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

Environmental

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

THR O UG H: Je ss Rowland

## ~ S

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

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

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

.

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

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

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

CHEMICAL: METHOMYL

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYMETHOMYL FINAL

ii TABLE OF CONTENTS

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

I. Introduction.......................................................................................3

II. Sources of Scientific Data and Technical Information......................................4

A. EDSP Tier 1 Screening Assays............................................................4

B. Other Scientifically Rele vant Information (OSRI)......................................4

III. Weight of Evidence (WoE) Evaluation.......................................................4

A. EDSP Tier 1 Screening Assays.............................................................7

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................8
1. Effects on Estrogen Pathway.....................................................8
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...............................................................................21

B. Estrogen Pathway...........................................................................23

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

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

E. Conclusions.................................................................................24

V. EDSP Tier 2 Testing Recommendations....................................................24

VI. References.......................................................................................25

LIST OF TABLES: Table 1. Tier 1 Assay for Methomyl...................................................................7
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Methomyl....................................10
Table 3. Androgenic/Anti-Andr ogenic Pathway for Methomyl..................................15
Table 4. Thyroid Pathway for Methomyl..........................................................19

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................29
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................39

APPENDIX 3. References Not Utilized in the Methomyl WoE Analysis............................52METHOMYL 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 EthanolMETHOMYL FINAL

iv Abbreviation Terminology

F Female
F1 First filial g eneration
F2 Second filial g eneration
Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Intera g enc y Coordinatin g Committee on the Validation of Alternative K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

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

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

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

v Abbreviation Terminology

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

OPP Office of Pesticide Programs

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

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

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

vi Abbreviation Terminology

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)

VC Vehicle Control VO Vaginal Opening

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

Page 1 of 54 Executive Summary

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

In determining whether a methomyl interacts with E, A or T hormone pathways, the number and type of effects induced, the magn itude and pattern of responses obs erved across studies, taxa, and
sexes were considered. Additiona lly, the conditions under which e ffects occur were considered, in particular, whether or not e ndocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On May 14, 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) analysis of the potential interaction of methomyl with the E, A or T signaling 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).

There was no convincing evidence of interaction of methomyl with the estrogen pathway either
in the in vitro ( i.e ., ER binding, ERTA, aromatase and steroidogenesis) or mammalian in vivo ( i.e . uterotrophic of female pubert al) Tier 1 assays or in th e mammalian or wildlife Part 158 studies. In the FSTRA, although decreases in GSI, fertility and fecundity were observed at the mid and high treatment (0.312 and 1.03 mg/L, respectiv ely) and increased incidence and severity of oocyte atresia at the high treatment were reported there were no effects observed on VTG. Although there were some complementary effects across the FSTRA, there was no effect on VTG (sex steroid hormones not measured), a nd there was limited redundancy across the other Tier 1 assays or Part 158 studies. Decreases in eggs laid (fecundity ) were observed in the bobwhite quail reproduction study at the highest concentration te sted (427 mg/kg-diet), but no other reproductive effects were observed. Decreases in fecundity alone are limited in the ability to inform about potential estrogen-related effect s. Additionally, potentially similar to the decrease in fish GSI, an increase in number of females with regressed ovaries was observed in the mallard duck reproduction study, but no other reproductive effects were observed and female body weight gains and food consumption were decrea sed at this concentration. Furthermore, in other toxicity testing with fathead minnow fish (early life-st age and chronic reproduction),METHOMYL FINAL

Page 2 of 54 effects on growth in young fry were observed at concentrations lower (0 .117 mg/L) or similar (0.243 or 0.280 mg/L) to the concen trations tested in the FSTRA. Therefore, while there were no clinical signs of toxici ty in female fish in the FSTRA (male body weights were significantly decreased), the concentrations tested in the FST RA were similar to concentrations which were shown to be overtly toxic in other fish assays; as such, the decreases observed in the FSTRA may be due to potential general stress on the fish fr om exposure to methomyl as oppose to potential
endocrine-related interactions.

There was no convincing evidence of interaction of methomyl with the androgen pathway. The
Tier 1 in vitro AR binding assay was negative, and while a decrease in testosterone production
was observed in the Tier 1 steroi dogenesis assay, there is uncertainty in this result based on the pattern of responses. No androgen-related effect s were observed in the Tier 1 Hershberger or male pubertal assays or Part 158 mammalian st udies. While androgen-related effects were observed in an OSRI study (El Medany et al . 1993), the results were questionable as the test doses overlapped with acute mortality values. In the FSTRA assay, there was a slight increase in the proportion of spermatogonia (mild to modera te) observed in male testes at the high concentration along with the de creases in fertility at the mid and high treatment; however, significant decreases in male body weight were observed at the mid and high treatment. Considering the body weight changes along with the discussion in the estrogen pathway regarding test concentrations and other obs ervations of overt toxicity in fish, the alterations/decreases observed may be due to gene ralized stress in the fish. Also, as in the estrogen pathway, there is a lack of redundancy in observed effects across the Tier 1 battery and
Part 158 studies.

There was no convincing evidence of interacti on of methomyl with th e thyroid pathway. No thyroid-related effects were observed in the ma mmalian studies and no effect on developmental endpoints or thyroid histopat hology were seen in the AMA.

Based on weight of evidence considerations, human health and wildlife EDSP Tier 2 testing is not recommended for methomyl since there was no convincing evidence of potential interaction with the estrogen, androgen or thyroid pathways.METHOMYL FINAL

Page 3 of 54 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 methomyl interacts with E, A or T hormone pa thways, the number and type of effects induced, the magn itude and pattern of responses observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whethe r or not endocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On May 14, 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) analysis of the potential interaction of methomyl 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).

Methomyl has a water solubility of 5.8 × 10 4 mg/L at 25°C and a vapor pressure of 5.4 × 10 -6 mm Hg at 25°C. The octanol/water partition coefficient is 0.6 (log K ow ) with a whole body
bioconcentration factor value in fish of approximately 3. Methomyl decomposes slowly in water but the rate of degradation is increased by temperature, aeration, and sunlight.

The available information considered in determin ing the potential interact ion of methomyl with the E, A, T pathways include submitted Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxicity stud ies and other published articles. These data are summarized in Section III.A through III.C. An an alysis of the data submitted to the agency, using the WoE approach outlined by the Agency (U SEPA, 2011), is presented in Section IV. The
EDSP Tier 2 studies r ecommendations are presen ted in Section V.METHOMYL FINAL

Page 4 of 54 II. Sources of Scientific Data and Technical Information

A. EDSP T1 Screening Assays

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the agency’s test order for methomyl 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 Methomyl (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any
additional relevant information. Su mmaries of the Tier 1 Assays and data submitted as OSRI are included in Appendix 1 and 2. Additi onally, literature/studies consid ered but not utilized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria, and approach used in the WoE determination on the potential of a
substance to interact with endocrine-related processes ( i.e., E, A, or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and 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.METHOMYL FINAL

Page 5 of 54 As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the concordance or consistency (complementarity) of the responses in the individual study is evaluated. For the
Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a speci fic endpoint for discerni ng whether or not the chemical has interacted with the endocrine syst em, are considered. The co llective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indi cation of potential interaction as measured by the study.

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

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

Page 6 of 54 systemic toxicity introduces uncertainty in the abili ty to distinguish effects specifically related to
an endocrine-related effect from a non-endocrine toxic response.

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

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

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

Page 7 of 54 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 or not a s ubstance has the potential to interact with the E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

A. EDSP T1 Screening Assays

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

.

Table 1. Tier 1 Assays for Methomyl

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48672003)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48685901)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48672001)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48685902)

Aromatase Assay (Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48672002)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48617701)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48617702)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID No. 48701404)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID No. 48701403)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48701401)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 486701402)METHOMYL FINAL

Page 8 of 54 B. Effects on Hypothalamic-Pit uitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for methomyl 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-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

Methomyl was negative (or not interactive) in the Tier 1 in vitro estrogen receptor (ER) binding assay, ER α transcriptional activation (ERTA), steroi dogenesis and aromatase assays. Methomyl was also negative in ERTA assays considered as OSRI (Anderson et al ., 2002 and Kojima et al

., 2004).

In the uterotrophic assay, no uter ine weight increases were see n. In the female pubertal assay, there were no estrogen-related effects observed.

In the FSTRA, decreases in fecundity of 23% and 77%, fertility of 1.6% and 33%, and GSI of
15% and 21% were noted in the females at the mid- and high-concentrations (0.312 and 1.03 mg/L), respectively. In addition, oocyte atresia wa s increased (mild to moderate) in females at the highest test concentration. However, no effect s were noted on VTG in females (or males) and sex hormone concentrations (17 β -estradiol) were not determined.

In the Part 158 mammalian toxicity studies, no es trogen-related effects we re observed, except for a decrease in ovarian weight in the F1 adults in the 2-generation ra t reproduction study which occurred in the presence of overt toxicity. An increase in pituitar y weight (absolute and relative) in the absence of histopathologi cal changes was observed in the subchronic rat study.

In a Part 158 avian reproduction study, reduction s in egg laid (40%) were observed in the bobwhite quail study at the highest dose tested (427 mg/kg-diet); however, reports of females with feather loss and toe lesions [(potentially tr eatment-related and due to male aggression (pen mate)] were reported at this concentration. No treatment-related reproductive effects were observed in the Part 158 mallard duck study; decr eases in female body wei ght gain was observed at the highest test concentra tion (427 mg/kg-diet). In anot her mallard duck reproduction study (MRID 49245301; preliminary review), an incr ease in the number of females with regressing/regressed ovaries (8/15 vs. 4/16 in control) was observed at 1120 mg/kg-diet, however, no other reproductive parameter was aff ected. Also, decreases in female body weight gain and food consumption was observed at 1120 mg/kg-dietMETHOMYL FINAL

Page 9 of 54 Reproductive effects were not observed in the Part 158 fish life-cycle study with fathead minnow; effects on parental surv ival and/or as well as parent al and offspring growth were reported at concentrations of ≥ 0.147 mg/L. Two Part 158 fathead minnow fish early-life stage
studies were available and in one study, decreases in growth (wei ght and length) were reported at

≥ 0.148 mg/L; in the other study, survival was decreased at ≥ 0.117 mg/L with decreases in growth at ≥ 0.243 mg/L.METHOMYLFINALPage 10 of 54Table 2. Estrogenic/Anti-Estrogenic Pathway for MethomylLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Methomyl1Study Type /Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone 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 2 Overt Toxicity 3EDSP Tier 1 AssayER Binding(MRID 48672003)NERTA(MRID 48685901)N4Steroidogenesis(MRID 48685902)NAromatase(MRID 48672002)NUterotrophic(MRID 48617701)NN NFemale Pubertal Rat(MRID 48701404)NNNNNNNNFSTRA(MRID 48701401)NE↓15,21%(M, H)P(H)5NFrt:↓1.6,33%;Fcd:↓23,77%(M, H)NNMETHOMYLFINALPage 11 of 54Table 2. Estrogenic/Anti-Estrogenic Pathway for MethomylLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Methomyl1Study Type /Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone 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 2 Overt Toxicity 3OSRIERTA assay(Andersenet al.,2002)NERTA assay(Kojimaet al., 2004)NSubchronic (13-week)toxicity(Rat; MRID 00007190)NENEN↑19%(H)X(H)NDevelopmental toxicity(Rat; MRID 00008621)NNNDevelopmentaltoxicity(Rabbit;MRID 00131257)NNX(H)X(H)Two-GenerationReproduction(Rat; MRIDs43250701, 43769401)N N NNNX(M,H)X(M, H)Chronic toxicity /carcinogenicity(Rat; MRID 00078361)NE NE N NX(H)NCarcinogenicity(Mouse;MRID 00078423)NE NE N NX(MH,H)X(MH,H)Chronic toxicity(Dog; MRID00007091)NE NE N NEX(H)X(H)METHOMYLFINALPage 12 of 54Table 2. Estrogenic/Anti-Estrogenic Pathway for MethomylLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Methomyl1Study Type /Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone 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 2 Overt Toxicity 3Fish Life-Cycle(MRID 43072101)NX(H)Avian Reproductivetoxicity(Quail;MRID 41898602)P6(H)NN7Avian Reproductivetoxicity(Duck;MRID 41898601)NNN8Avian Reproductivetoxicity(Duck;MRID 49245301)P9(H)NNN101. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in thisassay. Changes in weight are absolute unless otherwise indicated.2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by 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. The responsiveness of the cells to the very weak positive control 17α-methyltestosterone was lower than the expected values. Methomyl responses weresimilar to those of the negative control corticosterone and not comparable to the responses of 17α-methyltestosterone, which was able to reach amaximum of 24.3-26.6% PC. Methomyl was onlyable to reach a maximum of3.1-3.7% PC. Methomyl was considered negative in thistest system.5. Oocyte atresia was increased (mild to moderate) in females6. Reductions in egg laid (40%) were observed in the bobwhite quail study at the highest dose tested (427 mg/kg-diet)7. Increases in the incident of femaleswith feather loss and toe lesions [potentially treatment-related and due to male aggression (pen mate)] were reportedat mid and high treatment. No effects onsurvival or body weight were reported.8. Decreases in female body weight gain observed at highest concentration tested (427 mg/kg-diet); no other clinical signs oftoxicity observed.METHOMYLFINALPage 13 of 549. Increase in the number of females with regressing/regressed ovaries (8/15 vs. 4/16 in control) was observed at 1120 mg/kg-diet, however, no otherreproductive parameter was affected10. Decreases in female body weight gain (↓46.6%) and food consumption observed at 1120 mg/kg-diet; no other clinical signs of toxicity observed.P Positive findingsN Negative findings (in vitro) / No effect (in vivo)NE Not examinedNR Not reportedMETHOMYL FINAL

Page 14 of 54 2. Effects on Androgen Pathway

The potential for methomyl to interact w ith 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 androgeni c, anti-androgenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

Methomyl was negative (or a non-binder) in the Tier 1 in vitro androgen receptor (AR) binding assay. In the steroidogenesis assay, there were significant (p<0.05) decreases in mean testosterone levels at methomyl concentrations of 0.01 μ M (0.89-fold) and 100 μM (0.83-fold)
and the effect was consistent across all three ru ns. However, as a similar response was observed at two non-sequential concentrations (10,000-fold di fference), the confidence in these results are reduced. Methomyl was negative in the ARTA studies by Anderson et al . (2002) and Kojima et
al ., (2004).

