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 Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

Environmental Fat~~fec~~~n ~ Office of Pesticide Program 

THROUGH: Jess Rowland ~ S ~~~ Deputy Division Director 

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

EPA has completed its Weight of Evidence (WoE) assessment evaluating results of the 

Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. 

The WoE documents for the 52 chemicals are listed in Attachment A along with the chemical 

and report identifiers. 

Page I of2 

Page 1 of 62
Page 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 

Page 2 of 62
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

 

CHEMICAL: METHOMYL 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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METHOMYL 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 Relevant Information (OSRI)......................................4 

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

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

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................8 

1. Effects on Estrogen Pathway.....................................................8 

2. Effects on Androgen Pathway..................................................14 

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

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

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

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

2. OSRI...............................................................................21 

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

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

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

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

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

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

LIST OF TABLES: 

 Table 1. Tier 1 Assay for Methomyl...................................................................7 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Methomyl....................................10 

 Table 3. Androgenic/Anti-Androgenic Pathway for Methomyl..................................15 

 Table 4. Thyroid Pathway for Methomyl..........................................................19 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................29 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................39 

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

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METHOMYL 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 Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

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 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

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iv 

Abbreviation Terminology 

F Female 

F1 First filial generation 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

HPLC/MS/MS High Pressure Liquid Chromatography/Mass Spectroscopy

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

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 Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

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v 

Abbreviation Terminology 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

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vi 

Abbreviation Terminology 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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Page 1 of 54 

Executive Summary 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to 

provide the necessary empirical data to evaluate the potential of chemicals to interact with the 

estrogen (E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism 

and antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In 

addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality were considered 

in this weight of evidence (WoE) assessment. 

In determining whether a methomyl interacts with E, A or T hormone pathways, the number and 

type of effects induced, the magnitude and pattern 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 (WoE) analysis of the potential interaction of methomyl 

with the E, A or T signaling pathways. The T1WoERC conclusions from the WoE evaluation in 

this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in 

vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited 

OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature). 

There was no convincing evidence of interaction of methomyl with the estrogen pathway either 

in the in vitro (i.e., ER binding, ERTA, aromatase and steroidogenesis) or mammalian in vivo

(i.e. uterotrophic of female pubertal) Tier 1 assays or in the mammalian or wildlife Part 158 

studies. In the FSTRA, although decreases in GSI, fertility and fecundity were observed at the 

mid and high treatment (0.312 and 1.03 mg/L, respectively) and increased incidence and severity 

of oocyte atresia at the high treatment were reported there were no effects observed on VTG. 

Although there were some complementary effects across the FSTRA, there was no effect on 

VTG (sex steroid hormones not measured), and there was limited redundancy across the other 

Tier 1 assays or Part 158 studies. Decreases in eggs laid (fecundity) were observed in the 

bobwhite quail reproduction study at the highest concentration tested (427 mg/kg-diet), but no 

other reproductive effects were observed. Decreases in fecundity alone are limited in the ability 

to inform about potential estrogen-related effects. Additionally, potentially similar to the 

decrease in fish GSI, an increase in number of females with regressed ovaries was observed in 

the mallard duck reproduction study, but no other reproductive effects were observed and female 

body weight gains and food consumption were decreased at this concentration. Furthermore, in 

other toxicity testing with fathead minnow fish (early life-stage and chronic reproduction), 

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METHOMYL FINAL 

Page 2 of 54 

effects on growth in young fry were observed at concentrations lower (0.117 mg/L) or similar 

(0.243 or 0.280 mg/L) to the concentrations tested in the FSTRA. Therefore, while there were no 

clinical signs of toxicity in female fish in the FSTRA (male body weights were significantly 

decreased), the concentrations tested in the FSTRA were similar to concentrations which were 

shown to be overtly toxic in other fish assays; as such, the decreases observed in the FSTRA may 

be due to potential general stress on the fish from exposure to methomyl as oppose to potential 

endocrine-related interactions. 

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

Tier 1 in vitro AR binding assay was negative, and while a decrease in testosterone production 

was observed in the Tier 1 steroidogenesis assay, there is uncertainty in this result based on the 

pattern of responses. No androgen-related effects were observed in the Tier 1 Hershberger or 

male pubertal assays or Part 158 mammalian studies. While androgen-related effects were 

observed in an OSRI study (El Medany et al. 1993), the results were questionable as the test 

doses overlapped with acute mortality values. In the FSTRA assay, there was a slight increase in 

the proportion of spermatogonia (mild to moderate) observed in male testes at the high 

concentration along with the decreases in fertility at the mid and high treatment; however, 

significant decreases in male body weight were observed at the mid and high treatment. 

Considering the body weight changes along with the discussion in the estrogen pathway 

regarding test concentrations and other observations of overt toxicity in fish, the 

alterations/decreases observed may be due to generalized stress in the fish. Also, as in the 

estrogen pathway, there is a lack of redundancy in observed effects across the Tier 1 battery and 

Part 158 studies. 

There was no convincing evidence of interaction of methomyl with the thyroid pathway. No 

thyroid-related effects were observed in the mammalian studies and no effect on developmental 

endpoints or thyroid histopathology were seen in the AMA.

Based on weight of evidence considerations, human health and wildlife EDSP Tier 2 testing is 

not recommended for methomyl since there was no convincing evidence of potential interaction 

with the estrogen, androgen or thyroid pathways. 

 

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METHOMYL FINAL 

Page 3 of 54 

I. Introduction 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide 

the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen 

(E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism and 

antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In 

addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality were considered 

in this weight of evidence (WoE) assessment. 

In determining whether methomyl interacts with E, A or T hormone pathways, the number and 

type of effects induced, the magnitude and pattern 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 (WoE) analysis of the potential interaction of methomyl 

with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE evaluation in 

this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in 

vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited 

OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature). 

Methomyl has a water solubility of 5.8 × 104

 mg/L at 25°C and a vapor pressure of 5.4 × 10-6

mm Hg at 25°C. The octanol/water partition coefficient is 0.6 (log Kow) with a whole body 

bioconcentration factor value in fish of approximately 3. Methomyl decomposes slowly in water 

but the rate of degradation is increased by temperature, aeration, and sunlight. 

The available information considered in determining the potential interaction of methomyl with 

the E, A, T pathways include submitted Tier 1 assays and/or other scientifically relevant 

information (OSRI) such as general toxicity studies and other published articles. These data are 

summarized in Section III.A through III.C. An analysis of the data submitted to the agency, 

using the WoE approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The 

EDSP Tier 2 studies recommendations are presented in Section V. 

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II. Sources of Scientific Data and Technical Information 

A. EDSP T1 Screening Assays 

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the 

agency’s test order for methomyl are shown below in Table 1. Executive Summaries are 

presented in Appendix 1. 

B. Other Scientifically Relevant Information (OSRI) 

In response to the Agency’s Test Orders, data believed to be relevant to one or more of the Tier 1 

assays were submitted as OSRI by the Test Order recipients and/or the public. This included 

studies published in the open literature and/or data submitted to support pesticide registration 

(e.g., Part 158 guideline studies). The Agency’s review of the initial OSRI is provided in the 

Report of the Endocrine Disruptor Review Team on Methomyl (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 Tier 1 Assays and data submitted as OSRI are 

included in Appendix 1 and 2. Additionally, literature/studies considered but not utilized for the 

WoE analysis are listed in Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

The principles, criteria, and approach used in the WoE determination on the potential of a 

substance to interact with endocrine-related processes (i.e., E, A, or T hormone pathways) were 

as described in the WoE guidance document (USEPA, 2011) and presented at the 2013 FIFRA 

Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how 

the individual lines of evidence are assembled and integrated along two concepts (i.e., 

complementarity and redundancy) within the conceptual framework of an adverse outcome 

pathway). Broadly, there are four main steps outlined in the guidance which provide the 

foundation for WoE evaluations. The first step is to evaluate the individual 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 endpoints/responses across assays) 

in the observed responses across these different levels of biological organization. The third step 

is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to 

evaluate whether additional testing is needed based on the evidence and conclusions described 

above. 

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As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the 

EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other 

scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays 

consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were 

designed specifically to evaluate a number of key biological events including potential effects on 

receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other 

effects on the HPG and HPT axes. OSRI may include published literature studies as well as 

studies conducted under USEPA (often referred to as Part 158 data) or OECD guidelines 

submitted in support of pesticide registrations. Each study is evaluated for scientific quality and 

relevance for informing interactions with the E, A, or T pathway. Additionally, the concordance 

or consistency (complementarity) of the responses in the individual study is evaluated. For the 

Tier 1 in vivo assays, often multiple endpoints are measured in each assay. 

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the 

relative degree of diagnostic utility of a specific endpoint for discerning whether or not the 

chemical has interacted with the endocrine system, are considered. The collective response of the 

individual endpoints, as well as the conditions under which they were expressed, are considered 

when evaluating an overall indication of potential interaction as measured by the study. 

The second step in this WoE process is to formulate hypotheses and integrate 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 support a hypothesis that a chemical 

has the potential to interact E, A, or T pathways, are the concepts of complementarity and 

redundancy. These two concepts provide a basis for considering the plausibility, coherence, 

strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are 

meant to evaluate whether or not a chemical can interact with E, A and T consisting of different 

levels of biological organization from a molecular initiating event, such as receptor binding, 

through potential adverse effects in apical endpoints such as sexual development 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 relevant, robust and consistent evidence in terms of 

complementarity and redundancy as well as biological plausibility. Second, is at what level of 

biological organization were the responses observed and whether organisms exhibit 

compensatory responses at higher levels of biological organization? Finally, under what 

conditions did the responses occur including consideration of regarding whether the responses 

were observed in the presence of overt or systemic toxicity. The presence of overt and/or 

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systemic toxicity introduces uncertainty in the ability to distinguish effects specifically related to 

an endocrine-related effect from a non-endocrine toxic response. 

This uncertainty in distinguishing whether the responses were endocrine-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, weight should be placed on signs of endocrine disruption in the 

presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in terms 

of primary interactions with the endocrine system vs. secondary effects related to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

For these WoE analyses, overt toxicity was generally 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 included, but were not limited to: mortality; 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, especially 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/concentration 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 decreases 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 effects were generally considered as less severe 

forms of toxicity (e.g., changes in organ weights or blood chemistry). The circumstances for 

which a dose/concentration was considered overtly toxic for a particular study are described in 

Section IV.A. 

In summary, EPA considers multiple lines of evidence in including the observed responses in 

the Tier 1 assays and OSRI in the context of a chemical’s physical/chemical properties and its 

known modes of action in its overall 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 determining 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 evidence supporting a conclusion; and, 5) what, if any, 

additional data are needed and why. Assessing the need for additional data is based on a caseby-case analysis which took all available toxicity data into account. 

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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 biologically relevant with regard to a treatment-related effect 

which supports a conclusion of whether or not a substance has the potential to interact with the 

E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the 

text for each respective pathway (E, A or T) but are not reported in the table for E, A or T. 

A. EDSP T1 Screening Assays 

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

below in Table 1. 

Table 1. Tier 1 Assays for Methomyl 

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay 

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

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID No. 48685901) 

AR Binding Assay 

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

Steroidogenesis Assay 

(Human cell line H295R) 

OCSPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID No. 48685902) 

Aromatase Assay 

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

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID No. 48617701) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID No. 48617702) 

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied 

(MRID No. 48701404) 

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied 

(MRID No. 48701403) 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID No. 48701401) 

Amphibian Metamorphosis Assay 

(Frog) 

OCSPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID No. 486701402) 

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B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis 

1. Effects on Estrogen Pathway 

The potential for methomyl to interact with the estrogen pathway is summarized in Table 2. The 

various targets of the estrogen pathway across the relevant Tier 1 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 presence of overt toxicity are discussed in the text but are not reported in the table and not 

considered further in the WOE assessment. 

Methomyl was negative (or not interactive) in the Tier 1 in vitro estrogen receptor (ER) binding 

assay, ERα transcriptional activation (ERTA), steroidogenesis and aromatase assays. Methomyl 

was also negative in ERTA assays considered as OSRI (Anderson et al., 2002 and Kojima et al., 

2004). 

In the uterotrophic assay, no uterine weight increases were seen. In the female pubertal assay, 

there were no estrogen-related effects observed. 

In the FSTRA, decreases in fecundity of 23% and 77%, fertility of 1.6% and 33%, and GSI of 

15% and 21% were noted in the females at the mid- and high-concentrations (0.312 and 1.03 

mg/L), respectively. In addition, oocyte atresia was increased (mild to moderate) in females at 

the highest test concentration. However, no effects were noted on VTG in females (or males) and 

sex hormone concentrations (17β-estradiol) were not determined. 

In the Part 158 mammalian toxicity studies, no estrogen-related effects were observed, except for 

a decrease in ovarian weight in the F1 adults in the 2-generation rat reproduction study which 

occurred in the presence of overt toxicity. An increase in pituitary weight (absolute and relative) 

in the absence of histopathological changes was observed in the subchronic rat study. 

In a Part 158 avian reproduction study, reductions in egg laid (40%) were observed in the 

bobwhite quail study at the highest dose tested (427 mg/kg-diet); however, reports of females 

with feather loss and toe lesions [(potentially treatment-related and due to male aggression (pen 

mate)] were reported at this concentration. No treatment-related reproductive effects were 

observed in the Part 158 mallard duck study; decreases in female body weight gain was observed 

at the highest test concentration (427 mg/kg-diet). In another mallard duck reproduction study 

(MRID 49245301; preliminary review), an increase in the number of females with 

regressing/regressed ovaries (8/15 vs. 4/16 in control) was observed at 1120 mg/kg-diet, 

however, no other reproductive parameter was affected. Also, decreases in female body weight 

gain and food consumption was observed at 1120 mg/kg-diet 

Page 16 of 62
METHOMYL FINAL 

Page 9 of 54 

Reproductive effects were not observed in the Part 158 fish life-cycle study with fathead 

minnow; effects on parental survival and/or as well as parental and offspring growth were 

reported at concentrations of ≥0.147 mg/L. Two Part 158 fathead minnow fish early-life stage 

studies were available and in one study, decreases in growth (weight and length) were reported at 

≥0.148 mg/L; in the other study, survival was decreased at ≥0.117 mg/L with decreases in 

growth at ≥0.243 mg/L. 

