Unpublished study performed by ICI Central Toxicology Laboratory Report No.: CTL/P/3373: RB05045. (MRID 41962901).

Quinto, I., DeMarinis, E., Mall ardo, M., Arcucci, A., Della Morte, R., and Staiano, N. (1989). Effect of DNOC, Ferbam and Imidan Exposure on Mouse Sperm Morphology. Mutat Res 224:405–408. (MRID 48084301).

Staples, R.E., Kellam, R.G., and Haseman, J.K. (1976). Developmental toxi city in the rat after ingestion or gavage of or ganophosphate pesticides (Dipte rex, Imidan) during pregnancy. Environ. Health Perspect. 13:133–140. (MRID 00063192).

USEPA. (2010). Phosmet: Report of the Endocri ne Disruptor Review Team — Test Order #: EDSP-057801-36. August 5, 2010. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

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

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

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

Wagner, H. (2011). Phosmet: H295R Steroidog enesis Assay. Unpublished study conducted by CeeTox Inc., Kalamazoo, MI. Laborator y Study No. 9142V-100348STER, November 3, 2011. (MRID 48618701).

Wilga, P.C. and C. Toole. (2011). P hosmet: Human Recombinant Aromatase Assay. Unpublished study conducted by CeeTox Inc ., Kalamazoo, MI. Laboratory Study No. 9142V-100348AROM, November 3, 2011. (MRID 48618702).PHOSMET FINAL

Page 30 of 55 Willoughby, J.A (2011). Phosmet: Androgen Recept or Binding (Rat Prostate Cytosol). Unpublished study conducted by CeeTox, Ka lamazoo, MI. Laboratory Study #: 9142V-100348ARB, November 2, 2011. (MRID 48618703).

Willoughby, J.A. Sr. (2011). Phosmet: Estrogen Receptor Binding (Rat Uterine Cytosol). Unpublished study conducted by CeeTox, Ka lamazoo, MI. Laboratory Study #: 9142V-100348ERB, November 2, 2011. (MRID 48618704).

Willoughby, J.A. Sr. (2011). Phosmet: Estrogen R eceptor Transcriptiona l Activation (Human cell Line, HeLa-9903). Unpublished study condu cted by CeeTox, Kalamazoo, MI. Laboratory Study #: 9142V-100348ERTA, Nove mber 2, 2011. (MRID 48618705).

York, D.O. (2012). Phosmet — Short-term Reproduction Assay with the Fathead Minnow (Pimephales promelas) Following OPPTS 890.1350 and OECD 229 Guidelines. Unpublished study performed by Smithers Viscient, Wareha m, Massachusetts. Lab Study No.: 12791.6143. (MRID 48673002).

Zorrilla, L. (2011). The Hershberger assay with phosmet in castrated CRL:CD(SD) IGS Rats. Unpublished study conducted by Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study ID: C194-200, October 26, 2011. (MRID 48618706).

Zorrilla, L., and Codrea, E. (2011) . The Uterotrophic Assay with Phosmet in Ovariectomized CRL:CD(SD) IGS Rats. Unpublished study conducted by Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study ID : C194-100, October 19, 2011. (MRID 48618707).

Zorilla, L. (2012). Pubertal developmen tal and thyroid function with phosmet in juvenile/peripubertal CRL:CD(SD) IGS female and male rats. Unpublished study conducted by Integrated Laboratory Systems, Durham, NC , Laboratory Study No.: C194-300, February 24, 2012. (MRID 48673003).PHOSMET FINAL

Page 31 of 55 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100)

The 21-day assay (MRID 48673001) of phosmet on amphibian metamorphosis of African Clawed Frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae (80 tadpoles/treatment group, 20/re plicate) at Nieuwkoop-Faber (N F) stage 51 were exposed to phosmet (96.8% purity) at nominal concentra tions of 0 (negative control), 0.020, 0.20, and 2.0 mg a.i./L. Mean-measured concentrations were <0.0022 (<LOQ; control), 0.0081, 0.096, and 1.0 mg a.i./L The test system was maintained at 20 to 23°C and a pH of 7.4 to 8.2.

Phosmet significantly decreased normalized hi nd-limb length (HLL) in non-late stage tadpoles (NF stage < 60) at the mean-measured 0.091 and 1.0 mg a.i./L treatment groups on Days 7 and 21. Median Nieuwkoop-Faber (NF) stage was un affected on Day 7, though a significant delay was observed at on Day 21 at the mean-measured 0.096 and 1.0 mg a.i./L treatment groups. No asynchronous development was observed. Mild thyroid gland hypertrophy, follicular cell hyperplasia, and follicular cell hypertrophy were observed at all treatment levels, including controls; there was no apparent treatment-related pattern in hi stopathological findings. Tadpoles with spinal curvature were observed in 30 to 49% of tadpoles in the control and all treatment groups. Unless otherwise reported, all analyses are based on comparison to the negative (FETAX) control, and analyses of SVL, nor malized HLL, and body weight were performed excluding late stage (N F stage > 60) tadpoles.

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

In an androgen receptor (AR) binding assay (MRID 48618703), ventral prostate cytosol from Sprague Dawley rats was used as the source of the AR. 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 affi nity for the radiolabeled reference androgen (R1881).

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. The mean dissociation constant (K d ) for [ 3 H]-R1881 was 1.080±0.690 nM, and the estimated B max was 1.071±0.167 fmol/100 μg protein for the single batch of prostate cytosol that was used in the assay. Only one of the three K d values was within the range reported in the EPA validati on program (0.685 to 1.57 nM), and all B max values were below the recommended range (7 to 16 fmol/100 μg protein). Although the goodness of fit for individual runs was acceptable (R 2 = 0.880-0.986), confidence in th ese numbers is low based on the large variation in K d estimates among runs.PHOSMET FINAL

Page 32 of 55 The competitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-R1881 (10 nM) in the presence of increasing concentrations of phosmet (96.8% purity; logarithmic increase from 10 -10 to 10 -3 M). Dimethyl sulfoxide (DMSO) was
used as a vehicle at a final assay concentration of approxima tely 3.2%. The assay included dexamethasone as a weak positive control, and R1881 as the ligand reference standard. Three independent runs were conducted with 3 replicates per concentration per run. The estimated log

IC 50 s for R1881 and the weak positive control (dexamethasone) were in the ranges of -9.0 to -9.9 M and -4.3 to -4.6 M, respectively. Phosmet displ aced more than 50% of the radiolabeled R1181 from the AR in all three competitiv e binding runs. The estimated log IC 50 was -5.0 M in each run and averaged -5.0 M; therefore, phosmet is cla ssified as an AR “binder”. The mean relative binding affinity (RBA) for the positive cont rol dexamethasone was 0.0013%. The RBA for phosmet was calculated to be 0.0050%, indicating a stronger affinity for the AR than the weak positive control. Confidence in these numbers is high due to the small variation. The solvent control responses indicate d no drift in the study assa y. All performance crit eria were met, with the exception of the bottom (% bi nding) of dexamethasone in Assay #1, and the bottom plateau level could not be accurately calculated because of a deviation where the concentration range for dexamethasone was prepared slightly different th an that specified in the guideline. This was
considered a minor deviation.

Based on the results of the three competitive bindi ng runs, phosmet is classified as a Binder in the Androgen Receptor Binding Assay.

Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRI D 48618702), phosmet (96.8% a.i., batch # GQH9118000) in dimethyl sulfoxide (DMSO) was incubated with human recombinant
aromatase and tritiate d androstenedione ([1 β

3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]-ASDN) at concentrations of 0, 10

− 3 , 10

− 3.5 ,10

− 4 , 10

− 5 , 10

− 6 , 10

− 7 , 10

− 8 , 10

− 9 , or 10

− 10 M for 15 minutes at
37°C to assess the effect on aromat ase activity. Due to slight precipitation that was observed at the high concentration in the first two runs , the high concentration was decreased to 10 -3.5 M for
the third run.

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation period for each con centration of chemical. Tritiated water was quantified using liquid scintillation counting (L SC). Three independent runs were conducted,
and each run included a full activ ity control, a background activity control, a positive control series (10

− 10 to 10

− 5 M) using a known inhibitor (4-hydr oxyandrostenedione; 4-OH ASDN), and the test chemical series (10

− 10 to 10

− 3 ) with 3 replicates per concentration.PHOSMET FINAL

Page 33 of 55 Aromatase activity in the fu ll activity controls ranged from 0.150 to 0.376 nmol·mgprotein

− 1 ·min

− 1 for the 3 test runs, with a mean a nd standard deviation of 0.268±0.081 nmol·mgprotein

− 1 ·min

− 1 . Activity in the background controls ranged from 0.37% to 0.97% and averaged 0.56% of the full control activity. The respons e of the full activity controls and background controls were acceptable for each run. However, each full activity control was not generally within 10% of the average for the full activity co ntrols, as recommended by the EPA. Run 1 had roughly half the activity of the ot her runs and the 0.5 mL count fo r total activity was roughly half that of the other runs. This discrepancy was not explained in the methods of the study report, and applies to both the controls and phosme t in run 1 compared to runs 2 and 3.