The Hershberger assay was negative and in the male pubertal assay, no androgen-related effects
were observed.

In the FSTRA, significant decreases in fertilit y of 1.6% and 33% were noted at the mid- and high-concentrations, respectively. There was a slight increase in the proportion of spermatogonia (mild to moderate) in high-dose males. No othe r androgen-related effects were observed (sex hormone concentrations not measured). Body we ight was decreased approximately 10 and 13% in males from the mid- and high-concentrati on groups at study termination which may be suggestive of stress at the two highest test con centrations; no effects on mortality or clinical signs of toxicity were observed.

In the Part 158 mammalian toxicity data, th ere were no androgen-related effects observed. Mahgoub and El-Medany (1993/2001) investigat ed reproductive toxicity after oral administration of methomyl to male Wistar rats at 17 mg/kg/day by oral gavage for two months. Decreased (p<0.001) testosterone concentration was observed ( ↓ 35%) after methomyl administration for 60 days, as well as afte r a one-month recovery period (30%, p<0.001). Various degenerative changes in the seminife rous tubules, primary spermatocytes, and spermatids were also observed af ter treatment; histopathology afte r the recovery period was not presented. The results of this study are questiona ble because there was no reported mortality at doses that overlap the reported acute LD 50 range (17 to 45 mg/kg/day) for methomyl. In the Part 158 avian reproduction studies, ther e were no androgen-related eff ects reported. Similarly, in a fish life cycle toxicity test, there we re no androgen-relate d effects reported.METHOMYLFINALPage 15 of 54Table 3. Androgenic/Anti-Androgenic Pathway for MethomylLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Methomyl1Study Type/ LiteratureCitation AR Binding / AR activation Steroidogenesis Sex Steroid Hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed99999 3EDSP Tier 1 AssayAR Binding(MRID 48672001)NSteroidogenesis(MRID 48685902)P4Hershberger(MRID 48617702)NNN NMale Pubertal Rat(MRID 48701403)NNNNNNNNNNFSTRA(MRID 48701401)NENP5NFrt:↓1.6,33%;(M, H)NN6OSRIARTA assay(Andersenet al., 2002)NARTA assay(Kojimaet al., 2004)NSubchronic toxicity(13-week)(Rat; MRID 00007190)N N NE N N NEX(MH, H)X(MH)Two-generationReproductive toxicity(Rat; MRIDs43250701, 43769401)N N NE NENNX(M,H)X(M, H)Chronic toxicity /Carcinogenicity(Rat; MRID 00078361)NENNNENNNEX(H)NMETHOMYLFINALPage 16 of 54Table 3. Androgenic/Anti-Androgenic Pathway for MethomylLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Methomyl1Study Type/ LiteratureCitation AR Binding / AR activation Steroidogenesis Sex Steroid Hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed99999 3Carcinogenicity(Mouse;MRID 00078423)NENNNNNNEX(MH, H)X(MH, H)Chronic toxicity(Dog; MRID00007091)NENNNENENENEX(H)X(H)Testicular toxicity(Rat; El-Medanyet al.,1993 andMahgoubet al.,2001)↓35,30%7NE P8NE NENENFish life-cycle toxicity(MRID 43072101)NX(H)Avian reproductivetoxicity (Quail;MRID 41898602)NN N9Avian Reproductivetoxicity(Duck;MRID 41898601)NN N10Avian Reproductivetoxicity(Duck;MRID 49245301)NN N111. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay.Changes in weight are absolute unless otherwise indicated. LW=Liver weight2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. AMETHOMYLFINALPage 17 of 543. 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. Decreases in mean testosterone levels at 0.01μM (0.89-fold) and 100 μM (0.83-fold), and the effectwas consistent across all three runs. However, as asimilar response was observed at two non-sequential concentrations (10,000-fold difference), the confidence in these results are reduced.5. There was a slight increase in the proportion of spermatogonia (mild to moderate) in high-dose males6. Decreased body weight in mid-and high-dose fish (10% and 13%,respectively), however no effects on survival or clinical signs of toxicity were reported.7. Decreased concentrations aftermethomyl treatment for 60 days anda 30-day recovery, respectively.8. Degenerative changes in the seminiferous tubules, primary spermatocytes, spermatids, and Leydig cells.9. Increases in the incident of femaleswith feather loss and toe lesions (potentially treatment-related and due to male aggression (pen mate)) were reported atmid and high treatment. No effects on survival or body weight were reported.10. Decreases in male body weight gain observed at all test concentrations; no other signs of toxicity observed.11. Decreases in female body weight gain (↓46.6%) and food consumption observed at 1120 mg/kg-diet; no other clinical signs of toxicity observed. Thereduction in food consumption corresponded with slight reductions in mean body weight of both hens and drakes during the firsttwo weeks of the study:mean 0- to 2-week body weight changes were -104 versus -23 g for the control for drakes, and -72 versus -26 g for the control for hens.P Positive findingsN Negative findings (in vitro) / No effect (in vivo)NE Not examinedMETHOMYL FINAL

Page 18 of 54 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 methomyl 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 male and female pubertal a ssay and mammalian OSRI, there were no thyroidrelated effects observed. An increase in pituitary weight (absolute and rela tive) in the absence of histopathological changes was obs erved in the subchronic rat study.

In the amphibian metamorphosis assay, decrea sed HLL (normalized for SVL) values were observed on Day 7 ( ↓ 4.7%) and 21 ( ↓ 4%) in the high-dose group, and increased SVL ( ↑ 6%) was also seen on Day 21 in the high-dose group. Alt hough all of the surviving frogs were beyond NF stage 60 at test termination (termed “late-stag e”) which may confound the ability to adequately evaluate potential thyroid-related effects in th e AMA, there was no significant acceleration or delay of median NF developmental stage at a ny treatment concentration throughout the test and no effects on thyroid histopathology compared to the control. Therefore, the decrease in HLL and SVL on Day 7 and Day 21 in the absence of corroborating effects does not suggest potential
interaction with the HPT axis.METHOMYL FINAL

Page 19 of 54 Table 4. Thyroid Pathway for Methomyl

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

Study Type / Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmental stage(± or asynchronous,HLL)Growth(BW, SVL, HLL)Systemic ToxicityObsereved,2Overt ToxicityObserved3

EDSP Tier 1 Assays

Female Pubertal Rat (MRID 48701404) N N N N N N N N

Male Pubertal Rat (MRID 48701403) N N N N N N N N

AMA (Frog; MRID 48701402) N Days 7 and
21: ↓ 4.7 & 4% HLL (H) Day 21: ↑ 6% SVL (H) N

OSRI Subchronic (13-week) toxicity (Rat; MRID 00007190) NE N NE NE ↑ 19% (H) X (MH, H) X (MH)

Developmental toxicity (Rat; MRID 00008621) N
Developmental toxicity (Rabbit; MRID 00131257) X (H) X (H)

Two-generation Reproductive toxicity (Rat; MRIDs 43250701, 43769401) NE N N X (M, H) X (L, M, H)

Chronic toxicity / Carcinogenicity (Rat; MRID 00078361) NE N NE NE N X (H) N

Carcinogenicity (Mouse; MRID 00078423) NE N NE NE N X (MH,H) X (MH, H)

Chronic toxicity (Dog; MRID 00007091) N N NE NE NE X (H) X (H)

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

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A N Negative findings NE Not examinedMETHOMYL FINAL

Page 20 of 54 IV. Committee’s Assessment of Weight of Evidence Conclusions

This section of the document describes the we ight of evidence (WoE ) determination on the potential of methomyl to interact with endocrine related processes ( i.e., E, A or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated 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 ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence eval uation of the results of the Tire 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues shou ld be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l disruptions in homeostasis” (USEPA, 2013).

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 and OSRI
studies are described below. In addition to the endocrine-related effects described above for the

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

Page 21 of 54 1. Tier 1 in vivo Assays

Indications of systemic and/or overt toxicity were not obs erved in any of the Tier 1 in vivo mammalian studies with methomyl. In the FST RA study, decreased (p<0.05) body weights in
the male fish at the two hi ghest test concentrations ( ↓ 10 and 13%, respectively).

2. OSRI

In a subchronic toxicity study in rats,, no eff ects of treatment were observed on mortality or clinical signs (except for tremors in a singl e, high-dose female by the end of the study). Additionally, no adverse, treatment-related effects were not ed for plasma and red blood cell (RBC) ChE activity. Overall body we ight gain was decreased by 14% in the high-dose males, and body weight gains were decreased ( ↓ 13 and 17%) during Weeks 7 to 13 in the high-medium
dose animals when the dose was increased to 500 ppm. Liver weights were decreased in the high-medium and high-dose males, but no treatment-related histopathol ogical lesions were reported. In males, absolute and relative liver weights ( ↑ 17 and 7 to 11%, respectively) and absolute kidney weights ( ↑ 11 to 15%) were increased (p<0.05) at ≥ 480 ppm in males. Relative kidney weights were increased ( ↑ 8%, p<0.05) at 480 ppm only.

In a developmental toxicity study in rats, there were no mortalities or clinical signs of toxicity. In addition, there were no treatment-re lated decreases in body weights. In a developmental toxicity study in rabbits, seven rabbits in the high-dos e group died beginning w ith the initi ation of treatment and continuing throughout the tr eatment period (GD 7, 8, 9, 11, 17, 18, and 19). Central nervous system (CNS) stimulation occurred in the high-dose rabbits and was considered to be treatment related (dose-dependent in creases, p<0.05, in the incidence of tremors, hyperactivity, body jerks, excess salivation, aggressive behavior, hyperpnea and convulsions). Other dose-dependent, but not statistically sign ificant, increases in CNS stimulation were observed including ataxia, impaired or lost righting reflex, skin le sions (self-inflicted), dyspnea, hypersensitivity, pin-point pupils and vocalization. No treatment-re lated effects were noted for the low- and mid-dose groups.

In a two-generation reproduction study in rats , there was no evidence of a treatment-related
effect on survival. For the F 0 generation, mean body weights we re decreased (p<0.01) in the high-dose females (premating) and males (entir e study) by 10 and 16%, respectively. For the F 1 generation, mean body weights were decrease d (p<0.01) for the mid- and high-dose group females and males by 19 and 26%, respectivel y. During gestation and lactation, mean body weights were decreased (p<0.01) for the high-d ose females (and Lactation Week 1) of the

F 0 generation ( ↓ 11 to 18%) and for the mid- and high-dose females of the F 1 generation ( ↓ 11 to 27%). In the F 1 litters, mean pup weights were decreas ed (p<0.01) in the mid- and high-dose groups throughout lactation (approximately ↓ 10 to 30%) and decreased (p<0.05 or 0.01) in the low-dose group at LD 14 and 21 (approximately ↓ 10%). In the F 2 litters, mean pup weights were generally decreased (p<0.01) in the mid- and high-dose gr oups throughout lactationMETHOMYL FINAL

Page 22 of 54 (approximately ↓ 10 to 30%). The RBC and plasma cholinesterase concentrations were comparable between the treated and control groups.

In a chronic toxicity/carcinogeni city study in rats, the high-dos e group had decreased body weight gain during the time intervals 0 to 91, 0 to 182, and 0 to 364 days of treatment. High-dose male rats continued a similar reduction in body weight ga in through 0 to 560 days of treatment, but no differences between final body weight or body weight gain in the ra ts were observed compared to
control at the end of the study. No treatment-related morta lity was observed during the study. No significant differences were observed in RBC and brain ChE concentrations due to treatment in either sex. There were no differences in absolute organ weights for male or female rats in any treatment group. In a carcinogenicity study in mice, survival in the high-dose males and females was significantly decreased (per cent and significance not reported ) at Week 26, and survival in the mid-dose groups was also moderately decreased (percent and significance not reported). Due to these high mortality rates, the high and intermediate dose s were reduced. Survival was generally significantly decrease d (percent and significance no t reported) after 52, 80, and 104 weeks of treatment in mid- and high-dose animals. Decreased survival rates are considered to be treatment-related; surv ival exceeded 50% in all groups at 15 months of study. No treatmentrelated effects on body weight or body weight gain were observe d in this study. There were no differences in absolute organ weights for male or female rats in any treatment group, except for increased (percent and significance not reported) absolute adrenal weight in high-dose males.