Page 17 of 62
METHOMYL FINAL 

Page 10 of 54 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Methomyl

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Methomyl1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight / 

GSI 

Ovarian / Gonad 

Staging and 

Histo

pathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Vitellogenin 

EDSP Tier 1 Assay 

ER Binding (MRID 48672003) N ERTA (MRID 48685901) N4 Steroidogenesis (MRID 48685902) N Aromatase (MRID 48672002) N Uterotrophic (MRID 48617701) N Female Pubertal Rat (MRID 48701404) N N N N N N FSTRA (MRID 48701401) NE 

↓15, 

21% 

(M, H) 

P 

(H)5 N 

Frt: ↓1.6, 

33%; 

Fcd: 

↓23, 

77% 

(M, H) 

N 
METHOMYL FINAL 

Page 11 of 54 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Methomyl

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Methomyl1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight / 

GSI 

Ovarian / Gonad 

Staging and 

Histo

pathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Vitellogenin 

OSRI 

ERTA assay (Andersen et al., 2002) 

N 

ERTA assay (Kojima et al., 2004) N Subchronic (13-week) toxicity (Rat; MRID 00007190) 

NE NE N ↑19% (H) 

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

 N N 

Two-Generation Reproduction (Rat; MRIDs 43250701, 43769401) 

 N N N N N 

Chronic toxicity / carcinogenicity (Rat; MRID 00078361) 

 NE NE N N 

Carcinogenicity (Mouse; MRID 00078423) 

 NE NE N N 

Chronic toxicity (Dog; MRID 00007091) 

 NE NE N NE 
METHOMYL FINAL 

Page 12 of 54 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Methomyl

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Methomyl1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight / 

GSI 

Ovarian / Gonad 

Staging and 

Histo

pathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Vitellogenin 

Fish Life-Cycle (MRID 43072101) N Avian Reproductive toxicity (Quail; MRID 41898602) 

P6 (H) 

Avian Reproductive toxicity (Duck; MRID 41898601) 

N 

Avian Reproductive toxicity (Duck; MRID 49245301) 

P9

(H) N 

 1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=H

Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is 

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 th

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 

details see Section IV. A 

4. The responsiveness of the cells to the very weak positive control 17α-methyltestosterone was lower than the expected values. M

similar to those of the negative control corticosterone and not comparable to the responses of 17α-methyltestosterone, which wmaximum of 24.3-26.6% PC. Methomyl was only able to reach a maximum of 3.1-3.7% PC. Methomyl was considered nega

5. Oocyte atresia was increased (mild to moderate) in females 6. Reductions in egg laid (40%) were observed in the bobwhite quail study at the highest dose tested (427 mg/kg-diet) 

 7. Increases in the incident of females with feather loss and toe lesions [potentially treatment-related and due to male aggression 

at mid and high treatment. No effects on survival or body weight were reported. 

 8. Decreases in female body weight gain observed at highest concentration tested (427 mg/kg-diet); no other clinical signs of tox
METHOMYL FINAL 

Page 13 of 54 

 9. Increase in the number of females with regressing/regressed ovaries (8/15 vs. 4/16 in control) was observed at 1120 mg/kg-die

reproductive parameter was affected 

 10. Decreases in female body weight gain (↓46.6%) and food consumption observed at 1120 mg/kg-diet; no other clinical signs of 

 P Positive findings 

N Negative findings (in vitro) / No effect (in vivo) 

NE Not examined NR Not reported 
METHOMYL FINAL 

Page 14 of 54 

2. Effects on Androgen Pathway 

The potential for methomyl to interact with the androgen pathway is summarized in Table 3. The 

various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for androgenic, anti-androgenic, or HPG axis effects. This 

table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in 

the presence of overt toxicity are discussed in the text but are not reported in the table and not 

considered further in the WOE assessment. 

Methomyl was negative (or a non-binder) in the Tier 1 in vitro androgen receptor (AR) binding 

assay. In the steroidogenesis assay, there were significant (p<0.05) decreases in mean 

testosterone levels at methomyl concentrations of 0.01 μM (0.89-fold) and 100 μM (0.83-fold) 

and the effect was consistent across all three runs. However, as a similar response was observed 

at two non-sequential concentrations (10,000-fold difference), the confidence in these results are 

reduced. Methomyl was negative in the ARTA studies by Anderson et al. (2002) and Kojima et 

al., (2004). 

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

were observed. 

In the FSTRA, significant decreases in fertility of 1.6% and 33% were noted at the mid- and 

high-concentrations, respectively. There was a slight increase in the proportion of spermatogonia 

(mild to moderate) in high-dose males. No other androgen-related effects were observed (sex 

hormone concentrations not measured). Body weight was decreased approximately 10 and 13% 

in males from the mid- and high-concentration groups at study termination which may be 

suggestive of stress at the two highest test concentrations; no effects on mortality or clinical 

signs of toxicity were observed. 

In the Part 158 mammalian toxicity data, there were no androgen-related effects observed. 

Mahgoub and El-Medany (1993/2001) investigated reproductive toxicity after oral 

administration of methomyl to male Wistar rats at 17 mg/kg/day by oral gavage for two months. 

Decreased (p<0.001) testosterone concentration was observed (↓35%) after methomyl 

administration for 60 days, as well as after a one-month recovery period (30%, p<0.001). 

Various degenerative changes in the seminiferous tubules, primary spermatocytes, and 

spermatids were also observed after treatment; histopathology after the recovery period was not 

presented. The results of this study are questionable because there was no reported mortality at 

doses that overlap the reported acute LD50 range (17 to 45 mg/kg/day) for methomyl. In the Part 

158 avian reproduction studies, there were no androgen-related effects reported. Similarly, in a 

fish life cycle toxicity test, there were no androgen-related effects reported. 

Page 22 of 62
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Page 15 of 54 

Table 3. Androgenic/Anti-Androgenic Pathway for Methomyl

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Methomyl1Study Type/ Literature Citation 

AR Binding / 

AR activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight / GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Age and Weight at 

PPS 

EDSP Tier 1 Assay 

AR Binding (MRID 48672001) N Steroidogenesis (MRID 48685902) P4 Hershberger (MRID 48617702) N N Male Pubertal Rat (MRID 48701403) N N N N N N N N FSTRA (MRID 48701401) NE N P5 N 

Frt: ↓1.6, 

33%; 

(M, H) 

OSRI 

ARTA assay (Andersen et al., 2002) N ARTA assay (Kojima et al., 2004) N Subchronic toxicity (13-week) (Rat; MRID 00007190) 

 N N NE N N NE 

Two-generation Reproductive toxicity (Rat; MRIDs 43250701, 43769401) 

 N N NE NE N N 

Chronic toxicity / Carcinogenicity (Rat; MRID 00078361) 

NE N N NE N N NE 
METHOMYL FINAL 

Page 16 of 54 

Table 3. Androgenic/Anti-Androgenic Pathway for Methomyl

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Methomyl1Study Type/ Literature Citation 

AR Binding / 

AR activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight / GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Carcinogenicity (Mouse; MRID 00078423) 

NE N N N N N NE 

Chronic toxicity (Dog; MRID 00007091) 

NE N N NE NE NE NE 

Testicular toxicity (Rat; El-Medany et al., 1993 and Mahgoub et al., 2001) 

↓35, 

30%7 NE P8 NE NE NE 

Fish life-cycle toxicity (MRID 43072101) N Avian reproductive toxicity (Quail; MRID 41898602) 

 N 

Avian Reproductive toxicity (Duck; MRID 41898601) 

 N 

Avian Reproductive toxicity (Duck; MRID 49245301) 

 N 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High 

Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not a

Changes in weight are absolute unless otherwise indicated. LW=Liver weight 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OS

this column. For details see Section IV. A 
METHOMYL FINAL 

Page 17 of 54 

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 

details see Section IV. A 

4. Decreases in mean testosterone levels at 0.01 μM (0.89-fold) and 100 μM (0.83-fold), and the effect was consistent across all three 

similar response was observed at two non-sequential concentrations (10,000-fold difference), the confidence in these results are red

5. There was a slight increase in the proportion of spermatogonia (mild to moderate) in high-dose males 6. Decreased body weight in mid-and high-dose fish (10% and 13%, respectively), however no effects on survival or clinical signs of 7. Decreased concentrations after methomyl treatment for 60 days and a 30-day recovery, respectively. 8. Degenerative changes in the seminiferous tubules, primary spermatocytes, spermatids, and Leydig cells. 9. Increases in the incident of females with feather loss and toe lesions (potentially treatment-related and due to male aggression (pen 

mid and high treatment. No effects on survival or body weight were reported. 

10. Decreases in male body weight gain observed at all test concentrations; no other signs of toxicity observed. 11. Decreases in female body weight gain (↓46.6%) and food consumption observed at 1120 mg/kg-diet; no other clinical signs of toxi

reduction in food consumption corresponded with slight reductions in mean body weight of both hens and drakes during the first twmean 0- to 2-week body weight changes were -104 versus -23 g for the control for drakes, and -72 versus -26 g for the control for h

P Positive findings N Negative findings (in vitro) / No effect (in vivo) NE Not examined 
METHOMYL FINAL 

Page 18 of 54 

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

The current EDSP Tier 1 battery 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 detect changes in the HPT axis, i.e., the 

pubertal female and male (rat) assays, and the AMA (frog). 

The potential for methomyl to interact with thyroid regulation is summarized in Table 4. The 

various 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 includes 

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 considered 

further in the WOE assessment. 

In the Tier 1 male and female pubertal assay and mammalian OSRI, there were no thyroidrelated effects observed. An increase in pituitary weight (absolute and relative) in the absence of 

histopathological changes was observed in the subchronic rat study. 

In the amphibian metamorphosis assay, decreased HLL (normalized for SVL) values were 

observed on Day 7 (↓4.7%) and 21 (↓4%) in the high-dose group, and increased SVL (↑6%) was 

also seen on Day 21 in the high-dose group. Although all of the surviving frogs were beyond NF 

stage 60 at test termination (termed “late-stage”) which may confound the ability to adequately 

evaluate potential thyroid-related effects in the AMA, there was no significant acceleration or 

delay of median NF developmental stage at any treatment concentration throughout the test and 

no effects on thyroid histopathology compared to the control. Therefore, the decrease in HLL 

and SVL on Day 7 and Day 21 in the absence of corroborating effects does not suggest potential 

interaction with the HPT axis. 

 

Page 26 of 62
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Table 4. Thyroid Pathway for Methomyl

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Methomyl1

Study Type / Literature 

Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH Levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, SVL, HLL) Systemic Toxicity Obsereved,2

Overt Toxicity Observed3 

EDSP Tier 1 Assays 

Female Pubertal Rat 

(MRID 48701404) N N N N N N N N 

Male Pubertal Rat 

(MRID 48701403) N N N N N N N N 

AMA 

(Frog; 

MRID 48701402) 

 N 

Days 7 and 

21: ↓4.7 & 

4% HLL 

(H) 

Day 21: 

↑6% SVL 

(H) N 

OSRI

Subchronic (13-week) 

toxicity 

(Rat; MRID 00007190) 

NE N NE NE ↑19%

(H) 

X 

(MH, H) 

X 

(MH) 

Developmental toxicity 

(Rat; MRID 00008621) N 

Developmental toxicity 

(Rabbit; 

MRID 00131257) 

X 

(H) 

X 

(H) 

Two-generation 

Reproductive toxicity 

(Rat; MRIDs 

43250701, 43769401) 

NE N N X 

(M, H) 

X 

(L, M, H)

Chronic toxicity / 

Carcinogenicity 

(Rat; MRID 00078361) 

NE N NE NE N X 

(H) N 

Carcinogenicity 

(Mouse; 

MRID 00078423) 

NE N NE NE N X 

(MH,H) 

X 

(MH, H) 

Chronic toxicity 

(Dog; MRID 

00007091) 

N N NE NE NE X 

(H) 

X 

(H) 

 1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Mediumhigh treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the response. A shaded cell 

indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight 

are absolute unless otherwise indicated. 

 2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The 

systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 

 3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the 

OSRI is indicated by an X in this column. For details see Section IV. A 

 N Negative findings 

 NE Not examined 

Page 27 of 62
METHOMYL FINAL 

Page 20 of 54 

IV. Committee’s Assessment of Weight of Evidence Conclusions 

This section of the document describes the weight of evidence (WoE) determination on the 

potential of methomyl to interact with endocrine related processes (i.e., E, A or T hormonal 

pathways) as well as recommendations 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 peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (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 considerations of biological plausibility of the findings from the 

different lines of evidence by examining the consistency, 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 evaluation in helping elucidate if 

effects seen in the Tier 1 assay are related to perturbations of the endocrine system per se or 

alternatively a sequelae of systemic effects. Endocrine modes of action may elicit a number of 

phenotypic consequences other than those evaluated in the Tier 1 assays. 

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to 

whether or not the responses are 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 evaluation of the results of the Tire 1 assays stated that “In summary, 

the Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in 

the presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in 

terms of primary interactions with the endocrine system vs. secondary effects related to toxicity 

in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 and OSRI 

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

in vivo Tier 1 assays and OSRI, other effects 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, more than one parameter was needed to determine overt toxicity. 

Effects which were considered to be signs of systemic toxicity were generally less severe forms 

of toxicity (e.g., changes in organ weights or blood chemistry).

Page 28 of 62
METHOMYL FINAL 

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1. Tier 1 in vivo Assays 

Indications of systemic and/or overt toxicity were not observed in any of the Tier 1 in vivo

mammalian studies with methomyl. In the FSTRA study, decreased (p<0.05) body weights in 

the male fish at the two highest test concentrations (↓10 and 13%, respectively). 

