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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

# &

Health Effects Divison

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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: OXAMYL

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYOXAMYL 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..................................................13

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................16

IV. Committee’s Assessment of Weight of Evidence..........................................18

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................18

1. Tier 1 in vivo Assays.............................................................18

2. OSRI...............................................................................19

B. Estrogen Pathway...........................................................................21

C. Androgen Pathway.........................................................................21

D. Thyroid Pathway...........................................................................21

E. Conclusions.................................................................................22

V. EDSP Tier 2 Testing Recommendations....................................................22

VI. References.......................................................................................23

LIST OF TABLES: Table 1. Tier 1 Assays for Oxamyl....................................................................7
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Oxamyl .......................................10
Table 3. Androgenic/Anti-Andr ogenic Pathway for Oxamyl ....................................14
Table 4. Thyroid Pathway for Oxamyl ..............................................................17

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................26
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................38

APPENDIX 3. References Not Utilized in the Oxamyl WoE Analysis................................47OXAMYL FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

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

CG Cowper’s Gland

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

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

iv Abbreviation Terminology

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

K OW Octanol/Water Partition Coefficient

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

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

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

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated ConcentrationOXAMYL FINAL

v Abbreviation Terminology

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

OPP Office of Pesticide Programs

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

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

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

T4 Thyroxine (tetraiodothyronine)OXAMYL FINAL

vi Abbreviation Terminology

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

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

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

Page 1 of 48 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 oxamyl interacts with E, A or T hormone pathways, the number and type of effects induced, the magnitude and pattern of responses observed across studies, taxa and sexes were considered. Additionally, the conditi ons under which effects occur were considered, in particular, whether or not endoc rine-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 analysis of the potential interac tion of oxamyl with the E, A or T hormone pathways. The T1WoERC c onclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in

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

For oxamyl, there was no convincing evidence of interaction with the estrogen pathway. The
Tier 1 in vitro assays were negative (ER binding, ERTA, aromatase, and steroidogenesis assays), as well as the Tier 1 uterotrophic assay. In the female pubertal assay, while there were increases in uterine weights; however, the absence of complimentary data doe s not support estrogenrelated etiology for this eff ect. No treatment-related eff ects observed in the FSTRA.

For the androgen pathway, there was no convinc ing evidence of inte raction. The Tier 1 in vitro ( i.e , AR binding and steroidogenesi s) and Hershberger assays we re negative. No androgenrelated effects were observed in the Tier 1 male pubertal assays or ma mmalian Part 158 studies. In the FSTRA assay, while there was an increas e in GSI at high dose, no other effects were
observed.

There was no evidence of interaction of oxamyl with the thyroid pathway. Thyroid parameters
were not altered in Tier 1 mammalian or amphibian in vivo studies, except for a non-dosedependent increase in thyroid weights in the fe male pubertal rat (20% and 13% at the mid and high dose, respectively).OXAMYL FINAL

Page 2 of 48 Based on weight of evidence considerations, ma mmalian or wildlife Tier 2 testing is not recommended for oxamyl since there was no convi ncing evidence of poten tial interaction with the estrogen, androgen or thyroid pathways.OXAMYL FINAL

Page 3 of 48 I. Introduction

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 oxamyl interacts with E, A, or T hormone pathways, the number and type of effects induced, the magnitude and patt ern of responses observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whether 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 analysis of the potential interac tion of oxamyl with the E, A or T hormone pathways. The T1WoERC c onclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in

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

Oxamyl has a vapor pressure of 2.3 × 10 -4 mm Hg at 20 to 25°C, and will exist as a vapor if released to the atmosphere. Oxamyl is soluble in water (280 g/L at 25°C); it has an octanol/water partition coefficient (log K ow ) of -0.47. Oxamyl is susceptible to photolysis in the atmosphere, with a half-life of approximately 1.4 days. Volatilization from water is not expected to significantly contribute to the dissipation of oxamyl in the environment. The estimated
bioconcentration factor is 3.1.

The available information considered in determining the potential interaction of oxamyl with the E, A, or T pathways include submitted EDSP 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 Sections III.A through III.C. An analysis 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 study recommendations ar e presented in Section V.OXAMYL FINAL

Page 4 of 48 II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

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

1.

B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Order recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 Guideline studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team on OPP (USEPA, 2010 ). Since then, the Agency has also conducted a more recent search of available scientific literature for any
additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies c onsidered but not util ized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and approach used in the WoE determination on the potential of a
substance to interact with endocrine-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.

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data includes a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays wereOXAMYL FINAL

Page 5 of 48 designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data, as well as characterizing the extent to which the available data suppor t a hypothesis that a chemical has the potential to interact E, A, or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, coherence, strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are
meant to evaluate whether or not a chemical can in teract with E, A, and T consisting of different levels of biological organization from a mol ecular initiating event 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 where the responses occur including consideration of whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.OXAMYL FINAL

Page 6 of 48 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 availa ble toxicity data into account.

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

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, A,OXAMYL FINAL

Page 7 of 48 or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

A. EDSP Tier 1 Screening Assays

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

Table 1. Tier 1 Assays for Oxamyl

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48672702)

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

OECD 455 Requirement Satisfied (MRID No. 48686701)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48672703)
Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550;
OECD 456 Requirement Satisfied (MRID No. 48686702)
Aromatase Assay (Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48672701)

Uterotrophic Assay (Rat) OCSPP 890.1600;

OECD 440 Requirement Satisfied (MRID No. 48618401)

Hershberger Assay (Rat) OCSPP 890.1400;

OECD 441 Requirement Satisfied (MRID No. 48618402)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID No. 48702104)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID No. 48702103)

Fish Short-term Reproduction Assay OCSPP 890.1350;
OECD 229 Requirement Satisfied (MRID No. 48702101)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100;

OECD 231 Requirement Satisfied (MRID No. 48702102)OXAMYL FINAL

Page 8 of 48 B. Effects on Hypothalamic-Pit uitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for oxamyl to interact with the estrogen pathway is summarized in Table 2 . If an effect was observed but considered by WoE not to reflect a direct interact ion, then the effect is discussed in the text but not pr esented in the table. The variou s targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects. This table also includes HPG-relevant findings
from data evaluated as OSRI. Effects that occurred in the presen ce of overt toxicity are discussed in the text but are not reported in the table and not considered fu rther in the WOE assessment.

Oxamyl was negative (or not interactive) in the Tier 1 in vitro ER binding, ERTA aromatase, and steroidogenesis assays. Oxamyl was also negative in the in vivo uterotrophic assay. In the female pubertal rat assay, the average age at a ttainment of VO was decr eased by 1.8 days at the low dose, and by 1.7 days at the high dose. The av erage day of first estrus was increased 0.9 day (not statistically significant, NS). These change s in puberty attainment were not dose-dependent and not considered biologically significant. Comp ared to control, wet an d blotted uterus weights were increased by 62 and 43% at the low dos e and by 34 and 27–28% at the high dose. The overall changes in uterus weight were not dose-dependent, however when non-cycling animals are excluded from the analysis, the changes appe ar to be dose-dependent: wet uterus weight increased 59% and 54%, and blotte d uterus weight increased 38% and 45% at the low and high dose. After adjusting for stage of estrus, the change s in the uterine weights also appear to be dose dependent 1 : blotted weight ↑ 22.8% at the low dose and ↑ 34.0% at high dose for animals in the proliferative phase, a nd 22.7% at low dose and ↑ 27.1% at high dose for animals in the nonproliferative phase (concomitant increases in wet weight also a ppeared to be dose-dependent). No estrogen-related effects were obs erved in the mammalian Part 158 data.

In the FSTRA, there were no treatment-related e ffects on fecundity, fertilization, GSI or plasma VTG concentrations. While there were no reported treatment-related effects on gonadal stages or histopathology, oocyte atresia and granulomat ous inflammation was observed in both the controls and treatment groups with the inflam mation associated with presence of spores

1 For each dose groups, necropsied animals were estimated to e ither be in a proliferative or non-proliferative stage of estrus based on the latest cyclicity determination and assu ming 4 days per cycle. Animals determined not to be normally cycling were exclude d from the analysis. The changes in pro liferating animals were not statistically significant. Statistically anal ysis could not be performed in the non-pro liferating animal group s because the control group had only one animal.OXAMYL FINAL

Page 9 of 48 (presumed microsporidia). However, according to the report, it did not appear that the presence of either microsporidia or the inflammation ha d any effect on the study results. While effects on mortality or other clinical signs were not observed, female body weight was decreased 13% (p<0.05) in the high-dose group (0.989 mg a.i./L).

In the Part 158 avian reproduction study with ma llard ducks, mortality and observations of penmate aggression were observed in the contro l and all treatment groups. The study indicated a slight effect on egg production a nd a potentially higher ch ance of egg infertility at 50 mg/kg-diet. Due to increased mortality in the control (14%, al l females), there is conc ern with the ability to
discern endocrine-related effects seen in the ma llard study due to the pr esence of mortality and aggressive behavior in the pens. In the Northern bobwhite quail study, there were no reproductive impairments observed up to 50 mg/k g-diet. Also, no treatm ent-related effects on mortalities, bodyweight or clinical signs of toxicity were observe d in this study. In a Part 158 freshwater fish early life-stag e toxicity test with rainbow trout, at 1.5 mg/L (highest concentration tested), significant delays in the first day of embryo hatchi ng and larval swim-up were observed, as well as a significant decrease ( ↓ 96%) in the percentage hatching.OXAMYLFINALPage 10 of 48Table 2. Estrogenic/Anti-Estrogenic Pathway for OxamylLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Oxamyl1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt) / Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48672702)NERTA(MRID 48686701)N4Steroidogenesis(MRID 48686702)NAromatase(MRID 48672701)NUterotrophic(MRID 48618401)NN NFemale Pubertal Rat(MRID 48702104)NE↑27–34(L, H)5NNN N NNNFSTRA(MRID 48702101)NENNNNNN(H)6OSRIDevelopmental toxicity(Rat; MRID 40859201)NENX(H)X(H)Developmental toxicity(Rabbit;MRID 00063009)NNNNTwo-generationreproduction(Rat; MRID 41660801)NE NE NNENX(H)X(M, H)OXAMYLFINALPage 11 of 48Table 2. Estrogenic/Anti-Estrogenic Pathway for OxamylLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Oxamyl1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt) / Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Chronic toxicity /carcinogenicity(Rat; MRID 41963101)NENNNEX(MH,H)X(MH,H)Carcinogenicity(Mouse;MRID 00076813)NENNNENX(M, H)Chronic toxicity(Dog; MRIDs41697901/42052701)NENENNEX(L, M,H)X(H)Avian reproduction(Bobwhite Quail;MRID 00116610)NN N1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment..Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay.Changes in weight are absolute unless otherwise indicated.2. 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. 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. Although the conditions of this assay were not optimal to detect very weak activity, oxamyl responses were similar to thoseof the negative controlcorticosterone and not comparable to the responses of 17α-methyltestosterone (very weakpositive control). Oxamyl was only able to reach a maximum of2.9-4.4% PC when tested up to the highest concentration (10-5.3M). Because the RPCMax< PC10in both assay runs, oxamyl was considered negative forestrogen receptor transcriptional activation in this test system.5. Mean uterus weight changes (not corrected for estrus stageor cycling status) were not dose-dependent. Wet weight increased62% and blotted weightincreased 43% at the low dose, while wet weight increased 34% and blotted weight increased 27–28% (absolute-relative) at the high dose. When onlycycling animals are considered in the analysis, changes are dosedependent (Blotted: 38% at low, 45% at high dose; p =0.0031 for trend by ANOVA, p<0.01for pairwise by Dunnett’s. Wet: 59% at low, 54% at high dose; p=0.0043 for trend, p<0.05 and p<0.01 pairwise for low and high doses, respectively). Whenanalyzed by estimated estrus stage (basedon latest vaginal smear before termination and excluding non-cycling animals), changes also appear dosedependent (statistics not available).OXAMYLFINALPage 12 of 486. While effects on mortality or other clinical signs were not observed, female body weight was decreased 13% (p<0.05) in the high-dose group(0.989 mg a.i./L).P Positive findingsN Negative findings (in vitro) / No effect (in vivo)NE Not examinedOXAMYL FINAL

Page 13 of 48 2. Effects on Androgen Pathway

The potential for oxamyl to interact with the androgen pathway is summarized in Table 3. If an effect was observed but considered by WoE not to reflect a direct interact ion, then the effect is discussed in the text but not pres ented in the table. The various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for androgenic, anti-androgenic, or HP G axis effects. This table also includes HPG-relevant findings
from data evaluated as OSRI. Effects that occurred in the presen ce of overt toxicity are discussed in the text but are not reported in the table and not considered fu rther in the WOE assessment.