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.257±0.078 nmol·mg-protein

− 1 ·min

− 1 at the lowest tested concentration (10

− 10 M) and 0.002±0.001 nmol·mgprotein

− 1 ·min

− 1 at the highest tested concentration (10

− 5 M). The results were generally within the recommended ranges for the top of the curve, bottom curve, Hill slope, log IC 50 , and coefficient of variation for replicates of each concentration.

For phosmet, aromatase activity averaged 0.231±0.059 nmol·mg-protein

− 1 ·min

− 1 at the lowest
tested concentration of 10 -10 M and 0.033±0.002 nmol·mg-protein -1 ·min

− 1 at the highest tested
concentration of 10

− 3 M. The lowest portion of the res ponse curve across runs for phosmet was less than 50% activity, indi cating that phosmet is an inhibitor of aromatase activity at the highest
soluble concentration. Phosmet decreas ed aromatase activity to 34% at 10

− 4 M.

The estimated log IC 50 ranged from -4.37 to -4.89 M fo r phosmet and averaged -4.54 M compared to 4OH-ASDN, which averaged -7.21 M. The average slope of the concentration response curve was -1.20 for phosmet and -0.97 fo r 4OH-ASDN. Confidence in the mean log

IC 50 for both chemicals and the Hill slope for the positive control is high due to the small variation, but the variation was moderate (22.5% CV) for th e slope value of phosmet. At 10 -10 M in Run 2, aromatase activity was only 69-73% of control, as comp ared to 95-101% of control in the other runs; no explanation was provided.

Based on the data from the average response curv e, phosmet is classified as an Inhibitor of aromatase activity in this assay

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

In an estrogen receptor (ER) binding assa y (MRID 48618704), 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 radiolabeled reference estrog en prior to routinely conducting ER competitivePHOSMET FINAL

Page 34 of 55 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 th e presence of increasing concentrations of phosmet (96.8% pur ity; logarithmic increase from 10 -10 to 10 -3 M). DMSO
was used as a vehicle at a final concentration of <4%. The assa y included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17 β -estradiol as the natural ligand reference material.

Saturation binding data were not provided in the study report; however , summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency.

For the Competitive Binding Experiment, a log IC 50 could not be calculated for phosmet. The mean log IC 50 for 17 β -estradiol was

− 9.0 M and the average log IC 50 for 19-norethindrone was

− 5.5 M. The mean RBA was 0.03% for 19-norethi ndrone compared to the positive control (1

nM 17 β -estradiol), but a RBA could not be calculated for phosmet.

Based on the top % binding for 19-norethindrone at 49%, Run 2 should not be considered for evaluation of phosmet for binding to the ER. The Hill slope for 17 β -estradiol in Run 3 (

− 1.7) was outside of parameter limits. Precipitation was observed at 10 -4 M (but not 10 -3 M) in Run 1
and at the 10 -3 M in Runs 2 and 3. In these instances, data for those concentrations were disregarded by the study author despite indications of potential interaction with the estrogen receptor. The investigators failed to follow assay guidelines that recommend testing solubility of test substances in increasing solvent concentra tions (up to 10% DMSO) and including half log concentrations at the limit of solubility. Because of insufficient attempts to resolve issues with solubility of the test substance at concentrations that suggested an ER interaction, there is lack of
confidence in the results of the three runs; therefore the assay is considered to be technically inadequate.

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

In an estrogen receptor transcriptional ac tivation assay (ERTA; MRID 48618705) conducted by CeeTox, hER α -HeLa-9903 cells cultured in vitro were exposed to phosmet (96.8% a.i., lot# GQH91800) at concentrations incr easing logarithmically from 10 -11 to 10 -4 M mg/mL in DMSO (0.1%) for approximately 24 hours. The assay was performed twice using 96-well plates and each phosmet concentration was tested in 6 wells/plate in each assay. Cells were exposed to the phosmet for 24 ± 2 hours to induce reporter (luciferas e) gene products. Lu ciferase expression in response to activation of the estrogen recepto r by phosmet was measured using a proprietary CeeTox Standard Operating Protocol (SOP), which was not included in the report.PHOSMET FINAL

Page 35 of 55 Phosmet was tested up to the limit of cytotoxicity, 10 -4 M. The mean RPC Max for phosmet was 1.6-3.9%, the PC Max was 10 -7 M and the PC 10 could not be calculated as the RPC Max was less than 10%. Cytotoxicity of 20.1% was observed in the highest con centration in the first run. A similar trend of cytotoxicity was observed in the second run, with the highest concentration having cell viability of only 81.7%.

In the main assays, the responsiveness of th e cells to the very weak positive control 17 α

methyltestosterone was lower than the expected values, indicating a decrea sed sensitivity of the assay to very weak agonists. The PC 10 for 17 α -methyltestosterone should have been reached by

10 -6 M but its maximum RPC was 8.8%, at that concentration. A higher concentration (10 -5 M) reached 47% RPC but in the presence of cytotoxi city. Cytotoxicity was also observed at the highest concentrations of 17 α -estradiol and corticosterone. A lthough the conditions of this assay were not optimal to detect very weak activity, phosmet responses were similar to those of the negative control corticosterone and no t comparable to the responses of 17 α -methyltestosterone,
which was able to reach a maximum of 6.8-8.8% PC. Phosmet was only able to reach a maximum of 1.6-3.9% PC when tested up to the highest concentra tion possible based on cytotoxicity. Because the RPC Max < PC 10 in both assay runs, phosmet was considered negative for estrogen receptor transcriptional activation in this test system.

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

The 21-day short-term reproduction assay (M RID 48673002) of phosmet with fathead minnows ( Pimephales promelas ) was conducted under flow-through conditions. Sixteen groups of 6- month old fish (2 males and 4 females per group; 4 groups per treatment) were exposed to phosmet (96.8% purity) at nominal concentr ations of 0 (control), 0.0015, 0.015, and 0.150 mg a.i./L; mean-measured concentrations were <LOQ (<0.00021), 0.001, 0.0093, and 0.105 mg a.i./L. The test system was maintained at 24 to 25oC and a pH of 6.9 to 7.7.

Survival was low at the high treatment level (8%), with only one male and one female surviving to study termination; endpoints at this treatment le vel were not statistically analyzed or included
in the results. At the intermediate concentrati on, there was a 25% decrease in survival, compared to control, and generalized overt signs of toxic ity in both males and females, including lethargy in all surviving fish and loss of equilibrium in females. At the low concentration, survival was reduced by 13% compared to control. Body weight and length were not si gnificantly different in male or female fish at any treatment level.

In the control group, spawning occurred at least ev ery 4 days in two of the four replicates and fecundity was 16 eggs/female/day/replicate; fecundity was 15 eggs/female/day for three replicates and 6.4 eggs/female/da y for one replicate. Fertiliza tion success in the control group averaged 97%. At the intermediate concentr ation, phosmet significantly reduced fertilityPHOSMET FINAL

Page 36 of 55 (Jonckheere-Terpstra; p<0.05) by 7% and fecund ity (Jonckheere-Terpstra; p<0.05) by 81%, compared to the control.

Female gonado-somatic index (GSI) was significa ntly reduced by 34% at the intermediate concentration (Jonckheere-Terpst ra; p<0.05) relative to contro l; male GSI was unaffected by phosmet treatment (p>0.05). While not statistically significant (Mann-Whitney; p=0.07), median male nuptial tubercle scores were reduced by 42% at the intermediate concentration compared to the control; no tubercles were observed for female s. Male and female vitellogenin (VTG) levels were not significantly different from the cont rol, although there was an apparent 1175% increase
in male VTG at the intermediate concentration due solely to high levels in two males in this group. Plasma sex steroids were not measured.