2. OSRI 

In a subchronic toxicity study in rats,, no effects of treatment were observed on mortality or 

clinical signs (except for tremors in a single, high-dose female by the end of the study). 

Additionally, no adverse, treatment-related effects were noted for plasma and red blood cell 

(RBC) ChE activity. Overall body weight gain was decreased by 14% in the high-dose males, 

and body weight gains were decreased (↓13 and 17%) during Weeks 7 to 13 in the high-medium 

dose animals when the dose was increased to 500 ppm. Liver weights were decreased in the 

high-medium and high-dose males, but no treatment-related histopathological lesions were 

reported. In males, absolute and relative liver weights (↑17 and 7 to 11%, respectively) and 

absolute kidney weights (↑11 to 15%) were increased (p<0.05) at ≥480 ppm in males. Relative 

kidney weights were increased (↑8%, p<0.05) at 480 ppm only. 

In a developmental toxicity study in rats, there were no mortalities or clinical signs of toxicity. In 

addition, there were no treatment-related decreases in body weights. In a developmental toxicity 

study in rabbits, seven rabbits in the high-dose group died beginning with the initiation of 

treatment and continuing throughout the treatment period (GD 7, 8, 9, 11, 17, 18, and 19). 

Central nervous system (CNS) stimulation occurred in the high-dose rabbits and was considered 

to be treatment related (dose-dependent increases, p<0.05, in the incidence of tremors, 

hyperactivity, body jerks, excess salivation, aggressive behavior, hyperpnea and convulsions). 

Other dose-dependent, but not statistically significant, increases in CNS stimulation were 

observed including ataxia, impaired or lost righting reflex, skin lesions (self-inflicted), dyspnea, 

hypersensitivity, pin-point pupils and vocalization. No treatment-related effects were noted for 

the low- and mid-dose groups. 

In a two-generation reproduction study in rats, there was no evidence of a treatment-related 

effect on survival. For the F0 generation, mean body weights were decreased (p<0.01) in the 

high-dose females (premating) and males (entire study) by 10 and 16%, respectively. For the F1

generation, mean body weights were decreased (p<0.01) for the mid- and high-dose group 

females and males by 19 and 26%, respectively. During gestation and lactation, mean body 

weights were decreased (p<0.01) for the high-dose females (and Lactation Week 1) of the 

F0 generation (↓11 to 18%) and for the mid- and high-dose females of the F1 generation (↓11 to 

27%). In the F1 litters, mean pup weights were decreased (p<0.01) in the mid- and high-dose 

groups throughout lactation (approximately ↓10 to 30%) and decreased (p<0.05 or 0.01) in the 

low-dose group at LD 14 and 21 (approximately ↓10%). In the F2 litters, mean pup weights were 

generally decreased (p<0.01) in the mid- and high-dose groups throughout lactation 

Page 29 of 62
METHOMYL FINAL 

Page 22 of 54 

(approximately ↓10 to 30%). The RBC and plasma cholinesterase concentrations were comparable 

between the treated and control groups.

In a chronic toxicity/carcinogenicity study in rats, the high-dose group had decreased body weight 

gain during the time intervals 0 to 91, 0 to 182, and 0 to 364 days of treatment. High-dose male 

rats continued a similar reduction in body weight gain through 0 to 560 days of treatment, but no 

differences between final body weight or body weight gain in the rats were observed compared to 

control at the end of the study. No treatment-related mortality was observed during the study. No 

significant differences were observed in RBC and brain ChE concentrations due to treatment in 

either sex. There were no differences in absolute organ weights for male or female rats in any 

treatment group. In a carcinogenicity study in mice, survival in the high-dose males and females 

was significantly decreased (percent and significance not reported) at Week 26, and survival in 

the mid-dose groups was also moderately decreased (percent and significance not reported). Due 

to these high mortality rates, the high and intermediate doses were reduced. Survival was 

generally significantly decreased (percent and significance not reported) after 52, 80, and 104 

weeks of treatment in mid- and high-dose animals. Decreased survival rates are considered to be 

treatment-related; survival exceeded 50% in all groups at 15 months of study. No treatmentrelated effects on body weight or body weight gain were observed in this study. There were no 

differences in absolute organ weights for male or female rats in any treatment group, except for 

increased (percent and significance not reported) absolute adrenal weight in high-dose males. 

In a chronic toxicity study in dogs, one high-dose female was found dead in its cage on the first 

day of Week 9. A replacement female dog was started on treatment two weeks later but this 

animal had repeated seizures on Day 17 of treatment, followed by coma and death. Two highdose males exhibited tremors, salivation, incoordination and circling movements on a single 

afternoon during Week 13 of the study. Another high-dose male exhibited a higher incidence of 

diarrhea compared with other study animals and progressive anemia throughout the study with 

markedly declined hematology parameters at 18 months compared to baseline. Treatment was 

discontinued from Weeks 85 through 94, with improved hematological analyses. The animal was 

returned to study for the remainder of the dosing period (Weeks 95–104), with a concomitant 

return to severe anemia and moderate leucopenia by the end of treatment. Grossly, it was found 

to have an enlarged spleen, an enlarged liver, and increases in absolute and relative thyroid, 

heart, liver, and kidney weights. The remaining two high-dose males had increased absolute and 

relative kidney weights at study termination. No adverse effects of treatment were observed on 

body weight and body weight gain. Microscopic findings in the high-dose animals included 

changes in the kidneys and liver. 

In a flow-through, early life-stage toxicity test with the fathead minnow, egg hatchability and fry 

survival were not decreased at any test concentration. Both length and weight were decreased at 

≥0.143 mg/L. In another freshwater fish early life-stage toxicity test with the fathead minnow, 

percent hatch was decreased at concentrations ≥117 μg/L. Survival was decreased (p<0.05) by 

Page 30 of 62
METHOMYL FINAL 

Page 23 of 54 

10 to 20% at ≥117 μg/L, and mean length and wet weight were decreased (p<0.05) at 

concentrations ≥243 μg/L (medium-high concentration). In a freshwater fish full life-cycle 

toxicity test with the fathead minnow, survival was decreased (p<0.05) by 6 to 7% at the high 

concentration. Mean length (calculated at Days 28, 56, and 84) was decreased (p<0.05) at 

concentrations ≥142 μg/L, and wet weight were decreased (p<0.05) at the 280 μg/L 

concentration. The length of F1 larvae at 56 days post-hatch was decreased at 142 and 280 μg/L 

concentrations, and wet weight was decreased at the 280 μg/L concentration only. 

In an avian reproduction study in bobwhite quail, there were no treatment-related mortalities. 

Reports of females with feather loss and toe lesions [potentially treatment-related and due to 

male aggression (pen mate)] were reported at 427 mg/kg-diet. In the mallard duck reproduction 

study, significant reductions in male body weight gain were observed at all test concentrations, 

and in female body weight gain in the high treatment (427 mg/kg-diet). In another mallard duck 

reproduction study (MRID 49245301), decreases food consumption was observed at 1120 

mg/kg-diet (preliminary review). The reduction in food consumption corresponded with slight 

reductions in mean body weight of both hens and drakes during the first two weeks of the study: 

mean 0- to 2-week body weight changes were -104 versus -23 g for the control for drakes, and -

72 versus -26 g for the control for hens. In addition, there was a statistically-significant 

reduction in female body weights compared to the control (1135 versus 1228 g; p<0.05) at study 

termination. 

B. Estrogen Pathway 

There was no convincing evidence of interaction of methomyl with the estrogen pathway either 

in the in vitro (i.e., ER binding, ERTA, aromatase and steroidogenesis) or mammalian in vivo

(i.e. uterotrophic of female pubertal) Tier 1 assays or in the mammalian or wildlife Part 158 

studies. In the FSTRA, although decreases in GSI, fertility and fecundity were observed at the 

mid and high treatment (0.312 and 1.03 mg/L, respectively) and increased incidence and severity 

of oocyte atresia at the high treatment were reported there were no effects observed on VTG. 

Although there were some complementary effects across the FSTRA, there was no effect on 

VTG (sex steroid hormones not measured), and there was limited redundancy across the other 

Tier 1 assays or Part 158 studies. Decreases in eggs laid (fecundity) were observed in the 

bobwhite quail reproduction study at the highest concentration tested (427 mg/kg-diet), but no 

other reproductive effects were observed. Decreases in fecundity alone are limited in the ability 

to inform about potential estrogen-related effects. Additionally, potentially similar to the 

decrease in fish GSI, an increase in number of females with regressed ovaries was observed in 

the mallard duck reproduction study, but no other reproductive effects were observed and female 

body weight gains and food consumption were decreased at this concentration. Furthermore, in 

other toxicity testing with fathead minnow fish (early life-stage and chronic reproduction), 

effects on growth in young fry were observed at concentrations lower (0.117 mg/L) or similar 

(0.243 or 0.280 mg/L) to the concentrations tested in the FSTRA. Therefore, while there were no 

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clinical signs of toxicity in female fish in the FSTRA (male body weights were significantly 

decreased), the concentrations tested in the FSTRA were similar to concentrations which were 

shown to be overtly toxic in other fish assays; as such, the decreases observed in the FSTRA may 

be due to potential general stress on the fish from exposure to methomyl as oppose to potential 

endocrine-related interactions. 

C. Androgen Pathway 

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

Tier 1 in vitro AR binding assay was negative, and while a decrease in testosterone production 

was observed in the Tier 1 steroidogenesis assay, there is uncertainty in this result based on the 

pattern of responses. No androgen-related effects were observed in the Tier 1 Hershberger or 

male pubertal assays or Part 158 mammalian studies. While androgen-related effects were 

observed in an OSRI study (El Medany et al. 1993), the results were questionable as the test 

doses overlapped with acute mortality values. In the FSTRA assay, there was a slight increase in 

the proportion of spermatogonia (mild to moderate) observed in male testes at the high 

concentration along with the decreases in fertility at the mid and high treatment; however, 

significant decreases in male body weight were observed at the mid and high treatment. 

Considering the body weight changes along with the discussion in the estrogen pathway 

regarding test concentrations and other observations of overt toxicity in fish, the 

alterations/decreases observed may be due to generalized stress in the fish. Also, as in the 

estrogen pathway, there is a lack of redundancy in observed effects across the Tier 1 battery and 

Part 158 studies. 

D. Thyroid Pathway 

There was no convincing evidence of interaction of methomyl with the thyroid pathway. No 

thyroid-related effects were observed in the mammalian studies, and no effect on developmental 

endpoints or thyroid histopathology were seen in the AMA.

E. Conclusions 

Methomyl demonstrates no convincing evidence of potential interaction with the estrogen, 

androgen, or thyroid pathways. 

V. EDSP Tier 2 Testing Recommendations 

Based on weight of evidence considerations, human health and wildlife EDSP Tier 2 testing is 

not recommended for methomyl since there was no convincing evidence of potential interaction 

with the estrogen, androgen or thyroid pathways. 

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VI. References 

Andersen, H., Vinggaard, A., Rasmussen, T., Gjermandsen, I., and Bonefeld-Jorgensen. E. 

(2002). Effects of Currently Used Pesticides in Assays for Estrogenicity, Androgenicity, and 

Aromatase Activity In Vitro. Toxicol. Appl. Pharmacol. 179:1–12. 

Beavers, J.; Hawrot, R.; Lynn, S., and Jaber, M. (1991). H-17940: A One-Generation 

Reproduction Study with the Northern Bobwhite (Colinus virginianus): Lab Project No: 112-

227. Unpublished study prepared by Wildlife International Ltd. 168 p. (MRID 41898602). 

Busey, W.M. (1968). Final Report: Two-year Dietary Administration-Dogs: Project No. 201-

165. (Unpublished study including addendum, received Jun 5, 1969 under 9F0814; prepared by 

Hazleton Laboratories, Inc., submitted by E.I. duPont de Nemours & Co., Inc., Wilmington, 

Del.; CDL:091405-A). (MRID 00007091). 

Driscoll, R.R., and Muska, C.F. (1982). Early Life Stage Toxicity of Methomyl to Fathead 

Minnow: Haskell Laboratory Report No. 528-82; MR No. 0581-930. (Unpublished study 

received Oct 3, 1983 under 352-366; submitted by E.I. duPont de Nemours & Co., Inc., 

Wilmington, DE; CDL:251426-C). (MRID 00131255). 

El-Medany, A., El-Medany, G., and Rahmy, A.E. (1993). Evaluation of the effect of methomyl 

insecticide on the testicular function in normal albino rats (hormonal, histological and 

histochemical study). J. Med. Res. Inst. 14:165–187. 

Feussner, E.L., Hoberman, A.M., Christian, M.S., Lightkep, G., et al. (1983). Embryo-Fetal 

Toxicity and Teratogenicity Study of Methomyl in the Rabbit: Study No. 104-005. Final rept. 

(Unpublished study received Oct 3, 1983 under 352-366; prepared by Argus Research 

Laboratories, Inc., submitted by E.I. duPont de Nemours & Co., Inc., Wilmington, DE; 

CDL:251427-B). (MRID 00131257). 

Fort, D.J. (2012). Methomyl (DPX-X1179) Technical: 21-d Amphibian Metamorphosis Assay 

(AMA) with South African Clawed Frog, Xenopus laevis. Unpublished study performed by Fort 

Environmental Laboratories, Inc., Stillwater, OK. Laboratory report number DUPO01-00269. 

Study sponsored by E.I. du Pont de Nemours and Company (DuPont), Wilmington, DE. 

(MRID 48701402). 

Hicks, S.L. (2012). Methomyl Technical (DPX-X1179): Short-term Reproduction Assay with 

the Fathead Minnow, Pimephales promelas, Determined Under Flow-Through Conditions. 

Performed by ABC Laboratories, Inc., Columbia Missouri and Experimental Pathology 

Laboratories, Inc., Sterling, Virginia. Lab Study No.: 66795; Sponsor Project No.: DuPontPage 33 of 62
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32248. Submitted by E.I. du Pont de Nemours and Company, Wilmington, Delaware. (MRID 

48701401). 