In a chronic toxicity study in dogs, one high-dose female was found dead in its cage on the first day of Week 9. A replacement female dog was st arted on treatment two weeks later but this animal had repeated seizures on Day 17 of treatment, followed by coma and death. Two highdose males exhibited tremors, salivation, inco ordination and circling movements on a single afternoon during Week 13 of the study. Another hi gh-dose male exhibited a higher incidence of diarrhea compared with other study animals and progressive an emia throughout the study with markedly declined hematology parameters at 18 months compared to baseline. Treatment was discontinued from Weeks 85 through 94, with impr oved hematological analyses. The animal was returned to study for the remainder of the dosing period (Weeks 95–104), with a concomitant return to severe anemia and moderate leucope nia by the end of treatme nt. Grossly, it was found to have an enlarged spleen, an enlarged liver , and increases in absolu te and relative thyroid, heart, liver, and kidney weights. The remaining two high-dose males had increased absolute and relative kidney weights at study termination. No adverse effects of treatment were observed on body weight and body weight gai n. Microscopic findings in th e high-dose animals included changes in the kidneys and liver.

In a flow-through, early life-stage toxicity test with the fathead minnow, egg hatchability and fry survival were not decreased at any test concentr ation. Both length and weight were decreased at

≥ 0.143 mg/L. In another freshwater fish early life- stage toxicity test wi th the fathead minnow, percent hatch was decrea sed at concentrations ≥ 117 μg/L. Survival was decreased (p<0.05) byMETHOMYL FINAL

Page 23 of 54 10 to 20% at ≥ 117 μg/L, and mean length and wet we ight were decreased (p<0.05) at
concentrations ≥ 243 μg/L (medium-high concentration). In a freshwater fish full life-cycle toxicity test with the fathead minnow, survival was decreased (p<0.05) by 6 to 7% at the high concentration. Mean length (calculated at Da ys 28, 56, and 84) was decreased (p<0.05) at
concentrations ≥ 142 μg/L, and wet weight were d ecreased (p<0.05) at the 280 μg/L concentration. The length of F1 larvae at 56 da ys post-hatch was decreased at 142 and 280 μg/L concentrations, and wet weight was decrea sed at the 280 μg/L concentration only.

In an avian reproduction study in bobwhite quail, th ere were no treatment-related mortalities. Reports of females with feather loss and toe le sions [potentially treatment-related and due to male aggression (pen mate)] were reported at 427 mg/kg-diet. In the mallard duck reproduction study, significant reductions in male body weight gain were observed at all test concentrations, and in female body weight gain in the high treat ment (427 mg/kg-diet). In another mallard duck reproduction study (MRID 49245301), decreases food consumption was observed at 1120 mg/kg-diet (preliminary review ). The reduction in food consumption corresponded with slight reductions in mean body weight of both hens and dr akes during the first two weeks of the study: mean 0- to 2-week body weight changes were -104 versus -23 g for the control for drakes, and - 72 versus -26 g for the control for hens. In addition, there was a st atistically-significant reduction in female body weights compared to th e control (1135 versus 1228 g; p<0.05) at study
termination.

B. Estrogen Pathway

There was no convincing evidence of interaction of methomyl with the estrogen pathway either
in the in vitro ( i.e ., ER binding, ERTA, aromatase and steroidogenesis) or mammalian in vivo ( i.e . uterotrophic of female pubert al) Tier 1 assays or in th e mammalian or wildlife Part 158 studies. In the FSTRA, although decreases in GSI, fertility and fecundity were observed at the mid and high treatment (0.312 and 1.03 mg/L, respectiv ely) and increased incidence and severity of oocyte atresia at the high treatment were reported there were no effects observed on VTG. Although there were some complementary effects across the FSTRA, there was no effect on VTG (sex steroid hormones not measured), a nd there was limited redundancy across the other Tier 1 assays or Part 158 studies. Decreases in eggs laid (fecundity ) were observed in the bobwhite quail reproduction study at the highest concentration te sted (427 mg/kg-diet), but no other reproductive effects were observed. Decreases in fecundity alone are limited in the ability to inform about potential estrogen-related effect s. Additionally, potentially similar to the decrease in fish GSI, an increase in number of females with regressed ovaries was observed in the mallard duck reproduction study, but no other reproductive effects were observed and female body weight gains and food consumption were decrea sed at this concentration. Furthermore, in other toxicity testing with fathead minnow fish (early life-st age and chronic reproduction), effects on growth in young fry were observed at concentrations lower (0 .117 mg/L) or similar (0.243 or 0.280 mg/L) to the concen trations tested in the FST RA. Therefore, while there were noMETHOMYL FINAL

Page 24 of 54 clinical signs of toxici ty in female fish in the FSTRA (male body weights were significantly decreased), the concentrations tested in the FST RA were similar to concentrations which were shown to be overtly toxic in other fish assays; as such, the decreases observed in the FSTRA may be due to potential general stress on the fish fr om exposure to methomyl as oppose to potential
endocrine-related interactions.

C. Androgen Pathway

There was no convincing evidence of interaction of methomyl with the androgen pathway. The
Tier 1 in vitro AR binding assay was negative, and while a decrease in testosterone production
was observed in the Tier 1 steroi dogenesis assay, there is uncertainty in this result based on the pattern of responses. No androgen-related effect s were observed in the Tier 1 Hershberger or male pubertal assays or Part 158 mammalian st udies. While androgen-related effects were observed in an OSRI study (El Medany et al. 1993) , the results were questionable as the test doses overlapped with acute mortality values. In the FSTRA assay, there was a slight increase in the proportion of spermatogonia (mild to modera te) observed in male testes at the high concentration along with the de creases in fertility at the mid and high treatment; however, significant decreases in male body weight were observed at the mid and high treatment. Considering the body weight changes along with the discussion in the estrogen pathway regarding test concentrations and other obs ervations of overt toxicity in fish, the alterations/decreases observed may be due to gene ralized stress in the fish. Also, as in the estrogen pathway, there is a lack of redundancy in observed effects across the Tier 1 battery and
Part 158 studies.

D. Thyroid Pathway

There was no convincing evidence of interacti on of methomyl with th e thyroid pathway. No thyroid-related effects were observed in the ma mmalian studies, and no effect on developmental endpoints or thyroid histopat hology were seen in the AMA.

E. Conclusions

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

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations, human health and wildlife EDSP Tier 2 testing is not recommended for methomyl since there was no convincing evidence of potential interaction with the estrogen, androgen or thyroid pathways.METHOMYL FINAL

Page 25 of 54 VI. References

Andersen, H., Vinggaard, A., Rasmussen, T., Gjermandsen, I., and Bonefeld-Jorgensen. E. (2002). Effects of Currently Used Pesticides in Assays for Estrogeni city, Androgenicity, and Aromatase Activity In Vitro

. Toxicol. Appl. Pharmacol . 179:1–12.

Beavers, J.; Hawrot, R.; Lynn, S., and Jabe r, M. (1991). H-17940: A One-Generation Reproduction Study with the Northern Bobwhite ( Colinus virginianus ): Lab Project No: 112- 227. Unpublished study prepared by Wildlif e International Ltd. 168 p. (MRID 41898602).

Busey, W.M. (1968). Final Report: Two-year Dietary Administrati on-Dogs: Project No. 201- 165. (Unpublished study including addendum, r eceived Jun 5, 1969 under 9F0814; prepared by Hazleton Laboratories, Inc., submitted by E.I. duPont de Nemours & Co., Inc., Wilmington, Del.; CDL:091405-A). (MRID 00007091).

Driscoll, R.R., and Muska, C.F. (1982). Early Life Stage Toxicity of Methomyl to Fathead Minnow: Haskell Laboratory Re port No. 528-82; MR No. 0581-930

. (Unpublished study received Oct 3, 1983 under 352-366; submitted by E.I. duPont de Nemours & Co., Inc., Wilmington, DE; CDL: 251426-C). (MRID 00131255).

El-Medany, A., El-Medany, G., and Rahmy, A.E. ( 1993). Evaluation of the effect of methomyl
insecticide on the testicular function in normal albino rats (hormonal, histological and histochemical study). J. Med. Res. Inst. 14:165–187.

Feussner, E.L., Hoberman, A.M ., Christian, M.S., Lightkep, G., et al. (1983). Embryo-Fetal Toxicity and Teratogenicity Study of Methomyl in the Rabbi t: Study No. 104-005. Final rept. (Unpublished study received Oct 3, 1983 under 352-366; prepared by Argus Research Laboratories, Inc., submitted by E.I. duPont de Nemours & Co., Inc., Wilmington, DE; CDL:251427-B). (MRID 00131257).

Fort, D.J. (2012). Methomyl (DPX-X1179) T echnical: 21-d Amphibian Metamorphosis Assay (AMA) with South African Clawed Frog, Xenopus laevis . Unpublished study performed by Fort Environmental Laboratories, Inc., Stillwater, OK. Labor atory report number DUPO01-00269. Study sponsored by E.I. du Pont de Nemours and Company (DuPont), Wilmington, DE. (MRID 48701402).

Hicks, S.L. (2012). Methomyl Technical (DPX-X1179): Short-term Reproduction Assay with the Fathead Minnow, Pimephales promelas , Determined Under Flow-Through Conditions. Performed by ABC Laboratories, Inc., Colu mbia Missouri and Experimental Pathology Laboratories, Inc., Sterling, Virginia. Lab St udy No.: 66795; Sponsor Project No.: DuPontMETHOMYL FINAL

Page 26 of 54 32248. Submitted by E.I. du Pont de Nemours a nd Company, Wilmington , Delaware. (MRID 48701401).

Howard, T.; Rhodes, J.; Mihalik, R. (1991). Ea rly Life-Stage Toxicity of IN X1179-394 to the
Fathead Minnow (Pimephales promelas) Under Flow-Through Conditions: Final Report. Project Number: 39292, 9006/02, HL0/7052/91. Unpublished study prepared by Analytical BioChemistry Labs., Inc. 494 p. (MRID 46015305).

Hurtt, M. (1995). Nudrin-Two Generati on Reproduction Study in the Rat: Supplement No.1: Lab Project Number: RA274: WRC RIR-275: HLO 519-95. Unpublished study prepared by WIL Research Labs, Inc. 149 p. (MRID 43769401).

Kaplan, A.M., Hostetler, K.H., Dilworth, T., et al. (1981). Long-Term Feeding Study in Rats with S-Methyl N-[(Methylcarbamoyl) oxy]th ioacetimidate (Methomyl; INX-1179): Haskell Laboratory Report No. 235-81. Final rept. (U npublished study received Aug 28, 1981 under 1H5320; submitted by E.I. duPont de Nemours & Co., Inc., Wilmington, Del,; CDL:070242-A). (MRID 00078361).

Kojima, H., Katsura, E., Takeuchi, S., Niiyami, K., and Kobayashi, K. (2004) Screening for estrogen and androgen receptor activi ties in 200 pesticides by in vitr o reporter gene assays using Chinese hamster ovary cells. Environ. Health Perspect . 112 (5): 524-531.

Lu, C. (1983). Nudrin Two-Ge neration Reproduction Study in Rats: Lab Project Number: WRC RIR-275. Unpublished study prepared by WIL Re search Lab., Inc. 1144 p. (MRID 43250701).

Mahgoub, A.A. and El-Medany, A. H. (2001). Eval uation of chronic exposure of the male rat reproductive system to the insecticide methomyl. Pharmacological Research 44, 73–79.

Nabb, D.L. (2012). Methomyl (DPX-X1179) Technical: H295R Ster oidogenesis Assay. Unpublished study prepared by E.I. du Pont de Nemours and Company, Newark, DE. Laboratory Report No.: DuPont -32086, February 8, 2012. (MRID 48685902).

Paynter, O.E. (1966). Thr ee-Month Dietary Administra tion-Rats: Project No. 201-151. (Unpublished study including supplement, r eceived Feb 20,1966 under 352-342; prepared by Hazleton Laboratories, Inc., submitted by E.I. du Pont de Nemours & Co., Wilmington, Del.; CDL:050428-A) . (MRID 00007190).

Porter, W.P., Green, S.M., Debbink, N.L., and Ca rlson, I. (1993). Gr oundwater pesticides:
interactive effects of low concen tration of carbamates aldicarb and methomyl and the triazineMETHOMYL FINAL

Page 27 of 54 metribuzin on thyroxine and soma totropin levels in white rats. J Toxicol. Environ. Health 40:15–

34.

Rogers, A.S., Culik, R., Wood, R.A., et al. (1978). Oral Teratogenic Study in Rats with Lannate (INX-1179): Haskell Laboratory Report N o. 498-78. (Unpublished study received Oct 18, 1978 under 352-342; submitted by E.I. duPont de Nemours & Co., Wilmington, Del.; CDL:097449- A). (MRID 00008621).