Gonadal staging effects were indicated for female fish at the low treatmen t concentration, and for
males and females at the intermediate concentr ation. Effects on gonadal histopathology in male fish included an increased percen tage of spermatogonia (4/6) and mild diffuse thickening of the germinal epithelium lining by spermatogonia at the intermediate concentration; in female fish, incidences of decreased yolk synt hesis were observed at both low (2/11) and intermediate (7/12)
concentrations relative to the c ontrols (0/16). Occasional disrupti ons of VTG concentration were noted in the follicles of some females, and mi nimal interstitial fibrosis was observed at the
intermediate concentration. In a ddition, incidences of increased ooc yte atresia were observed in the control and treatment groups, with 11/16, 4/11, and 9/12 of the fish e xhibiting this finding in the control and low and mid treatments, respectively. In 44% of these cases, granulomatous inflammation was observed; evidence of mi crosporidia infection was also observed.

In summary, at the mid-concentration, fec undity, fertilization, and female were GSI were decreased compared to controls. Gonadal hist opathology was affected as shown by decreased yolk synthesis in the low- and mid- concentr ation females, and occasional disruptions of follicular VTG concentration. The proportion of sper matogonia in the testes was increased at the high concentration. High mortality and clinical signs of toxicity in this study may confound the interpretation of these parameters.

Hershberger Assay; (OCSPP 890.1400)

The Hershberger Assay consists of androgenic a nd anti-androgenic components. To screen for potential androgenic activity, phosmet (96.8% a.i., Batch # 6QH9118000) in 0.5% aqueous carboxymethyl cellulose was administered daily via oral gavage to 59- to 60-day old, castrated male Sprague Dawley rats (8/dose group) at dose levels of 0 (vehicle), 5 or 25 mg/kg/day. An androgenic positive control group consisted of 10 cas trated rats dosed by subcutaneous injection with 0.4 mg/kg/day testosterone propi onate (TP) prepared in corn oil.PHOSMET FINAL

Page 37 of 55 To screen for potential anti-a ndrogenic activity phosmet (96.8% a.i., Batch # 6QH9118000) in 0.5% aqueous carboxymethyl cellulose was administer ed daily via oral gavage to 59 to 60-day old, castrated male Sprague Dawley rats (8/dose group) at dose le vels of 0 (vehicle), 5, 15, or 25 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by subcutaneous injection. An anti-androgenic posit ive control group consisted of 10 castrated rats exposed to 0.4 mg/kg/day TP and 3.0 mg/kg/da y flutamide (FT). Flutamide and TP were prepared in corn oil.

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

All animals survived until scheduled termina tion. Cholinergic signs (tremors) and a 12% decrease in terminal bodyweight was seen at 25 mg/kg/day suggesting th at the high dose may
have been excessive.

In the androgen agonist assay, clinical signs in the 25 mg/kg/day group included tremors, uncoordinated movements and porphyrin staining around the eyes. No clinical signs were observed in the 5 mg/kg/day group. Body wei ghts in the 5 and 25 mg/kg/day groups were comparable to controls throughout the duration of the assay. At 25 mg/kg/day, mean overall (Days 1-11) body weights gains were decreased (p ≤ 0.05) by 69%. Animals dosed with 0.4 mg/kg/day TP had increased (p ≤ 0.05) mean body weights of 12% on Day 11, and increased mean overall body weight gains (p ≤ 0.05) of 105%. There were no si gnificant incr eases in the accessory sex organ weights in animals dosed with the test compound. Animals in the 0.4 mg/kg/day TP group had accessory sex organ weight increases (p ≤ 0.05) as follows: 1926% in seminal vesicles; 1264% in ventral prostate; 251% in levator ani-bulbocavernosus (LABC); 794% in Cowper’s gland; and 72% in glans pe nis. All tissue weight CVs were below the maximum values recommended by the test guideline. The performance criteria indicated that this assay was performing as expected.

In the anti-androgen assay, clinical signs in the 25 mg/kg/day group included tremors, and uncoordinated movements. No clinical signs were observed in the 5 or 15 mg/kg/day groups. At 25 mg/kg/day, mean body wei ghts were decreased (p ≤ 0.05) by 12% on Day 11, and overall body weight gains were decreased (p ≤ 0.05) by 60%. There were no effects on body weights or body weight gains in the 5 and 15 mg/kg/day group, or the positive control gr oup. At 15 mg/kg/day, the weights of seminal vesi cles were decreased 25% (p ≤ 0.05). At 25 mg/kg/day, accessory sex organ weight decreases (p ≤ 0.05) were noted as follows: 50% in seminal vesicles; 40% in ventral prostate; 27% in LABC; 34% in Cowpe r’s gland; and 10% in glans penis. Animals dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p ≤ 0.05) accessory sex organ weights as follows: 85% in seminal vesicles; 76% in vent ral prostate; 50% in LABC; 66% in Cowper’s gland; and 21% in glans penis. All tissue weight CVs were below the maximum valuesPHOSMET FINAL

Page 38 of 55 recommended by the test guideline. The perfor mance criteria indicated that this assay was performing as expected.

Phosmet was negative for androgenic activity as no statistically signifi cant increases in organ weights were seen in two or more of the five androgen sensitive tissues in the androgen agonist assay. However, phosmet was positive for anti-androgenicity in the Hershberger assay as statistically significant decreases in organ weights were seen in al l five androgen se nsitive tissues in the androgen antagonist assay. However, the decreases in androgen-sensitive tissue weights were seen at a dose (25 mg/kg/day) that resulted in overt toxicity (12% decrease in body weight and clinical signs), which confounds th e interpretation of the results.

Female Pubertal Assay; (OCSPP 890.1450)

In a female pubertal assay (MRID 48673003), 16 Sprague-Dawley (Crl:CD ® (SD) IGS) rats/dose group were treated daily by oral gavage with phosmet (96.8% a.i., Batch #: GQH9118000) in aqueous 0.5% carboxymethylcellulose (CMC) at doses of 0, 10, or 20 mg/kg/day (5 mL/kg dosing volume) from post-natal day (PND) 22 to 42 or 43. The animals were examined for vaginal opening (VO) daily be ginning on PND 22, and age and body we ight at day of attainment was recorded. After sacrifice on PND 42/43, serum thyroxine (T 4 ) and thyroid stimulating hormone (TSH) concentrations were analyzed using radioimmunoassays . Various clinical chemistry parameters were measured in the seru m. Liver, kidney, pituitary, adrenals, thyroid, ovaries, and uterus were weighe d and examined macroscopically; microscopic examination was also conducted on the thyroid, ova ries, uterus, and kidneys.

No treatment-related effects were observed on mortality, organ weights, serum TSH concentrations, clinical chemistry, or gross pa thology. Changes (NS) in ovary, uterus, thyroid, and pituitary weights were notedin the low- and high-dose animals. There were no treatmentrelated histopathological findings noted in the ov aries, uterus, thyroid, or kidneys. In addition, no treatment-related effects were noted on cycle status, mean age at first estrus, mean cycle length, percent cycling, or percent regularly cycling.

One 10-mg/kg/day rat exhibited localized head tremors on a single day, and tremors were observed at various times post-dose in up to 10/16 10-mg/kg/day rats on 10 days and up to sixteen 20-mg/kg/day rats at 18 da ys. No other treatment-related clin ical signs were observed in animals from either dose group. Decreased body weights (p<0.05; ↓ 9% to 10%) were observed at 20-mg/kg/day during PND 26 to 36. No significant change in mean final body weight or body weight gain was observed in either treatment group compared to the vehicle control group.

The day of VO was comparable among the groups. Unadjusted and adjusted body weights at VO were decreased (p<0.05) by 9% each in the 20 mg /kg/day group compared to control. No treatment-related effect was noted on cycle stat us, mean age at first estrus, mean cycle length,PHOSMET FINAL

Page 39 of 55 percent cycling, or percent regularly cycling. At 20 mg/kg/day, the number of ovarian antral follicles was increased ( ↑ 93%), and the number of atretic follicles was decreased ( ↓ 29%). All other values in the treated gr oups were similar to control.

Serum T 4 concentrations were increased ( ↑ 24 to 38%) in the treated gr oups. A marginal decrease in the thyroid colloid area and increase in the follicular cell height was observed in animals administered phosmet (10 and 20 mg/kg) compared to control.

There were no effects on liver, ki dney, pituitary, adrenal, uterus , or thyroid organ weights at either dose level compared to the control.