Oxamyl was negative in the Tier 1 in vitro AR binding and steroidogenesis assays. The Hershberger assay was negative and no androgen-re lated effects were obs erved in the Tier 1 male pubertal assay.

In the FSTRA, there was an increase in male gonado-somatic index (GSI) of 30% in the highdose group (0.989 mg a.i./L), but no other effects ( e.g. , plasma VTG concentration, gonadal stages, and histopathology) were observed. There were no treatme nt-related effects on mortality, male body weight or clini cal signs of toxicity.

No androgen-related effects have been reported in oxamyl Part 158 mammalian toxicity studies, except in the two-generation reproductive study. In that study, increased average relative testes weights were observed (F 0 high-dose, ↑ 19%; F 1 medium and high-dose, ↑ 12 and 33%, respectively) and co-occurred w ith overt toxicity [(decreased average male body weight at the mid ( ↓ 7–20% F 1 ) and high dose ( ↓ 10–18% F 0 , ↓ 17–37% F 1 )]. In the Part 158 northern bobwhite quail study, there were no androgen-related effect s observed up to 50 mg/kg-diet; in the mallard duck study, potentially higher chance of egg infe rtility at 50 mg/kg-diet. Due to increased mortality in the control (14%, all females), there is concern with the ability to discern endocrinerelated effects seen in the mallard study.OXAMYLFINALPage 14 of 48Table 3. Androgenic/Anti-Androgenic Pathway for OxamylLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Oxamyl1Study Type / LiteratureCitation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histo p atholo gy Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex C h a r acte ri st i cs Fertility (Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48672703)NSteroidogenesis(MRID 48686702)NHershberger(MRID 48618402)NENN NMale Pubertal Rat(MRID 48702103)NNNNNNNNNNFSTRA(MRID 48702101)NEGSI:↑30%(H)NNNNNOSRITwo-generationreproduction(Rat; MRID 41660801)NNNENNENE NX(H)X(M, H)Chronic toxicity /carcinogenicity(Rat; MRID 41963101)N N NENNE NEX(MH,H)X(MH,H)Carcinogenicity(Mouse;MRID 00076813)N N NE N NR NRXX(M, H)Chronic toxicity(Dog; MRIDs41697901/ 42052701)NNNENNENEX(L, M,H)X(H)OXAMYLFINALPage 15 of 48Table 3. Androgenic/Anti-Androgenic Pathway for OxamylLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Oxamyl1Study Type / LiteratureCitation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histo p atholo gy Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex C h a r acte ri st i cs Fertility (Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Avian reproduction(Quail;MRID 00116610)NN N1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑)indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weightare absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate(VP), Dorsal prostate (DP),Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), Glans penis (GP).2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1 assays are presented in this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. AP Positive findingsN Negative findings (in vitro) / No effect (in vivo)NE Not examinedNR Not reportedOXAMYL FINAL

Page 16 of 48 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 oxamyl to interact w ith thyroid regulation are summarized in Table 4. If an effect was observed but considered by WoE not to reflect a direct interact ion, then the effect is discussed in the text but not pr esented in the table. The variou s targets of the thyroid 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 in cludes HPT-relevant findings from data evaluated

as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reported in the table and not consider ed further in the WOE assessment.

In both the female and male pubertal rat assays, no treatment-related effects were noted for thyroid histopathology, or serum T 4 or TSH levels. A non-dose-depe ndent increase in thyroid weight was observed in low- (0.25 mg/kg/day) and high- (5 mg/kg/ day) dose pubertal female rats (20 and 13%, respectively). There were no thyr oid-related effects obs erved in the Part 158
mammalian studies.

In the AMA, oxamyl did not significantly alter 7- or 21-day hind-limb length (HLL), snout-vent length (SVL) or body weight at a ny treatment concentration. No tr eatment-related mortality was observed during the study, and no clinical signs of toxicity were noted. Although all of the surviving frogs were beyond NF stage 61 at test termination (termed “l ate-stage”) which may confound the ability to adequately evaluate potential thyroid-rela ted effects in the AMA, there was no significant acceleration or delay of median NF developm ental stage at any treatment concentration throughout the test nor effects on thyroid histopathol ogy compared to the control. Therefore, there is no convinci ng evidence of potential thyroid-re lated interaction in the AMA for oxamyl.OXAMYL FINAL

Page 17 of 48 Table 4. Thyroid Pathway for Oxamyl

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

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

EDSP Tier 1 Assays

Female Pubertal Rat (MRID 48702104) N N N N N N N N

Male Pubertal Rat (MRID 48702103) N N N N N N N N

AMA (Frog; MRID 48702102) N N N N

OSRI Developmental toxicity (Rat; MRID 40859201) X (H) X (H) Developmental toxicity (Rabbit; MRID 00063009) N N

Two-generation reproduction (Rat; MRID 41660801) NE X (H) X (M, H)

Chronic toxicity / carcinogenicity (Rat; MRID 41963101) NE N NE X (MH, H) X (MH, H)

Carcinogenicity (Mouse; MRID 00076813) NE N NE N X (M, H) Chronic toxicity (Dog; MRIDs 41697901/ 42052701) N N NE X (L, M, H) X (M, 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 examinedOXAMYL FINAL

Page 18 of 48 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of oxamyl to interact w ith 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 Tier 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues shou ld be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l disruptions in homeostasis” (USEPA, 2013).

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. In addition to the endocrine -related effects described above
for the in vivo Tier 1 assays and OSRI, other eff ects observed which may be considered overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. However, in other instances, mo re than one parameter was needed to determine overt toxicity. Effects which were considered to be signs of system ic toxicity were generally less severe forms of toxicity (e.g., or gan weights, blood chem istry).

1. Tier 1 in vivo AssaysOXAMYL FINAL

Page 19 of 48 No overt toxicity was observed in any of the mammalian in vivo Ti er 1 assays. In the female and male pubertal rat studies, there were generally no tr eatment-related changes in organ weights, macroscopic observations or histopathological ob servations in any examined organs. The only exception was an increased absolute and adjusted thyroid weights in the female pubertal rat study, without concomitant histopathological or hormonal effects. Although ChE activity was
not determined in these studies and no clini cal signs were observed at oxamyl doses up to 0.5 mg/kg/day, this dose approximates the maximu m tolerated dose (MTD) based on data from earlier studies showing decrease s in brain and RBC ChE activity.

In the FSTRA study, no effects on survival or clini cal signs of toxicity we re observed, but there was a significant decrease in female body weight in the high-concentration group (0.989 mg a.i./L) of 13% (body length was not de termined). No overt or systemic signs of toxicity were observed in the AMA study at test concentratio ns up to 0.12 mg/L.

2. OSRI

In the developmental study with rats, there were no mortalities, but incide nce of clinical signs was increased, including diarrh ea (6/25), eye discharge (12/25 ), salivation (20/25), tremors (25/25), and wet legs - perine al and underbody (5–11/25) - during the treatment period in high dose animals, with a lower incidence (NS) of tremors at the medium-high dose (4/25). Although ChE activity was not determined in this study, th ese effects are consistent with ChE inhibition. Decreased body weight gain of 30 to 55% and de creases in food consumption were noted during treatment in most treatment groups except at the low dose. A body weight loss of 28% in the high dose group on GD 7 to 9.

In the developmental study with rabbits, matern al absolute body weights and food consumption were generally comparable between the treated and control groups; however, during GD 6 to 19, the medium- and high-dose groups had decrease d body weight gains compared to control ( ↓ 61.5 and 67.2%, respectively).

In a two-generation reproduction study with rats, in creased incidences of a variety of clinical signs (alopecia, stained fur, hyperactivity and skin sores) in different se xes/generations at the high dose, but only the following were statisti cally significant (p<0.05): skin sores in F 0 males, hyperactivity in F 1 males, and alopecia in F 1 females (also significant at medium dose). Treatment-related decreases in body weight we re observed in the mid- and high dose groups during the premating phase, as we ll as gestation and lactation phases for females. Body weights
for males and females in the F 0 and F 1 generations were decreased at the medium and high doses during the premating phase (70 or 105 days, resp ectively, of treated diet) in a dose-dependent manner. Body weights females in the F 0 and F 1 generations were decrea sed at the medium and high doses during the gestation a nd lactation phases of the study, w ith weight recovery observed during the lactation period. Sim ilar decreases were noted for food consumption for high-doseOXAMYL FINAL

Page 20 of 48 animals at the majority of the sampling interval s, and for medium-dose males; food consumption was generally comparable to control for medium-dose females.

In the chronic toxicity study w ith rats, clinical signs includ ed increased incidences ofhyperactivity in the mid-high- and high-dose males/females, swollen legs/paws in mid- and highdose males and high-dose females, and skin sore s or scabs in the medium-high- and high-dose females. Body weights were decreased in males in the medium-high dose (Day 7-470) and high dose (Day 7–680), and in females in the mi d-high and high dose groups throughout the study (Day 7–722); decreases were consistently >10% in the high dose animals and in the mediumhigh females by Day 84 until study termination. Body weight in mid-high dose males was >10% during a portion of the first year, but later decreases ranged from 7–9% through Day 470. Mean body weight gains were decreased by 10–25%, resp ectively, in males, and female body weight gains were decreased by 27 to 37%, respectively, during the first year only. The decreased body weights and body weight gains were consider ed secondary to hyperactivity, because food consumption and food efficiency were not affect ed. In the medium-high- and high-dose groups, plasma ChE activities were decr eased by 4 to 37% and 18 to 48%, respectively, in males, and by 38% and 69%, respectively, in females after the first month of treatment only. Plasma ChE
activities were not affected in the lower dose groups, and RBC and brain ChE activities were not decreased in any treated animals.