Snajdr, S. (2011). Methomyl (DPX-X1179) Tec hnical: 3-Day Uterotrophic Assay for Detecting Estrogenic Activity. Unpublished study prepared by DuPont Haske ll Global Centers for Health & Environmental Sciences, Newark, DE. Laboratory Study ID: DuPont-32084, November 8, 2011. (MRID 48617701).

Snajdr, S. (2011). Methomyl (DPX-X 11 79) Technical: 10-Day Hershberger Bioassay for Detecting Androgenic Activity. Unpublished stu dy prepared by DuPont Haskell Global Centers for Health & Environmental Sciences, Newa rk, DE. Laboratory Study ID: DuPont-32085, November 8, 2011. (MRID 48617702).

Snajdr, S. (2012). Methomyl (DPX-X1179) technical: in vitro androgen receptor binding assay using rat prostate cytosol. Unpublished study prepared by DuPont Haskell Global Centers for Health & Environmental Sciences, Newark, DE. Laboratory Project ID number: DuPont-32080, January 6, 2012. (MRID 48672001).

Snajdr, S. I. (2011). Methomyl (DPX-X 1179) aromatase (human recombinant) assay. Unpublished study prepared by E.I. du Pont de Nemours and Company, DuPont Haskell Global
Centers for Health & Environmental Scien ces, P.O. Box 50, Newark, Delaware, 19714. Laboratory Report No.: DuPont- 32081. January 5, 2012. (MRID 48672002).

Snajdr, S.I. (2012). Meth omyl (DPX-X1179) technical: in vitro estrogen receptor binding assay using rat uterine cytosol (ER-RU C). Unpublished study prepared by E.I. du Pont de Nemours and Company, DuPont Haskell Global Centers fo r Health & Environmental Sciences, Newark, DE. Laboratory Project ID.: Du Pont-32083, January 5, 2012. (MRID 48672003).

Snyder, F.G. (1981). 104-week Chronic Toxicity and Carcinogenicity Study in Mice: Project No. 201-510. (Unpublished study received Aug 28, 1981 under 1E2556; submitted by E.I. duPont de Nemours & Co., Inc., Wilm ington, Del.; CDL:070247-C; 070246. (MRID 00078423).

Strawn, T., Rhodes, J., and Leak, T. (1993). Full Life-Cycle Toxi city of DPX-X1179-394 (Methomyl) to the Fathead Minnow (Pimephales promelas) Under Flow-Through Conditions:METHOMYL FINAL

Page 28 of 54 Final Report: Lab Project Nu mber: 39293: HLO 47-93. Unpublishe d study prepared by ABC Laboratories, Inc. 3582 p. (MRID 43072101).

Toot, J.D. (2012). Pubertal Development and Th yroid Function in Intact Juvenile/Peripubertal Male Rats Following Oral Administration of Methomyl (DPX-X1179) Technical. Unpublished study prepared by WIL Research Laboratories, LLC, Ashland, OH, Laboratory Project ID: WIL-189230, June 5, 2012. (MRID 48701403).

Toot, J.D. (2012). Pubertal Development and Th yroid Function in Intact Juvenile/Peripubertal Female Rats Following Oral Administrati on of Methomyl (DPX -X1179) Technical. Unpublished study prepared by WIL Research La boratories, LLC, Ashland, OH. Laboratory Project ID: WIL-189227, June 5, 2012. (MRID 48701404).

USEPA. (2010). Methomyl — Report of the Endocrine Disruptor Review Team — Test Order #: EDSP- 090301-62. December 6, 2010. Office of Ch emical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

USEPA. (2011). Endocrine Disruptor Screenin g 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.

Willoughby, J.A (2012). Methomyl (DPX-X117 9) Technical: Estrogen Receptor Transcriptional Activation (H uman Cell Line (HeLa-9903)). Unpublished study prepared by CeeTox, Inc., Kalamazoo, MI. Laboratory Report No.: 9144V-100360ERTA, Feb 7, 2012. (MRID 48685901).METHOMYL FINAL

Page 29 of 54 APPENDIX 1. EDSP T1 Screening Assays

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100)

The 21-day assay (MRID 48701402) of methomyl on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was studied under flow-through c onditions. Amphibian larvae (80 larvae per group) at Nieuwkoop-Faber (NF) stage 51 were exposed to nominal concentrations of 0 (negative control), 0.0183, 0.056, and 0.168 mg a.i. /L; mean-measured concentrations were <0.0124 (<LOD; control), 0.0170, 0.0522, and 0.186 mg a.i. /L. The test system was maintained at 21.9 to 22.4°C and a pH of 6.9 to 7.2.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. One mortality (1.7%) was obser ved in the highest test concentration. All surviving tadpoles appeared normal and healthy with no apparent c linical signs present at test
termination.

There was no significant acceleration or delay of median NF developmental stage at Day 7 or Day 21 in any treatment group. Furthermore, no asynchronous development was observed. All individuals developed beyond NF st age 60 by Day 21; the NF stage ranged from 61 to 65 in all treatment groups and in the control. The reviewe r’s analysis detected significantly decreased normalized (for snout vent length) hind-limb length (HLL) at both Day 7 ( ⇘ 4.7%) and Day 21 (

⇘ 4%) at high treatment level relative to th e negative control (p<0.05). Additionally, Day 21 snout vent length (SVL) was significantly increased ( ⇖ 6%) at the high treatment level relative to the negative control (p<0.05). Thyroid gland histopa thology revealed mild to moderate follicular cell hypertrophy in the negative control and all treatment groups, although there was no treatment-related pattern. There were no inci dences of thyroid gl and hypertrophy, atrophy, or follicular cell hyperplasia.

At Day 21, all tadpoles were greater than NF stage 60 (late stage) which may confound the ability to adequately interpre t the Day 21 results, however, th ere was no significant acceleration or delay of median NF developmental stage at any treatment concentra tion throughout the test nor effects on thyroid histopath ology compared to the control.

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

In an androgen receptor (AR) binding assay (MRID 48672001), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a saturation binding experiment and a competitive binding experiment. The sa turation binding experiment was conducted to demonstrate that the AR in the rat prostate cy tosol was present in reasonable numbers and was functioning with appropriate affi nity for the radio-labeled refe rence androgen (R1881) prior to conducting AR competitive binding experiments. The competitive binding assay was conductedMETHOMYL FINAL

Page 30 of 54 to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (10

− 10 to 10

− 3 M) of methomyl (99.4% a.i., Lot # JUL06LP276).
Water was used as the solvent vehicle for methom yl, and ethanol was used as the solvent vehicle for the reference compounds at a final assay con centration of 3%. A total of three independent runs were performed, and each run included dexa methasone as a weak positive control, and R1881 as the ligand reference standard.

In the saturation binding experime nt, the maximum binding capacity (B max ) was 4.5 fmol/100 μ g protein and the disso ciation constant (K d ) was 0.903 nM. The K d was within the range reported in the EPA validation program, but the B max was below the EPA values (7-16 fmol/100 μ g protein). The Scatchard plots for th e three saturation assays were linear.

In the competitive binding experiment, the estimated average log IC 50 was

− 9.15 M for R1881
and

− 4.41 M for dexamethasone. Compared to R1881, the mean RBA for the dexamethasone was 0.0019%. The performance criteria for R1881 and dexamethasone were not met across the three runs. In the first and third runs, the bottom % binding and Hill slope were outside the
tolerance limits for R1881. For dexamethasone a ll of the three cr iteria were met, except for the top % binding in the third run which was hi gher than the toleran ce limits. Although, the performance criteria for both R1881 and dexameth asone were not achieved across the three runs,
based on the estimated IC 50 s for R1881 and the weak positive c ontrol, the androgen receptor was functioning with appropriate affinity for the ligand in the competitive binding assays. It is unlikely that information from additional competitive binding runs would change the interpretation of this study.

The percent [ 3 H]-ligand bound to the AR was >75% at a ll concentrations of methomyl in all three runs. Therefore, a log IC 50 and RBA could not be calculated for methomyl.

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

Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48672002), methomyl (99.4% a.i., Lot # JUL06LP276) was incubated with human recombin ant aromatase and tritiated androstenedione (1- β [ 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) in water at l og concentrations of 10

− 10 to
10

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

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC). A total of four runs were conducted and each runMETHOMYL FINAL

Page 31 of 54 included full enzyme activity controls, background activity controls, a positive control series (10

− 10 to 10

− 5 M) using a known inhibitor, 4-hydroxya ndrostenedione (4-OH ASDN), and the methomyl series (10

− 10 to 10

− 3 M) with three repetitions per c oncentration. Because the solvent vehicle was water for methomyl and dimethyl su lfoxide (DMSO) for 4-OH ASDN, separate sets of the background activity and full enzyme activit y controls were run using only water or 1% DMSO (final concentration in assay). In the second run, the full activity controls (water only) had aromatase activity below the performance criteria specified in the guideline; therefore, the
second run was excluded from the calcula tions and a fourth run was conducted.

Aromatase activity in the full activity controls for the three successful test runs averaged 0.381±0.117 nmol·mg-protein

− 1 ·min

− 1 in water only, and 0.431±0.141 nmol·mg-protein

− 1 ·min

− 1 in 1% DMSO. Activity in the background controls averaged 0.59% of the full activity controls in water and 0.54% of full activity controls in 1% DMSO. The response of each full activity
control within a run wa s between 90 to 110% of the average full control activity, except for the average initial value of 117% in 1% DMSO and the average ending value of 83% in 1% DMSO
for Run 1.

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.431±0.090 nmol·mg-protein

− 1 ·min

− 1 at the lowest tested concentration (10

− 10 M) and 0.004±0.001 nmol·mgprotein

− 1 ·min

− 1 at the highest tested concentration (10

− 5 M). As a percent of the control, aromatase activity for 4-OH ASDN was 1.02% at 10

− 5 M and 103.14% at 10

− 10 M.

For methomyl, aromatase activity averaged 0.358±0.096 nmol·mg-protein

− 1 ·min

− 1 at the lowest tested concentration (10

− 10 M) and 0.366±0.118 nmol·mg-protein

− 1 ·min

− 1 at the highest tested concentration (10

− 3 M). The lowest portion of the respons e curve was greater than 75% activity
at concentrations from 10

− 10 M to 10

− 3 M methomyl across all runs.

Based on the data from the average response curve, methomyl is classified as a Non-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 48672003) for methomyl (99.4% purity, Lot #JUL06LP276), uterine cytosol from Sprague Dawl ey rats was used as the source of ER to conduct saturation binding experiments and compet itive binding experiments. The saturation binding experiment was conducted to demonstrate that the ER in the ra t uterine cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radiolabeled reference estrogen prior to routinely conducti ng ER competitive binding experiments. The competitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of incr easing concentrations (logarithmic increaseMETHOMYL FINAL

Page 32 of 54 from 10

− 10 to 10

− 3 M) of methomyl. Water was used as the solvent vehicle for methomyl, and
ethanol was used for the reference chemicals at a final concentration of 2%. A total of 3 runs were performed, and each run included 19- norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17 β -estradiol as the na tural ligand reference
material.

In the saturation binding experime nts, the maximum binding capacity (B max ) was 32.5 ± 2.25 fmol/100 μg protein and th e dissociation constant (K d ) was 0.081 ± 0.022 nM. The K d for each run was within the expected range of 0.03 to 1.5 nM. The saturation binding experiment demonstrated that the estrogen receptor was present in a reasonable concentration and functioning with appropriate affi nity for the reference ligand 17 β -estradiol.

In the competitive binding experiments, the thre e reference chemicals performed as expected. The average log IC 50 was

− 9.16 M for 17 β -estradiol and

− 4.85 M for the weak positive control, 19-norethindrone, and the mean RBA was 0.0054% for the weak positive control. The negative control, octyltriethoxysilane, had no effect on specific binding of the [ 3 H]-ligand (85-105% binding).

Methomyl had no appreciable effect on specific binding (88 to 113%) of the [ 3 H]-ligand to the ER in the three runs; therefore, a log IC 50 and RBA could not be calcu lated for methomyl. Based on the results from the three runs, methomyl is classified as Not Interactive in the Estrogen Receptor Assay.

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

In an estrogen receptor transcript ional activation assay (MRID 999), hER α -HeLa-9903 cells
cultured in vitro were exposed to methomyl (99.4% a.i ., Lot # JUL06LP276) at logarithmically increasing concentrations from 10

− 10.8 to 10

− 3.8 M in water for approximately 24 hours in two independent runs. Experiments were performed using 96-well plates and each concentration was tested in 6 wells/plate in each run. Cells were exposed to the test agent for 24±2 hours at 37±1°C to induce reporter (luciferase) ge ne products. Luciferase expressi on in response to activation of the estrogen receptor was measured usi ng a proprietary luciferase assay.