Thyroid effects (increased serum T4 levels and th yroid histopathology) were seen at both doses. However, these doses (10 and 20 mg/kg/day) were determined to be excessive based on persistent tremors seen at both doses and seve re body weight loss at the high dose. Additionally, data from the existing toxicity database show th at lower doses should have been tested in this assay. Following the agency’s determination, the registrant submitted additional information (MRID 49610501) in response to the agency’s review of this assay. The submitted data show
that tremors were seen in 10/16 rats at the 10 mg/kg/day dose level ( during 10 days) and all animals at the 20 mg/kg/day dose (during 18 days). The severity of the tremors ranged from “not
characterized to moderate.” U pon review of these data, the ag ency reaffirms its previous
conclusion that both doses tested in this assay were excessive.

Male Pubertal A ssay; (OCSPP 890.1500)

In a male pubertal assay (MRID 48673003), 16 Sprague-Dawley (Crl:CD ® (SD) IGS) rats/dose group were treated daily via oral gavage with phosmet (96.8% a.i., Batch #: 6QH9118000) in aqueous 0.5% carboxymethylcellulose (5 mL/kg) atdoses of 0, 10, or 20 mg/kg/day from postnatal day (PND) 23 to 53 or 54. The animals were examined for preputial separation (PPS) daily beginning on PND 30, and age and body weight at day of attainment was recorded. Following sacrifice on PND 53/54, total seru m testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were analyzed by ra dioimmunoassay. Various clinical chemistry parameters were measured in the serum. Liver, kidney, pituitary, adrenals, seminal vesicles with coagulating glands, and ventral and dorsolate ral prostate were weighed and examined macroscopically; in addition, microscopic examin ations were conducted on the epididymides, testis, thyroid, and kidneys.

All rats survived until study termin ation. Tremors were observed peri odically in the majority of the rats at 20 mg/kg/day. No other clinical signs of toxicity were observe d in the study at either
dose.PHOSMET FINAL

Page 40 of 55 The decreases in body weight gains and fina l body weights were minimal (<10%) and not biologically significant. The sma ll variations in clinical chem istry parameters observed at 20 mg/kg/day were not considered to be indicative of toxic e ffects on the kidney or liver.

There was no treatment-related effect on prep utial separation at ei ther dose level.

The adjusted ventral prostate weights showed a li near trend of decreased weight with increasing dose; however, no statistically significant effect was detected between the treated groups and
control. Both means were lower than historical control mean ve ntral weight for Sprague-Dawley male rats but were within the acceptable range. There were no effects on liver, kidney, pituitary, adrenal, testis, or other sex organ weights at either dose level compared to the control.

Testosterone levels were lower in both dose gr oups compared to the control, but statistical significance was not attained and the means were within the expected ra nge for Sprague-Dawley
rats. It is to be noted that the standard deviation for testoste rone in the 20 mg/kg/day dose group (1.62) was similar to the mean value (1.80).

Serum T 4 levels were increased in both treatment gr oups compared to the c oncurrent control and the mean value for Sprague-Dawley male rats, but they were within the acceptable range in the performance criteria. Additionally, these increas es were within the range of control values observed at the analytical laborat ory (3-7 μg/dL). TSH levels we re slightly higher at both dose levels compared to the control, but the cont rol value was below the acceptable range and the Guideline criteria for this parameter were not met.

During the agency’s initial review, the doses ( 10 and 20 mg/kg/day) used in this study were considered excessive, based on the dose-selectio n rationale and the periodic occurrence of tremors in the majority of the males at 20 mg/kg/day. Subsequently, the registrant submitted additional data (MRID 4961502) in response to the agency’s review of this assay. The submitted data show at the 10 mg/kg/day dose level, mild tremors were seen during one day only in 6/16 animals. In contrast, the severity of tremors at the 20 mg/kg/day dose level ranged from mild to moderate and were seen on multiple days in 11/16 animals. Based on this additional information, the low dose was determined to be adequate with only the high dose showing evidence of overt toxicity.

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48618701), H295R cells cultured in vitro in 24-well plates were incubated with phosmet (96.8% purity, Batch # GQH9118000) at concentrations of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 μM for 48 hours in triplicate for four independent experiments. Run 1 was not analyzed because on e of the forskolin concentrations on the quality control plate was different than the proposed co ncentration outlined in the test guideline (3.33PHOSMET FINAL

Page 41 of 55 μ M was used instead of 1 μ M). Dimethyl sulfoxide (DMSO) wa s used as a vehicle, at a final
concentration of 0.05% v/v.

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

All guideline acceptability recomm endations and requirements we re met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptabl e reproducibility (low %CV), and appropriate induction and i nhibition with posit ive controls.

Neither testosterone nor estradio l concentrations were affected by phosmet. Decreases (p<0.05)
of 10-11% in estradiol concentration were observed at 0.0001, 0.001, and 0.01 μM of phosmet in Run 2, but not at higher concentrat ions nor at any concentration in the other runs. An increase (p ≤ 0.05) of 10% in estradiol concentration was obs erved at 100 μM of phosmet in only one of
the three runs.

Based on hormone responses in each of three i ndependent runs, phosmet treatment did not result in statistically significant and re producible alterations in testosterone or estradiol production.

Uterotrophic Assay; (OCSPP 890.1600)

In an uterotrophic assay (MRID 48618707) conducted to screen for potential estrogenic activity, phosmet (96.8% a.i., Batch # 6QH9118000) in 0.5% carboxymethyl cellulose was administered daily via oral gavage to groups of eight ovariectomized female Sprague-Dawley rats at dose levels of 0 (vehicle), 5, or 20 mg/kg/day on post-natal days (PND) 63-65. A positive control group was treated with 17 α -ethynyl estradiol (EE) by daily or al gavage at a dose level of 0.1 mg/kg/day. All animals were terminated and necropsied on PND 66 approximately 24 hours
after the final dose to determine wet and blotted ut erine weights.

All animals survived until sc heduled termination. Two 20 mg /kg/day rats had decreased movements at 1-4 h post-dosing on Days 1 and 2. Body weights in the dose groups were comparable to the controls throughout the durati on of the assay. Uterine weights in the dose groups were comparable to the controls. Abso lute wet and blotted uterus weights for the 0.1 mg/kg/day EE group were increased (p< 0.05) by 407% and 231%, respectively.PHOSMET FINAL

Page 42 of 55 The dose levels selected for this study are adeq uate based on previously conducted studies which showed no mortality at doses up to 27 mg/kg/day and significant ch olinesterase inhibition at 10 mg/kg/day.

No statistically significant changes were seen in uterine weight in th is assay. Phosmet was negative in the uterotrophic assay.PHOSMET FINAL

Page 43 of 55 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 studies consid ered relevant for dete rmining the potential interaction of phosmet with the endocrine system are summarized below. Given the breadth of toxicity data available on phosmet, the only Part 158 studies discussed in this appendix are those that fulfill current guideline requirements and are directly re levant to assessing potential endocrine effects ( i.e. , multiple-generation rodent reproduc tive studies, development studies, chronic toxicity studies, etc.).

In vitro transactivation assays

In a published literature study, Kojima et al. ( MRID 48033008 ) screened 200 pesticides, covering nine chemical classes, for their potentia l to interact with two human estrogen receptor subtypes (hER α and hER β ) and the human androgen receptor (hAR ) in transactivation assays in Chinese hamster ovary cells (CHO-K1 cells). In this study phosmet was reportedly negative for ER and AR transactivation up to the highest concentration tested was 10 -5 M (range 10 -8 to 10 -5 M).

21-Day Dermal Toxicity in Rodents (Rats)

In a subchronic dermal toxicity study ( MRID 44795801 ), 10 SD (Crl:CD VAF/Plus)
rats/sex/dose were administer ed phosmet (71.2% purity as Imidan 70-WSB) in deionized distilled water by dermal application (once a day for 6 hours, 5 days/week) for 15 days at concentrations of 0, 15, 22.5, or 60 mg/kg/day. Hair was clipped from 20 to 25% of the body to allow an application area of approximately 30 cm 2

.
There were no deaths, and clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights, macro- and microsco pic lesions were comparable among the groups (both sexes), except as noted. Dermal irritation wa s not observed in either sex at any dose. The only effects observed were on inhi bition of ChE activity. In high- dose males, brain ChE activity was decreased (p ≤ 0.01, ↓ 36%) at study termination. Plas ma and RBC ChE activities were unaffected by treatment in males. In high-dose fe males, ChE activity was decreased in plasma (p ≤ 0.01, ↓ 38%) on Day 22 and decreased in RBC (p ≤ 0.05, ↓ 29%) on Day 8, and brain ChE activity was inhibited (p ≤ 0.01) in a dose-dependent manner ( ↓ 13, 20, and 61%) in the low-, mid-, and high-dose groups, respectively.