In the carcinogenicity study with mice, mortality was increased early in the study (2 males, 9 females at the medium dose: 14 males, 11 fema les at the high dose through Week 4). Due to these high mortality rates, the dose for the high-dose group was decreased at Week 6 from 100 to 75 ppm, and extra animals were added to most of the dose groups. Overall survival at study termination was not significan tly different for any treatment group compared to control. Apparent treatment-related effects on body weight were observed in this study in the mid- and high-dose groups, but body weight data were not presented in the available reviews; it was reported that decreased body weight in females was sporadic but persis tent in males throughout the study. No treatment-related effect on food consumption was noted. Determination of ChE activity was not conduc ted in this study.

In the chronic toxicity study with dogs, one fe male dog in the high-dose group died on study, but
the cause of death was not determined. Increased incidences of clinical signs including tremors and vomiting were observed in animals in th e medium- and high-dose groups. Mean body weights/body weight gains in high-dose male s and females were decreased 23%/81% and 17%/49%, respectively, at study te rmination. Body weight decrease s in males and females were

≥ 10% by Week 7, but decreases we re only significant for the la st 7 weeks of the study (male body weight) and for overall male body weight gain. In low-dose males, plasma ChE was inhibited at 6, 9, and 12 months by approximately 30% throughout the study; females exhibitedminor decreases in plasma ChE at the same sampling times. Plasma ChE activity in the mediumand high-dose groups in males was decreased, bu t generally not dose-dependently, from Week 1OXAMYL FINAL

Page 21 of 48 through study termination (approximately 50 to 70 %). Decreases in plasma ChE activity in the medium- and high-dose females also were obser ved from Week 1 through study termination (approximately 30 to 60%). RBC ChE activity was not affected, and brain ChE activity was only
decreased in males. No treatment-related effect s were noted for any absolute organ weights.

In a follow-up chronic dog study with lower doses to determine a NOAEL, no treatment-related clinical signs were present, and no mortality occurred. In addition, there were no treatmentrelated effects on body weight, bod y weight gain, food consumpti on, or organ weights. At study termination, plasma ChE at the high dose ( 50 ppm) was comparable to control and RBC ChE activity was decreased ( ↓ 22%), although RBC ChE activity th roughout the study was variable. A decrease ( ↓ 20%, NS) in brain (cerebellum with medu lla) ChE activity was noted in the high-dose group and is considered relevant , as tremors were observed at 150 and 250 ppm in males, and at all doses in females, in the previous study. In the medium-high-dose group, plasma, RBC, and
brain ChE activities were decrease d 18%, 5%, and 2%, respectively.

In the Part 158 avian reproduction study with ma llard ducks, mortality and observations of penmate aggression were observed in the control and all treatment groups.

In a freshwater fish early life-stage test in rainbow trout, no treatment -related effects on embryo hatching, larval survival, first day of larval sw im-up or larval growth was observed at test
concentrations ≤ 0.77 mg/L. At 1.5 mg/L (the highest concentration tested ), statistically significant (p<0.05) delays in the first day of embryo hatching (+2 days) and larval swim-up (+5 days) were observed, as well as a decrease ( ↓ 96%) in the percentage embryo hatching.

B. Estrogen Pathway

There was no convincing evidence of interaction of oxamyl with the estrogen pathway. The Tier

1 in vitro assays were negative (ER binding, ERTA, ar omatase and steroidogenesis assays), as well as the Tier 1 uterotrophic assay. In the fe male pubertal assay, while there were increases in uterine weights, the absence of complimentary data does not support estrogen-re lated etiology
for this effect. There were also no trea tment-related effects observed in the FSTRA.

C. Androgen Pathway

There was no convincing evidence of interaction of oxamyl with the androgen pathway. The Tier

1 in vitro ( i.e , AR binding and steroidogene sis) and Hershberger assa ys were negative. No androgen-related effects were observed in the Ti er 1 male pubertal assa ys or mammalian Part 158 studies. In the FSTRA assay, while there wa s an increase in GSI at high dose, no other
effects were observed.

D. Thyroid PathwayOXAMYL FINAL

Page 22 of 48 There was no evidence of interaction of oxamyl with the thyroid pathway. Thyroid parameters
were not altered in Tier 1 mammalian or amphibian in vivo studies, except for a non-dosedependent increase in thyroid weights in the fe male pubertal rat (20% and 13% at the mid and high dose, respectively).

E. Conclusions

Oxamyl demonstrates no convincing evidence of potential interacti on with the estrogen, androgen, or thyroid pathways.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations, ma mmalian or wildlife Tier 2 testing is not recommended for oxamyl since there was no convi ncing evidence of poten tial interaction with the estrogen, androgen or thyroid pathways.OXAMYL FINAL

Page 23 of 48 VI. References

Adamik, E.R., Criswell, M.K., Mahler, S.C., et al . (1981). Long Term Feeding Study in Mice with Oxamyl: Project No. WIL-77033: HL 0-252-81. (Unpublished study received May 29, 1981 under 352-372: Unpublished study conducted by WIL Research Laboratories, Inc., submitted by E. I. du Pont de Nemours and Co., Wilmington, Delaware: COL: 070136-A, 070137, 070138, 070139, 070140, 070141, 070142, 070143). (MRID 00076813).

Dickrell, L. (1991). 52-Week dietary toxicity study with IN 01410-196 (oxamyl) in male dogs. Unpublished study conducted by Hazleton Laborat ories, Inc.; Study No. HLO 555-90; Submitted to EPA by E. I. du Pont de Nemours and Co.; 10/3/91. (MRID 42052701).

Fort, D.J. (2012). Oxamyl (DPX-D1410) Tec hnical: 21-d Amphibian Metamorphosis Assay (AMA) with South African Clawed Frog, Xenopus laevis. Unpublished study conducted by Fort Environmental Laboratories, Inc., Stillwater, OK 74074. Laborat ory report number DUPO01- 00270. Study sponsored by E. I. du Pont de Ne mours and Company (DuPont), Wilmington, Delaware. May 31, 2012. (MRID 48702102).

Hicks, S.L. (2012). Oxamyl Technical (DPX -D1410): Short-term Reproduction Assay with the Fathead Minnow, Pimephales promelas . Determined Under Flow-Through Conditions. Unpublished study conducted by Performed by ABC Laboratories, Inc., Columbia, Missouri and Experimental Pathology Laboratories, Inc., Sterling, Virginia, Report No.: 66763. Submitted by E. I. du Pont de Nemours and Comp any, Wilmington, Delaware. May 14, 2012. (MRID 48702101).

Hobermam, A.M., Mossburg, P.A., Wolfe, G.W., et al . (1980). Teratogenicity Study in Rabbits. Unpublished study conducted by Hazleton Laborator y of America, Inc., Vienna, VA. Study No. 201-405. October 1980. (MRID 00063009).

Hurtt, M. (1990). Reproductive and Fertility Effects with Oxam yl (IN D1410): Multigeneration Reproduction Study in Rats: Lab Project Number: 423-90: 8433-88- 001. Unpublished study prepared by Haskell Laboratory / E. I. du Pont de Nemours and Co . (MRID 41660801).

Hutton, D. G. (1988). Early Life Stage Toxicity of IN 01410-196 (Oxamyl) to Rainbow Trout·. Report No. 468-88. Unpublished study conducted by E. I. du Pont de Nemours and Company, Inc., Newark, Delaware; Submitted by E. I. du Pont de Nemours and Company, Inc., Newark, Delaware. (MRIDs 40901101 and 41472301).

Malley, L. A. (1991). Combined chronic t oxicity/ oncogenicity study with oxamyl (IN 01410- 196) long term feeding study in rats. Unpublis hed study conducted by Haskell Lab.; Study No. 278-91. (July 11, 1991). Submitted to EPA by DuPont Agricultural Products. (MRID 41963101).OXAMYL FINAL

Page 24 of 48 Mebus, C.A. (1990). Chronic t oxicity study with oxamyl (IN 01410-196) one year feeding study in dogs. Unpublished study conducted by Haskell Lab.; Study No. 381-90. Submitted to EPA by E. I. du Pont de Nemours and Co. (MRID 41697901).

Munley, S.M. (1998). DuPont’s Position on Fetal Weight Changes in Rats Following Developmental Toxicity Testing with Oxamyl. Unpublished study conducted by E. I. du Pont de Nemours and Company, Newark, Delaware . Medical Research No. 8428-001. December 16, 1998. (MRID 44737501).

Nabb, D.L. (2012). Oxamyl (DPX-D1410) Tec hnical (98% w/w): H 295R Steroidogenesis Assay. Unpublished study conducted by E. I. du Pont de Nemours and Company, Newark, DE. Laboratory report number DuPont-32077, Feb 8 2012. (MRID 48686702).

Rickard, L. B. (1988). Terat ogenicity Study of IN D1410-196 in the rat. Unpublished study conducted by Haskell Laboratory for Toxicology & Industrial Medicine. Medical Research No. 8424-001; Lab. Project. No. 473-88. October 3, 1988. (MRID 40859201).

Roberts, N.L., Phillips, C.N.K., Chanter, D.O., Mc Allister, A. (1982a). The effects of dietary inclusion of oxamyl on reproduction in the ma llard duck. DuPont Report No.: HLO 337-82, USEPA guideline 163.71-4 (1978). (MRID 00116609).

Roberts, N.L., Phillips, C.N.K., Chanter, D.O., McAllister, A. (1982b). The effects of dietary inclusion of oxamyl on reproduction in the bobw hite quail. DuPont Report No.: HLO 453-82, USEPA guideline 163.71-4 (1978). (MRID 00116610).

Snajdr, S. (2011). Oxamyl (DPX-D1410) Techni cal (98% w/w): 10-Day Hershberger Bioassay for Detecting Androgenic Activity. Unpublished study conducted by E. I. du Pont de Nemours and Company, DuPont Haskell Global Centers fo r Health & Environmental Sciences, Newark, DE. Laboratory Project ID: DuPont -32076, November 8, 2011. (MRID 48618402).

Snajdr, S.I. (2011). Oxamyl (D PX-D1410) Technical (98% w/w): In Vitro Aromatase Inhibition Using Human Recombinant Microsomes. U npublished study conducted by DuPont Haskell
Global Centers for Health and Environmental Sciences, Newark, DE. Laboratory Project ID: DuPont-32072, January 5, 2012. (MRID 48672701).

Snajdr, S.I. (2011). Oxamyl (DPX-D1410) Techni cal (98% w/w): 3-Day Uterotrophic Assay for Detecting Estrogenic Activity. Unpublished stud y conducted by DuPont Haskell Global Centers for Health & Environmental Sciences, Newar k, DE. Laboratory Project ID: DuPont-32075, November 8, 2011. (MRID 48618401).OXAMYL FINAL

Page 25 of 48 Snajdr, S.I. (2012). Oxamyl (D PX-D1410) Technical (98% w/w): In Vitro Androgen Receptor Binding Assay Using Rat Prostate Cytosol. Unpublished study conducted by DuPont Haskell
Global Centers for Health & Environmental Scien ces, E. I. du Pont de Nemours and Company, Newark, DE. Laboratory Project ID: DuPont-32153, January 6, 2012. (MRID 48672703).

Snajdr, S.I. (2012). Oxamyl (D PX-D1410) Technical (98% w/w): In Vitro Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RU C). Unpublished study conducted by DuPont
Haskell Global Centers for Health & Environmen tal Sciences, E. I. du Pont de Nemours and Company, Newark, DE. La boratory Project ID: Du Pont-32074, January 5, 2012. (MRID 48672702).