No cytotoxicity or precipitation were noted at the highest concentration of methomyl tested, 10

-

3.8 M. In the main assays, the responsiveness of the 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. Although the conditi ons of this assay were not optimal to detect very weak activity, methomyl responses were similar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to reach a maximum of 24.3-26.6% PC. Methomyl was only able to reach a maximum of 3.1-3.7%METHOMYL FINAL

Page 33 of 54 PC when tested up to the highest concentrati on possible based on cytotoxicity. Because the

RPC Max < PC 10 in two assay runs, methomyl was cons idered negative for estrogen receptor transcriptional activation in this test system.

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

The 21-day short-term reproduction assay (MRID 48701401) of methomyl with fathead minnows ( Pimephales promelas ) was conducted under continuous flow-through conditions. Adult fish (16 spawning groups, 2 males and 4 fe males in each group; ca. 6 months old) were exposed to methomyl (99.4% purity) at nominal concentrations of 0 (control), 0.090, 0.30, and 1.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.0124, negative control), 0.0947, 0.312, and 1.03 mg a.i./L. The test system was main tained at 24.2 to 24.8oC and a pH of 7.88 to

8.33.

Adult male and female survival was unaffected by treatment with methomyl and was 100% for
all test levels. There were no notable observations with regard s to behavior or changes in appearance, specifically color/bandi ng, ovipositor appearance, or si ze of dorsal nape pad in the control or treated groups, and no clinical signs of toxicity were observed. Male body weights were reduced (p<0.05; Jonckheere-Terpstra test) by 9.8 and 13% at the mid and high concentrations, respectiv ely, relative to the negative c ontrol; female body weights were not affected. Body lengths at test termination were not reported.

Spawning occurred at least every three days, an d mean fecundity in negative control was 54 eggs/female/day/replicate (replicate range was 50-62 eggs/female/day); fertilization success in the negative control group averaged 92%. There were significant reductions in fecundity of 23 and 77% and in fertility of 1.6 and 33% at the mid and high concentrations, respectively, compared to the negative control (p <0.05; Jonckheere-Terpstra test).

Additionally, female gonadal-somatic index (GSI ) was significantly reduced by 15 and 21% at the mid and high concentrations , respectively, compared to th e negative control (p<0.05; Jonckheere-Terpstra test). Male GSI was not significantly different from the control. Histopathology showed treatment -related effects at the high c oncentration, consisting of increased prevalence and severity of increased spermatogonia in males and increased prevalence and severity of increased oocyte atresia in females. There were no differences in gonadal stages between treated groups and the negative c ontrols. Plasma vitellogenin (VTG) was not significantly different from controls for either sex. Sex steroids we re not measured in this study.

Hershberger Assay; (OCSPP 890.1400)

In a Hershberger assay (MRID 48617702) testing fo r androgenic activity, me thomyl (99.4% a.i., lot # JUL06LP276) in deionized water was administ ered daily via oral gavage to six 56-day oldMETHOMYL FINAL

Page 34 of 54 (approximate age), castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 0.25, 0.5, or 1.0 mg/kg/day. An androgenic positive control group consisted of 6 castrated rats exposed to 0.4 mg/kg/day testosterone propionate (TP) in corn oil with 1% ethanol by subcutaneous (s.c.) injection.

To screen for potential anti-a ndrogenic activity, methomyl (99.4% a.i., lot # JUL06LP276) in
deionized water was administered daily via oral gavage to six 56- day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 0.25, 0.5, or 1.0 mg/kg/day in conjunction with a daily dose of TP at 0.4 mg/kg/day in corn oil with 1% ethanol by s.c. injecti on. The anti-androgenic positive control group consisted of 6 castrated rats dosed with 0.4 mg/kg/day TP in corn oil with 1% ethanol by s.c. injection and flutamide (FT) in 0.1% Tween® 80 in 0.5% methylcellulose at 3 mg/kg/day by oral gavage. TP alone was used as the anti-a ndrogenic negative control.

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 liver and the five androgen-dependent tissu es. Body weights, body weight gain and food consumption were measured throughout the dosing period.

All animals survived until schedul ed termination. There were no c linical signs of toxicity and no treatment-related gross pathological findings.

In the androgen agonist assay, there were no e ffects on body weights, body weight gains, or food efficiency in the methomyl treated groups. Ov erall food consumption was increased (p<0.05) in the 0.5 mg/kg/day group (9%), but the effect wa s not dose dependent. Rats dosed with TP (positive control) had incr eases (p<0.05) in overall body we ight gains (23%) and food consumption (18%), but there was no effect on food efficiency. There were no significant increases in the weights of the five accessory sex organs in any of the methomyl-treated groups. Liver weights were comparable to the vehicle control in all dose groups. Rats in the positive control group responded appropriately with signif icant increases in all five accessory sex organ weights (p<0.05). The performance criteria indicat ed that the assay was performing as expected.

In the anti-androgen assay, there were no e ffects on body weights, body weight gains, food consumption, or food efficiency in the methom yl groups or positive control (TP + FT) group when compared to the TP control. There wa s a 31% decrease (p<0.05) in Cowper’s glands weight in the 0.5 mg/kg/day methomyl group; however, no significant decreases in organ weights were seen in the remaining four target accessory sex tissues. The liver weights in the methomyl-treated groups were comparable to the TP control. Rats in the positive (TP + FT) control group responded with significant decreases in all five accessory sex organ weights. The performance criteria indicated that the assay was performing as expected.METHOMYL FINAL

Page 35 of 54 The dose levels tested in this study are considered to be adequate based on data from earlier studies showing toxicologically-a dverse clinical signs of cholinesterase inhibition at doses ≥ 2.0 mg/kg/day.

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

Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48701404), 15 Crl:CD(SD) Sprague-Dawley rats/dose group were treated daily by oral gavage with met homyl technical (99.4%; Lot # JUL06LP276) in
deionized water at doses of 0, 0.5 or 1.0 mg/kg/day from post-natal day (PND) 22 to PND 42. Animals were examined for vaginal opening (V O) daily beginning on PND 22, and the age and weight at day of attainment was recorded. Following sacrifice on PND 42, blood was collected for clinical chemistry analyses, including total thyroxine (T 4 ) and thyroid stimulating hormone (TSH), which were analyzed with electrochemiluminescent immunoassays or radioimmunoassays, respectively. Kidney, liver, adrenal gland, t hyroid, pituitary, uterus, and ovary weights were recorded, and microscopi c examinations were conducted on the thyroid, kidney, ovary, and uterus.

All animals survived until scheduled termination and no treatment-related c linical findings were observed. No effects were observed on body weight s or body weight gain in the study. No significant differences were observed in mean age at VO, body weight at VO, or final body weights in the treated groups relative to control. There were no dose-related effects on mean age at first vaginal estrus, mean cycle length, percen tage of females cycling, percentage of females regularly cycling, or cycle status at necropsy. Serum T 4 and TSH were not affected by treatment.

There were no treatment-related microscopic findings in the thyroid, ovaries, uterus, or kidneys. No effects on organ weights were observed durin g the study. All clinical chemistry parameters in the treated groups were similar to control.

The dose levels tested in this assay are consider ed to be adequate based on the cholinesterase
inhibition seen at doses ≥ 2.0 mg/kg/day in the toxicology database for this chemical.

Male Pubertal A ssay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48701403), 15 Crl:CD(SD) Sprague-Dawley rats/dose group were treated daily via oral gavage (5.0 mL/kg) with meth omyl technical (99.4%; Lot #METHOMYL FINAL

Page 36 of 54 JUL06LP276) in deionized water at doses of 0, 0.5 or 1.0 mg/kg/ day from post-natal day (PND) 23 to PND 53. Animals were examined for pr eputial separation (PPS) daily beginning on PND 30, and their age and weight at day of attainment was recorded. Following sacrifice on PND 53,
blood was collected for clinical chemistr y analyses, including total thyroxine (T 4 ), testosterone, and thyroid stimulating hormone (TSH). Testosterone and T 4 were analyzed with an electrochemiluminescent assay and TSH levels were analyzed by using radioimmunoassay. Liver, adrenals, pituitary, and urogenital or gan weights were reco rded, and microscopic examinations were performed on the thyr oid, kidney, testis and epididymis.

Red material around the mouth or ear was observed infrequently dur ing the daily examinations. No treatment-related effects were observed on mortality, body weights, b ody weight gain, organ weights, or gross pathology. Ag e and body weight at PPS in the tr eated groups were similar to controls. Thyroid hormone levels, colloid area, and follicular cell height scores for the thyroid were similar in the treated groups to the control group. Serum testosterone levels were similar in the treated groups to the control group.

The dose levels tested in this study are considered to be adequate based on data from earlier studies showing toxicologically-a dverse clinical signs of cholinesterase inhibition at doses ≥ 2.0 mg/kg/day.

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (MRID 48685902), H295R cells cultured in vitro in 24-well plates were incubated with methomyl (99.4% a.i., Lo t # JUL06LP276) at log concentrations from

0.0001 to 100 μ M for 48 hours in triplicate in three i ndependent experiments. Dimethyl sulfoxide (DMSO) was used as the vehicle for the positive controls and water was used as the vehicle for methomyl.

Testosterone and estradiol leve ls were measured using HPLC/MS/MS method with internal standards. A Quality Control (QC) plate was ru n concurrently with each independent run of a test chemical plate to demonstrate that the assa y responded properly to positive control agents at two concentration levels; positive controls included the known inhi bitor (prochloraz) and inducer (forskolin) of estradiol a nd testosterone production.

The minimum basal hormone production levels for estradiol were below the recommended concentration of 40 pg/mL, except in the blank and DMSO cells in Run 1. Although the basal estradiol production was generally less than 40 pg/mL, all values were >12.5 pg/mL (2.5-fold greater than the minimum detection limit (MDL ) as per the Guideline recommendation). Estradiol production in the pr esence of 10 μM forskolin me t induction requirements ( ≥ 7.5-fold). However, induction of test osterone production at 10 μ M forskolin was 1.76- to 1.80-fold, whichMETHOMYL FINAL

Page 37 of 54 is slightly less than the ≥ 2-fold requirement, and induction pr oduction in the pr esence of 1 μM forskolin was slightly higher th an expected (1.59- to 1.63-fold). Testosterone and estradiol inhibition were both greater than the required 50% in the presence of 1 μM prochloraz. Because all other guideline requirements for basal con centrations and hormone induction and inhibition were met for testosterone and estradiol, these results were deemed acceptable.

The %CVs within each plate fo r the SC on the test chemical plates analyzed for hormone production ranged from 3.12 to 12.37% for testoste rone and 0.78 to 12.45% for estradiol. The between-plate %CVs for the test plates were 6.93% and 7.0%, resp ectively, for testosterone and
estradiol. These values are within the Guideline limit of ≤ 30%.

Precipitation was not observed at any tested me thomyl concentration, and methomyl was not found to be cytotoxic to the H295R cells at concentrations up to 100 μ M. No effect on estradiol production was observed at any tested methomyl concentration. Across the three runs, there were significant (p<0.05) decreases in mean testosterone levels at methomyl concentrations of

0.01 μ M (0.89-fold) and 100 μM (0.83-fold) methomyl. The effect was consistent across all three runs, but it was not dose-dependent, as it occurred only at two non-sequential
concentrations.

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

Uterotrophic Assay; (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48617701 ) conducted to screen for pot ential estrogenic activity, methomyl (99.4% a.i., lot # JUL06LP276) in wate r was administered daily via oral gavage to groups of six ovariectomized female Sprague Dawl ey rats at dose levels of 0 (vehicle), 0.25, 0.5 or 1.0 mg/kg/day on post-natal da ys (PND) 63-65 (approximate). A positive control group was
treated with 17 α -ethynyl estradiol (EE) by daily oral ga vage at a dose level of 0.1 mg/kg/day on
PND 63-65. All animals were terminated and n ecropsied approximately 24 hours after the final
dose administration to determine we t and blotted uterine weights.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in any treatment group. Body weights, food consum ption, and food efficiency in the methomyl treated groups were comparable to the cont rol throughout the study. In the positive control group, body weights were decreased (p<0.05) by 10% on Day 4 compared to the vehicle control group, resulting in an overall we ight loss (p<0.05) of -4.0 g, comp ared to the vehicle control group (23.1 g weight gain). Over all food consumption and effici ency were decreased (p<0.05) by 28% and 128%, respectively, in the positive co ntrol group compared to the vehicle control group. Uterine weights in the methomyl treated groups were comparable to the controls.METHOMYL FINAL

Page 38 of 54 The mean blotted vehicle control uterine weig ht was <0.031% of the mean vehicle control terminal body weight, which meets the Guide line requirement (<0.04%). Absolute wet and blotted uterine weights for the EE group were increased (p<0.001 and p<0.05, respectively) by 213% and 166%, respectively. The increased uter ine weights were in the expected range.

The dose levels selected for this study ar e adequate based on red blood cell and brain
cholinesterase inhibition seen at dose levels ≥ 2.0 mg/kg/day in the toxicology database.