Because historical control data were unavail able, an expanded control study was initiated immediately after completion of the 21-day derm al study. The expanded c ontrol group consisted of 20 rats/sex of the same strain and ag e under identical test conditions. Decreases (p ≤ 0.01, ↓ 33%) were observed in plasma ChE activity in males in the mid- and high-dose groups atPHOSMET FINAL

Page 44 of 55 Day 22 compared to the expanded control group. Brain ChE activity was decreased (p ≤ 0.01) by 25% and 53% in high-dose males and females, respectively, compared to the expanded control

group.

The NOAEL for systemic effects is 60 mg/kg/da y, the highest dose tested. The NOAEL for ChE inhibition is 15 mg/kg/day, and the LOAEL is 22.5 mg/kg/day ba sed on decreased plasma ChE activity in males and decreased brain ChE activity in females.

Prenatal Development in Rodents (Rat)- Guideline

In a rat teratology study ( MRID 41962902 ), randomized groups of 24 assumed pregnant Alpk:APfSD Wistar rats were ad ministered phosmet (96.4% purity) in corn oil at 0 (vehicle only), 5, 10, and l5 mg/kg/day by gavage during GD 7 to 16. All da ms survived until sacrifice on GD 22, except for one control animal killed in extremis on GD 14. Food-consumption was decreased (p<0.01) GD 7 to 16 ( ↓ 16) and GD 16 to 22 ( ↓ 14) in the high-dose dams. Body weightgain was decreased (p<0.01 and 0.05) for the dosing period (GD 7 to 16) in the mid- and highdose females ( ↓ 12 and 47%, respectively); decreases were also noted for the entire gestation period (p<0.01, ↓ 20%) and corrected body we ight gain (p<0.05, ↓ 56%) at the high dose. No treatment-related gross pathology findings were note d. Clinical signs of toxi city were seen in the high dose group including one or more of the following symptoms: piloerection, tremors, salivation, urinary incontinence, and subdued mood.

Comparable incidences between control and treated groups were observed for corpora lutea/dam, implantations/dam, and the number of live fetuses. In addition, no differences were noted in the incidences of intrauterine death, pre- a nd post-implantation loss, and fetal sex ratio. Mean fetal weight was decreased (p<0.05) for the high-dose group, but the value was within the historical control range. No treatment-rela ted findings or malformations were noted in the fetal external examinations, and visceral examination findings were incidental. There were no apparent treatment-related fetal skeletal defects due to phosmet treatment; the total percentage of fetuses displaying major and minor defects in the treatment groups were sim ilar to or less than control.

The maternal toxicity LOAEL is 15 mg/kg based on clinical signs , and decreased food consumption and body weight gain. The NOAEL is 10 mg/kg/day. Developmental toxicity was not observed in this study, so the NOAEL is 15 mg/kg/day, and the LOAEL is not determined.

Prenatal Development in Rodents (Rat) — Non-guideline

In a non-Guideline developmental toxicity study ( MRID 00063192 ; Staples et al. ), Imidan (phosmet; 95.8% purity) administered to assumed-pr egnant female CD rats in the diet or by gavage. Nominal dietary concentrations were 0, 10, 25, 40, and 50 mg/kg/day (prepared in 23 g of ground Wayne Lab Blox; 25 g of treated feed were provided daily/dam on GD 6 through 15). Mean phosmet doses consumed were 0, 10, 22, 27, and 29 mg/kg/day. Gavage dosing ofPHOSMET FINAL

Page 45 of 55 phosmet was conducted at doses of 0 (0.5% me thylcellulose), 5, 10, 20, 25, and 30 mg/kg/day on GD 6 through 15. All dams were weighted and sacrificed on GD 21; reproductive status was determined and general fetal examinations were conducted (live fetuses weighed, determination
of external and visceral malforma tions, and skeletal alterations).

Selection of dietary and gavage phosmet doses to achieve a MTD acknowledged the tendency of phosmet effects to accumulate and a lessened suscep tibility of pregnant females compared to non-pregnant females. Food intake was decreas ed at higher doses of phosmet; therefore, achieved doses at the low-medium, high-medium, and high concentrations were similar (22, 27, and 29 mg/kg/day, respectively). No parental mortality occurred in the dietary portion of the study, and the percentage pregnant was not affected (89 to 100 %). Food intake and body weight gain were decreased (p<0.05) in a dose-dependent manner (up to ↓ 46 and 102%, respectively) in all but the low-dose dietary group, with no fetal effects noted af ter dietary administration of phosmet. In the gavage administ ration portion, five dams died at the high-medium dose (16%) and both dams died in the high-dose group (100%). The number of pregnant dams in the highmedium group was decreased (p<0.05, ↓ 25%), but dams at the lower doses were not affected (83 to 100%). Food intake was decreased (p<0.05) in all but the low-dose group in a dose-dependent manner ( ↓ 8 to 38%) and body weight gain was decr eased (p<0.05) in th e medium and highmedium dose groups ( ↓ 88 to 73%, respectively). Fetal weight s were decreased (p<0.05) in the low- and low-medium-dose groups ( ↓ 8%) and the medium- and high-medium-dose groups ( ↓ 12%). No increases in stunted or malformed fetuses were observed.

Phosmet administration (dietary or gavage) wa s not teratogenic even up to demonstrating toxicity/lethality to dams ( ≥ 25 mg/kg/day by gavage). Decreased fetal weights ( ↓ 12%) at gavage
doses ≥ 20 mg/kg/day further demonstr ate systemic toxicity.

Prenatal Development in Non-Rodents (Rabbit)

In a rabbit tera tology study ( MRID 41962901 ), randomized groups of 20 assumed pregnant New Zealand White rabbits were administered phosmet ( 96.4% purity) in corn o il at 0 (vehicle only), 2, 5, and l5 mg/kg/day by gavage during GD 7 to 19. All does were sacrificed on GD 30. One high-dose doe died on GD 10, one low- and on e high-dose animal were killed due to moribundity, and three animals in the mid-dose gr oup were sacrificed on GD 27 due to signs of abortion. Two high-dose does exhibited multip le signs of clinical toxicity ( e.g. , salivation, shaking, irregular breathing, or unsteady gait) and two additiona l animals exhibited salivation only. Body weight and food-consumption were no t affected by phosmet treatment. Body weight gain was decreased (p<0.0001) for the dosing pe riod (GD 7 to 19) in the high-dose females ( ↓ 74%); decreases were also noted for the entire gesta tion period (p<0.05, ↓ 23%). No trea tmentrelated gross pathology findings were noted.PHOSMET FINAL

Page 46 of 55 Comparable incidences between control and treated groups were observed for corpora lutea/doe, implantations/doe, and the number of live fetuses, and mean litter and fetal weights. In addition,
no differences were noted in the incidences of in trauterine death, pre- and post-implantation loss,
and fetal sex ratio. No treatment-related findings or malf ormations were noted in the fetal external examinations, and visceral examinati on findings were incident al. Several treatmentrelated increases (p<0.05) in skel etal variations and minor defects were observed in the highdose group, along with an increased (p<0.05) pes score. Overall, the total percentage of fetuses displaying major and minor defects in the treatment groups was sim ilar to or less than control.

The maternal toxicity LOAEL is 15 mg/kg base d on clinical signs a nd decreased body weight gain. The NOAEL is 5 mg/kg/da y. Developmental toxicity was present in the increased
incidence of fetal skeletal va riations at the high dose; the LOAEL is 15 mg/kg/day, and the NOAEL is 5 mg/kg/day.

Prenatal Development in Non-Rodents (Monkey)

In a developmental toxicity study ( MRID 00043397 ), randomized groups of 7 pregnant rhesus monkeys ( Macaca mulatta ) were administered phosmet (pur ity not reported) in a 50% honey solution at doses of 2.0, 4.0 or 8.0 mg/kg/day on GD 22 to 32. Pregnancies were terminated by cesarean section on GD 84. Also investigated in th is study were various doses or regimens of a positive control (thalidomide) and another pestic ide (captan); the results from these compounds
are not discussed further.

Six of seven monkeys at in the high-dose group produced anatomically normal fetuses, and one monkey aborted. All seven monkeys at the inte rmediate dose produced anatomically normal fetuses. Finally, five of seven monkeys in the low-dose group produced anatomically normal fetuses; one monkey in the lo w-dose group aborted and one monkey resorbed the fetus. Fetal mortalities present in th is study were not dose-related and were within the historical range for fetal mortality. The body weights, x-rays, organ we ights and organ to body weight ratios, as well as crown-rump lengths of the fetuses were w ithin normal limits after exposure to phosmet. Maternal body weights and hematology result s indicated no compound-related effects. The LOAELs for maternal and developmental toxicity are not determined. The NOAELs for maternal and developmental toxicity are equal to or greater than 8.0 mg/kg/day.