Toot, J. (2012). Pubertal Development and T hyroid Function in Intact Juvenile/Peripubertal Female Rats following Oral Administration of Oxamyl (DPX-D1410) T echnical (98% w/w). Unpublished study conducted by WIL Research, Ashland, OH. Laboratory Project ID: WIL189243, June 5, 2012. (MRID 48702104).

Toot, J. (2012). Pubertal Development and T hyroid Function in Intact Juvenile/Peripubertal Male Rats following Oral Administration of Oxamyl (DPX-D1410) T echnical (98% w/w). Unpublished study conducted by WIL Research, Ashland, OH. Laboratory Project ID: WIL189244, June 5, 2012. (MRID 48702103).

USEPA (2010). Oxamyl — Report of the Endoc rine Disruptor Review Team; Test Order # EDSP-103801-63. December 6, 2010. Office of Chem ical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, D.C.

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

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). Oxamyl (DPX-X1410) Tec hnical: Estrogen Receptor Transcriptional Activation (Human Cell Line (HeLa-9903 )). Unpublished study conducted by CeeTox, Kalamazoo, MI. Laboratory Report No.: 9144V-100359ERTA, Feb 7, 2012. (MRID 48686701).OXAMYL FINAL

Page 26 of 48 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA): (OCSPP 890.1100)

The 21-day assay (MRID 48702102) of oxamyl (DPX -D1410) on amphibian metamorphosis of South African clawed frogs ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) Stage 51 (80/control and treatment group; 20/replicate) were exposed to oxamyl (98.0% pur ity) at nominal concentr ations of 0 (negative control), 0.013, 0.040, and 0.120 mg a.i./L; time-weighted, mean-measured concentrations were <0.0085 (<LOD; negative control), 0.0106, 0.0399, a nd 0.1301 mg a.i./L. The test system was maintained at 22.4 to 22.9°C and a pH of 6.8 to 7.7.

No treatment-related mortality was observed dur ing the study, with mortalities of 1.7, 3.3, 0, and 3.3% in the control, lo w, mid, and high treatment groups, re spectively. No clinical signs of toxicity were noted in any tadpol es at test termination. Ther e were no effects (p>0.05) on body weight or snout-vent length at any of the treatme nt levels compared to the negative control.

Oxamyl did not significantly increase or d ecrease (p>0.05) Day 7 or Day 21 normalized (to snout-vent length) hind-limb length (HLL) at any treatment level. Furthermore, there was no significant acceleration or delay (p>0.05) of median NF developm ental stage at any treatment level throughout the test, t hough all surviving tadpoles at Day 21 were late-stage (NF ≥ 61). No asynchronous development was observed. Mild fo llicular cell hyperplasi a and hypertrophy were observed in the negative cont rol and all treatment groups, though there was no apparent treatment-related effect in incidence. Follicular cell height and area were not determined.

Although all of the surviving frogs were beyond NF stage 61 at test termination which may confound the ability to adequately evaluate potential thyroid-rela ted effects in the AMA, there was no significant acceleration or delay of median NF developm ental stage at any treatment concentration throughout the test , and there were no effects on thyroid histopathology compared
to the control.

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

In an androgen receptor (AR) binding assay (MRID 48672703), ventral prostate cytosol from castrated Sprague Dawley rats was used as the source of AR to c onduct a saturation binding experiment and a competitive binding experime nt. The saturation binding experiment was
conducted to demonstrate that the AR in the ra t prostate cytosol was present in reasonable numbers and was functioning with appropriate affi nity for the radiolabeled reference androgen (R1881) prior to routinely conducting AR compe titive binding experiments. The competitive binding assay was conducted to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (logarithmic increase from 10 -10 to 10 -3 M) of oxamyl (98.0% purity, Lot #6689-176-5). Deionized water was used as the vehicle for oxamyl,OXAMYL FINAL

Page 27 of 48 and ethanol was used for the reference chemicals, at a final concentration of 3%. A total of four 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. The Scatchard plots for the three saturation assays were linear.

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

− 9.05 M for R1881
and

− 4.37 M for the weak positive c ontrol (dexamethasone). Compared to R1881, the mean RBA for the positive control was 0.0022%. One run was repeated. Although the performance
criteria for both R1881 and dexamethasone were not achieved across the three runs, based on the
estimated IC 50 s for R1881 and the weak positive control, the androgen receptor was present in a reasonable concentration and 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 interp retation of this study.

Oxamyl had no effect on specific binding of the [ 3 H]-ligand; therefore, a log IC 50 and RBA could not be calculated for oxamyl as it did not re sult in 50% reduc tion in ligand binding at any
concentration.

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 401) , oxamyl (98.0% a.i., Lot#: 6689-176-5)
was incubated with human recombinant arom atase 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 oxamy l 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 run included full enzyme activity controls, background activity controls, a positive control series (10

− 10 to 10

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

− 10 to 10

− 3 M) with three replicates per co ncentration. Because the solvent vehicle was water for oxamyl and dimethyl sulfoxi de (DMSO) for 4-OH ASDN, separate sets of the background activity and full enzyme activity controls were run using only water or 1%OXAMYL FINAL

Page 28 of 48 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 calculatio ns and a fourth run was performed.

Aromatase activity in the full act ivity controls for the three successful test runs ranged 0.201-
0.532 nmol

· mg-protein

− 1

· min

− 1 in water only with a mean and standard deviation of 0.344±0.112
nmol

· mg-protein

− 1

· min

− 1 , and 0.242-0.631 nmol

· mg-protein

− 1

· min

− 1 in 1% DMSO with a mean
and standard deviation of 0.437±0.119 nmol

· mg-protein

− 1

· min

− 1 . Activity in the background controls averaged 0.62% of full activity controls in water and 0.54% of full activity controls in 1% DMSO. The response of each full activity cont rol was generally between 90 to 110% of the average full activity, with only a few minor exceptions.

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.436±0.096
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). These results were generally within the recommended ranges for the top of the curve, bottom curve, Hill Slope, log IC 50 , and coefficient of variation (CV) for replicates of each concentration, with the following exceptions:
the overall %CV for concentrations between 10

− 7.5 M and 10

− 5 M (16.5 to 21.5%) exceeded the acceptable limit of 15%, and the top of the curve in Run 1 ( 115%) exceeded the recommended range (90 to 110%).

For oxamyl, aromatase activity averaged 0.370±0.111 nmol

· mg-protein

− 1

· min

− 1 at the lowest tested concentration (10

− 10 M) and 0.351±0.115 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 oxamyl across all runs.

Based on the data from the average response curve, oxamyl 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 a ssay (MRID 48672702) for oxamyl (98.0% purity, Lot #6689-176-5), uterine cytosol from ovariectomized Sp rague Dawley rats was used as the source of estrogen receptors (ER) to conduct satu ration binding experiments and competitive binding experiments in this assay. The saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was functioning with appropriate affinity for the ra diolabeled reference estrogen prior to routin ely conducting ER competitive binding experiments. The compe titive binding experiment was conducted to measure the binding of a si ngle concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of increasing concentrations (logarithmic increase from 10

− 10 to 10

− 3 M) of oxamyl. DeionizedOXAMYL FINAL

Page 29 of 48 water was used as the vehicle for oxamyl, and etha nol was used for the reference chemicals, at a
final concentration of 2%. A total of three 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 natural ligand reference material.

In the saturation binding experiment , the mean maximum binding capacity (B max ) was 32.5 fmol/100 μg protein and the m ean dissociation constant (K d ) was 0.081 nM. These values were within the recommended ranges, and the Scatchar d plots for the three saturation assays were linear. The specific binding reached a platea u, and non-specific bindin g (NSB) was less than 20% of total binding (4 to 12%). The satura tion binding experiment de monstrated that the estrogen receptor was present in a reasonable concentration and functio ning with appropriate affinity for the reference ligand 17 β -estradiol.

In the competitive binding experiment, the estimated mean log IC 50 was -9.16 M for 17 β

estradiol and -4.85 M for the weak positive control. The mean RBA was 0.0054% for the weak positive control. Performance criteria were not met for 17 β -estradiol top % binding (76% for Run 1) and the bottom % binding for 19-norethind rone was considerably lower in Run 2 (-22.1%) compared to the values for the other tw o runs. However, the Hill slope values were consistent in the three competitive binding experiments and were within the range of the values classically obtained during the validation program of the assay, indicating one-site competitive binding.

Oxamyl had no effect on specific binding of the [ 3 H]-ligand; therefore, a log IC 50 and RBA could not be calculated for oxamyl; th e percent labeled estr adiol bound to the ER was >75% at all concentrations of oxamyl tested. Based on the resu lts from the three runs, oxamyl is classified as Not Interactive in the Estrogen Receptor Binding Assay.

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

In an estrogen receptor transcript ional activation assay (MRID 48686701), hER α -HeLa-9903
cells cultured in vitro were exposed to oxamyl (98.0% a. i. Lot # 16995) at logarithmically increasing concentrations from 10

− 10.3 to 10

− 3.3 M in water for 24 hours in two independent runs. The experiments were performed using 96-well pl ates and each oxamyl 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) gene products. Lucife rase expression in response to activation of the estrogen receptor by oxamyl was measured upon using a proprietary luciferase assay.

Oxamyl was tested up to the limit of cytotoxicity, which was observed at 10 -4.3 M in the first run. In the main assays, the responsiveness of th e cells to the very weak positive control 17 α

methyltestosterone was lower than the expected values, indicating a decrea sed sensitivity of theOXAMYL FINAL

Page 30 of 48 assay to very weak agonists. Although the conditi ons of this assay were not optimal to detect very weak activity, oxamyl 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. Oxamyl was only able to reach a maximum of 2.9-4.4% PC when tested up to the highest concentration possible based on cytotoxicity (10 -5.3 M). Because the

RPC Max < PC 10 in both assay runs, oxamyl was considered negative for estrogen receptor transcriptional activation in this test system.

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

The 21-day short-term reproduction assay (M RID 48702101) of oxamyl with fathead minnows ( Pimephales promelas) was conducted under continuous flow-through conditions. Adult fish (16 spawning groups, 2 males and 4 females in each gr oup; 5.5 months old) were exposed to oxamyl (98.0% purity) at nominal concentrations of 0 (negative control), 0.090, 0.30, and 1.0 mg a.i./L;
mean-measured concentrations were <LOD (<0.0085, negative control), 0.0866, 0.305, and 0.989 mg a.i./L. The test system was maintained at 24.6 to 25.1 o C and a pH of 7.64 to 8.27.

Adult male and female survival were unaffected by treatment. In the negative control, low, mid, and high treatment groups, male survival was 88, 88, 100, and 88%, respectively, and female survival was 100, 94, 94, and 88%, respectively. Th ere were no notable observations with regards to behavior or changes in ovipositor app earance or size of dorsal nape pad in the negative control or treated groups. One male in the high treatment group periodica lly displayed signs of spinal curvature during the chemical exposure, and a second male in this treatment exhibited edema at test termination. Starting after Day 9 of the exposure, females displaying vertical banding were occasionally observed in the nega tive control and all treated groups, but the
number of these observed females was not mo re than three per treatment. There was a significant decrease (p<0.05) in female body we ight of 13.3% at the high treatment level compared to the negative control. Fish lengths at test termination were not reported.