No statistically significant changes were seen in uterine weight in th is assay. Methomyl is negative in the uterotrophic assay.METHOMYL FINAL

Page 39 of 54 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and general toxicity studies (Part 158 studie s) considered relevant for determining the potential interaction of methomyl with the endocrine system are summarized below. Given the breadth of toxicity data available on methomyl, the only general toxicity studies discussed in this appendix are those th at fulfill current guideline requirements and are directly relevant to assessi ng potential endocrine effects ( i.e. , multiple-generation rodent reproductive studies, development studies , chronic toxicity studies, etc.).

Transcriptional Activation

In a study by Andersen et al. (2002), methomyl was tested for in teractions with the androgen and estrogen receptors in in vitro transactivation assays at concentration from 10 -8 to 10 -5 M, and effects on aromatase were also investigated at a single concentration (50 μ M). In the androgen receptor transcriptional activation (ARTA) assay, methomyl was tested in transfected Chinese hamster ovary (CHO) cells in triplicate or qua druplicate at each concentration in two runs. Methomyl did not show agonist or antagonist activity at the androgen receptor at any concentration. In the ERTA assay, methomyl was tested using transfected the MCF-7 cells in triplicate or quadruplicate at ea ch concentration in two runs. Methomyl was negative in the ERTA assay at each concentration tested. Methomyl was not cytotoxi c in either cellular assay at concentrations up to 100 μ M. Methomyl also did not inhib it aromatase activity at the single
concentration tested.

In a published, non-Guideline study ( Kojima et al ., 2004) , the potential of 200 chemicals (including methomyl; purity 95-100%) to interact with two human ER subtypes (hER α and
hER β ) and one human androgen receptor (hAR) in tr ansactivation assays with Chinese hamster ovary cells (CHO-K1 cells) was inve stigated. For detection of hER α or hER β activity, cells were transfected with pcDNAER α or pcDNAER β , pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were transfected with pZ eoSV2AR, pIND-ARE, an d pRL-SV40. Flutolanil was tested over a concentration range from 10

− 8 to 10

− 5 M, and the responses were compared to the activity of 17 β -estradiol (E 2 ; purity >97%) or 5 α -dihydrotestosterone (DHT; purity 95%) for the ER or AR assays, respectively. For meas urement of antagonistic activity to hER α , hER β and hAR, either 10

− 11 or 10

− 10 M E 2 or 10

− 10 M DHT was added to the cell cultures along with the test compound, respectively. A gonistic activities were eval uated by the relative activity expressed as 20% relative effective concentration (REC 20 ), the concentration of the compound demonstrating 20% of the activity of 10

− 10 M E 2 , 10

− 9 M E 2 , or 10

− 9 M DHT for ER α , ER β , and AR, respectively. Antagonistic activities were evaluated similarly by the relative activity expressed as 20% relative i nhibitory concentration (RIC 20 ), the concentrat ion of the compound showing 20% inhibition of the activity of E 2 or DHT against ER α , ER β , and AR, respectively. Methomyl did not show agonist or antagonist activity for ER α , ER β , or AR in this study.METHOMYL FINAL

Page 40 of 54 90-Day (Subchronic) Oral T oxicity in Rodents (Rat)

In a non-Guideline, subchronic toxicity study ( MRID 00007190 ), Crl:CD rats (10 rats/sex/dose) were administered methomyl (100% purity) at nominal concentrations of 0, 10, 50, and 250 ppm in the diet for 13 weeks; an additional group (m id-high) was administer ed 125 ppm for 6 weeks and 500 ppm for the remaining 7 weeks. In this latt er group, five animals/se x were sacrificed at two months and the remaining animals were sa crificed at study termination. The estimated maximum dose equivalence was 0, 1.0, 5.0, 12.5/50.0, and 25.0 mg/kg/day (calculated by reviewers); due to assumed changes in food cons umption with increased age/weight, actual dose equivalence may be slightly lower (0, 0.8, 3.1, 18.3, and 19.2 mg/kg/day). Organ weights
evaluated included the adrenals , liver, kidneys, pituitary, and testes. Gross and microscopic pathology was conducted on tissues, including the adrenals, testes, epid idymis, liver, kidneys, ovary, uterus, pituit ary, and thyroid.

No effects of treatment were observed on mortality, clinical signs (except for tremors in a single, high-dose female at the end of the study), food consumption, hematology, urinalysis, or macroscopic pathology. Additionally, no adverse, treatment-related effects were noted for plasma and RBC ChE concentrations. Overall (Weeks 0–13) body weight gain (calculated by reviewers) was decreased by 14% in the high-d ose males. Additionally, overall body weight gains were decreased ( ↓ 13 and 17%; females and males, resp ectively) in the high-medium dose animals; body weights in males were d ecreased (p<0.05) at Weeks 7 and 13 ( ↓ 14%) after the dose was increased from 125 to 500 ppm. No treatme nt-related changes were seen in absolute or relative weights of the testes, thyr oid or adrenal glands at any dos e. Liver weights were increased (p<0.05) in the high-medium and high-dose ma les at the end of the study (13 and 17%, respectively), and absolute and relative pituitar y weight was increased (p<0.05) in the high-dose females (19 and 20%, respectively). No treatment-re lated histopathological lesions were reported for the liver, kidneys, testes, ovari es, uterus, thyroid, adrenal or pituitary glands at any dose.

The LOAEL is 250 ppm (high dose) based on decr eased body weight gai n, moderate erythroid hyperplasia, and decreased RBC counts observed in the 250 and/or the 125/500 ppm dose groups. Although a NOAEL of 125 ppm (medium-high dose) was proposed (due to no adverse effects observed at this dose during the first si x weeks), this is confounded because the dose was increased to 500 ppm at Week 7 and all observati ons (except clinical) we re not conducted until 2 months (5 animals/sex) or 3 months (end of study). A demonstrable NOAEL is 50 ppm (medium-low dose) in male and female rats.METHOMYL FINAL

Page 41 of 54 Prenatal Development in Rodents (Rat); (OCSPP 870.3700)

In a developmental toxicity study ( MRID 00008621 ), methomyl (>99% purity) was administered to 25 presumed-pregnant female CD ® (ChR-CD) rats/dose in the diet at nominal concentrations of 0, 50, 100, or 400 ppm (mean-measured doses of 0, 4.9, 9.4, and 33.9 mg/kg/day, respectively) from ge station day (GD) 6 to 15. On GD 21, all dams were euthanized and underwent a gross necropsy a nd cesarean section. Fetuses were examined for external, visceral, and skeletal anomalies and malformations.

There were no mortalities or clin ical signs of toxicity, and no treatment-related effects were observed in gross pathology. At the high dose, mean maternal body weight was decreased by 8% (NS) on GD 16 and by 5% (p<0.05) on GD 21. Body we ight gain in this group was decreased by 22% for the treatment period (GD 6 to 16). Food consumption in the high-dose group was decreased by 18% during treatment.

There were no abortions, prematur e deliveries, or complete litte r resorptions, and no effects of treatment on the numbers of litters, live fetuses, or resorptions. A decrease (NS) in the pregnancy rate was observed in the high-dose group ( ↓ 16%) relative to control, bu t no dose-dependent trend was present. There were no treatment-related e ffects on growth or development as measured by fetal weight, crown-rump lengt h, or skeletal ossification. Th ere were no treatment-related external, visceral, or skeletal malformations or variations.

The maternal LOAEL is the high dose (400 ppm , equivalent to 33.9 mg/kg/day) based on decreased body weight, body weight gain, and food consumption. The maternal NOAEL is the intermediate dose (100 ppm, equivalent to 9.4 mg/kg/day). The developmental LOAEL was not observed. The developmental NOAEL is the high dose (33.9 mg/kg/day).

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

In a developmental toxicity study ( MRID 00131257 ), methomyl (98.7% purity) was administered by oral gavage to 20 New Zealand White (DLI:NZW) rabbits/dose at doses of 0, 2, 6, or 16 mg/kg/day from GDs 7 to 19. On GD 29, all surviving rabbits were euthanized and subjected to a gross necropsy. A cesarean section was performed, and the fetuses were examined for external, visceral, and skeletal malformations and variations.

In the high-dose group, seven rabbits died begi nning with the initiation of treatment and continuing throughout the treatment period (G Ds 7, 8, 9, 11, 17, 18, and 19). Central nervous system (CNS) stimulation occurred in the high-dose rabbits and wa s considered to be treatment related. Dose-dependent increases (p<0.05) in the incidence of tr emors, hyperactivity, body jerks, excess salivation, aggressi ve behavior, hyperpnea, and c onvulsions were observed in the rabbits administered the high dose. Other dose- dependent, but not statistically significant,METHOMYL FINAL

Page 42 of 54 increases in CNS stimulation were observed in the high-dose animals, including ataxia, impaired or lost righting reflex, skin le sions (self-inflicted), dyspnea, h ypersensitivity, pin-point pupils, and vocalization. A single deat h in the mid-dose group was attr ibuted to gavage error.

A decrease in the number of litters at the high dose, resu lting in a lower number of live fetuses, was due to the high maternal mortality in this group. After exclusion of th ese mortalities, there
were no treatment-related effects on the numbers of resorptions or fetuses on a per doe basis. Sex ratio and post-implantation losse s in the treated groups also were comparable to control. Abortions in the low- and intermediate-dose grou ps were considered spontaneous events because they occurred in a sing le animal/dose group and did not occu r at the high dose. A dose-related increase (p<0.05) in mean fetal body weight was observed. There was no adverse basis for increased fetal weight; the increased weight was attributed to the decreased maternal anorexia (increased feed consumption) in the latter pa rt of gestation after treatment cessation. No treatment-related skeletal retardations were noted during the fetal examinations, and there were no treatment-related external, visceral, or skeletal variations or malformations.

The maternal LOAEL is 16 mg/kg/day (high dose) based on mortality an d clinical signs of toxicity indicative of CNS stimulation. The maternal NOAEL is 6 mg/kg/day (intermediate dose). The LOAEL for developmental toxicity was not observed. The developmental NOAEL is 16 mg/kg/day (high dose).

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

In a two-generation reproductive study ( MRIDs 43250701 and 43769401 ), male and female SD rats received methomyl (98 to 100% purity) in the diet to 13 male and 26 female rats in the F 0 parental generation at nominal concentra tions of 0, 75, 600, or 1200 ppm; 20 males and 40 females (38 in the 1200 ppm group) from the F 1 offspring were treated at the same dosages. The

F 0 generation was treated for 100 days prior to mating; the F 1 generation was treated for 120 days prior to mating. Mean -measured doses were 4.6, 36.9, and 73.7 mg/kg/day and 4.8, 38.8, and 75.7 mg/kg/day, respectively, in F 0 males and females; and 6.9, 56.5, and 116.7 mg/kg/day and 7.2, 59.2, and 128.0 mg/kg/day, respectively, in F 1 males and females).

There was no evidence of a treatment-related effect on survival. For the F 0 generation, mean body weights were decreased (p<0.01) in the hi gh-dose females (premating) and males (entire study) by 10 and 16%, respectively. For the F 1 generation, mean body weights were decreased (p<0.01) for the mid- and high-dose group females and males by ↓ 19 and 26%, respectively. During gestation and lactation, mean body weights were decreased (p<0.01) for the high-dose females (and Lactation Week 1) of the F 0 generation ( ↓ 11 to 18%) and for the mid- and highdose females of the F 1 generation ( ↓ 11 to 27%). Mean food consumption was generally decreased (NS or p<0.05 or 0.01) concomitant with body weight decreases in dose groups. No evidence of a treatment-related effect on fertility, gestation or lactation indices. Viability indexMETHOMYL FINAL

Page 43 of 54 was decreased (p<0.05) in the F 0 (68%) and F 1 (68%) generations at the high dose. In the F 1 litters, the number of live pups was decreased (p<0.01) on and after Da y 4 in the high-dose group. Mean pup weights were decreased (p<0.01) in the mid- and high-dose groups throughout lactation (approximately ↓ 10 to 30%), and decreased (p<0.05 or 0.01) in the low-dose group at LD 14 and 21 (approximately ↓ 10%). In the F 2 litters, the number of live pups was decreased (p<0.01) on and after Day 1 in the high-dose gro up, and decreased (p<0.05 or 0.01) at early time points in the low- and mid-dose groups. Mean pu p weights were generally decreased (p<0.01) in the mid- and high-dose groups throughout lactation (approximately ↓ 10 to 30%). There also was
a decrease in litter size of the F 2 pups in all the treated grou ps (p<0.05; mid-dose group only). The RBC and plasma cholinesterase concentrations were comparable between the treated and control

groups. No differences in testicular sperm head counts were observed between the treatment groups and control. Differences in organ wei ghts between control and the mid- and high-dose groups in the F 0 and F 1 adults (and F 1 and F 2 pups) were observed. No tr eatment-related changes
were seen in absolute or rela tive weights of the ovaries or ut erus (except decreased absolute weights in the F 1 adults). Occasional, inconsistent decreas es (p<0.05 or 0.01) in absolute and/or relative weights were noted for adrenal glands, te stes, liver, and kidneys in the mid- and highdose adults and pups. No treatment-related histopat hological lesions were seen in any examined
tissues.