Subchronic Neurotoxicity in Rodents (Rat)

In this subchronic oral neurotoxicity study ( MRID 44811801 ), phosmet (97.3% purity) was administered for 90 days to 32 Crl:CD(SD )IGS BR rats/sex/dose at nominal dietary concentrations of 0, 25, 50, or 150 ppm. Actual mean dietary concentrations of phosmet were 0, 21.6, 39.7, and 136 ppm [equivalent to achie ved doses of 0/0, 1.5/1.6, 2.7/3.1, or 9.4/11.0 mg/kg/day (M/F)]. Cholinesterase activity was de termined with a modified Ellman method in thePHOSMET FINAL

Page 47 of 55 blood and brains of 2 rats/sex/dose during the pr etest and 6 rats/sex/dos e during Study Weeks 3, 7, and 13. The remaining 12 rats/sex/dose were subjected to the functiona l observational battery (FOB) and motor activity measurements during the pretest period and Study Weeks 3, 7, and 12, and were perfused in situ at study termination. Five animals/ sex from the control and high-dose groups were used for neurohistological examin ation. No animals died during the study. No changes in appearance or beha vior, body weights or body weight gains, and food consumption were observed in any of the treated groups compar ed to control. Results of the FOB indicated no treatment-related findings, and there were no gross ne urological findings. No treatment-related
effects were observed on brain we ight in non-perfused and perfused animals. An increase in the incidence of neuropathological ch anges (characterized by digesti on chambers in the sciatic and peroneal nerves) was observed in the high-dose perfused males.

In general, whole blood and RBC ChE activities were decreased in all treated animals, and plasma and regional brain ChE activities were decr eased in all treated females. The details are
summarized below:

In the low-dose group, mean RBC ChE activity wa s decreased (p<0.01) in males at Week 13 ( ↓ 19%) and females at Week 7 ( ↓ 42%). Decreases (p<0.05 or 0.01) were also observed at Week 13 in mean plasma ChE activity in females ( ↓ 29%), mean whole blood ChE activity in both sexes ( ↓ 16–19%), and ChE activity of the olfactory bulb ( ↓ 36%) and brain stem ( ↓ 21%) regions in females.

In the mid-dose group, the following decreased (p<0.05 or 0.01) mean ChE activities were observed: (i) plasma ChE in the males at Week 3 ( ↓ 21%) and females at Weeks 3 and 13 ( ↓ 27– 46%); (ii) RBC ChE in the males at Weeks 3, 7, and 13 ( ↓ 26–39%) and females at Week 7 ( ↓ 38%); (iii) whole blood ChE at Weeks 3, 7, and 13 in both sexes ( ↓ 24–36%); and (iv) whole brain ChE at Week 7 in the females ( ↓ 20%) and at Weeks 3 and 7 in the males ( ↓ 11–17%).

In the high-dose group, the following decreased (p<0.05 or 0.01) mean ChE activities were observed: (i) plasma ChE at W eeks 3, 7, and 13 in both sexes ( ↓ 23–71%); (ii) RBC ChE at Weeks 3, 7, and 13 in males ( ↓ 65–70%) and at Weeks 3 and 7 in females ( ↓ 66-89%); (iii) whole blood ChE in both sexes ( ↓ 59–74%) at Weeks 3, 7, and 13; (iv) whole brain ChE in both sexes ( ↓ 43–68%) at Weeks 3 and 7; and (v) in a ll six brain regions in the females ( ↓ 36–67%) and in all brain regions (except for olf actory region) in the males ( ↓ 27–52%) at Week 13.

The LOAEL is 22 ppm (equivalent to 1.5/1.6 mg /kg/day (Male/Female; LDT) based on doserelated decreases in plasma, RBC, whole bl ood, and brain ChE activities. The NOAEL is not
established.

Two-Generation Reproduction in Rodents (Rat)

In a Two-Generation Reproduction study in the rat ( MRID 41520001 ), phosmet (95.2% purity) was administered at target doses of 0, 20, 80, and 300 ppm in the diet to SD rats (Crl:CD(SD)Br; 25/sex/dose) for two generations. Compound inta ke values were determined for malesPHOSMET FINAL

Page 48 of 55 (premating) and females (premating, ge station, and lactation) in the F 0 and F 1 generations: the
overall intake values for both sexes duri ng the entire study period were 0, 1.5, 6.1, and 23.4 mg/kg/day. The F 0 rats were fed the diet for 8 weeks prior to mating, through gestation and
lactation of the F 1a litter, and three weeks after weaning prior to re -mating to produce the F 1b litters. After weaning, F 1b rats were selected as F 1 parents and treated for 11 weeks prior to mating. As previously, the F 1 parents were allowed three weeks after weaning the F 2a litters prior to re-mating to produce the F 2b litters. Blood samples were collect ed at terminal sacrifice for the
determination of serum and RBC ChE activities.

No treatment-related mortality was observed; three and two females, respectively, in the F 0 and

F 1 high-dose groups had tremors. Body wei ghts during the premating periods for F 0 and F 1 highdose animals were occasionally decreased (p<0.01 or 0.05) by 3 to 8%, with a decrease (p<0.01) of 10% observed on Day 21 in high-dose F 0 females. Effects on premating body weight gain
were observed in the F 0 animals, but observed decreases ( ↓ 8 to 10%) were NS. Maternal body weights during gestation were slightly d ecreased (NS) in the high-dose animals ( ↓≤ 8%), with one decrease (p<0.01) of 8% in F 1 dams on GD 20. Mean body weight gains were generally decreased (NS) only in the high -dose animals. Maternal body weights during lactation were decreased (p<0.01 or 0.05) in the high-dose dams ( ↓ 9 to 13%; LD 7 to 21) with the F 1a and F 2a litters; decreased body weights (p<0.05 or NS) with the F 1b and F 2b litters were ≤ 8%. Mean body weight gains (losses) were ge nerally greater (NS) in the mi d- and high-dose animals. Food consumption was decreased (NS; ↓ 7 to 21%) or (p<0.01; ↓ 18 to 40%) in high-dose dams during
lactation.

Mean absolute and relative ovarian weights were decreased ( ↓ 15 and 21%, respectively; p<0.05) in high-dose F 0 parents , but no histopathological lesions were seen in the ovaries in either parental group . Absolute testis weights were decreased ( ↓ 10%; p<0.05) in high-dose F 0 and F 1 parents, but relative testis weights were comparable to control, and no treatment-related histopathological lesi ons were seen in the testes in either parental group.

Serum ChE activity was d ecreased in mid-dose F 0 females (p<0.01, ↓ 24%) and high-dose F 0 /F 1 males (p<0.01, ↓ 30/41%) and females (p<0.01, ↓ 65/63%). RBC ChE activity was decreased in
low-dose F 0 males (p<0.05, ↓ 8%), low-dose F 0 /F 1 females (p<0.01, ↓ 11/16%), mid-dose F 0 /F 1 males (p<0.01, ↓ 37/48%) and females (p<0.01, ↓ 48/59%), and high-dose F 0 /F 1 males (p<0.01, ↓ 74/85%) and females (p<0.01, ↓ 81/80%).

Fertility index and gestation index were decreas ed (NS, or p<0.05 or 0.01) in all mid- and highdose parental groups, with a generally greater impact on the F2 litters. Lactation index was decreased (p<0.01) for litters from all high-dose parents.

The mean number of live pups per litter generall y were decreased (NS, or p<0.05 or 0.01), and mean pup weight per litter was decreased (p<0.0 5 or 0.01) for all litters from high-dose parents after Day 0. Pup sex ratio was not affected by treatment. No definitive treatment-related gross orPHOSMET FINAL

Page 49 of 55 histopathological findings were obser ved in pups culled on PND 4 or any F 1 -generation weanlings.

The NOAEL for parental toxicity is not determin ed and the LOAEL is 20 ppm (1.5 mg/kg/day), based on inhibition of RBC ChE activity. The NOAEL for reproductive toxicity is 20 ppm, and the LOAEL is 80 ppm (6.1 mg/kg/da y) based on decreased fertilit y and decreased fertility and gestation indexes. The NOAEL for offspring t oxicity is 80 ppm and the LOAEL is 300 ppm (23.4 mg/kg/day), based on decreased pup body weight gain and survival.