Fecundity in the negative c ontrol was 51 eggs/female/day/ replicate (range: 45-59), and fertilization success averaged 91%. There was a significant increase (p<0. 05) of 30% in male gonado-somatic index (GSI) at the high treatment le vel compared to the negative control. There were no apparent differences between gonadal stages in treated groups compared to the negative control. There were no significan t differences between the treatment levels and negative control for any other endpoint. Plasma sex st eroid hormones were not measured.

Histopathological findings in males were unrem arkable; minimal testicular degeneration was reported in one male fish in the negative c ontrol and high treatment group. Increased oocyte atresia was observed in 44, 20, 33, a nd 29% of females in the negative control, low, mid, and high concentration groups, respec tively. This lesion was characterized as minimal to mild with moderate severity in one female from the ne gative control and mid treatment group and twoOXAMYL FINAL

Page 31 of 48 females from the low and high treatment groups. Minimal to mild granulomatous inflammation of the ovary was present in the negative cont rol (25%), low (27%), mid (13%), and high (7%) treatment groups. In some instances, granulomatous inflammation was associated spatially with the presence of microsporidia fungi, although it is suspected that most, if not all, of the inflammation was induced by these organisms. Acco rding to the histologi cal report, it did not appear that the presence of either microsporid ia or inflammation had any effect on the study
results.

Hershberger Assay; (OCSPP 890.1400)

The Hershberger Assay consists of androgenic a nd anti-androgenic components. To screen for potential androgenic activity, oxamyl (98.0% a. i., lot # 6689-176-5) in deionized water was administered daily via oral gavage to groups of 56-day old, castrated male Sprague Dawley rats (6/group) at dose levels of 0 (vehicle), 0.1, 0.25, or 0.5 mg/kg/day. An androgenic positive control group consisted of 6 castr ated rats exposed to 0.4 mg/kg/da y testosterone propionate (TP) in corn oil by subcutaneous injection.

To screen for potential anti- androgenic activity oxamyl ( 98.0% a.i., lot # 6689-176-5) in
deionized water was administered daily via oral gavage to 56-d ay old, castrated male Sprague Dawley rats (6/group) at dose levels of 0.1, 0.25, or 0.5 mg/kg/day in conjunction with a daily dose of reference androgen TP in corn oil at 0.4 mg/kg/day by subcutaneous injection. An antiandrogenic positive control group consisted of 6 cas trated rats exposed to 0.4 mg/kg/day TP in corn oil and 3.0 mg/kg/day flutamide (FT) in 0.5 % methylcellulose. TP alone was used as the anti-androgenic ne gative 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 tissues.

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, body weight and body weight gains for the 0.1, 0.25, and 0.5 mg/kg/day treatment groups were comparable to controls throughout the duration of the assay. Animals dosed with TP had increased body weights on days 10-11 ( ↑ 7%) and increased cumulative body weight gains (p<0.05) for days 1-11 ( ↑ 25%). No effects on mean daily food consumption or food efficiency were observe d in the 0.1, 0.25, and 0.5 mg/kg/day groups.
Animals dosed with TP had increased mean daily food consumption (p<0.05) for days 1-11OXAMYL FINAL

Page 32 of 48 ( ↑ 10%). No significant increases in the organ weights were observed in any of the five accessory sex tissues of animals dosed with oxamyl. Animals in the TP group displayed the expected increases (p<0.05) in accessory sex organ weights as follows: 699% in seminal vesicles; 768% in ventral prostate; 196% in levator ani-bulbocavernosus (LABC); 465% in Cowper’s gland; and 37% in glans penis. The pe rformance criteria indica ted that this assay was performing as expected.

In the anti-androgen assay, there were no sign ificant effects on body weights or body weight gains for any dose group. For the positive control group (TP + FT), cumulative body weight gains were decreased for days 1-11 ( ↓ 16%). No effects on mean daily food consumption or food efficiency were observed in the 0.1 and 0.5 mg/kg/ day groups, or the posi tive control group. At 0.25 mg/kg/day, mean daily food consumption was decreased (p<0.05) for days 1-11 ( ↓ 10%) however, food efficiency for the 0.25 mg/kg/day group was comparable to the controls. No significant decreases in organ weights were observed in any of the accessory sex tissues of the oxamyl-treated animals. Animals dosed with TP + FT displayed the expected decreases (p<0.05) in accessory sex organ weights as follows: 73% in seminal vesicles; 48% in LABC; 59% in Cowper’s gland; and 25% in glans penis.

The maximum guideline-specified CVs were exceeded for the ventral prostate CV in the 0.1 mg/kg/day group (46% vs. 40%), the LABC in 0.25 mg/kg/day (26% vs. 20%) and the positive control (28% vs. 20%) groups, and the glans peni s in the positive control (21% vs. 17%) group.
All other CV values were less than the maximu m recommended values as indicated in the test guideline.

The high dose of oxamyl selected for this study (0.5 mg/kg/day) is adequate based on data from earlier studies showing decreases in brain and red blood cell cholines terase activity at dose levels ranging from 0.75-2.0 mg/kg/day.

Statistically significant changes we re not seen in two or more of the androgen sensitive tissue weights. Oxamyl was negative for androgenicity and anti-androgenicity in the Hershberger

assay.

Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48702104), 15 Sprague Dawley rats/dose group were treated daily by oral gavage with oxamyl (98.0% a.i., Lo t #6689-176-5) in deionized water at doses of 0 (vehicle), 0.25, or 0.5 mg/kg/day from post-nata l day (PND) 22 to 42 or 43. Animals were examined for vaginal opening (VO) daily begi nning on PND 22, and age and weight at day ofOXAMYL FINAL

Page 33 of 48 attainment were recorded. Af ter sacrifice on PND 42 or 43, blood was collected for creatinine, urea nitrogen, and thyroid hormone analyses. Total thyroxine (T 4 ) levels were determined using an electrochemiluminescent immunoassay and th yroid stimulating hormone (TSH) levels were determined using a radioimmunoassay. Adrenal, liv er, pituitary, thyroid, and urogenital organs were weighed, and microscopic examinations were performed on the ovaries, uterus, thyroid, and kidneys.

All animals survived until scheduled sacrifice. There were no trea tment-related clinical signs of toxicity, and no treatment-rela ted effects were noted on fina l body weights, body weight gain, mean age at first vaginal estrus, percent cycling, or the concentrations of serum T 4 , TSH, creatinine, or urea nitrogen. There were no treatment-related macroscopic observations or histopathological changes in th e ovaries, uterus, t hyroid glands, or kidneys. There were no treatment-related effects on the weights of liver , kidneys, pituitary, adrenals, or ovaries.

The age at attainment of VO was decreased (p <0.05) by 1.8 and 1.7 to 1.8 days at 0.25 and 0.5 mg/kg/day, respectively. However, these decreases were not dose-dependent. In addition, there was a 9% decrease (p<0.05) in body we ight at VO at 0.5 mg/kg/day.

Compared to the vehicle control, wet and blotte d uterus weights were increased by 62 and 43% (p<0.01) at 0.25 mg/kg/day and by 34 and 28% (p<0.05) at 0.5 mg /kg/day. These differences were not dose-dependent and were attributed to di fferences in the animal’s stage of estrus at necropsy. Considering only the regularly cyc ling animals, the number of females in the proliferative phase of estrus (diestrus or proe strus) was one in the c ontrol, eight in the 0.25 mg/kg/day group, and three in the 0.5 mg/day group, and the number of animals in the nonproliferative phase was ten in the control, six in the 0.25 mg/ kg/day group, and seven in the 0.5 mg/day group. Animals in the proliferative phase in each group were shown to have uterine weights that were greater than animals in the non-proliferative phase of the same group; this result is expected based on the increase in ce ll proliferation and accumulation of luminal fluid that occurs during the pr oliferative phase. Accordingly, the lo w number of control animals in the proliferative phase bias ed the results.

When the phase of estrus at necropsy is take n into account, wet and bl otted uterine weights (unadjusted) for animals in the proliferative phase were increased by 48 and 23%, respectively, in the 0.25 mg/kg/day group and by 54 and 34% for the 0.5 mg/kg/ day group, compared to the control (single animal). For animals in the non-proliferative phase, wet and blotted uterine weights were increased respectively by 23 a nd 23% in the 0.25 mg/kg/day group and 26 and 27% in the 0.5 mg/kg/day group, compared to the control (10 animals). Although there was an increase in uterine weights that appears to be dose-dependent, the differenc es in uterus weights between groups was not significant (NS). Therefore, the observed differences were attributed to normal biological variation in the animals.OXAMYL FINAL

Page 34 of 48 Although the differences were not significant, at 0.5 mg/kg/day, the mean cycle length was also increased to 5.8 days from 4.9 day (control), the percent of females regularly cycling was 54.5% compared to 69.2% in controls, and the number of females in estrus was six compared to two in control group. In the absence of any other clear effect on the re productive system, these differences may have been incidental.

Absolute and adjusted thyroid weights were in creased by 19 to 20% (p<0.05) at 0.25 mg/kg/day and by 12 to 13% (NS) at 0.5 mg/kg/day. Although these changes are substantial, the relevance of these weight changes are unclear as they were not dose-dependent and there were no associated changes in thyroid hist ology or hormone concentrations.

The high dose selected for this study (0.5 mg/ kg/day) was considered adequate based on data from earlier studies showing decrea ses in brain and red blood cell cholinesterase (ChE) activity at dose levels ranging from 0.75 to 2.0 mg/kg/day.

Male Pubertal A ssay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48702103), 15 Sp rague Dawley rats/dose group were treated daily via oral gavage with oxamyl (98.0% a.i., Lot #6689-176-5) in deionized water at doses of 0 (vehicle), 0.25, or 0.5 mg/kg/day from post-nata l day (PND) 23 to 53 or 54. Animals were examined for preputial separation (PPS) daily beginning on PND 30 and age and weight at day of attainment were recorded. Following s acrifice on PND 53 or 54, blood was collected for creatinine, urea nitrogen, and thyroid hormone analyses. Total thyroxine (T 4 ) and testosterone levels were determined using an electroche miluminescent immunoassay and thyroid stimulating hormone (TSH) levels were determined by radioi mmunoassay. Adrenal, liver, pituitary, thyroid, and urogenital organs were collected and we ighed, and microscopic examinations were performed on the testes, epid idymides, thyroid, and kidneys.

All animals survived until scheduled sacrifice. There were no trea tment-related clinical signs of toxicity, and no treatment-rela ted effects were noted on fina l body weights, body weight gain, age at PPS, body weight at PPS, or the levels of serum T 4 , TSH, testosterone, creatinine, or urea nitrogen. There were no treatment-related macroscopic observations or histopathological changes in the testes, epididymides, thyroid glan ds, or kidneys. There were no treatment-related effects on the weights of liver, p ituitary, adrenals, seminal vesicles , ventral prostate, dorsolateral prostate, LABC, epididymis , testes, or thyroid.

At 0.25 mg/kg/day, relative kidne y weight was increased (p<0.05) by 6% compared to the controls, but the unadjusted and adjusted kidney weights at 0.25 and 0.5 mg /kg/day were similarOXAMYL FINAL

Page 35 of 48 to controls. This minor change in relative kidney weight is not considered treatment-related as it occurred only at the low dose and was not associat ed with any macro- or microscopic finding.