The NOAEL for parental systemic toxici ty is 75 ppm (4.6 mg/kg/day for F 0 males, 4.8 mg/kg/day for F 0 females, 6.7 mg/kg/day for F 1 males, and 6.3 mg/kg/day for F 1 females); the LOAEL is 600 ppm based on decreased body we ight and food consumption, and altered hematology parameters. The reproductive NOAEL is 1200 ppm (73.7 mg/kg/day for F 0 males, 75.7 mg/kg/day for F 0 females, 116.7 mg/kg/day for F 1 males, and 128 mg/kg/day for F 1 females) due to no effects in either genera tion; the LOAEL is not observed. The NOAEL for offspring toxicity is 75 ppm (4.6 mg/kg/day for F 0 males, 4.8 mg/kg/day for F 0 females, 6.7 mg/kg/day for F 1 males, and 6.3 mg/kg/day for F 1 females); the LOAEL is 600 ppm based on
decreases in both the mean number of liv e pups and mean body weights of offspring.

Testicular Toxicity (Rodent)

In a published study, El-Medany et al . (1993) [see also Mahgoub and El-Medany (2001)] evaluated the testicular toxicity of methomyl after oral administra tion to male Wistar rats at 17 mg/kg/day in saline by oral gavage for tw o months. Decreased (p<0.001) testosterone concentration ( ↓ 35%) and increased (p<0.01 to p<0.001) FSH ( ↑ 55%), luteinizing hormone ( ↑ LH; 59%) and prolactin ( ↑ 2.2-fold) hormone concentrations were observed after methomyl administration for 60 days. One month after cessation of treatment, testosterone was still decreased (30%, p<0.001) and FSH, LH and prolac tin concentrations were still elevated 64%, 59%, and 2.2-fold, respectively. Re ductions in succinic dehydrogena se activity were observed in the degenerated spermatogenic cells, as well as increased acid phospha tase activity, and noMETHOMYL FINAL

Page 44 of 54 differences in alpha esterase and alkaline phos phatase activities. In Leydig cells, succinic dehydrogenase activity was vari able, acid phosphatase and alpha esterase activities were increased, and no change was observed in alkali ne phosphatase activity. Variable grades of degenerative changes in the seminiferous tubul es were observed after methomyl treatment; primary spermatocytes showed variable degrees of degenerative changes, and spermatids displayed the highest degree of degeneration am ong the spermatogenic cells. There were several issues regarding this study: The initial citati on reportedly states that a methomyl dosage of 17 mg/kg is 1/30 of the methomyl LD 50 ; the actual range of methomyl LD 50 values is 17 to 45 mg/kg. The second citation correc tly cites the demonstrated LD 50 of methomyl as 17 to 45 mg/kg, but incorrectly cites meth omyl dose units in general toxi city studies (3-month subchronic oral toxicity in the rat and dog, developmental toxicology in the ra t) as mg/kg/day instead of as parts per million. Although animals were administered methomyl at or close to the LD 50 for 2 months, no mortality was reported in this study.

Thyroid Hormone Levels (Rodent)

In a published study, Porter et al. (1993) investigated the individual and interactive effects of methomyl, aldicarb, and triazine on concentrations of thyroxine (T 4 ) and somatotropin in female and male SD rats (n=6/sex/dose). Animals were ad ministered control (distilled water) or one of eight treatments in the drinking water for 6 weeks (females) or 16 weeks (males). T 4 and somatotropin concentrations, and T 3 uptake, were determined after Weeks 2 and 6 in female rats, and after Weeks 7, 13, and 16 in male rats. A fu ll-factorial experimental design was used to
assess interactions of co mbinations of two or all three pest icides compared to responses after
individual treatments of each chemical.

The results of this investigation indicate that treatment with methomyl alone (1 ppm) did not increase (p>0.05) T 4 or somatotropin concentrations in fema le or male rats at any time point (up to 6 weeks in females and 16 weeks in males).

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

In a combined chronic toxicity/carcinogenicity study ( MRID 00078361 ), male and female Crl:CD rats (80 rats/sex/dose) were administered methomyl (>99% purity) 0, 50, 100, and 400 ppm for 104 weeks (equivalent to 0, 2.5, 5 or 20 mg/kg/day). In addition, 20 rats/s ex/dose were
included in the control and tr eatment groups for erythrocyte and brain cholinesterase determinations. After 52 weeks, 10 rats from each group were sacrificed for gross and histopathological evaluati on. After 104 weeks, all surviving rats in all groups were sacrificed for gross and histopathological evaluati on. Select organs were weighe d at each sacrifice interval (including liver, kidneys, testes, adrenals, a nd pituitary gland). Gross and microscopic pathological examination included the liver, kidn ey, pituitary, thyroid, adrenals, mammary gland, testis, epididymis, pros tate, uterus, and ovary.METHOMYL FINAL

Page 45 of 54 Male and female rats in the high-dose group had decreased body weight gain during the time intervals 0 to 91, 0 to 182, and 0 to 364 days of treatment. High-dose male rats continued a similar reduction in body weight gain through 0 to 560 days of treatment. Male and female highdose rats appeared to adapt to the dietary ad ministration of methomyl after 2 years; no differences between final body wei ght or body weight gain in the rats were observed compared to control at the end of the study. No treatme nt-related mortality was observed during the study.
Survival exceeded 50% in a ll groups at 18 months, and was ≥ 50% in all groups after 2 years except for the control females (45%). No signifi cant differences were observed in RBC and brain
ChE concentrations due to treatment in either sex.

There were no differences in absolute organ wei ghts for male or female rats in any treatment group, or relative organ weights for male rats compared to control. Mid- and high-dose female rats had increased relative liver weights comp ared to control; however, no histopathological changes were observed. No treatment-related neopla stic or non-neoplastic lesions were observed that could be attributed to the dietary admini stration of methomyl up to 400 ppm. The NOAEL in the chronic toxicity/carcinogenicity study in rats is 100 ppm (intermediate dose) for males and females (approximately 5.0 mg/kg/ day for males and females), ba sed on decreased weight gain and hematology changes at the LOAEL (high dose, 400 ppm).

Carcinogenicity in Rodents (Mouse); (OCSPP 870.4200)

In a carcinogenicity study ( MRID 00078423 ), male and female CD-1 mice (80 mice/sex/dose) were administered methomyl (99% purity) at no minal concentrations in the diet of 0, 50, 100, and 800 ppm for 104 weeks. Due to high mortality, the high dose was reduced to 400 ppm beginning Week 28, and was further reduced to 200 ppm at Week 39 (the intermediate dose was also reduced to 75 ppm at Week 39). Mean-m easured doses were 0, 8.7, 15.3 or 93.3 mg/kg/day for males and 10.6, 19.0 or 118.5 mg/kg/day for female s. After 104 weeks, a ll surviving mice in all groups were sacrificed for gross and hist opathological evaluation. Select organs were weighed at terminal sacrifice, including liver, ki dneys, testes with epididymides, adrenals, and pituitary gland. Gross and microscopic pathol ogical examination included the liver, kidney, pituitary, thyroid, adrenals, mamm ary gland, testes with epidi dymides, prostate, uterus, and
ovaries.

Survival in the high-dose males and female s was significantly decreased (percent and significance not reported) at Week 26, and survival in the mid-dose groups was also moderately decreased (percent and significance not reported). Due to these high mortality rates, the doses for the high- and mid-dose groups were decreased as described previ ously. Survival was generally significantly decreased (percent and significance not reported) after 52, 80, and 104 weeks of treatment in mid- and high-dose animals. Decr eased survival rates are considered to beMETHOMYL FINAL

Page 46 of 54 treatment-related; surv ival exceeded 50% in all groups at 15 months of study. No treatmentrelated effects on body weight or body wei ght gain were observed in this study.

No treatment-related gross or microscopic pa thological effects were noted. There were no differences in absolute or relative organ weight s for male or female rats in any treatment group, except for increased (percent and significance not reported) absolute adre nal weight in high-dose males. Relative adrenal weight in this group wa s reported to be slightly elevated; however, no histopathological changes were observed. No treatment-relate d neoplastic or non-neoplastic
lesions were observed that could be attributed to the dietary admi nistration of methomyl in this study. The NOAEL in the carcinogenicity study in is 50 ppm (low dose) for males and females (8.7 mg/kg/day for males and 10.6 mg/kg/day for females). This NOAEL is based on increased mortality doses ≥ 75 ppm (15.3 mg/kg/day for males and 19.0 mg/kg/day for females).

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

In a chronic toxicity study ( MRID 00007091 ) , methomyl; (90 or 100% purity; possibly different batches) was administered in the diet to 4 Beag le dogs/sex/dose at nominal concentrations of 0, 50, 100, 400, or 1000 ppm (mean-measured doses 0/0, 1.5/1.6, 3.0/3.3, 11.5/13.8, and 30.6/31.7 mg/kg/day in males/females were calculated by the reviewer; similar, calculated doses of 1.25, 2.5, 10, and 25 mg/kg/day were determined a ssuming 0.025mg/kg/day per 1 ppm compound in the diet) for up to two years. On e dog/sex/dose was sacrificed afte r 12 months of treatment, and the remaining 3 dogs/sex/dose were treated for up to 24 months. Select organs were weighed at terminal sacrifice (including liv er, kidneys, testes, and adrenal and thyroid glands). Gross and microscopic pathological examination included the liver, kidney, pituitary, thyroid, adrenals, testes, uterus, and ovaries.

One high-dose female was found dead in its cag e on the first day of Week 9. A replacement female dog was started on treatment two weeks late r, but this animal had repeated seizures on Day 17 of treatment, followed by coma and death. Two high-dose males exhibited tremors, salivation, incoordination, and ci rcling movements on a single af ternoon during Week 13 of the study. Another high-dose male exhibited a higher incidence of diarrhea compared with other study animals and progressive an emia throughout the st udy with markedly declined hematology parameters at 18 months compared to baseli ne. Treatment was discontinued from Weeks 85 through 94, with improved hematological analyses. The animal was returned to study for the remainder of the dosing period (Weeks 95–104), with a concomitant return to severe anemia and moderate leucopenia by the end of treatment. Gro ssly, it was found to have an enlarged spleen, an enlarged liver, and increases in absolute and relative thyroid, heart, liver, and kidney weights. The remaining two high-dose males had increased absolute and relative kidney weights at study
termination.METHOMYL FINAL

Page 47 of 54 No adverse effects of treatment were obs erved on body weight, body weight gain, food consumption, clinical chemistry, or urinalysis . Microscopic findings in the high-dose animals included changes in the kidneys and liver, with extramedullary hematopoiesis and hematogenic activity in the spleen and bone marrow. The LO AEL is 1000 ppm (approximately equivalent to 30.6/31.7 mg/kg/day in males/females) , based on anemia (with asso ciated gross and microscopic pathological findings) and increase d absolute and relative kidney weights in the males, mortality in the females, and clinical signs of toxicity and microscopic findings in the kidney, liver, spleen,
and bone marrow as described above in both sexes. The NOAEL is 400 ppm (approximately equivalent to 11.5/13.8 mg/kg/ day in males/females).

Avian Reproductive Toxicity (OCSPP 850.2300)

In an avian reproduction study ( MRID 41898602 ), 20-week old Bobwhite quail ( Colinus virginianus ) were dosed with methomyl (98.35% purity) in the feed at nominal concentrations of 0 (acetone and corn oil, 1:1.67 ratio), 50, 150, and 500 ppm for approximately 20 weeks. Quails in the 50 ppm group were dosed at 150 ppm fo r the first four weeks in error; this error was corrected and this group was administered 50 ppm for the remainder of the study. Mean
achieved dose concentrations of methomyl in the study were 0 (basal diet), 50 (67; timeweighted average including first four weeks of dosing at 150 ppm), 150, and 458 ppm. Methomyl concentrations in the diet were re ported over the first 4 w eeks of the study, with variability from nominal concen trations of approximately 20 to 30%. The mean dietary
concentrations over the firs t 4 weeks were 123, 137, and 427 pp m in the treated groups.

Each dose group consisted of 32 quails, with a si ngle cock and hen paired in 16 pens/dose group. The photoperiod was eight hours of light per day dur ing the acclimation period (pre egg-laying) and the first seven weeks on study, and was incr eased to 17 hours of light per day for the remainder of the study. All adult birds were obser ved daily for mortality, signs of toxicity, and abnormal behavior. Body weights were record ed at study initiation, Weeks 2, 4, 6, 8, and at study termination. Eggs were collected daily, soaked and washed in a chlo rine-based detergent, stored in a cold room, and counted weekly. Meas urements were taken of the average thickness of air-dried shells (with membrane ) after collection of one egg on alternating weeks from odd- and even-numbered pens. Remaining eggs were candl ed to detect abnormalities and cracks. Normal eggs were set for incubation, and candled on Da ys 11 and 21 of incubation to determine embryo viability and survival, respectiv ely. Hatchlings and unhatched e ggs were removed on Day 25 or 26 of incubation. Feed consumption was measured every week. The study room temperature was
maintained at 19.8 ± 2.9°C and 36 ± 12% relative humidity. The hatchling room temperature was maintained at 25.8 ± 2.4°C; surviving hatchlings were weighed on Day 14.METHOMYL FINAL

Page 48 of 54 There were no treatment-related mortalities, effects on body weight and feed consumption. According to the study review, the percentage of females with feather lo ss/ and or toe lesions increased dose-responsively, likely due to increased aggression in males and were statistically different from controls at the mid and high treat ment. Incidental mortalities and findings were observed across all study groups. In general, there were no treatment-related effects on reproductive parameters at ≤ 137 ppm. The number of eggs laid and set per hen were decreased (p<0.05) by 40% at the high-dose (4 27 ppm). The percentage of eggs cracked/eggs laid was decreased (p<0.05) at the intermediate dose, but eggshell thickness displayed no treatmentrelated effects. No differences due to treatment were observed in body weights of hatchlings or 14-day survivors. The NOAEL is 150 ppm and the LOAEL is 427 based on the decreases in eggs laid and set per hen.