Howard, T.; Rhodes, J.; Mihalik, R. (1991). Early Life-Stage Toxicity of IN X1179-394 to the 

Fathead Minnow (Pimephales promelas) Under Flow-Through Conditions: Final Report. Project 

Number: 39292, 9006/02, HL0/7052/91. Unpublished study prepared by Analytical BioChemistry Labs., Inc. 494 p. (MRID 46015305). 

Hurtt, M. (1995). Nudrin-Two Generation Reproduction Study in the Rat: Supplement No.1: 

Lab Project Number: RA274: WRC RIR-275: HLO 519-95. Unpublished study prepared by WIL 

Research Labs, Inc. 149 p. (MRID 43769401). 

Kaplan, A.M., Hostetler, K.H., Dilworth, T., et al. (1981). Long-Term Feeding Study in Rats 

with S-Methyl N-[(Methylcarbamoyl) oxy]thioacetimidate (Methomyl; INX-1179): Haskell 

Laboratory Report No. 235-81. Final rept. (Unpublished study received Aug 28, 1981 under 

1H5320; submitted by E.I. duPont de Nemours & Co., Inc., Wilmington, Del,; CDL:070242-A). 

(MRID 00078361). 

Kojima, H., Katsura, E., Takeuchi, S., Niiyami, K., and Kobayashi, K. (2004) Screening for 

estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using 

Chinese hamster ovary cells. Environ. Health Perspect. 112 (5): 524-531. 

Lu, C. (1983). Nudrin Two-Generation Reproduction Study in Rats: Lab Project Number: WRC 

RIR-275. Unpublished study prepared by WIL Research Lab., Inc. 1144 p. (MRID 43250701). 

Mahgoub, A.A. and El-Medany, A. H. (2001). Evaluation of chronic exposure of the male rat 

reproductive system to the insecticide methomyl. Pharmacological Research 44, 73–79. 

Nabb, D.L. (2012). Methomyl (DPX-X1179) Technical: H295R Steroidogenesis Assay. 

Unpublished study prepared by E.I. du Pont de Nemours and Company, Newark, DE. 

Laboratory Report No.: DuPont-32086, February 8, 2012. (MRID 48685902). 

Paynter, O.E. (1966). Three-Month Dietary Administration-Rats: Project No. 201-151. 

(Unpublished study including supplement, received Feb 20,1966 under 352-342; prepared by 

Hazleton Laboratories, Inc., submitted by E.I. du Pont de Nemours & Co., Wilmington, Del.; 

CDL:050428-A). (MRID 00007190). 

Porter, W.P., Green, S.M., Debbink, N.L., and Carlson, I. (1993). Groundwater pesticides: 

interactive effects of low concentration of carbamates aldicarb and methomyl and the triazine 

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metribuzin on thyroxine and somatotropin levels in white rats. J Toxicol. Environ. Health 40:15–

34. 

Rogers, A.S., Culik, R., Wood, R.A., et al. (1978). Oral Teratogenic Study in Rats with Lannate 

(INX-1179): Haskell Laboratory Report No. 498-78. (Unpublished study received Oct 18, 1978 

under 352-342; submitted by E.I. duPont de Nemours & Co., Wilmington, Del.; CDL:097449-

A). (MRID 00008621). 

Snajdr, S. (2011). Methomyl (DPX-X1179) Technical: 3-Day Uterotrophic Assay for Detecting 

Estrogenic Activity. Unpublished study prepared by DuPont Haskell Global Centers for Health 

& Environmental Sciences, Newark, DE. Laboratory Study ID: DuPont-32084, November 8, 

2011. (MRID 48617701). 

Snajdr, S. (2011). Methomyl (DPX-X 11 79) Technical: 10-Day Hershberger Bioassay for 

Detecting Androgenic Activity. Unpublished study prepared by DuPont Haskell Global Centers 

for Health & Environmental Sciences, Newark, DE. Laboratory Study ID: DuPont-32085, 

November 8, 2011. (MRID 48617702). 

Snajdr, S. (2012). Methomyl (DPX-X1179) technical: in vitro androgen receptor binding assay 

using rat prostate cytosol. Unpublished study prepared by DuPont Haskell Global Centers for 

Health & Environmental Sciences, Newark, DE. Laboratory Project ID number: DuPont-32080, 

January 6, 2012. (MRID 48672001). 

Snajdr, S. I. (2011). Methomyl (DPX-X1179) aromatase (human recombinant) assay. 

Unpublished study prepared by E.I. du Pont de Nemours and Company, DuPont Haskell Global 

Centers for Health & Environmental Sciences, P.O. Box 50, Newark, Delaware, 19714. 

Laboratory Report No.: DuPont-32081. January 5, 2012. (MRID 48672002). 

Snajdr, S.I. (2012). Methomyl (DPX-X1179) technical: in vitro estrogen receptor binding assay 

using rat uterine cytosol (ER-RUC). Unpublished study prepared by E.I. du Pont de Nemours 

and Company, DuPont Haskell Global Centers for Health & Environmental Sciences, Newark, 

DE. Laboratory Project ID.: DuPont-32083, January 5, 2012. (MRID 48672003). 

Snyder, F.G. (1981). 104-week Chronic Toxicity and Carcinogenicity Study in Mice: Project 

No. 201-510. (Unpublished study received Aug 28, 1981 under 1E2556; submitted by E.I. 

duPont de Nemours & Co., Inc., Wilmington, Del.; CDL:070247-C; 070246. (MRID 00078423). 

Strawn, T., Rhodes, J., and Leak, T. (1993). Full Life-Cycle Toxicity of DPX-X1179-394 

(Methomyl) to the Fathead Minnow (Pimephales promelas) Under Flow-Through Conditions: 

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Final Report: Lab Project Number: 39293: HLO 47-93. Unpublished study prepared by ABC 

Laboratories, Inc. 3582 p. (MRID 43072101). 

Toot, J.D. (2012). Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal 

Male Rats Following Oral Administration of Methomyl (DPX-X1179) Technical. Unpublished 

study prepared by WIL Research Laboratories, LLC, Ashland, OH, Laboratory Project ID: 

WIL-189230, June 5, 2012. (MRID 48701403). 

Toot, J.D. (2012). Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal 

Female Rats Following Oral Administration of Methomyl (DPX-X1179) Technical. 

Unpublished study prepared by WIL Research Laboratories, LLC, Ashland, OH. Laboratory 

Project ID: WIL-189227, June 5, 2012. (MRID 48701404). 

USEPA. (2010). Methomyl — Report of the Endocrine Disruptor Review Team — Test Order 

#: EDSP- 090301-62. December 6, 2010. Office of Chemical Safety and Pollution Prevention. 

U.S. Environmental Protection Agency, Washington, DC. 

USEPA. (2011). Endocrine Disruptor Screening Program — Weight of Evidence: Evaluating 

Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical 

Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC. 

USEPA (2013) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting 

held July 30 - August 1, 2013 on Weight-of-Evidence: Evaluating Results of EDSP Tier 1 

Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection 

Agency, Washington, DC. 

Willoughby, J.A (2012). Methomyl (DPX-X1179) Technical: Estrogen Receptor 

Transcriptional Activation (Human Cell Line (HeLa-9903)). Unpublished study prepared by 

CeeTox, Inc., Kalamazoo, MI. Laboratory Report No.: 9144V-100360ERTA, Feb 7, 2012. 

(MRID 48685901).

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APPENDIX 1. EDSP T1 Screening Assays 

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100) 

The 21-day assay (MRID 48701402) of methomyl on amphibian metamorphosis of African 

clawed frog (Xenopus laevis) was studied under flow-through conditions. Amphibian larvae (80 

larvae per group) at Nieuwkoop-Faber (NF) stage 51 were exposed to nominal concentrations of 

0 (negative control), 0.0183, 0.056, and 0.168 mg a.i./L; mean-measured concentrations were 

<0.0124 (<LOD; control), 0.0170, 0.0522, and 0.186 mg a.i./L. The test system was maintained 

at 21.9 to 22.4°C and a pH of 6.9 to 7.2. 

Unless otherwise indicated, all effects are reported based on comparison to the negative (clean 

water) control. One mortality (1.7%) was observed in the highest test concentration. All 

surviving tadpoles appeared normal and healthy with no apparent clinical signs present at test 

termination. 

There was no significant acceleration or delay of median NF developmental stage at Day 7 or 

Day 21 in any treatment group. Furthermore, no asynchronous development was observed. All 

individuals developed beyond NF stage 60 by Day 21; the NF stage ranged from 61 to 65 in all 

treatment groups and in the control. The reviewer’s analysis detected significantly decreased 

normalized (for snout vent length) hind-limb length (HLL) at both Day 7 (⇘ 4.7%) and Day 21 (

⇘ 4%) at high treatment level relative to the negative control (p<0.05). Additionally, Day 21 

snout vent length (SVL) was significantly increased (⇖ 6%) at the high treatment level relative to 

the negative control (p<0.05). Thyroid gland histopathology revealed mild to moderate follicular 

cell hypertrophy in the negative control and all treatment groups, although there was no 

treatment-related pattern. There were no incidences of thyroid gland hypertrophy, atrophy, or 

follicular cell hyperplasia. 

At Day 21, all tadpoles were greater than NF stage 60 (late stage) which may confound the 

ability to adequately interpret the Day 21 results, however, there was no significant acceleration 

or delay of median NF developmental stage at any treatment concentration throughout the test 

nor effects on thyroid histopathology compared to the control. 

Androgen Receptor (AR) Binding Assay; (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48672001), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of AR to conduct a saturation binding experiment 

and a competitive binding experiment. The saturation binding experiment was conducted to 

demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and was 

functioning with appropriate affinity for the radio-labeled reference androgen (R1881) prior to 

conducting AR competitive binding experiments. The competitive binding assay was conducted 

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to measure the binding of a single concentration of [3H]-R1881 (1 nM) in the presence of 

increasing concentrations (10−10 to 10−3 M) of methomyl (99.4% a.i., Lot # JUL06LP276). 

Water was used as the solvent vehicle for methomyl, and ethanol was used as the solvent vehicle 

for the reference compounds at a final assay concentration of 3%. A total of three independent 

runs were performed, and each run included dexamethasone as a weak positive control, and 

R1881 as the ligand reference standard. 

In the saturation binding experiment, the maximum binding capacity (Bmax) was 4.5 fmol/100 μg 

protein and the dissociation constant (Kd) was 0.903 nM. The Kd was within the range reported 

in the EPA validation program, but the Bmax was below the EPA values (7-16 fmol/100 μg 

protein). The Scatchard plots for the three saturation assays were linear. 

In the competitive binding experiment, the estimated average log IC50 was −9.15 M for R1881 

and −4.41 M for dexamethasone. Compared to R1881, the mean RBA for the dexamethasone 

was 0.0019%. The performance criteria for R1881 and dexamethasone were not met across the 

three runs. In the first and third runs, the bottom % binding and Hill slope were outside the 

tolerance limits for R1881. For dexamethasone all of the three criteria were met, except for the 

top % binding in the third run which was higher than the tolerance limits. Although, the 

performance criteria for both R1881 and dexamethasone were not achieved across the three runs, 

based on the estimated IC50s for R1881 and the weak positive control, the androgen receptor was 

functioning with appropriate affinity for the ligand in the competitive binding assays. It is 

unlikely that information from additional competitive binding runs would change the 

interpretation of this study. 

The percent [3

H]-ligand bound to the AR was >75% at all concentrations of methomyl in all 

three runs. Therefore, a log IC50 and RBA could not be calculated for methomyl. 

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

Receptor Binding Assay. 

Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48672002), methomyl (99.4% a.i., Lot # 

JUL06LP276) was incubated with human recombinant aromatase and tritiated androstenedione 

(1-β [3H(N)]-androst-4-ene-3,17-dione; [3H]ASDN) in water at log concentrations of 10−10 to 

10−3 M for 15 minutes to assess the potential of methomyl to inhibit aromatase activity. 

 

Aromatase activity was determined by measuring the amount of tritiated water produced at the 

end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified 

using liquid scintillation counting (LSC). A total of four runs were conducted and each run 

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included full enzyme activity controls, background activity controls, a positive control series 

(10−10 to 10−5 M) using a known inhibitor, 4-hydroxyandrostenedione (4-OH ASDN), and the 

methomyl series (10−10 to 10−3 M) with three repetitions per concentration. Because the solvent 

vehicle was water for methomyl and dimethyl sulfoxide (DMSO) for 4-OH ASDN, separate sets 

of the background activity and full enzyme activity controls were run using only water or 1% 

DMSO (final concentration in assay). In the second run, the full activity controls (water only) 

had aromatase activity below the performance criteria specified in the guideline; therefore, the 

second run was excluded from the calculations and a fourth run was conducted. 

Aromatase activity in the full activity controls for the three successful test runs averaged 

0.381±0.117 nmol·mg-protein−1

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

in 1% DMSO. Activity in the background controls averaged 0.59% of the full activity controls 

in water and 0.54% of full activity controls in 1% DMSO. The response of each full activity 

control within a run was between 90 to 110% of the average full control activity, except for the 

average initial value of 117% in 1% DMSO and the average ending value of 83% in 1% DMSO 

for Run 1. 

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

nmol·mg-protein−1·min−1 at the lowest tested concentration (10−10 M) and 0.004±0.001 nmol·mgprotein−1

·min−1 at the highest tested concentration (10−5 M). As a percent of the control, 

aromatase activity for 4-OH ASDN was 1.02% at 10−5 M and 103.14% at 10−10 M. 

For methomyl, aromatase activity averaged 0.358±0.096 nmol·mg-protein−1·min−1 at the lowest 

tested concentration (10−10 M) and 0.366±0.118 nmol·mg-protein−1

·min−1 at the highest tested 

concentration (10−3 M). The lowest portion of the response curve was greater than 75% activity 

at concentrations from 10−10 M to 10−3 M methomyl across all runs. 

Based on the data from the average response curve, methomyl is classified as a Non-inhibitor of 

aromatase activity in this assay. 