The high dose of oxamyl selected for this study (0.5 mg/kg/day) was adequate based on data from earlier studies showing decrea ses in brain and red blood cell c holinesterase activity at dose levels ranging from 0.75-2.0 mg/kg/day.

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48686702), H295R cells cultured in vitro in 24-well plates were incubated with oxamyl (98.0% a.i., Lot # 6689-176-5) at log concen trations from of 0.0001

to 100 μ M for 48 hours in triplicate for three indepe ndent experiments. Dimethyl sulfoxide (DMSO) was used as the vehicle for the positive controls and water was used as the vehicle for oxamyl.

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

The minimum basal hormone production levels for testosterone (500 pg/mL) were met in both blank and solvent control (SC) wells for testos terone. However, the minimum basal production
for estradiol was below the recommended concen tration of 40 pg/mL, except for the blank and SC in Run 1. Although the basal estradiol produc tion as generally <40 pg/mL, all values were >12.5 pg/mL (2.5-fold greater than the minimu m detection limit (MDL) as per the Guideline recommendation). Estradiol production was induced by forskolin by ≥ 7.5-fold, but induction of testosterone production (1.76- to 1.80-fold) was slightly below the Guideline specified ≥ 2-fold requirement. Testosterone and estradiol inhibition by prochloraz was >50% as required. Because all other guideline requirements for ba sal concentrations and hormone induction and
inhibition were met for testosterone and estr adiol, the performance criteria were deemed acceptable.

The variability (%CV) between th e runs based on the absolute concentrations of the SC was 20.9% for testosterone and 23.5% for estradiol, which is within the recommended limit of ≤ 30%.
The %CVs within each run for the QC plates were 0.04-15.64%.

Precipitation was not observed at any tested oxa myl concentration, and oxamyl was not overtly cytotoxic to the H295R cells. Cell viab ility for the QC and test plates was ≥ 91% at ≤ 10μM of oxamyl, and 86.3-93.5% at 100 μM. No effect on estradiol production was observed at oxamylOXAMYL FINAL

Page 36 of 48 concentrations ≤ 100 μ M. Slightly decreased testoster one levels were observed at 100μM oxamyl (approximately 0.8- to 0.9-fold; p ≤ 0.05). This decrease was accompanied by a decrease in viability that could account for the s lightly lower testosterone production at 100 μ M. Adjusting for the 86.3-93.5% viability, the fold change in testosterone production at 100 μ M oxamyl becomes 0.87-, 0.94- and 0.93-fold for Runs 1-3, respectively, and averaged 0.91-fold.

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

Uterotrophic Assay; (OCSPP 890.1600)

In an uterotrophic assay (MRID 48618401) conducted to screen for potential estrogenic activity, oxamyl (98% a.i., Batch # DPX-D1 410-196) in deionized water was administered daily via oral gavage to groups of ovariectomized female Spra gue-Dawley rats (6/group ) at dose levels of 0 (vehicle), 0.1, 0.25, or 0.5 mg/kg/day on post-natal days (PND) 67-69. A positive control group
was treated with 17 α -ethynyl estradiol (EE) by daily oral gavage at a dose level of 0.1 mg/kg/day. All animals were terminated and necropsied on PND 70 approximately 24 hours
after the final dose to determine wet and blotted ut erine weights.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in any dose group. No treatment-related effects on the stage of estrous over the duration of the study were observed for oxamyl. All rats in th e negative control and oxa myl treated groups were determined to be in diestrus during Days 1-4. Rats in the positive control group (EE) showed effects on the stage of estrus on Days 3 and 4. By Day 4, two of six rats in the positive control group showed cytological markers indicative of es trus and three of six rats showed markers indicative of proestrus.

Body weights in the oxamyl trea ted groups were comparable to the controls throughout the duration of the assay. In the positive control group (EE), body we ights were decreased (p<0.05) by 7% on Study Day 4 compared to controls and these animals actually lost weight (-7.6 g, p<0.05) compared to controls (12.2 g).

No treatment-related effects on food consumption or food efficiency were noted in the oxamyl treated groups. In the positive control group (EE), overall (Days 1-4) food consumption was decreased (p<0.05) by 29% compared to cont rols and food efficiency was -0.14%.

Uterine weights in the oxamyl treated groups were comparable to controls. The mean blotted vehicle control uterine weight wa s 0.032% of the mean vehicle c ontrol terminal body weight. As expected, the absolute wet and blotted uterus weights for the positive control (EE) group were increased (p<0.05) by 3.1- and 2.4-fold, respectively.OXAMYL FINAL

Page 37 of 48 The dose levels selected for this study are adeq uate based on marked decreases in brain and red
blood cell cholinesterase activity at doses ≥ 0.75 mg/kg/day in studies in the database.

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

Page 38 of 48 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

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

Prenatal Development in Rodents (Rat)

In a developmental toxicity study ( MRID 40859201 and 44737501 ), oxamyl (97.2% purity) was administered by gavage to groups of pregnant CD BR rats (25/dose) at doses of 0, 0.2, 0.5, 0.8, and 1.5 mg/kg/day from GD 7 to 16, inclusive. On GD 22, the livers and gravid uteri were removed, and the dams were sacrificed. There were no mortalities, but clinical signs were increased (p<0.05) including dia rrhea (6/25), eye discharge (12/ 25), salivation (20/25), tremors (25/25), and wet legs, perineal, and underbody (5 –11/25) during the treatment period in the highdose animals, with a lower incidence (NS) of tr emors at the medium-high dose (4/25), associated
with ChE inhibition. While ther e were no significant dose-rela ted changes in body weight or overall body weight gain, dams 17–3 0% less weight during GD 7–17 at ≥ 0.8 mg/kg/day. Decreases in food consumption were noted between GD 7 and 17 in the medium-high- (10%) and high-dose (16%) animals. Treatment had no effect on the reproductive parameters and/or fetal malformations, or visceral/skeleta l variations. A dose-related decrease (p ≤ 0.05) in fetal body weights was observed at the medium-low dose and above ( ↓ 3.9, 6.8, and 6.9%, respectively). The maternal toxicity NOAEL is 0.5 mg/kg/day, and the LOAEL is 0.8 mg/kg/day, based on decreased body weight gain, decreased food consumption, and the increased incidence of tremors. The developmental toxicity NOAEL is 0.2 mg/kg/day, and the LOAEL is 0.5 mg/kg/day, based on dose-relate d decreases in fetal body weight.

Prenatal Development in Non-Rodents (Rabbit)

In a developmental toxicity study ( MRID 00063009 ), pregnant New Zealand White rabbits (17/dose) were administered oxamyl (97.1% purity) by gavage at doses of 0, 1, 2, or 4 mg/kg/day on GD 6 to 19, inclusive. Ovulation was induced by i.v. injection of HCG (100 IU), followed by artificial insemination (GD 0). On GD 29, all surviving does were sa crificed and all fetuses were weighed, measured for crown-rump distance, and examined for external malformation/variations. Each fetus was examined viscerally by fresh di ssection and the sex determined. The heads from one-third of the fetuses were removed, fixed in Bouin’s solution, a nd sectioned by Wilson’s freehand razor technique. All carcasses were eviscer ated and processed for skeletal examination.

One doe each in the low- and high- dose groups died prior to sche duled sacrifice; these deaths were attributed to gavage error. All other an imals survived to terminal sacrifice, and noOXAMYL FINAL

Page 39 of 48 treatment-related clinical signs of toxicity were observed in a ny treated group. Maternal absolute body weights and food consumption were comparab le between the treated and control groups throughout the study; however, during the treatme nt interval (GD 6 to 19), the medium- and high-dose groups had decreased (p<0.05) body weight gains compared to control ( ↓ 61.5 and 67.2%, respectively). Slight decreases (NS) in ov arian and uterine weight s were noted in the medium- and high-dose groups, but data were not included. Therefore, th e maternal toxicity LOAEL was 2 mg/kg/day based on reduced body we ight gain, and the maternal toxicity NOAEL is 1 mg/kg/day.

No treatment-related differences were observe d between the treated and control groups for number of corpora luteal/doe, implantations/ doe, pre-implantation loss, fetal body weights and lengths, or fetal sex ratios. Dose-related incr eased resorption rates fo r the medium- and highdose groups resulted in increased (NS) post-implan tation losses and decrease d (NS) litter sizes. For the control, low-, medium-, and high-dose groups, the post-implantation losses were 10.4%, 8.7%, 15.9%, and 24.8%, respectivel y, and the number of live fe tuses/litter was 6.6, 6.6, 5.9, and 5.8. In the high-dose group only early resorptions were observed, and two animals had whole litter resorption consisting of early resorptions of 1 and 7 implantation sites, respectively. These
were not considered treatment-related because a complete resorption in the rabbit is not uncommon. Furthermore, resorptions in the hi gh-dose group may have been a consequence of the maternal toxicity at this dose. No treatment-related external, visceral, or skeletal
malformations/variations were observed in the fe tuses. Therefore, the developmental toxicity NOAEL was 4 mg/kg/day and a developm ental LOAEL was not determined.

Multi-Generation Reproduc tion in Rodents (Rat)

In a two-generation reproduction study ( MRID 41660801 ), Crl:CD ® BR rats (30/sex/dose) were fed oxamyl (97.1 % purity) in the diet at nominal dosages of 0, 25, 75, or 150 ppm (approximately 0, 1.7, 5.2, or 11.6 mg/kg/day fo r males and 0, 2.0, 6.6, or 15.8 mg/kg/day for females, respectively). After 74 days of oxamyl dietary treatment, the F 0 females were mated with males from the same group in a ratio of 1: 1 until evidence of mating was obtained, or for a maximum of three weeks. The day on which mating was confirmed was designated GD 0. The F 1 animals were mated in a similar fashion after 105 days (post-weaning) on the test diet. Study measurements included body weight/body weight gain, food consumption, and determination of reproductive parameters, organ weight (testes), and gross and microsc opic pathology (including pituitary, testes, epididymis, prostate, seminal vesicles, coagulating gland, ovary, uterus, and vagina).

No treatment-related mortality was observed; deaths of one high-dose female (F 0 ) and three high-dose males (F 1 ) were not considered due to treatme nt. Increased incidences of several clinical signs were observed in high-dose animals in each generation: F 0 males, skin sores (p<0.05); F 0 females, alopecia and stained fur (NS); F 1 males, hyperactivity and skin soresOXAMYL FINAL

Page 40 of 48 (p<0.05 and NS, respectively); F 1 females, alopecia (also seen at the medium dose), hyperactivity, and skin sores (p<0.05, NS, and NS, respectively).

Treatment-related decreases (p<0.05) in body wei ght were observed for both sexes in the medium- and high-dose groups during the prema ting phase, as well as during gestation and lactation phases in females. In the F 0 generation, body weight for males and females was decreased at the medium ( ↓ 5 to 9%) and high doses ( ↓ 10 to 18% for males and ↓ 10 to 12% for females) during the premating phase ( 70 days of treated diet). In the F 1 generation, body weight for males and females was decreased at the medium ( ↓ 7 to 20%) and high doses ( ↓ 17 to 37%) during the premating phase (105 days post-weaning on treated diet). Body weights were also decreased consistently during the gestation phase for the F 0 females at the medium ( ↓ 6 to 8%) and high doses ( ↓ 13 to 15%) and for F 1 females at the medium ( ↓ 12 to 13%) and high doses ( ↓ 19 to 20%). Body weights recovered somewhat duri ng the lactation phase with decreases for F 0 females at the medium ( ↓ 2 to 8%) and high doses ( ↓ 7 to 15%) and F 1 females at the medium ( ↓ 8 to 12%) and high doses ( ↓ 15 to 21%). Similar decreases were noted for food consumption for high-dose animals and medium-dose males at a majority of the sampling intervals; food consumption was generally comparable to control for medium-dose females.