In an avian reproduction study ( MRID 41898601 ), mallard ducks ( Anas platyrhychos ) were dosed with methomyl (98.35% purity) in the feed at nominal concentrations of 0 (acetone and corn oil, 1:1.67 ratio), 50, 150, and 500 ppm for approximately 20 weeks. Ducks in the 50 ppm group were dosed at 150 ppm for the first four week s in error; this error was corrected and this group was administered 50 ppm for the remainder of the study. Mean achieved dose concentrations of methomyl in the study were 0 (basal diet), 50 (67; time-weighted average including first four weeks of dosing at 150 ppm ), 150, and 458 ppm. Methomyl concentrations in the diet were reported over the first 4 week s of the study, with variability from nominal concentrations of approximately 20 to 30%. The mean dietary concentrations over the first 4 weeks were 123, 137, and 427 ppm in the treated gr oups. Due to the dosing error in the 50 ppm group, the reviewer could not make definitive conc lusions about this treatment level. There were significant differences from the control for all levels tested for male body weight gain, and at 427 ppm, female weight gain was also sign ificantly decreased. At 137 ppm, there was a significantly higher number of cracked eggs when compared to the control. Since 427 ppm was statistically equivalent to the control, this occurrence is not believed to be treatment related. No other reproductive parameters were affected.

In another mallard duck reproduction study (M RID 49245301; preliminary review only), a onegeneration reproductive toxicity of methomyl to 16 pairs per level of 19-week old mallard duck ( Anas platyrhynchos ) was assessed over 21 weeks. DPX-X1179-571 (99.4% methomyl) was administered to the birds in the diet at nominal concentrations of 0 (control), 41, 122, 367 and 1100 mg ai/kg. Mean-measured concentrations (reviewer-calculated) were <10 (<LOQ, control), 41, 121, 373 and 1120 mg ai/kg diet, respectively.

There were no treatment-related effects on any adult parameter at the (mean-measured) 41, 121 or 373 mg ai/kg diet levels and no treatment-re lated effect on any reproductive or offspring parameter at all levels. At the 1200 mg ai/kg di et level, there was a marked reduction in food consumption during Week 1 (40 versus 103 g/bird /day; p<0.01) that cont inued through Week 2 (82 versus 108 g/bird/day; p<0.05) that was consid ered related to treatme nt. The reduction inMETHOMYL FINAL

Page 49 of 54 food consumption corresponded with slight reduc tions in mean body weight of both hens and drakes during the first two weeks of the study: mean 0- to 2-week body weight changes were - 104 versus -23 g for the control for drakes, and - 72 versus -26 g for the control for hens. In addition, there was a statistically -significant reduction in female body weights compared to the control (1135 versus 1228 g; p< 0.05) at study termination. Necrops y also revealed an increase
in the number of hens with a re gressing/regressed ovary (8/15 ve rsus 4/16 for the control group)
that was considered related to treatment . The NOAEC and LOAEC were 373 and 1120 mg ai/kg diet, respectively.

Fish Early Life-Stage Toxicity; (OCSPP 850.1400)

The effects of methomyl ( 98.4% purity) on eggs and fr y of the fathead minnow ( P. promelas) were determined in a flow-through, earl y life-stage 35-day toxicity test ( MRID 46015305 ). Treatments consisted of a dilution water (n egative) control and nominal methomyl concentrations of 19, 38, 75, 150, and 300 μg/L. Tw o replicates containing 25 eggs/replicate were exposed to each treatment concentration and control in incubation cups. All incubation cups were observed daily, and dead eggs and fungal- infected eggs were removed. The 29-day posthatch growth period began on Study Day 6, after 95% hatch was attained . Observations of abnormal behavior, physical change s, and mortality were recorded daily. At the end of the study, standard length and blotted wet weight were determined.

Mean measured concentrations of methomyl were 18.0, 39.3, 73.0, 145, and 261 μg/L, and ranged from 87 to 103% of nominal concentrations . Egg hatchability and fry survival were not decreased (p>0.05) at any test concentration. All chemical and physical parameters for the 35- day study were within acceptable ranges. The following endpoints were determined for the methomyl test concentrations and control (in pare nthesis): mean percentage survival ranged from 86 to 94% (92% for water control); mean percentage egg hatched ranged from 88 to 96% (94% for water control); mean standard length ranged from 18.56 to 21.17 mm (21.15 mm for water control); and mean wet weight ranged from 0.112 to 0.148 g (0.146 g for water control). The NOAEC for percentage hatch and percentage surv ival was 261 μg/L (hi gh dose); the LOAEC is
not determined.

The overall NOAEC for the Fathead minnow wa s 73.0 μg/L, and the LOAEC was 145 μg/L based on decreased weight and length.

Fish Early Life-Stage Toxicity; (OCSPP 850.1400)

In a freshwater fish early life-stage toxicity test ( MRID 00131255 ), Fathead minnow ( P. promelas ) embryos were exposed to methomyl (>99% purity) at nominal concentrations of <1METHOMYL FINAL

Page 50 of 54 (<LOQ, negative control), 31, 62, 125, 250, and 500 μg /L in a flow-through system for 35 days. Achieved exposure concentrations were < 1, 27, 57, 117, 243, and 491 μg/L for the treated groups. This study was scientifica lly sound. Although the study author’s analysis failed to detect a significant reduction in percent hatch at any le vel, when compared to control, the reviewer visually determined the NOAEC fo r percent hatch to be 57 μg/L given an apparent reduction at
concentrations ≥ 117 μg/L. Survival was decreased (p< 0.05) by 10 to 20% at the 117 μg/L concentration. Mean length and wet weight were decreased (p<0.05) at concentrations ≥ 243 μg/L.

Fish Life Cycle Toxicity; (OCSPP 850.1500)

In a freshwater fish full li fe-cycle toxicity test ( MRID 43072101 ), Fathead minnow ( P. promelas ) embryos were exposed to methomyl (98.35% purity) at nominal concentrations of <1 (<LOQ, negative control), 19, 38, 75, 150, and 300 μg /L in a flow-through system for 193 days. Mean measured concentrations were <1, 18, 40.7, 76, 142, and 280 μg/L for the treated groups. This study was scientifically sound. In-house spawned juvenile fathead minnows were initially exposed in two hatching/growth chambers per a quarium (duplicate aquaria; 200/treatment) in a nested experimental design. Larvae were thinned to 25/replicate chamber on Day 7 (post-hatch). On Days 28 and 58, length measurements we re determined; lengths and weights were determined after anesthetizing fish on Day 84. Fi sh in each treatment group were collected into two groups/treatment on Day 100, and on Day 101, eight spawning pairs/treatment were selected and placed into spawning aquaria. The spawning phase was terminated on Day 154, and adult survivors were weighed and measured. The m ean reproduction parameters examined included the number of days to first spawn, number of spawning days, number of spawns per pair, and number of eggs per spawn. Sublots of 35 embr yos were collected from random spawns to determine hatchability, survival, and growth in the F1 generation. At 56 days post-hatch, the F1 fish were anesthetized, weighed, and measured. Fish were observ ed for signs of toxicity and mortality throughout the study.

Scattered mortality was observed through 48 hours after embryo placement, with only one additional mortality after 48 hours. Hatching succ ess (%) of the F0 gene ration calculated from Day 2 determined the NOAEC for percent hatch to be 280 μg/L [hatching success is decreased (p<0.05) at 142 μg/L only if calculated from Day 0]. Survival (calculate d at Days 28, 56, and 84) was decreased (p<0.05) by 6 to 7% at the hi gh concentration (280 μg /L). Mean length (calculated at Days 28, 56, and 84) was decreased (p<0.05) at concentrations ≥ 142 μg/L, and wet weight were decreased (p<0.05) at the 280 μg /L concentration. No significant effect on reproduction was demonstrated at any test concentration compared to control; the number of eggs and number of eggs/spawn (fecundity) were highly variable. Hatching success (%) of the F1 generation calculated from Day 2 determined the NOAEC for percent hatch to be 280 μg/L [hatching success is decreased (p<0.05) at 40.7 and 280 μg/L if calculated from Day 0]. TheMETHOMYL FINAL

Page 51 of 54 length of F1 larvae at 56 days post-hatch was decreased at 142 and 280 μg /L concentrations, and wet weight was decreased at th e 280 μg/L concentration only.METHOMYL FINAL

Page 52 of 54 APPENDIX 3: References Not Utiliz ed in the Methomyl 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 Methomyl 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 data on invertebrates.

Crisp, T.M. et al . (1997). Special Report on Environmen tal Endocrine Disruption: An Effects Assessment and Analysis. Report Numb er EPA/630/R-96/012 February 1997. http://www.epa.gov/ORD/WebPubs/endocrine/endocrine.pdf

Celik, L., Lund, J., and Schiott, B. (2008). E xploring interactions of endocrine-disrupting compounds with different confirmations of th e human estrogen receptor a ligand binding domain: A molecular docking study. Chem. Res. Toxicol. 21:2195–2206.

Crisp, T.M. et al . (1998). Environmental Endocrine Disruption: An Effects Assessment and Analysis. Environ Health Perspectives 106 , Supplement 1: 1156.METHOMYL FINAL

Page 53 of 54 Gruggs, C. J., Perkham, J.C., and Cato, K.D. (1983). Mammary carcinogenesis in rats in relation to the age at time of N-nitroso-N-methylurea administration. 1. Natl.Cancer Institute

. 70:209–212.

Grue, C.E., Hart, A.D.M., and Mineau, P. (1991) . Biological consequences of depressed brain cholinesterase activity in wildlife in: Cholines terase-inhibiting insecticides: Their impact on
wildlife and the environment. ed. P. Mineau .. Elsevier Science Publishers B.V., Amsterdam. pp. 151–209.

Hoffman, D.J and Albers, P.H. (1984). Evaluation of Potentia l Embryotoxicity and Teratogenicity of 42 Herbicides, Insecticides, and Petroleum Contaminants to Mallard Eggs.
Arch. Environ. Contam. Toxicol . 13: 15–27.

Hseuh, AJ.W., Dufau, M.L., and Cart, KJ. (19 77). Gonadotrophin induced hormone receptors and desensitization of testicular 3' ,5'-c yclic AMP and testosterone responses. Proc. Natl. Acad.

Sci. USA 74:592–595.

Jiao, B. et al . (2008). Establishment of a Transgenic Yeast Screening System for Estrogenicity and Identification of the Anti-Estrog enic Activity of Malachite Green . J. Cell. Biochem

. 105:1399-1409.

Montague, B. (1997). Brian.dbf database, US Environmental Protec tion Agency, Office of Pesticide Programs, August 13, 1997.

National Research Council. (1999). Hormonally Active Agents in the Environment. National Academy Press. Washington DC. 430 pages. http://www.nap.eduibooks/0309064198/htnV

Nishihara, T. et al. (2000). Estrogenic ac tivities of 517 chemicals by yeast two-hybrid assay. J. of Health Sci . 46: 282–298.

Nendza, M. and Wenzel, A. (2006). Discrimi nating Toxicant Classes by Mode of Action, 1) (Eco)toxicity Profiles). Environ. Sci. & Pollut. Res . 13:192–203.

Roncaglioni, A., Piclin, N., Pintor e, M., and Benfenati, E. (2008) . Binary classification models for endocrine disrupter effect s related-through the estrogen receptor. SAR and QSAR.
Environmental Research 19:697–733.

Shalaby, M.A., El Zorba, H.Y., and Ziada, R.M. (2010). Reproductive toxicity of methomyl insecticide in male rats and pr otective effect of folic acid. Food Chem Toxicol . 48(11):3221–

3226.

Shanthalatha, A., Madhuranath, B.N., and Yajurvedi, H.N. (2012). Effect of methomyl formulation, a carbamate pesticide on ovarian follicular development and fertility in mice.
J. Environ. Biol. 33(1):33–37.METHOMYL FINAL

Page 54 of 54 Stevens, J.T., Tobia, A., Lamb, J.C., Tellone, C ., and O’Neil, F. (1997) . FIFRA Subdivision F testing guidelines: Are these tests adequate to detect potential hor monal activity for crop protection chemicals? Toxicol. Environ. Health 50:415–431.

van Ravenzwaay, B. (1992). Discussion of pren atal and reproduction toxi city study of Reg. No. 83-258 (Vinclozolin). Data submission to US EPA from BASF Corporation, 425813-02.