Estrogen Receptor (ER) Binding Assay; (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48672003) for methomyl (99.4% purity, Lot 

#JUL06LP276), uterine cytosol from Sprague Dawley rats was used as the source of ER to 

conduct saturation binding experiments and competitive binding experiments. 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 radiolabeled 

reference estrogen prior to routinely conducting ER competitive binding experiments. The 

competitive binding experiment was conducted to measure the binding of a single concentration 

of [3

H]-17β-estradiol (1 nM) in the presence of increasing concentrations (logarithmic increase 

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from 10−10 to 10−3 M) of methomyl. Water was used as the solvent vehicle for methomyl, and 

ethanol was used for the reference chemicals at a final concentration of 2%. A total of 3 runs 

were performed, and each run included 19-norethindrone as a weak positive control, 

octyltriethoxysilane as a negative control, and 17β-estradiol as the natural ligand reference 

material. 

In the saturation binding experiments, the maximum binding capacity (Bmax) was 32.5 ± 2.25 

fmol/100 μg protein and the dissociation constant (Kd) was 0.081 ± 0.022 nM. The Kd for each 

run was within the expected range of 0.03 to 1.5 nM. The saturation binding experiment 

demonstrated that the estrogen receptor was present in a reasonable concentration and 

functioning with appropriate affinity for the reference ligand 17β-estradiol. 

In the competitive binding experiments, the three reference chemicals performed as expected. 

The average log IC50 was −9.16 M for 17β-estradiol and −4.85 M for the weak positive control, 

19-norethindrone, and the mean RBA was 0.0054% for the weak positive control. The negative 

control, octyltriethoxysilane, had no effect on specific binding of the [3

H]-ligand (85-105% 

binding). 

Methomyl had no appreciable effect on specific binding (88 to 113%) of the [3

H]-ligand to the 

ER in the three runs; therefore, a log IC50 and RBA could not be calculated for methomyl. Based 

on the results from the three runs, methomyl is classified as Not Interactive in the Estrogen 

Receptor Assay. 

ERα Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 999), hERα-HeLa-9903 cells 

cultured in vitro were exposed to methomyl (99.4% a.i., Lot # JUL06LP276) at logarithmically 

increasing concentrations from 10−10.8 to 10−3.8 M in water for approximately 24 hours in two 

independent runs. Experiments were performed using 96-well plates and each concentration was 

tested in 6 wells/plate in each run. Cells were exposed to the test agent for 24±2 hours at 37±1°C 

to induce reporter (luciferase) gene products. Luciferase expression in response to activation of 

the estrogen receptor was measured using a proprietary luciferase assay. 

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

3.8 M. In the main assays, the responsiveness of the cells to the very weak positive control 17αmethyltestosterone was lower than the expected values, indicating a decreased sensitivity of the 

assay to very weak agonists. Although the conditions of this assay were not optimal to detect 

very weak activity, methomyl responses were similar to those of the negative control 

corticosterone and not comparable to the responses of 17α-methyltestosterone, which was able to 

reach a maximum of 24.3-26.6% PC. Methomyl was only able to reach a maximum of 3.1-3.7% 

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PC when tested up to the highest concentration possible based on cytotoxicity. Because the 

RPCMax < PC10 in two assay runs, methomyl was considered negative for estrogen receptor 

transcriptional activation in this test system. 

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

The 21-day short-term reproduction assay (MRID 48701401) of methomyl with fathead 

minnows (Pimephales promelas) was conducted under continuous flow-through conditions. 

Adult fish (16 spawning groups, 2 males and 4 females in each group; ca. 6 months old) were 

exposed to methomyl (99.4% purity) at nominal concentrations of 0 (control), 0.090, 0.30, and 

1.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.0124, negative control), 0.0947, 

0.312, and 1.03 mg a.i./L. The test system was maintained at 24.2 to 24.8oC and a pH of 7.88 to 

8.33. 

Adult male and female survival was unaffected by treatment with methomyl and was 100% for 

all test levels. There were no notable observations with regards to behavior or changes in 

appearance, specifically color/banding, ovipositor appearance, or size of dorsal nape pad in the 

control or treated groups, and no clinical signs of toxicity were observed. Male body weights 

were reduced (p<0.05; Jonckheere-Terpstra test) by 9.8 and 13% at the mid and high 

concentrations, respectively, relative to the negative control; female body weights were not 

affected. Body lengths at test termination were not reported. 

Spawning occurred at least every three days, and mean fecundity in negative control was 54 

eggs/female/day/replicate (replicate range was 50-62 eggs/female/day); fertilization success in 

the negative control group averaged 92%. There were significant reductions in fecundity of 23 

and 77% and in fertility of 1.6 and 33% at the mid and high concentrations, respectively, 

compared to the negative control (p<0.05; Jonckheere-Terpstra test). 

Additionally, female gonadal-somatic index (GSI) was significantly reduced by 15 and 21% at 

the mid and high concentrations, respectively, compared to the negative control (p<0.05; 

Jonckheere-Terpstra test). Male GSI was not significantly different from the control. 

Histopathology showed treatment-related effects at the high concentration, consisting of 

increased prevalence and severity of increased spermatogonia in males and increased prevalence 

and severity of increased oocyte atresia in females. There were no differences in gonadal stages 

between treated groups and the negative controls. Plasma vitellogenin (VTG) was not 

significantly different from controls for either sex. Sex steroids were not measured in this study. 

Hershberger Assay; (OCSPP 890.1400) 

In a Hershberger assay (MRID 48617702) testing for androgenic activity, methomyl (99.4% a.i., 

lot # JUL06LP276) in deionized water was administered daily via oral gavage to six 56-day old 

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(approximate age), castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 0.25, 0.5, or 

1.0 mg/kg/day. An androgenic positive control group consisted of 6 castrated rats exposed to 0.4 

mg/kg/day testosterone propionate (TP) in corn oil with 1% ethanol by subcutaneous (s.c.) 

injection. 

To screen for potential anti-androgenic activity, methomyl (99.4% a.i., lot # JUL06LP276) in 

deionized water was administered daily via oral gavage to six 56-day old, castrated male Sprague 

Dawley rats at dose levels of 0 (vehicle), 0.25, 0.5, or 1.0 mg/kg/day in conjunction with a daily 

dose of TP at 0.4 mg/kg/day in corn oil with 1% ethanol by s.c. injection. The anti-androgenic 

positive control group consisted of 6 castrated rats dosed with 0.4 mg/kg/day TP in corn oil with 

1% ethanol by s.c. injection and flutamide (FT) in 0.1% Tween® 80 in 0.5% methylcellulose at 3 

mg/kg/day by oral gavage. TP alone was used as the anti-androgenic negative control. 

For both components of the assay, the animals 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. Body weights, body weight gain and food 

consumption were measured throughout the dosing period. 

All animals survived until scheduled termination. There were no clinical signs of toxicity and no 

treatment-related gross pathological findings. 

In the androgen agonist assay, there were no effects on body weights, body weight gains, or food 

efficiency in the methomyl treated groups. Overall food consumption was increased (p<0.05) in 

the 0.5 mg/kg/day group (9%), but the effect was not dose dependent. Rats dosed with TP 

(positive control) had increases (p<0.05) in overall body weight gains (23%) and food 

consumption (18%), but there was no effect on food efficiency. There were no significant 

increases in the weights of the five accessory sex organs in any of the methomyl-treated groups. 

Liver weights were comparable to the vehicle control in all dose groups. Rats in the positive 

control group responded appropriately with significant increases in all five accessory sex organ 

weights (p<0.05). The performance criteria indicated that the assay was performing as expected. 

In the anti-androgen assay, there were no effects on body weights, body weight gains, food 

consumption, or food efficiency in the methomyl groups or positive control (TP + FT) group 

when compared to the TP control. There was a 31% decrease (p<0.05) in Cowper’s glands 

weight in the 0.5 mg/kg/day methomyl group; however, no significant decreases in organ 

weights were seen in the remaining four target accessory sex tissues. The liver weights in the 

methomyl-treated groups were comparable to the TP control. Rats in the positive (TP + FT) 

control group responded with significant decreases in all five accessory sex organ weights. The 

performance criteria indicated that the assay was performing as expected. 

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The dose levels tested in this study are considered to be adequate based on data from earlier 

studies showing toxicologically-adverse clinical signs of cholinesterase inhibition at doses ≥2.0 

mg/kg/day. 

Statistically significant changes were not seen in two or more of the five androgen sensitive 

tissue weights. Methomyl was negative for androgenicity and anti-androgenicity in the 

Hershberger assay 

Female Pubertal Assay; (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48701404), 15 Crl:CD(SD) Sprague-Dawley rats/dose group 

were treated daily by oral gavage with methomyl technical (99.4%; Lot # JUL06LP276) in 

deionized water at doses of 0, 0.5 or 1.0 mg/kg/day from post-natal day (PND) 22 to PND 42. 

Animals were examined for vaginal opening (VO) daily beginning on PND 22, and the age and 

weight at day of attainment was recorded. Following sacrifice on PND 42, blood was collected 

for clinical chemistry analyses, including total thyroxine (T4) and thyroid stimulating hormone 

(TSH), which were analyzed with electrochemiluminescent immunoassays or 

radioimmunoassays, respectively. Kidney, liver, adrenal gland, thyroid, pituitary, uterus, and 

ovary weights were recorded, and microscopic examinations were conducted on the thyroid, 

kidney, ovary, and uterus. 

All animals survived until scheduled termination and no treatment-related clinical findings were 

observed. No effects were observed on body weights or body weight gain in the study. No 

significant differences were observed in mean age at VO, body weight at VO, or final body 

weights in the treated groups relative to control. There were no dose-related effects on mean age 

at first vaginal estrus, mean cycle length, percentage of females cycling, percentage of females 

regularly cycling, or cycle status at necropsy. Serum T4 and TSH were not affected by treatment. 

 

There were no treatment-related microscopic findings in the thyroid, ovaries, uterus, or kidneys. 

No effects on organ weights were observed during the study. All clinical chemistry parameters 

in the treated groups were similar to control. 

The dose levels tested in this assay are considered to be adequate based on the cholinesterase 

inhibition seen at doses ≥2.0 mg/kg/day in the toxicology database for this chemical. 

Male Pubertal Assay; (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48701403), 15 Crl:CD(SD) Sprague-Dawley rats/dose group 

were treated daily via oral gavage (5.0 mL/kg) with methomyl technical (99.4%; Lot # 

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JUL06LP276) in deionized water at doses of 0, 0.5 or 1.0 mg/kg/day from post-natal day (PND) 

23 to PND 53. Animals were examined for preputial separation (PPS) daily beginning on PND 

30, and their age and weight at day of attainment was recorded. Following sacrifice on PND 53, 

blood was collected for clinical chemistry analyses, including total thyroxine (T4), testosterone, 

and thyroid stimulating hormone (TSH). Testosterone and T4 were analyzed with an 

electrochemiluminescent assay and TSH levels were analyzed by using radioimmunoassay. 

Liver, adrenals, pituitary, and urogenital organ weights were recorded, and microscopic 

examinations were performed on the thyroid, kidney, testis and epididymis. 

Red material around the mouth or ear was observed infrequently during the daily examinations. 

No treatment-related effects were observed on mortality, body weights, body weight gain, organ 

weights, or gross pathology. Age and body weight at PPS in the treated groups were similar to 

controls. Thyroid hormone levels, colloid area, and follicular cell height scores for the thyroid 

were similar in the treated groups to the control group. Serum testosterone levels were similar in 

the treated groups to the control group. 

The dose levels tested in this study are considered to be adequate based on data from earlier 

studies showing toxicologically-adverse clinical signs of cholinesterase inhibition at doses ≥2.0 

mg/kg/day. 

Steroidogenesis Assay; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48685902), H295R cells cultured in vitro in 24-well plates 

were incubated with methomyl (99.4% a.i., Lot # JUL06LP276) at log concentrations from 

0.0001 to 100 μM for 48 hours in triplicate in three independent experiments. Dimethyl 

sulfoxide (DMSO) was used as the vehicle for the positive controls and water was used as the 

vehicle for methomyl. 

Testosterone and estradiol levels were measured using HPLC/MS/MS method with internal 

standards. A Quality Control (QC) plate was run concurrently with each independent run of a 

test chemical plate to demonstrate that the assay responded properly to positive control agents at 

two concentration levels; positive controls included the known inhibitor (prochloraz) and inducer 

(forskolin) of estradiol and testosterone production. 

The minimum basal hormone production levels for estradiol were below the recommended 

concentration of 40 pg/mL, except in the blank and DMSO cells in Run 1. Although the basal 

estradiol production was generally less than 40 pg/mL, all values were >12.5 pg/mL (2.5-fold 

greater than the minimum detection limit (MDL) as per the Guideline recommendation). 

Estradiol production in the presence of 10 μM forskolin met induction requirements (≥7.5-fold). 

However, induction of testosterone production at 10 μM forskolin was 1.76- to 1.80-fold, which 

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is slightly less than the ≥2-fold requirement, and induction production in the presence of 1 μM 

forskolin was slightly higher than expected (1.59- to 1.63-fold). Testosterone and estradiol 

inhibition were both greater than the required 50% in the presence of 1 μM prochloraz. Because 

all other guideline requirements for basal concentrations and hormone induction and inhibition 

were met for testosterone and estradiol, these results were deemed acceptable. 

The %CVs within each plate for the SC on the test chemical plates analyzed for hormone 

production ranged from 3.12 to 12.37% for testosterone and 0.78 to 12.45% for estradiol. The 

between-plate %CVs for the test plates were 6.93% and 7.0%, respectively, for testosterone and 

estradiol. These values are within the Guideline limit of ≤30%. 

Precipitation was not observed at any tested methomyl concentration, and methomyl was not 

found to be cytotoxic to the H295R cells at concentrations up to 100 μM. No effect on estradiol 

production was observed at any tested methomyl concentration. Across the three runs, there 

were significant (p<0.05) decreases in mean testosterone levels at methomyl concentrations of 

0.01 μM (0.89-fold) and 100 μM (0.83-fold) methomyl. The effect was consistent across all 

three runs, but it was not dose-dependent, as it occurred only at two non-sequential 

concentrations. 