No treatment-related effect was noted on absolute te stes weights, but relati ve testes weights were increased (p<0.05) in high-dose F 0 males ( ↑ 19%) and medium- and high-dose F 1 males ( ↑ 12 and 33%); however, these weight increases were not associated with any microscopic finding. In addition, no treatment-related micros copic findings were noted in F 0 or F 1 adults in the pituitary, epididymis, prostate, seminal vesicles, co agulating gland, ovary, uterus, or vagina.

Based on clinical signs, and decreased body weights and food consumption, the LOAEL is 75 ppm (approximately 5.2 and 6.6 mg/kg/day for males and females, re spectively), and the NOAEL for systemic/developmental toxicity is 25 ppm (approximately 1.7 and 2.0 mg/kg/day for males and females, respectively).

Fertility, mating, and gestational indices were not affected by oxamyl treatment at any dose in either generation. Therefore, the reproducti ve NOAEL is 150 ppm (approximately 11.6 and 15.8 mg/kg/day for males and females, respectivel y). Offspring toxicity was observed in the medium- and high-dose groups in both genera tions based on significan tly decreased pup body weights during lactation in both generations; in the F 0 generation, pup body weights were decreased at the medium ( ↓ 7 to 14%) and high doses ( ↓ 13 to 25%), and decreased in the F 1 generation at the medium ( ↓ 16 and 19%) and high doses ( ↓ 20 to 32%). In addition, at the high dose, the number of live pups per litter was ge nerally decreased in both generations and the viability index was decreased 21 and 43%, resp ectively. Based on these results, the NOAEL for offspring toxicity is 25 ppm (approximately 1.7 and 2.0 mg/kg/day for males and females, respectively), and the LOAEL is 75 ppm (appr oximately 5.2 and 6.6 mg/kg/day for males and females, respectively) based on decr eased pup body weights during lactation.OXAMYL FINAL

Page 41 of 48 Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat)

In a combined chronic toxicity/carcinogenicity study ( MRID 41963101 ) oxamyl (97.1%. purity)
was administered in the diet to Crl:CD ® BR rats (62 rats/s ex/dose) at nominal doses of 0, 25, 50, 100 or 150 ppm (mean-measured intake of oxamyl was calculated as 0, 0.992, 1.97, 4.19, or 6.99 mg/kg/day for males and 0, 1.32, 2.69, 6.73 or 11.1 mg/ kg/day for females, respectively) for two years. An interim sacrifice of 10 rats/sex/ dose was conducted at 12 months. Body weight, food consumption, food efficiency, hematology, clinical chemistry, urinalysis, and organ weights were recorded. Plasma and RBC ChE activity wa s assessed at 1, 3, 6, 12, 18, and 24 months. After 104 weeks, all surviving rats in all groups were sacrificed for gros s and histopathological evaluation. Select organs weighed at each sacrifice interval included liver, kidneys, testes, and
ovaries. Brains were collected at the interim a nd final sacrifices for a ssessment of brain ChE activity. Gross and microscopic pathological ex amination included the liver, kidney, pituitary, thyroid/parathyroids, adrenals, mammary gland, testes, epididymides, prostate, seminal vesicles, uterus, vagina, and ovaries.

Treatment with oxamyl did not affect mort ality, food consumption, food efficiency, or hematology, clinical chemistry, and urinalysis parameters. The incidence of hyperactivity was increased (p<0.05) in the medium-high and high-dose groups (males/females, 52%/50% and 63%/73%, respectively; control, 27%/17%). The incidence of sw ollen legs/paws in mid- and high-dose males was increased (p<0.05) by 29% and 34%, respectively, compared to 11% in control; in high-dose females, the incidence wa s increased (p<0.05) 23% compared to 5% in control. Females also exhibited an increased (p<0 .05) incidence of skin sores or scabs in the medium-high and high-dose groups (61% and 82%, respectively) compared to 39% in control.
Survival rates in treated males and females were similar to better than in control animals. Mean body weights and body weight gains were decrease d in males and females at the medium-high and high doses during the first ye ar of study. Body weights were de creased (p<0.05) in males atthe medium-high (Day 7–470) and high doses (Day 7–680), and in females in the medium-highand high-dose groups throughout th e study (Day 7–722); decreases we re consistently >10% in the high-dose animals and >10% in the medium-high females by Day 84 until study termination. Body weight in medium-high dose males was >10% during a portion of the first year, but later decreases ranged from 7–9% through to Day 470. During the first year, the mean body weight gains in medium-high- and high-dose male s were decreased 10% and 25% (p<0.05), respectively, compared to cont rol, and medium-high- and high -dose female body weight gains were decreased by 27% and 37%, respectiv ely. Although weekly body weights generally remained decreased (p<0.05) during the second y ear of the study, the overall body weight gain during this period was not significantly decrease d. The decreased body weights and body weight gains in males and females were considered secondary to hyperactivity, since neither food consumption nor food efficiency (only slight decr eases) was affected in these test groups. In medium-high- and high-dose males, plasma ChE ac tivities were decreased (p<0.05) by 4 to 37% and 18 to 48%, respectively, during the study, comp ared to control. In medium-high- and highOXAMYL FINAL

Page 42 of 48 dose females, plasma ChE was inhibited (p<0.0 5) by 38% and 69%, respectively, after the first month of treatment only. Plasma Ch E activity in males and females was not affected in the lower dose groups, and RBC and brain ChE activities were not decreased in treated animals. No histopathological findings were noted in the eval uated tissues, and a treatment-related increase in tumor incidence was not observed in this study.

Based on the results of this study (body weights, hyperactivity, plasma ChE inhibition, and eye lesions), the highest dose tested (150 ppm) appeared to be a MT D, and was sufficiently high to evaluate the chronic toxicity and carcinogenicity of oxamyl. Th e systemic toxicity NOAEL is 50 ppm (1.97 mg/kg/day for male s and 2.69 mg/kg/day for females), and the LOAEL is 100 ppm (4.19 mg/kg/day for males and 6.73 mg/kg/day fo r females) based on hyperactivity, swollen legs/paws, and skin sores, decreased body wei ghts and body weight gains, and inhibition of plasma ChE activity in male rats.

Carcinogenicity in Rodents (Mouse)

In a carcinogenicity study ( MRID 00076813 ), CD-1 mice were administered oxamyl (97.1% purity) at nominal concentrations in the diet of 0, 25, 50, and 100 ppm for 24 months (calculated equivalency of 0, 3.75, 7.5 or 15/11.2 5 mg/kg/day). Initially, 80 mice/ sex were included in each group, but extra mice were added to most treatmen t groups due to initial high mortality. The high dose was reduced to 75 ppm at Week 6 and extr a mice were added to the treatment groups as follows: low-dose females and mid-dose males, n=81; mid-dose females, n=84; and high-dose animals, n=88. Mortality, clinical signs of t oxicity, body weights and food consumption were monitored at appropriate times during the study. After 24 months, surviving mice in all groups were sacrificed for gross and histopathological evaluation. Sel ect organs were weighed at terminal sacrifice (liver, kidneys, testes and heart; unknown if additiona l organ weights were determined). Gross and microscopic patholog ical examination included the liver, kidney, pituitary, thyroid, adrenals, mamm ary gland, testes, epididymides , prostate, seminal vesicles, uterus, and ovaries.

Mortality was increased early in the study (2 male s, 9 females at the medium dose, and 14 males and 11 females at the high dose through Week 4). Due to these high mortality rates, the dose for the high-dose group was decrease d at Week 6 from 100 to 75 ppm. Overall survival at study termination was not significan tly different for any treatment group compared to control.Apparent treatment-related effects on body weight were observed in this study in the mediumand high-dose groups. It was reported that decrea sed body weight was sporadic in females, but persistent in males, throughout the study. At Week 11, the male body weight was decreased by 5%; no other body weight data was included in the Agency review . No treatment-related effect on food consumption was noted. Clinical chemis try assessment was not required or conducted, so ChE activity was not determined.OXAMYL FINAL

Page 43 of 48 Decreases in liver weights in the medium- and high-dose males ( ↓ 23 and 16%, respectively) were not significant or dose-dependent. Similarly, increases (NS) in kidney weights in low- and high-dose males ( ↑ 6 and 9%, respectively) were not dose- dependent, and there was no change at the medium dose. No effects were noted in orga n weights in female mice. No treatment-related gross pathological effects were not ed. Apparent increases in glom erulosclerosis in all treated male mice, and pulmonary tumors in all treated females, were investigat ed against historical
control data and determined to not be trea tment-related. The NOAEL in the carcinogenicity study is 25 ppm (low dose; approximately 3.75 mg /kg/day), and the LOAEL is 50 ppm based on decreased body weight a nd increased mortality.

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

In a one-year chronic feeding study ( MRID 41697901 ) oxamyl (99% purity) was administered in the diet to groups of male and female beagle dogs (5/sex/dose) at nominal doses of 0, 50, 150, or 250 ppm (equivalent to 0, 1.56, 4.60, or 8.00 mg/kg/day for males and 0, 1.46, 4.50, or 7.84 mg/kg/day for females, respectively). The dogs were offered the food once daily (approximately 350 g/dog). Select organs were weighed at terminal sa crifice (liver, kidneys, thyroid/parathyroids, and testes ). Gross and microscopic pathological examination included the liver, kidney, pituitary, thyroid/ parathyroids, adrenals, mammar y gland, testes, epididymides, prostate, vagina, uterus, and ova ries. ChE activity was determined in several brain sections ( e.g. , caudate nucleus, cerebellum/medulla, and cerebrum).

One female dog in the high-dose group died on st udy, but the cause of death was not determined.
Increased incidences of clinical signs including tremors and vo miting were observed in animals in the medium- and high-dose groups. Mean body weights/body weight gain s in high-dose males
and females were decreased 23%/81% and 17%/49% , respectively, at termination compared to control. Body weight decreases in males and females were ≥ 10% by Week 7, but decreases were only significant (p<0.05) for the last 7 weeks of the study (male body weight) and for male body weight gain. The body weight changes were conc omitant with decrease d food consumption and food efficiency. The food consumption/food e fficiency in high-dose males was decreased (p<0.05) by 11%/75% compared to the control; in high-dose females, decreases of 7%/42% were NS. Administration of oxamyl did not pr oduce any adverse eff ects in urinalysis, ophthalmological examinations, and gross pathology at any doses. In the low-dose males, plasma ChE was inhibited (p<0.05) at 6, 9, and 12 m onths by 33, 34, and 32%, respectively, compared to control; females exhibited minor decreases in plasma ChE at the same sampling times. Plasma ChE activity in the medium- and high-dose groups in males was decreased (p<0.05), but generally not dose-dependently, from Week 1 through study termination (approximately ↓ 50 to 70%). Plasma ChE activity in the medium- and high-dose groups in females was decreased (p<0.05 or NS), but generally dose-dependent ly, from Week 1 through study termination (approximately ↓ 30 to 60%). Treatment-related effects were not noted for RBC, and brain ChEOXAMYL FINAL

Page 44 of 48 activity was only decreased in the low-, medium-, and high-dos e males; caudate nucleus: ↓ 31, 47, and 30%, respectively, and cerebrum: ↓ 23, 46, and 34%.