Reproductive Toxicity in Male Rodents (Mouse)

In a published non-Guideline study, Quinto et al. ( MRID 48084301 ) examined the effects of selected pesticides on male mouse sperm morphology. Imidan (phosmet; 99.8% purity) was dissolved in distilled water; male C3H×C57BL/6 F1 mice were administered phosmet by p.o. or i.p. at doses of 0, 12.5, 25, and 50 mg/kg/day for 5 days. On Study Day 35, animals were killed by cervical dislocation and te stes weight, sperm count, and sperm abnormalities were
determined.

No treatment-related effects on testes wei ght, sperm count, and sperm abnormalities were observed after phosmet treatment by either th e p.o. or i.p. routes of administration.

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat)

In a chronic two-year oral toxicity study in rats ( MRID 41916401 ), phosmet (R-1504 Technical, 94.3/95.2% purity) was administered as 0 (control) , 20, 40, 200, or 400 ppm in the diet to Crl:CD (SD) Br rats (70/sex in the control group, 60/sex in all other dose groups except for the high-dose group) for 104 weeks. An interi m-sacrifice at Week 53 includ ed 20/sex in the control group, 10/sex in the 20-, 40-, and 200-ppm dose groups ; the high-dose group (400 ppm; 20/sex) was
sacrificed in toto after Week 52. For the first six week s of the study, the low-medium-dose group (40 ppm) was administered a 100-ppm diet. Excludi ng this error, the mean concentrations of phosmet in the treatment diets were ca lculated as 19.3±1.1, 39.4±2.9, 194.1±5.6, and 389.5±17.9 ppm. Calculated mean intake values for ma les and females were 1.1, 1.8, 9.4, and 23 mg/kg/day and 1.1, 2.1, 10.9, and 27 mg/kg/day, respectively.

Parameters examined in all rats were t hose routinely examined in a chronic oral toxicity/carcinogenicity study. Abso lute and relative (% of body a nd brain) weights of organs were determined for adrenals, kidneys, liver, testes/ovaries, and brain for both scheduled sacrifices; histopathology was conducted on thes e tissues as well as epididymides, mammary gland, pituitary, prostate, seminal vesicles, thyr oid/parathyroid, vagina, and uterus with cervix.
Serum and RBC ChE activities were determined at 6, 12, 18, and 24 month. Brain ChE activity was determined at Weeks 52 (10 rats/sex/dose gr oup) and 104 (animals designated for laboratory tests).PHOSMET FINAL

Page 50 of 55 No clinical signs of toxicity co uld be attributed to administration of the test material. Survival was comparable among the control and the treated groups (84 to 100% at Week 52, and 20 to 42% at termination). Although decreases (p<0.05 or 0.01; up to 10%) in mean body weight were observed in the mid-high (Week 1) and high-dose (Weeks 1 to 13) females, no other treatmentrelated weight effects were noted. Mean compound intake was not affected by treatment and, in general, no relevant, treatment-re lated effects were noted in a ny hematology, clinical chemistry, or urinalysis parameters (except for ChE activity). Dose-related inhibition (p ≤ 0.01 or 0.05) of serum ChE activity occurred in males in the high- dose group at all sample times and in the highmedium group at 18 months, and in females in the high-medium- and high-dose groups at all sample times and in the low-medium gr oup at 6 months. RBC ChE was inhibited (p ≤ 0.01 or 0.05) in males and females in the low-medium -, high-medium-, and high-dose groups at all sample times, except at 12 months in the low-me dium animals. RBC ChE also was inhibited in the low-dose males only at the 6-month sample time. Brain ChE was inhibited (p ≤ 0.01) in males and females in the high-medium- and high-dose gr oups at all sample times, and in the 24-month low-dose samples in males (p<0.05). Organ weights, gross pathology, and neoplastic/nonneoplastic microscopic pathology were generall y unremarkable. Absolute kidney weight was decreased (p<0.05, ↓ 12%) in the high-dose females at Week 53. An increased (p<0.05) incidence of fatty liver was observed in the high-medium- a nd high-dose males at the interim sacrifice, and males in all remaining treatment groups at st udy termination. Females in the high-medium-dose group had increased (p<0.05) incide nces of fatty liver and minera lization of the thyroid at study
termination. No evidence of treatment-related carcinogenicity was observed in the present study at phosmet exposures of ≤ 400 ppm. A NOAEL of <20 ppm is determined in this chronic study, with a LOAEL of 20 ppm based on decreased RBC and brain ChE activity. The systemic NOAEL and LOAEL are also <20 ppm and 20 ppm, respectively, based on the increased incidence of fatty liver.

Carcinogenicity in Rodents (Mouse)

In a two-year oral oncogenicity study in mice ( MRID 00141659 ), phosmet (R-1504 Technical, 94.7% purity) was administered as 0 (control), 5, 25, or 100 ppm in the diet to B6C3F1-Crl-BR (60/sex/dose) for 104 weeks. At 12 months, 10 mice/sex/dose were selected for an interim sacrifice. Calculated mean intake values for males and females were 1.0, 4, and 14 mg/kg/day and 1.2, 5, and 18 mg/kg/day, respectively.

Parameters examined in all mice were those r outinely examined in a chronic carcinogenicity study. Absolute and relative (% of body) weights of organs were determined for heart, kidneys, liver, testes with epididymides, and brain; hi stopathology was conducted on these tissues as well as adrenals, mammary gland, ovari es, pituitary, prostate, seminal vesicles, thyroi ds, vagina, and uterus. Plasma, RBC, and brain Ch E activities were determined at 12 (interim sacrifice animals only) and 24 months (surviving an imals at study termination). Due to reported reagent problems, RBC ChE activity was not determined at the interim sacrifice.PHOSMET FINAL

Page 51 of 55 A dose-related trend in the incidence of convulsi ons was attributed to administration of phosmet in male mice only. Survival was comparable am ong the control and the treated groups with unscheduled deaths ranging from 4 to 10 males an d 11 to 15 females, respectively. No treatmentrelated effects on body weight were observed; f ood consumption was decreased in all treated males and females in the mid- and high-dose groups (data not provided). No relevant, treatmentrelated effects were noted in hematology parame ters; clinical chemistry determinations were limited to ChE activity. Inhibition (s ignificance not reported) of pl asma ChE activity occurred in males in the low- and mid-dose groups and mid-dose females ( ↓ 11 to 13%) at the interim sacrifice, with lower inhibition ( ↓ 4 to 10%) observed at study termination. Greater inhibition (significance not reported) of plasma ChE ac tivity was noted in high-dose males and females ( ↓ 54 to 56%) at the interim sacrifice, with similar inhibition ( ↓ 47 to 52%) observed at study
termination. RBC ChE activities could not be dete rmined at the interim sacrifice due to a technical issue, but were not di fferent from control at study term ination. Brain ChE was inhibited (significance not reported) in males and fema les in all dose groups to a similar degree ( ↓ 22 to 34%) at the interim sacrifice; at study termination, brain ChE activity in males was not different from control and was decreased only in mid- and high-dose females ( ↓ 14 and 22%, respectively). At the interim sacrifice, there were increases (significance not reported) in absolute kidney and liver weights in high-dose females; at termina tion, liver weights were increased (significance not reported) in mid- and high-dose females and high-dose males. Gross pathology findings in the livers of high-dose males included increased incidences of masses, nodules, or cysts and areas of focal discoloration/depression. Mi croscopic examinations at study termination noted increases in high-dose animals of testicular atrophy (19/50 vs . 11/49) and myometrial/uterine atrophy (8/50 vs. 2/49). Hepatocellular adenomas and carcinoma s were occasionally present in treated male mice at the interim sacrifice, and in all contro l and treated mice at study termination. Increased (p=0.05 and 0.06, respectively) hepa tocellular adenomas and carcinom as were observed in highdose males and females 35/60 vs. 23/60 and 18/60 vs. 10/60, respectively). Further analyses of the tumor data indicated significant increases (p<0.05) for high-dose males and females. A NOAEL of <5 ppm is determined in this oncog enicity study, with a LOAEL of 5 ppm based on inhibition of brain ChE activity in males and fe males. The systemic NOAEL is 5 ppm and the LOAEL is 25 ppm based on convulsions in male mice.

Chronic Oral Toxicity/Carcinogenicity in Non-Rodents (Dog)

In a chronic two-year oral toxicity study in dogs ( MRID 00076436 ), phosmet (purity unknown) was administered as 0, 20, 40, and 400 ppm in th e diet to Beagle dogs (3/sex) for 104 weeks. Phosmet doses were calculated as 0, 0.5, 1.0, and 10 mg/kg/day with the conversion factor of 0.025 mg/kg/day per ppm.