Based on hormone responses in three independent runs, methomyl treatment did not result in 

statistically significant and reproducible alterations in testosterone or estradiol production. 

Uterotrophic Assay; (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48617701) conducted to screen for potential estrogenic activity, 

methomyl (99.4% a.i., lot # JUL06LP276) in water was administered daily via oral gavage to 

groups of six ovariectomized female Sprague Dawley rats at dose levels of 0 (vehicle), 0.25, 0.5 

or 1.0 mg/kg/day on post-natal days (PND) 63-65 (approximate). A positive control group was 

treated with 17α-ethynyl estradiol (EE) by daily oral gavage at a dose level of 0.1 mg/kg/day on 

PND 63-65. All animals were terminated and necropsied approximately 24 hours after the final 

dose administration to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. No clinical signs of toxicity were observed in 

any treatment group. Body weights, food consumption, and food efficiency in the methomyl 

treated groups were comparable to the control throughout the study. In the positive control 

group, body weights were decreased (p<0.05) by 10% on Day 4 compared to the vehicle control 

group, resulting in an overall weight loss (p<0.05) of -4.0 g, compared to the vehicle control 

group (23.1 g weight gain). Overall food consumption and efficiency were decreased (p<0.05) 

by 28% and 128%, respectively, in the positive control group compared to the vehicle control 

group. Uterine weights in the methomyl treated groups were comparable to the controls. 

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The mean blotted vehicle control uterine weight was <0.031% of the mean vehicle control 

terminal body weight, which meets the Guideline requirement (<0.04%). Absolute wet and 

blotted uterine weights for the EE group were increased (p<0.001 and p<0.05, respectively) by 

213% and 166%, respectively. The increased uterine weights were in the expected range. 

The dose levels selected for this study are adequate based on red blood cell and brain 

cholinesterase inhibition seen at dose levels ≥2.0 mg/kg/day in the toxicology database. 

No statistically significant changes were seen in uterine weight in this assay. Methomyl is 

negative in the uterotrophic assay. 

 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Published literature and general toxicity studies (Part 158 studies) considered relevant for 

determining the potential interaction of methomyl with the endocrine system are summarized 

below. Given the breadth of toxicity data available on methomyl, the only general toxicity 

studies discussed in this appendix are those 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.). 

Transcriptional Activation 

In a study by Andersen et al. (2002), methomyl was tested for interactions with the androgen 

and estrogen receptors in in vitro transactivation assays at concentration from 10-8 to 10-5 M, and 

effects on aromatase were also investigated at a single concentration (50 μM). In the androgen 

receptor transcriptional activation (ARTA) assay, methomyl was tested in transfected Chinese 

hamster ovary (CHO) cells in triplicate or quadruplicate at each concentration in two runs. 

Methomyl did not show agonist or antagonist activity at the androgen receptor at any 

concentration. In the ERTA assay, methomyl was tested using transfected the MCF-7 cells in 

triplicate or quadruplicate at each concentration in two runs. Methomyl was negative in the 

ERTA assay at each concentration tested. Methomyl was not cytotoxic in either cellular assay at 

concentrations up to 100 μM. Methomyl also did not inhibit aromatase activity at the single 

concentration tested. 

In a published, non-Guideline study (Kojima et al., 2004), the potential of 200 chemicals 

(including methomyl; purity 95-100%) to interact with two human ER subtypes (hERα and 

hERβ) and one human androgen receptor (hAR) in transactivation assays with Chinese hamster 

ovary cells (CHO-K1 cells) was investigated. For detection of hERα or hERβ activity, cells were 

transfected with pcDNAERα or pcDNAERβ, pGL3-tkERE, and pRL-SV40. For detection of 

hAR activity, cells were transfected with pZeoSV2AR, pIND-ARE, and pRL-SV40. Flutolanil 

was tested over a concentration range from 10−8

 to 10−5 M, and the responses were compared to 

the activity of 17β-estradiol (E2; purity >97%) or 5α-dihydrotestosterone (DHT; purity 95%) for 

the ER or AR assays, respectively. For measurement of antagonistic activity to hERα, hERβ and 

hAR, either 10−11 or 10−10 M E2 or 10−10 M DHT was added to the cell cultures along with the 

test compound, respectively. Agonistic activities were evaluated by the relative activity 

expressed as 20% relative effective concentration (REC20), the concentration of the compound 

demonstrating 20% of the activity of 10−10 M E2, 10−9 M E2, or 10−9

 M DHT for ERα, ERβ, and 

AR, respectively. Antagonistic activities were evaluated similarly by the relative activity 

expressed as 20% relative inhibitory concentration (RIC20), the concentration of the compound 

showing 20% inhibition of the activity of E2 or DHT against ERα, ERβ, and AR, respectively. 

Methomyl did not show agonist or antagonist activity for ERα, ERβ, or AR in this study. 

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90-Day (Subchronic) Oral Toxicity in Rodents (Rat) 

In a non-Guideline, subchronic toxicity study (MRID 00007190), Crl:CD rats (10 rats/sex/dose) 

were administered methomyl (100% purity) at nominal concentrations of 0, 10, 50, and 250 ppm 

in the diet for 13 weeks; an additional group (mid-high) was administered 125 ppm for 6 weeks 

and 500 ppm for the remaining 7 weeks. In this latter group, five animals/sex were sacrificed at 

two months and the remaining animals were sacrificed at study termination. The estimated 

maximum dose equivalence was 0, 1.0, 5.0, 12.5/50.0, and 25.0 mg/kg/day (calculated by 

reviewers); due to assumed changes in food consumption with increased age/weight, actual dose 

equivalence may be slightly lower (0, 0.8, 3.1, 18.3, and 19.2 mg/kg/day). Organ weights 

evaluated included the adrenals, liver, kidneys, pituitary, and testes. Gross and microscopic 

pathology was conducted on tissues, including the adrenals, testes, epididymis, liver, kidneys, 

ovary, uterus, pituitary, and thyroid. 

No effects of treatment were observed on mortality, clinical signs (except for tremors in a single, 

high-dose female at the end of the study), food consumption, hematology, urinalysis, or 

macroscopic pathology. Additionally, no adverse, treatment-related effects were noted for 

plasma and RBC ChE concentrations. Overall (Weeks 0–13) body weight gain (calculated by 

reviewers) was decreased by 14% in the high-dose males. Additionally, overall body weight 

gains were decreased (↓13 and 17%; females and males, respectively) in the high-medium dose 

animals; body weights in males were decreased (p<0.05) at Weeks 7 and 13 (↓14%) after the 

dose was increased from 125 to 500 ppm. No treatment-related changes were seen in absolute or 

relative weights of the testes, thyroid or adrenal glands at any dose. Liver weights were increased 

(p<0.05) in the high-medium and high-dose males at the end of the study (13 and 17%, 

respectively), and absolute and relative pituitary weight was increased (p<0.05) in the high-dose 

females (19 and 20%, respectively). No treatment-related histopathological lesions were reported 

for the liver, kidneys, testes, ovaries, uterus, thyroid, adrenal or pituitary glands at any dose. 

The LOAEL is 250 ppm (high dose) based on decreased body weight gain, moderate erythroid 

hyperplasia, and decreased RBC counts observed in the 250 and/or the 125/500 ppm dose 

groups. Although a NOAEL of 125 ppm (medium-high dose) was proposed (due to no adverse 

effects observed at this dose during the first six weeks), this is confounded because the dose was 

increased to 500 ppm at Week 7 and all observations (except clinical) were not conducted until 2 

months (5 animals/sex) or 3 months (end of study). A demonstrable NOAEL is 50 ppm 

(medium-low dose) in male and female rats. 

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Prenatal Development in Rodents (Rat); (OCSPP 870.3700) 

In a developmental toxicity study (MRID 00008621), methomyl (>99% purity) was 

administered to 25 presumed-pregnant female CD®(ChR-CD) rats/dose in the diet at nominal 

concentrations of 0, 50, 100, or 400 ppm (mean-measured doses of 0, 4.9, 9.4, and 33.9 

mg/kg/day, respectively) from gestation day (GD) 6 to 15. On GD 21, all dams were euthanized 

and underwent a gross necropsy and cesarean section. Fetuses were examined for external, 

visceral, and skeletal anomalies and malformations. 

There were no mortalities or clinical signs of toxicity, and no treatment-related effects were 

observed in gross pathology. At the high dose, mean maternal body weight was decreased by 8% 

(NS) on GD 16 and by 5% (p<0.05) on GD 21. Body weight gain in this group was decreased by 

22% for the treatment period (GD 6 to 16). Food consumption in the high-dose group was 

decreased by 18% during treatment. 

There were no abortions, premature deliveries, or complete litter resorptions, and no effects of 

treatment on the numbers of litters, live fetuses, or resorptions. A decrease (NS) in the pregnancy 

rate was observed in the high-dose group (↓16%) relative to control, but no dose-dependent trend 

was present. There were no treatment-related effects on growth or development as measured by 

fetal weight, crown-rump length, or skeletal ossification. There were no treatment-related 

external, visceral, or skeletal malformations or variations. 

The maternal LOAEL is the high dose (400 ppm, equivalent to 33.9 mg/kg/day) based on 

decreased body weight, body weight gain, and food consumption. The maternal NOAEL is the 

intermediate dose (100 ppm, equivalent to 9.4 mg/kg/day). The developmental LOAEL was not 

observed. The developmental NOAEL is the high dose (33.9 mg/kg/day). 

Prenatal Development in Non-Rodents (Rabbit); (OCSPP 870.3700) 

In a developmental toxicity study (MRID 00131257), methomyl (98.7% purity) was 

administered by oral gavage to 20 New Zealand White (DLI:NZW) rabbits/dose at doses of 0, 2, 

6, or 16 mg/kg/day from GDs 7 to 19. On GD 29, all surviving rabbits were euthanized and 

subjected to a gross necropsy. A cesarean section was performed, and the fetuses were examined 

for external, visceral, and skeletal malformations and variations. 

In the high-dose group, seven rabbits died beginning with the initiation of treatment and 

continuing throughout the treatment period (GDs 7, 8, 9, 11, 17, 18, and 19). Central nervous 

system (CNS) stimulation occurred in the high-dose rabbits and was considered to be treatment 

related. Dose-dependent increases (p<0.05) in the incidence of tremors, hyperactivity, body 

jerks, excess salivation, aggressive behavior, hyperpnea, and convulsions were observed in the 

rabbits administered the high dose. Other dose-dependent, but not statistically significant, 

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increases in CNS stimulation were observed in the high-dose animals, including ataxia, impaired 

or lost righting reflex, skin lesions (self-inflicted), dyspnea, hypersensitivity, pin-point pupils, 

and vocalization. A single death in the mid-dose group was attributed to gavage error. 

A decrease in the number of litters at the high dose, resulting in a lower number of live fetuses, 

was due to the high maternal mortality in this group. After exclusion of these mortalities, there 

were no treatment-related effects on the numbers of resorptions or fetuses on a per doe basis. Sex 

ratio and post-implantation losses in the treated groups also were comparable to control. 

Abortions in the low- and intermediate-dose groups were considered spontaneous events because 

they occurred in a single animal/dose group and did not occur at the high dose. A dose-related 

increase (p<0.05) in mean fetal body weight was observed. There was no adverse basis for 

increased fetal weight; the increased weight was attributed to the decreased maternal anorexia 

(increased feed consumption) in the latter part of gestation after treatment cessation. No 

treatment-related skeletal retardations were noted during the fetal examinations, and there were 

no treatment-related external, visceral, or skeletal variations or malformations. 

The maternal LOAEL is 16 mg/kg/day (high dose) based on mortality and clinical signs of 

toxicity indicative of CNS stimulation. The maternal NOAEL is 6 mg/kg/day (intermediate 

dose). The LOAEL for developmental toxicity was not observed. The developmental NOAEL is 

16 mg/kg/day (high dose). 

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

In a two-generation reproductive study (MRIDs 43250701 and 43769401), male and female SD 

rats received methomyl (98 to 100% purity) in the diet to 13 male and 26 female rats in the F0

parental generation at nominal concentrations of 0, 75, 600, or 1200 ppm; 20 males and 40 

females (38 in the 1200 ppm group) from the F1 offspring were treated at the same dosages. The 

F0 generation was treated for 100 days prior to mating; the F1 generation was treated for 120 

days prior to mating. Mean-measured doses were 4.6, 36.9, and 73.7 mg/kg/day and 4.8, 38.8, 

and 75.7 mg/kg/day, respectively, in F0 males and females; and 6.9, 56.5, and 116.7 mg/kg/day 

and 7.2, 59.2, and 128.0 mg/kg/day, respectively, in F1 males and females). 

There was no evidence of a treatment-related effect on survival. For the F0 generation, mean 

body weights were decreased (p<0.01) in the high-dose females (premating) and males (entire 

study) by 10 and 16%, respectively. For the F1 generation, mean body weights were decreased 

(p<0.01) for the mid- and high-dose group females and males by ↓19 and 26%, respectively. 

During gestation and lactation, mean body weights were decreased (p<0.01) for the high-dose 

females (and Lactation Week 1) of the F0 generation (↓11 to 18%) and for the mid- and highdose females of the F1 generation (↓11 to 27%). Mean food consumption was generally 

decreased (NS or p<0.05 or 0.01) concomitant with body weight decreases in dose groups. No 

evidence of a treatment-related effect on fertility, gestation or lactation indices. Viability index 

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was decreased (p<0.05) in the F0 (68%) and F1 (68%) generations at the high dose. In the F1

litters, the number of live pups was decreased (p<0.01) on and after Day 4 in the high-dose 

group. Mean pup weights were decreased (p<0.01) in the mid- and high-dose groups throughout 

lactation (approximately ↓10 to 30%), and decreased (p<0.05 or 0.01) in the low-dose group at 

LD 14 and 21 (approximately ↓10%). In the F2 litters, the number of live pups was decreased 

(p<0.01) on and after Day 1 in the high-dose group, and decreased (p<0.05 or 0.01) at early time 

points in the low- and mid-dose groups. Mean pup weights were generally decreased (p<0.01) in 

the mid- and high-dose groups throughout lactation (approximately ↓10 to 30%). There also was 

a decrease in litter size of the F2 pups in all the treated groups (p<0.05; mid-dose group only). 