No treatment-related effects were noted for any ab solute organ weights. Histologically, 3/5 highdose males exhibited increased regenerative re nal tubular epithelial alterations. A NOAEL for male dogs was not established in this study due to decreased ChE activity in plasma and brain at
all doses.

Subsequently, a second one-year study ( MRID 42052701 ) was repeated in male dogs (5/dose) at nominal oxamyl (98% purity) doses of 0, 12.5, 20, 35, or 50 ppm (equi valent to 0, 0.372, 0.577, 0.930 or 1.36 mg/kg/day). The oxamyl exposure at the 50 ppm dose in this study was slightly lower than the exposure in the first study ( 1.56 mg/kg/day). In this study, food was offered

ad libitum . All other study design parameters were sim ilar to the previous chronic toxicity study in the dog.

No treatment-related clin ical signs were present in this study, and no mortality occurred. In addition, no treatment-related effects were observed on body weight, body weight gain, food consumption, clinical biochemistry/hemat ology, gross pathology, or organ weights. Histopathological examinations we re not conducted for this study. At study termination, plasma ChE at the high dose was comparable to the control and RBC ChE activity was decreased (p<0.05, ↓ 22%), although RBC ChE activity throughout the study was vari able. A decrease (NS, ↓ 20%) in brain (cerebellum with medulla) ChE ac tivity was noted in the high-dose group and is considered relevant, since tremors were observed at 150 and 250 ppm in males and at all doses in females in the previous study. In the medium-high dose group, plasma, RBC, and brain ChE activities were decreased 18%, 5%, and 2%, and may be cons idered a true NOAEL. Although the study was not replicated iden tically to the previous study in order to establish a NOAEL in male dogs, the results suggest that the plasma Ch E activities are comparable and the information from this study can be utilized to derive a NOAEL in male dogs.

The systemic toxicity NOAEL is 50 ppm ( 1.56 mg/kg/day for males and 1.46 mg/kg/day for females) and the LOAEL is 150 ppm (4.60 mg /kg/day for males and 4.50 mg/kg/day for females), based on decreased body weights and body weight gains. The ChE NOAEL is 35 ppm (0.930 mg/kg/day for males) a nd 50 ppm (1.56 mg/kg/day for fe males), and LOAEL is 50 ppm (1.36 mg/kg/day for males and 4.50 mg/kg/day for females), based on brain ChE activities in males and vomiting, tremors, and plasma and brain ChE inhibition in females.

Avian Reproduction (Duck) (OCSPP 850.2300)

In an avian reproduction study ( MRID 00116609 ), wild-caught young ad ult Mallard ducks ( Anas platyrhynchos ) approaching their firs t laying season were administered oxamyl (97.1% purity) in the feed at co ncentrations of 0 (control), 10, or 50 ppm for 22 weeks (ten weeks prior to the start of egg production and during twelve weeks of egg production). Each treatment groupOXAMYL FINAL

Page 45 of 48 comprised six replicates, with two drakes and five hens per pen. A progressive photoperiod was employed in this study: 7 hours/day from Day 1 to 44; 8 hours/day from Day 45 to 51; 9 hours/day from Day 52 to 65; 12 hours/day from Day 66 to 72; 13 hours/day from Day 73 to 79; and 14 hours/day from Day 80 to 154. Temper ature ranged from 8 to 24°C throughout the study; humidity was not reported. Individual ad ult body weights were recorded on Days 0, 14, 28, 42, 56, and 154; food consumption in each rep licate was measured weekly throughout the pre-treatment and treatment pe riods of the adult phase. Indi vidual chick body weights were recorded within 24 hours of hatching and on Da y 14 after hatching; e gg shell thickness was measured on the first two days of Weeks 1, 3, 5, 7, 9, and 11 of the egg production period. Adult
birds and chicks were observed daily for mortality and clinical signs; all adult birds were subjected to post-mortem examination. Ch E determinations were not conducted.

Seven birds died or were sacrif iced during the pre-egg production period primarily attributed to pen-mate aggression (two in the low-dose and five in the high-dose groups), all of which were replaced with spare birds. During the egg produc tion period, six birds died in the control group (14%), two birds died in the low-dose group (5%), and nine bi rds died in the high-dose group (21%) (one was sacrificed). Pe n-mate aggression (bullying) continued during the egg production period (Days 71–154) and was listed as an observation in five of the 14 bird s that died during the study. Post mortem examination showed that a nu mber of females were not in egg production at the end of the 12-week laying period as their ova ries were underdeveloped. According to the reviewer’s report, the study i ndicated a slight effect on egg production at 50 ppm and a potentially higher chance of in fertility in the eggs. Although th ere were no treatment-related effects on adult mortality, food consumption, or b ody weight, there is concer n with the ability to
discern endocrine-related effects seen in the ma llard study due to the pr esence of mortality and aggressive behavi or in the pens.

In another avian reproduction study ( MRID 00116610 ), young adult Bobwhite quail ( Colinus virginianus ), were offered diet containing technical oxamyl (97.1% purity) at concentrations of 0, 10, or 50 ppm diet for 28 weeks (sixteen weeks prior to the start of egg production and twelve weeks during egg production). Each treatment group was comprised of 14 replicates of one male and two females. Individual adult body wei ghts were recorded on Days 0, 14, 28, 42, 56, 70, 84, and 196; food consumption in each replicate was measured weekly throughout the pre-treatment and treatment periods of the adult phase. Individu al chick body weights were recorded within, 24 hours of hatching and on Day 14 after hatching; egg shell thickness was measured on the first two days of Weeks 1, 3, 5, 7, 9, and 11 of the egg-production period. Adult birds and chicks were observed daily for mortalit y and clinical signs; all adul t birds were subjected to postmortem examination. ChE determinations were not conducted during this study.

Four birds died or were sacrificed (two in the control group and two in the high-dose group) during the pre-egg production period; all were replaced with spare birds. There was no treatment-related effect on adult mortality; clin ical observations and post mortem changes wereOXAMYL FINAL

Page 46 of 48 consistent with long-term housing of birds in ca ges, and were not related to treatment. Adult body weight changes over the treatment period were similar in all groups, and there was no
evidence of a treatment-related effect. Adult food consumption was decreased (significance NR) in the two oxamyl-treated groups during both pre-egg production and egg production periods (data NR). Initial chick body weights were simila r overall in all treatmen t groups; chicks that died after hatching during the 14-day observation period were examined and no abnormalities were detected. Of the reproductive parameters me asured, few differences were detected between the control and treatment groups, with two exceptions: the proportion of damaged eggs was lower in the low-dose group compared to control (15 vs. 28 control), and the proportion of chicks surviving to 14 days was signifi cantly higher in the 10 ppm diet treatment compared to control (Williams test, p<0.05; 79.5 vs. 80.1%).

The reproductive NOAEC for bobwhite quail expose d to oxamyl in the diet for 28 weeks was 50 ppm diet, based on the absence of adverse re productive effects at th e highest dose tested.

Fish Early Life-Stage Toxicity; (OCSPP 850.1400)

In a freshwater fish early life-stage toxicity test ( MRIDs 40901101 and 41472301 ), Rainbow

trout (Salmo gairdnerii, (O ncorhynchus mykiss)) embryos and larvae were continuously exposed to oxamyl (97.2% purity) at nominal concentrations of 0 (water control), 0.06, 0.12, 0.25, 0.50, 1.0, and 2.0 mg/L (mean-measured concentrations of 0.06, 0.10, 0.23, 0.40, 0.77, and 1.5 mg/L) in a flow-through system for 61 days. Observations were made on embryo hatching and larval survival and growth. Twenty embryos were added to each cup (80 embryos/concentration). The first larvae ha tched on Day 20 and hatching was considered complete by Day 29. As the larvae hatched, they were transferred from the embryo cup to the appropriate aquarium, with up to 15 larvae per re plicate; excess larvae we re counted and then discarded. Survival of the remaining larvae was assessed for 32 days, and at the end of the study all surviving larvae were weighed and measured.

The mean measured concentratio ns of oxamyl in the test vesse ls ranged from 75% to 100% of
the nominal values. No treatment-related effects on embryo hatching, larval survival, first day of larval swim-up, or larval growth was observed at measured test concentrations of 0.06 to 0.77 mg/L. At 1.5 mg/L (highest con centration tested), delays (p<0.05) in the first day of embryo hatching and larval swim-up were observed comp ared to control, as well as a decrease ( ↓ 96%; p<0.05) in the percentage ha tching. The NOAEC for rainbow trout exposed to oxamyl was 0.77 mg/L, based on effects on embryo hatching and larval swim-up at the high dose (1.5 mg/L).OXAMYL FINAL

Page 47 of 48 APPENDIX 3: References Not Utiliz ed in the Oxamyl 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 Oxamyl 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.

Andersen, H.R., Vinggaard, A.M., Rasmussen, T.H., Gjermandsen, I.M., and BonefeldJorgensen, E.C. (2002). Effects of Currently Us ed Pesticides in Assays for Estrogenicity, Androgenicity, and Aromatase Activity In Vitro. Toxicol. Appl. Pharmacol . 179:1–12.

Grubbs, C.J., Perkham, J.C., and Cato, K.D. (1983) . Mammary carcinogenesis in rats in relation to the age at time of N-nitr oso-N-methylurea administration . J. Natl. Cancer Institute

. 70:209-212.

Hellwig, J., van Ravenzwaay, B., Mayer, M., and Gembardt, C. ( 2000). Pre- and postnatal oral toxicity of vinclo zolin in Wistar and Long-Evans rats. Regulatory Toxicology and Pharmacolgy.

32:42–50.OXAMYL FINAL

Page 48 of 48 Hseuh, A.J.W., Dufau, M.L., and Catt, K.J. (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., Yeung, E.K.C., Chan, C.B., and Cheng, C.H. K. (2008). Establishment of a Transgenic Yeast Screening System for Estrogenicity and Id entification of the Anti-Estrogenic Activity of
Malachite Green. J. Cell. Biochem . 105:1399–1409.

Klotz, D. M., Arnold, S.F., and McLachlan, J.A. (1997). Inhibition of 17 Beta-Estradiol and Progesterone Activity in Human Breast Cancer and Endometria l Cancer Cells by Carbamate
Insecticides. Life Sci. 60:1467–1475.

Kojima, H., Katsura, E., Takeuchi, S., Niiyama, K., and Koba yashi, K. (2004). Screening for Estrogen and Androgen Receptor Ac tivities in 200 Pesticides by In Vitro Reporter Gene Assays Using Chinese Hamster Ovary Cells. Environ

. Health. Perspec. 112 : 524–531.

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

Nishihara, T., Nishikawa, J., Kanayama, T., Dakeyama, F., Saito, K., Imagawa, M., Takatori, S., Kitagawa, Y., Hori, S., and Utsumi, H. (2000). Estrogenic activities of 517 chemicals by yeast two-hybrid assay. J. of Health Sci . 46: 282–298.

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 hormonal activity for crop protection chemicals? J. Toxicol. Environ. Health 50:415–431.