Organs weighed at study termination included adrenals, liver, kidneys , ovaries, pituitary, prostate, testes, thyroid, and ut erus, and organs examined micr oscopically included adrenals, liver, kidneys, mammary gland, ovaries, pituitary, pr ostate, testes, thyroid, and uterus. Gross and microscopic pathology were unremarkable (no treatment-related finding s). Brain and RBC ChEPHOSMET FINAL

Page 52 of 55 inhibition was observed in the high-dose animals; no additional treatment -related effects on ChE activity were observed at the low and intermediate doses. No other systemic toxicity effects were observed due to phosmet treatment in this study.

Avian Reproduction (OCSPP 850.2300)

In an avian reproduction study ( MRID 00105999 ), 14 to 16-week old Mallard ducks ( Anas platyrhynchos ) were administered phosmet (94.7% purity) in the feed at concentrations of 0 (acetone; vehicle control), 25, 60, and 150 ppm for up to 27 weeks (includes 13-week administration prior to the initia tion of egg laying). Mean measured phosmet concentrations in the control and treated diets were <2.0, 22.1 to 27.3, 62.0 to 95.0, and 144.0 to 183.0 ppm. Each dose group consisted of 12 ducks/sex, with a sing le drake and hen being paired in 12 pens/dose group. It was noted that photoperi od was gradually lengthened according to 1978 Guidelines. No treatment-related effect on body weight was observed. Weekly egg production data was provided. Eggs were candled during fourteen 7-day periods and eggshell thickness was determined. Feed consumption was measured every two weeks. Surviving ducklings were weighed on Day 14, and viability was reported on Day 21. ChE determinations were not conducted during this study.

No adult mortalities occurred in any test group, a nd no overt signs of toxicity or clinical signs were observed. No effects were seen at any te st dose on female body weight or average feed consumption in males and females. Male body wei ght was increased (significance not reported) in the low-dose group for Week 27; no other diff erences were noted. In general, no reproductive impairment (p>0.05) was seen at any dose. Ther e were no apparent trea tment-related effects on the total number of eggs lai d, shell thickness, or egg qualit y. There were also no treatmentrelated effects on live 21-day old embryos or pe rcentage viability. Occasional differences in duckling body weight (Day 1 or 14) and Day 14 viability were not considered to be treatment related. Overall, fewer total eggs were laid in the high-dose group, but this may be in part due to the failure of a single pen/mating pair (No. 8) to produce (only 1 egg laid over the 13-week period).

In another avian reproduction study ( MRID 00125786 ), 16 to 20-week old Bobwhite Quail (species not identified) were ad ministered phosmet (97.4% purity) in the feed at concentrations of 0 (acetone; vehicle control), 25, 60, or 150 ppm during the laying season (includes 14-week administration prior to the initia tion of egg laying). Each dose gr oup consisted of 24 quails, with a single cock and hen paired in 12 pens/dose gr oup. It was noted that photoperiod was gradually lengthened according to 1978 Guidelines. Eggs we re collected during 7- day collection periods, stored at 16°C and 65% relative humidity, and turned twice daily. On Day 21, embryo survival was determined and eggs were placed in a ha tcher (37.2 to 37.8°C). Hatchling were weighed on Day 14.PHOSMET FINAL

Page 53 of 55 Mortality occurred in all treatm ent groups (low dose: one female; intermediate dose: one male and five females; high dose: three females). Thes e deaths were considered to be due to open sores attributed to increased activity and stress during the 16-hour light period for egg promotion. Adult body weights and feed consump tion in the treated groups generally were comparable to control, although significant decrea ses were observed at Week 26 in the low- and high-dose groups. High incidences of reduced fluid in the gall bladder with diffuse discoloration of bile were noted in males and females (femal es>males) in all treatment groups; this response was dose dependent in females. There was no tr eatment-related effect on the prevalence of cracked eggs; the number of eggs cracked in th e vehicle-control group was approximately 2-fold greater compared to treatment groups. Decreases in eggs laid (p<0.05) were observed in the high-dose group (NS). Reproductive parameters in the low- and mid-dose groups generally were similar to, or better, compared to control values.

Fish Early Life-Stage Test (Freshwater) (OPPTS 850.1400)

In a freshwater fish early life-stage test ( MRID 40938701 ), Rainbow trout ( Oncorhynchus mykiss; previously Salmo gairdneri ) embryos were exposed to [ 14 C]-phosmet (97 to 99% purity) at nominal concentrations of 0 [negative c ontrol and dimethylformamide (DMF), vehicle control], 3.0, 6.0, 12, 25, and 50 μg/L. Achieved exposure concentrations were 3.2, 6.1, 12, 24, and 51 μg/L for the treated groups. Eggs were ex posed from Day 1 (fertilization) until hatch ( ≥ 95% by Day 36); exposure was continued for 60 days post-hatch until termination on Day 96. Four replicates of 30 eggs/replicate were incl uded in each treatment group on Day 1. On Day 39, unhatched eggs were removed, and the number of fr y was reduced to 15/replicate. Fry survival, and length and weight, were determ ined on Day 35 and 60 post-hatch.

No solvent effects on study parameters were observed with DMF. Egg viability determined on Day 11 ranged from 86 to 96%, with a mean value of 92.5%. The percent hatch was 99% (negative control), 98% (vehicle control), 99% (low, low-medium, and medium concentrations), and 97% (high-medium and high concentrations); no treatment -related effect on hatching was noted. Fry survival on post-hatch Day 35 wa s decreased (p<0.05) in the intermediate concentration group by 7%, but no significant decr ease was noted at the higher concentrations. Mean length on post-hatch Day 35 was decreased (p<0.05) in the medium, high-medium, and high concentrations in a treatment-related manner ( ↓ 4, 7, and 15%, respectively). Fry survival on post-hatch Day 60 was decreased (p<0.05) at the high-medium and high concentrations by 16 and 57%, respectively. From the low-medium to the high concentration, mean length was decreased (p<0.05) by 5, 13, 20, and 28%, respectivel y, compared to the negative control. Mean weight on post-hatch Day 60 also was decrea sed (p<0.05) in the medium, high-medium, and high concentrations in a treatment-related manner ( ↓ 35, 50, and 62%, respectively).PHOSMET FINAL

Page 54 of 55 APPENDIX 3: References Not Utilized in the Phosmet 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 Phosmet 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.

Burgess, D. (1988) Chronic toxicity of 14C-Imidan® to Daphnia magna under flow-through test conditions. Analytical Bio-Chemistry Laborator ies, Inc. Laboratory report no. 35778. (MRID 40652801)

Cappon, G. (1999) A Dietary Subchr onic (90-Day) Neurotoxicity Study of Phosmet in Rats. WIl Research Laboratories, Inc. Report No.: WIL-331002. (MRID 44811801)

Fernandez-Perea, M. T., Prados, E. A., Villajos, A. N., Prados, J. L., and Baudin, J. M. (2009) Influence of avian reproduction ecotoxicological endpoints in the assessm ent of plant protection products . J Environ Sci Health B . 44(2):106-112.PHOSMET FINAL

Page 55 of 55 Gajdova, M., Vargova, M., Jakubovsky, J., Grunt , J., Valky, J., and Galbavy, S. (1988) Estrogenic effects of Phosmet on the Uterus of Neonatal Rats. Bratisl. Lek. Listy . 89: 843-847.

Guiñazú, N., Rena, V., Genti-Raimondi, S., Rivero, V., and Magnarelli, G. (2012) Effects of the organophosphate insecticides phosmet and chlorpyrifos on trophoblast JEG-3 cell death, proliferation and inflamma tory molecule production. Toxicol In Vitro . 26(3):406-413.

Hollingsworth, R., Johnston, C.D., Woodard, G., and. Cronin, M.T.I (1965) Three-Generation Reproduction Study in Rats. Woodard Re search Corporation. (MRID 00081432)

Jablonika, A. and Kobzova, D. (1988) Morphologi cal Changes in Sperm in Experimental Mice
after Administration of Phosmet and Mancozeb. Bratisl. Lek. Listy . 89: 611-614.

Johnston, C.D. and Cronin, M.T.I. (1962) Imidan: An Evaluation of Safety of Imidan in the Rat and Dog. Woodard Research Cor poration. (MRIDs 00081426, 00081428, and 00081429)

Kenwood, S.M., (1988) 21-Day Dermal Toxicity Study with lmidan Technical in Rabbits. Hazleton Laboratories America, Inc. Report No.: HLA 6142-103. (MRID 40538101)