The RBC and plasma cholinesterase concentrations were comparable between the treated and control 

groups. No differences in testicular sperm head counts were observed between the treatment 

groups and control. Differences in organ weights between control and the mid- and high-dose 

groups in the F0 and F1 adults (and F1 and F2 pups) were observed. No treatment-related changes 

were seen in absolute or relative weights of the ovaries or uterus (except decreased absolute 

weights in the F1 adults). Occasional, inconsistent decreases (p<0.05 or 0.01) in absolute and/or 

relative weights were noted for adrenal glands, testes, liver, and kidneys in the mid- and highdose adults and pups. No treatment-related histopathological lesions were seen in any examined 

tissues. 

The NOAEL for parental systemic toxicity is 75 ppm (4.6 mg/kg/day for F0 males, 4.8 

mg/kg/day for F0 females, 6.7 mg/kg/day for F1 males, and 6.3 mg/kg/day for F1 females); the 

LOAEL is 600 ppm based on decreased body weight and food consumption, and altered 

hematology parameters. The reproductive NOAEL is 1200 ppm (73.7 mg/kg/day for F0 males, 

75.7 mg/kg/day for F0 females, 116.7 mg/kg/day for F1 males, and 128 mg/kg/day for F1

females) due to no effects in either generation; the LOAEL is not observed. The NOAEL for 

offspring toxicity is 75 ppm (4.6 mg/kg/day for F0 males, 4.8 mg/kg/day for F0 females, 

6.7 mg/kg/day for F1 males, and 6.3 mg/kg/day for F1 females); the LOAEL is 600 ppm based on 

decreases in both the mean number of live pups and mean body weights of offspring. 

Testicular Toxicity (Rodent) 

In a published study, El-Medany et al. (1993) [see also Mahgoub and El-Medany (2001)] 

evaluated the testicular toxicity of methomyl after oral administration to male Wistar rats at 17 

mg/kg/day in saline by oral gavage for two months. Decreased (p<0.001) testosterone 

concentration (↓35%) and increased (p<0.01 to p<0.001) FSH (↑55%), luteinizing hormone 

(↑LH; 59%) and prolactin (↑2.2-fold) hormone concentrations were observed after methomyl 

administration for 60 days. One month after cessation of treatment, testosterone was still 

decreased (30%, p<0.001) and FSH, LH and prolactin concentrations were still elevated 64%, 

59%, and 2.2-fold, respectively. Reductions in succinic dehydrogenase activity were observed in 

the degenerated spermatogenic cells, as well as increased acid phosphatase activity, and no 

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differences in alpha esterase and alkaline phosphatase activities. In Leydig cells, succinic 

dehydrogenase activity was variable, acid phosphatase and alpha esterase activities were 

increased, and no change was observed in alkaline phosphatase activity. Variable grades of 

degenerative changes in the seminiferous tubules were observed after methomyl treatment; 

primary spermatocytes showed variable degrees of degenerative changes, and spermatids 

displayed the highest degree of degeneration among the spermatogenic cells. There were several 

issues regarding this study: The initial citation reportedly states that a methomyl dosage of 17 

mg/kg is 1/30 of the methomyl LD50; the actual range of methomyl LD50 values is 17 to 45 

mg/kg. The second citation correctly cites the demonstrated LD50 of methomyl as 17 to 45 

mg/kg, but incorrectly cites methomyl dose units in general toxicity studies (3-month subchronic 

oral toxicity in the rat and dog, developmental toxicology in the rat) as mg/kg/day instead of as 

parts per million. Although animals were administered methomyl at or close to the LD50 for 2 

months, no mortality was reported in this study. 

Thyroid Hormone Levels (Rodent) 

In a published study, Porter et al. (1993) investigated the individual and interactive effects of 

methomyl, aldicarb, and triazine on concentrations of thyroxine (T4) and somatotropin in female 

and male SD rats (n=6/sex/dose). Animals were administered control (distilled water) or one of 

eight treatments in the drinking water for 6 weeks (females) or 16 weeks (males). T4 and 

somatotropin concentrations, and T3 uptake, were determined after Weeks 2 and 6 in female rats, 

and after Weeks 7, 13, and 16 in male rats. A full-factorial experimental design was used to 

assess interactions of combinations of two or all three pesticides compared to responses after 

individual treatments of each chemical. 

The results of this investigation indicate that treatment with methomyl alone (1 ppm) did not 

increase (p>0.05) T4 or somatotropin concentrations in female or male rats at any time point (up 

to 6 weeks in females and 16 weeks in males). 

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

In a combined chronic toxicity/carcinogenicity study (MRID 00078361), male and female 

Crl:CD rats (80 rats/sex/dose) were administered methomyl (>99% purity) 0, 50, 100, and 400 

ppm for 104 weeks (equivalent to 0, 2.5, 5 or 20 mg/kg/day). In addition, 20 rats/sex/dose were 

included in the control and treatment groups for erythrocyte and brain cholinesterase 

determinations. After 52 weeks, 10 rats from each group were sacrificed for gross and 

histopathological evaluation. After 104 weeks, all surviving rats in all groups were sacrificed for 

gross and histopathological evaluation. Select organs were weighed at each sacrifice interval 

(including liver, kidneys, testes, adrenals, and pituitary gland). Gross and microscopic 

pathological examination included the liver, kidney, pituitary, thyroid, adrenals, mammary gland, 

testis, epididymis, prostate, uterus, and ovary. 

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Male and female rats in the high-dose group had decreased body weight gain during the time 

intervals 0 to 91, 0 to 182, and 0 to 364 days of treatment. High-dose male rats continued a 

similar reduction in body weight gain through 0 to 560 days of treatment. Male and female highdose rats appeared to adapt to the dietary administration of methomyl after 2 years; no 

differences between final body weight or body weight gain in the rats were observed compared 

to control at the end of the study. No treatment-related mortality was observed during the study. 

Survival exceeded 50% in all groups at 18 months, and was ≥50% in all groups after 2 years 

except for the control females (45%). No significant differences were observed in RBC and brain 

ChE concentrations due to treatment in either sex. 

There were no differences in absolute organ weights for male or female rats in any treatment 

group, or relative organ weights for male rats compared to control. Mid- and high-dose female 

rats had increased relative liver weights compared to control; however, no histopathological 

changes were observed. No treatment-related neoplastic or non-neoplastic lesions were observed 

that could be attributed to the dietary administration of methomyl up to 400 ppm. The NOAEL in 

the chronic toxicity/carcinogenicity study in rats is 100 ppm (intermediate dose) for males and 

females (approximately 5.0 mg/kg/day for males and females), based on decreased weight gain 

and hematology changes at the LOAEL (high dose, 400 ppm). 

Carcinogenicity in Rodents (Mouse); (OCSPP 870.4200) 

In a carcinogenicity study (MRID 00078423), male and female CD-1 mice (80 mice/sex/dose) 

were administered methomyl (99% purity) at nominal concentrations in the diet of 0, 50, 100, 

and 800 ppm for 104 weeks. Due to high mortality, the high dose was reduced to 400 ppm 

beginning Week 28, and was further reduced to 200 ppm at Week 39 (the intermediate dose was 

also reduced to 75 ppm at Week 39). Mean-measured doses were 0, 8.7, 15.3 or 93.3 mg/kg/day 

for males and 10.6, 19.0 or 118.5 mg/kg/day for females. After 104 weeks, all surviving mice in 

all groups were sacrificed for gross and histopathological evaluation. Select organs were 

weighed at terminal sacrifice, including liver, kidneys, testes with epididymides, adrenals, and 

pituitary gland. Gross and microscopic pathological examination included the liver, kidney, 

pituitary, thyroid, adrenals, mammary gland, testes with epididymides, prostate, uterus, and 

ovaries. 

Survival in the high-dose males and females was significantly decreased (percent and 

significance not reported) at Week 26, and survival in the mid-dose groups was also moderately 

decreased (percent and significance not reported). Due to these high mortality rates, the doses for 

the high- and mid-dose groups were decreased as described previously. Survival was generally 

significantly decreased (percent and significance not reported) after 52, 80, and 104 weeks of 

treatment in mid- and high-dose animals. Decreased survival rates are considered to be 

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treatment-related; survival exceeded 50% in all groups at 15 months of study. No treatmentrelated effects on body weight or body weight gain were observed in this study. 

No treatment-related gross or microscopic pathological effects were noted. There were no 

differences in absolute or relative organ weights for male or female rats in any treatment group, 

except for increased (percent and significance not reported) absolute adrenal weight in high-dose 

males. Relative adrenal weight in this group was reported to be slightly elevated; however, no 

histopathological changes were observed. No treatment-related neoplastic or non-neoplastic 

lesions were observed that could be attributed to the dietary administration of methomyl in this 

study. The NOAEL in the carcinogenicity study in is 50 ppm (low dose) for males and females 

(8.7 mg/kg/day for males and 10.6 mg/kg/day for females). This NOAEL is based on increased 

mortality doses ≥75 ppm (15.3 mg/kg/day for males and 19.0 mg/kg/day for females). 

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

In a chronic toxicity study (MRID 00007091), methomyl; (90 or 100% purity; possibly different 

batches) was administered in the diet to 4 Beagle dogs/sex/dose at nominal concentrations of 0, 

50, 100, 400, or 1000 ppm (mean-measured doses 0/0, 1.5/1.6, 3.0/3.3, 11.5/13.8, and 30.6/31.7 

mg/kg/day in males/females were calculated by the reviewer; similar, calculated doses of 1.25, 

2.5, 10, and 25 mg/kg/day were determined assuming 0.025mg/kg/day per 1 ppm compound in 

the diet) for up to two years. One dog/sex/dose was sacrificed after 12 months of treatment, and 

the remaining 3 dogs/sex/dose were treated for up to 24 months. Select organs were weighed at 

terminal sacrifice (including liver, kidneys, testes, and adrenal and thyroid glands). Gross and 

microscopic pathological examination included the liver, kidney, pituitary, thyroid, adrenals, 

testes, uterus, and ovaries. 

One high-dose female was found dead in its cage on the first day of Week 9. A replacement 

female dog was started on treatment two weeks later, but this animal had repeated seizures on 

Day 17 of treatment, followed by coma and death. Two high-dose males exhibited tremors, 

salivation, incoordination, and circling movements on a single afternoon during Week 13 of the 

study. Another high-dose male exhibited a higher incidence of diarrhea compared with other 

study animals and progressive anemia throughout the study with markedly declined hematology 

parameters at 18 months compared to baseline. Treatment was discontinued from Weeks 85 

through 94, with improved hematological analyses. The animal was returned to study for the 

remainder of the dosing period (Weeks 95–104), with a concomitant return to severe anemia and 

moderate leucopenia by the end of treatment. Grossly, it was found to have an enlarged spleen, 

an enlarged liver, and increases in absolute and relative thyroid, heart, liver, and kidney weights. 

The remaining two high-dose males had increased absolute and relative kidney weights at study 

termination. 

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No adverse effects of treatment were observed on body weight, body weight gain, food 

consumption, clinical chemistry, or urinalysis. Microscopic findings in the high-dose animals 

included changes in the kidneys and liver, with extramedullary hematopoiesis and hematogenic 

activity in the spleen and bone marrow. The LOAEL is 1000 ppm (approximately equivalent to 

30.6/31.7 mg/kg/day in males/females), based on anemia (with associated gross and microscopic 

pathological findings) and increased absolute and relative kidney weights in the males, mortality 

in the females, and clinical signs of toxicity and microscopic findings in the kidney, liver, spleen, 

and bone marrow as described above in both sexes. The NOAEL is 400 ppm (approximately 

equivalent to 11.5/13.8 mg/kg/day in males/females). 

Avian Reproductive Toxicity (OCSPP 850.2300) 

In an avian reproduction study (MRID 41898602), 20-week old Bobwhite quail (Colinus 

virginianus) were dosed with methomyl (98.35% purity) in the feed at nominal concentrations 

of 0 (acetone and corn oil, 1:1.67 ratio), 50, 150, and 500 ppm for approximately 20 weeks. 

Quails in the 50 ppm group were dosed at 150 ppm for the first four weeks in error; this error 

was corrected and this group was administered 50 ppm for the remainder of the study. Mean 

achieved dose concentrations of methomyl in the study were 0 (basal diet), 50 (67; timeweighted average including first four weeks of dosing at 150 ppm), 150, and 458 ppm. 

Methomyl concentrations in the diet were reported over the first 4 weeks of the study, with 

variability from nominal concentrations of approximately 20 to 30%. The mean dietary 

concentrations over the first 4 weeks were 123, 137, and 427 ppm in the treated groups. 

Each dose group consisted of 32 quails, with a single cock and hen paired in 16 pens/dose group. 

The photoperiod was eight hours of light per day during the acclimation period (pre egg-laying) 

and the first seven weeks on study, and was increased to 17 hours of light per day for the 

remainder of the study. All adult birds were observed daily for mortality, signs of toxicity, and 

abnormal behavior. Body weights were recorded at study initiation, Weeks 2, 4, 6, 8, and at 

study termination. Eggs were collected daily, soaked and washed in a chlorine-based detergent, 

stored in a cold room, and counted weekly. Measurements were taken of the average thickness of 

air-dried shells (with membrane) after collection of one egg on alternating weeks from odd- and 

even-numbered pens. Remaining eggs were candled to detect abnormalities and cracks. Normal 

eggs were set for incubation, and candled on Days 11 and 21 of incubation to determine embryo 

viability and survival, respectively. Hatchlings and unhatched eggs were removed on Day 25 or 

26 of incubation. Feed consumption was measured every week. The study room temperature was 

maintained at 19.8 ± 2.9°C and 36 ± 12% relative humidity. The hatchling room temperature was 

maintained at 25.8 ± 2.4°C; surviving hatchlings were weighed on Day 14. 

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There were no treatment-related mortalities, effects on body weight and feed consumption. 

According to the study review, the percentage of females with feather loss/ and or toe lesions 

increased dose-responsively, likely due to increased aggression in males and were statistically 

different from controls at the mid and high treatment. Incidental mortalities and findings were 

observed across all study groups. In general, there were no treatment-related effects on 

reproductive parameters at ≤137 ppm. The number of eggs laid and set per hen were decreased 

(p<0.05) by 40% at the high-dose (427 ppm). The percentage of eggs cracked/eggs laid was 

decreased (p<0.05) at the intermediate dose, but eggshell thickness displayed no treatmentrelated effects. No differences due to treatment were observed in body weights of hatchlings or 

14-day survivors. The NOAEL is 150 ppm and the LOAEL is 427 based on the decreases in eggs 

laid and set per hen. 

In an avian reproduction study (MRID 41898601), mallard ducks (Anas platyrhychos) were 

dosed with methomyl (98.35% purity) in the feed at nominal concentrations of 0 (acetone and 

corn oil, 1:1.67 ratio), 50, 150, and 500 ppm for approximately 20 weeks. Ducks in the 50 ppm 

group were dosed at 150 ppm for the first four weeks in error; this error was corrected and this 

group was administered 50 ppm for the remainder of the study. Mean achieved dose 

concentrations of methomyl in the study were 0 (basal diet), 50 (67; time-weighted average 

including first four weeks of dosing at 150 ppm), 150, and 458 ppm. Methomyl concentrations 

in the diet were reported over the first 4 weeks of the study, with variability from nominal 

concentrations of approximately 20 to 30%. The mean dietary concentrations over the first 4 

weeks were 123, 137, and 427 ppm in the treated groups. Due to the dosing error in the 50 ppm 

group, the reviewer could not make definitive conclusions about this treatment level. There 

were significant differences from the control for all levels tested for male body weight gain, and 

at 427 ppm, female weight gain was also significantly decreased. At 137 ppm, there was a 

significantly higher number of cracked eggs when compared to the control. Since 427 ppm was 

statistically equivalent to the control, this occurrence is not believed to be treatment related. No 

other reproductive parameters were affected. 

In another mallard duck reproduction study (MRID 49245301; preliminary review only), a onegeneration reproductive toxicity of methomyl to 16 pairs per level of 19-week old mallard duck 

(Anas platyrhynchos) was assessed over 21 weeks. DPX-X1179-571 (99.4% methomyl) was 

administered to the birds in the diet at nominal concentrations of 0 (control), 41, 122, 367 and 

1100 mg ai/kg. Mean-measured concentrations (reviewer-calculated) were <10 (<LOQ, 

control), 41, 121, 373 and 1120 mg ai/kg diet, respectively. 

There were no treatment-related effects on any adult parameter at the (mean-measured) 41, 121 

or 373 mg ai/kg diet levels and no treatment-related effect on any reproductive or offspring 

parameter at all levels. At the 1200 mg ai/kg diet level, there was a marked reduction in food 

consumption during Week 1 (40 versus 103 g/bird/day; p<0.01) that continued through Week 2 

(82 versus 108 g/bird/day; p<0.05) that was considered related to treatment. The reduction in 

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food consumption corresponded with slight reductions in mean body weight of both hens and 

drakes during the first two weeks of the study: mean 0- to 2-week body weight changes were -

104 versus -23 g for the control for drakes, and -72 versus -26 g for the control for hens. In 

addition, there was a statistically-significant reduction in female body weights compared to the 

control (1135 versus 1228 g; p<0.05) at study termination. Necropsy also revealed an increase 

in the number of hens with a regressing/regressed ovary (8/15 versus 4/16 for the control group) 

that was considered related to treatment. The NOAEC and LOAEC were 373 and 1120 mg 

ai/kg diet, respectively. 

Fish Early Life-Stage Toxicity; (OCSPP 850.1400) 

The effects of methomyl (98.4% purity) on eggs and fry of the fathead minnow (P. promelas) 

were determined in a flow-through, early life-stage 35-day toxicity test (MRID 46015305). 

Treatments consisted of a dilution water (negative) control and nominal methomyl 

concentrations of 19, 38, 75, 150, and 300 μg/L. Two replicates containing 25 eggs/replicate 

were exposed to each treatment concentration and control in incubation cups. All incubation cups 

were observed daily, and dead eggs and fungal-infected eggs were removed. The 29-day posthatch growth period began on Study Day 6, after 95% hatch was attained. Observations of 

abnormal behavior, physical changes, and mortality were recorded daily. At the end of the study, 

standard length and blotted wet weight were determined. 

Mean measured concentrations of methomyl were 18.0, 39.3, 73.0, 145, and 261 μg/L, and 

ranged from 87 to 103% of nominal concentrations. Egg hatchability and fry survival were not 

decreased (p>0.05) at any test concentration. All chemical and physical parameters for the 35-

day study were within acceptable ranges. The following endpoints were determined for the 

methomyl test concentrations and control (in parenthesis): mean percentage survival ranged from 

86 to 94% (92% for water control); mean percentage egg hatched ranged from 88 to 96% (94% 

for water control); mean standard length ranged from 18.56 to 21.17 mm (21.15 mm for water 

control); and mean wet weight ranged from 0.112 to 0.148 g (0.146 g for water control). The 

NOAEC for percentage hatch and percentage survival was 261 μg/L (high dose); the LOAEC is 

not determined. 

The overall NOAEC for the Fathead minnow was 73.0 μg/L, and the LOAEC was 145 μg/L 

based on decreased weight and length. 

Fish Early Life-Stage Toxicity; (OCSPP 850.1400) 

In a freshwater fish early life-stage toxicity test (MRID 00131255), Fathead minnow (P. 

promelas) embryos were exposed to methomyl (>99% purity) at nominal concentrations of <1 

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(<LOQ, negative control), 31, 62, 125, 250, and 500 μg/L in a flow-through system for 35 days. 

Achieved exposure concentrations were <1, 27, 57, 117, 243, and 491 μg/L for the treated 

groups. This study was scientifically sound. Although the study author’s analysis failed to detect 

a significant reduction in percent hatch at any level, when compared to control, the reviewer 

visually determined the NOAEC for percent hatch to be 57 μg/L given an apparent reduction at 

concentrations ≥117 μg/L. Survival was decreased (p<0.05) by 10 to 20% at the 117 μg/L 

concentration. Mean length and wet weight were decreased (p<0.05) at concentrations ≥243 

μg/L. 

Fish Life Cycle Toxicity; (OCSPP 850.1500) 

In a freshwater fish full life-cycle toxicity test (MRID 43072101), Fathead minnow (P. 

promelas) embryos were exposed to methomyl (98.35% purity) at nominal concentrations of <1 

(<LOQ, negative control), 19, 38, 75, 150, and 300 μg/L in a flow-through system for 193 days. 

Mean measured concentrations were <1, 18, 40.7, 76, 142, and 280 μg/L for the treated groups. 

This study was scientifically sound. In-house spawned juvenile fathead minnows were initially 

exposed in two hatching/growth chambers per aquarium (duplicate aquaria; 200/treatment) in a 

nested experimental design. Larvae were thinned to 25/replicate chamber on Day 7 (post-hatch). 

On Days 28 and 58, length measurements were determined; lengths and weights were 

determined after anesthetizing fish on Day 84. Fish in each treatment group were collected into 

two groups/treatment on Day 100, and on Day 101, eight spawning pairs/treatment were selected 

and placed into spawning aquaria. The spawning phase was terminated on Day 154, and adult 

survivors were weighed and measured. The mean reproduction parameters examined included 

the number of days to first spawn, number of spawning days, number of spawns per pair, and 

number of eggs per spawn. Sublots of 35 embryos were collected from random spawns to 

determine hatchability, survival, and growth in the F1 generation. At 56 days post-hatch, the F1 

fish were anesthetized, weighed, and measured. Fish were observed for signs of toxicity and 

mortality throughout the study. 

Scattered mortality was observed through 48 hours after embryo placement, with only one 

additional mortality after 48 hours. Hatching success (%) of the F0 generation calculated from 

Day 2 determined the NOAEC for percent hatch to be 280 μg/L [hatching success is decreased 

(p<0.05) at 142 μg/L only if calculated from Day 0]. Survival (calculated at Days 28, 56, and 84) 

was decreased (p<0.05) by 6 to 7% at the high concentration (280 μg/L). Mean length 

(calculated at Days 28, 56, and 84) was decreased (p<0.05) at concentrations ≥142 μg/L, and wet 

weight were decreased (p<0.05) at the 280 μg/L concentration. No significant effect on 

reproduction was demonstrated at any test concentration compared to control; the number of 

eggs and number of eggs/spawn (fecundity) were highly variable. Hatching success (%) of the 

F1 generation calculated from Day 2 determined the NOAEC for percent hatch to be 280 μg/L 

[hatching success is decreased (p<0.05) at 40.7 and 280 μg/L if calculated from Day 0]. The 

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length of F1 larvae at 56 days post-hatch was decreased at 142 and 280 μg/L concentrations, and 

wet weight was decreased at the 280 μg/L concentration only. 

 

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APPENDIX 3: References Not Utilized in the Methomyl WoE Analysis 

In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing 

Test Orders for the EDSP Tier 1 screening battery was made available in a Federal Register 

Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted 

as “other scientifically relevant 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 registration/reregistration, published literature articles 

and/or Tier 1 assays. The agency also conducted a more recent search (2009 to 2014) of 

available scientific literature for any additional relevant information for their weight of evidence 

(WoE) evaluations. These articles were evaluated in accordance with the agencies Evaluation 

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

(http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species

_reregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance 

for considering and Using Open Literature Toxicity Studies to Support Human Health Risk 

Assessment (http://www.epa.gov/pesticides/science/lit-studies.pdf). 

The following published and unpublished references were considered for use in the WoE 

analysis for Methomyl but were not utilized due to one or more of the following reasons: 1) the 

article was not available in English; 2) the compound of interest was not used 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 adequately described; 7) only an abstract of the study 

was available; 8) the reference is a review article or book chapter and does not contain primary 

study data; 9) insufficient information was available to adequately assess the validity of the study 

results; 10) the 40 CFR Part 158 guideline study was classified as unacceptable/inadequate; 11) 

the study dealt only with non-EDSP assay development; 12) no specific endocrine-related 

endpoints were assessed in the study; and 13) the study contained only data on invertebrates. 

Crisp, T.M. et al. (1997). Special Report on Environmental Endocrine Disruption: An Effects 

Assessment and Analysis. Report Number EPA/630/R-96/012 February 1997. 

http://www.epa.gov/ORD/WebPubs/endocrine/endocrine.pdf 

Celik, L., Lund, J., and Schiott, B. (2008). Exploring interactions of endocrine-disrupting 

compounds with different confirmations of the human estrogen receptor a ligand binding 

domain: A molecular docking study. Chem. Res. Toxicol. 21:2195–2206. 

Crisp, T.M. et al. (1998). Environmental Endocrine Disruption: An Effects Assessment and 

Analysis. Environ Health Perspectives 106, Supplement 1: 1156. 

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Gruggs, C. J., Perkham, J.C., and Cato, K.D. (1983). Mammary carcinogenesis in rats in 

relation to the age at time of N-nitroso-N-methylurea administration. 1. Natl.Cancer Institute. 

70:209–212. 

Grue, C.E., Hart, A.D.M., and Mineau, P. (1991). Biological consequences of depressed brain 

cholinesterase activity in wildlife in: Cholinesterase-inhibiting insecticides: Their impact on 

wildlife and the environment. ed. P. Mineau .. Elsevier Science Publishers B.V., Amsterdam. 

pp. 151–209. 

Hoffman, D.J and Albers, P.H. (1984). Evaluation of Potential Embryotoxicity and 

Teratogenicity of 42 Herbicides, Insecticides, and Petroleum Contaminants to Mallard Eggs. 

Arch. Environ. Contam. Toxicol. 13: 15–27. 

Hseuh, AJ.W., Dufau, M.L., and Cart, KJ. (1977). Gonadotrophin induced hormone receptors 

and desensitization of testicular 3' ,5'-cyclic AMP and testosterone responses. Proc. Natl. Acad. 

Sci. USA 74:592–595. 

Jiao, B. et al. (2008). Establishment of a Transgenic Yeast Screening System for Estrogenicity 

and Identification of the Anti-Estrogenic Activity of Malachite Green. J. Cell. Biochem. 

105:1399-1409. 

Montague, B. (1997). Brian.dbf database, US Environmental Protection Agency, Office of 

Pesticide Programs, August 13, 1997. 

National Research Council. (1999). Hormonally Active Agents in the Environment. National 

Academy Press. Washington DC. 430 pages. http://www.nap.eduibooks/0309064198/htnV

Nishihara, T. et al. (2000). Estrogenic activities of 517 chemicals by yeast two-hybrid assay. 

J. of Health Sci. 46: 282–298. 

Nendza, M. and Wenzel, A. (2006). Discriminating Toxicant Classes by Mode of Action, 1) 

(Eco)toxicity Profiles). Environ. Sci. & Pollut. Res. 13:192–203. 

Roncaglioni, A., Piclin, N., Pintore, M., and Benfenati, E. (2008). Binary classification models 

for endocrine disrupter effects related-through the estrogen receptor. SAR and QSAR. 

Environmental Research 19:697–733. 

Shalaby, M.A., El Zorba, H.Y., and Ziada, R.M. (2010). Reproductive toxicity of methomyl 

insecticide in male rats and protective effect of folic acid. Food Chem Toxicol. 48(11):3221–

3226. 

Shanthalatha, A., Madhuranath, B.N., and Yajurvedi, H.N. (2012). Effect of methomyl 

formulation, a carbamate pesticide on ovarian follicular development and fertility in mice. 

J. Environ. Biol. 33(1):33–37. 

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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? Toxicol. Environ. Health 50:415–431. 

van Ravenzwaay, B. (1992). Discussion of prenatal and reproduction toxicity study of Reg. No. 

83-258 (Vinclozolin). Data submission to US EPA from BASF Corporation, 425813-02. 

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