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 63
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 63
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHAWAYS 

CHEMICAL: PHOSMET 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

Page 3 of 63
PHOSMET FINAL 

ii 

TABLE OF CONTENTS 

Abbreviations...................................................................................................iii 

Executive Summary..........................................................................................1 

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

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

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

B. Other Scientifically Relevant Information (OSRI)......................................5 

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

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

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9 

1. Effects on Estrogen Pathway.....................................................9 

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

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

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

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

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

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

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

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

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

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

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

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

LIST OF TABLES: 

 Table 1. Tier 1 Assay for Phosmet.....................................................................8 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Phosmet ....................................11 

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

 Table 4. Thyroid Pathway for Phosmet ............................................................19 

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

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

APPENDIX 3. References Not Utilized in the Phosmet WoE Analysis...............................53 

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PHOSMET 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 

F Female 

F1 First filial generation 

F2 Second filial generation 

Fcd Fecundity

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PHOSMET FINAL 

iv 

Abbreviation Terminology 

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 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

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PHOSMET FINAL 

v 

Abbreviation Terminology 

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 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

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PHOSMET FINAL 

vi 

Abbreviation Terminology 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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PHOSMET FINAL 

Page 1 of 55 

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 phosmet 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 April 9, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) 

of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy 

(OSCP) conducted a weight-of-evidence (WoE) analysis of the potential interaction of phosmet 

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).

Phosmet is a member of the organophosphate class of insecticides whose primary mode of toxic 

action is cholinesterase inhibition leading to neurotoxicity (e.g., cholinergic signs). 

For the estrogen pathway, while the Tier 1 estrogen receptor (ER) binding assay was considered 

inadequate (due to solubility issues), the Tier 1 ER transactivation assay (ERTA), as well as an 

additional ERTA study which evaluated both agonism and antagonism was negative. 

Furthermore, the Tier 1 uterotrophic assay was negative. While the Tier 1 in vitro aromatase 

assay was positive, the steroidogenesis assay was negative. There were no estrogen-related 

effects observed in the Part 158 mammalian and wildlife studies, except for an increased 

incidence of dilation and myometrial atrophy of the uterus observed at 12 and 24 months, 

respectively, in the mouse carcinogenicity study. In the female pubertal assay, although all test 

doses were considered to be overtly toxic, no estrogen-related effects were observed in this 

study. Similarly, the mid and high treatment groups in the fish short-term reproduction assay 

(FSTRA) were considered to be overtly toxic, and the information obtained from the low 

concentration was limited (essentially due to the loss of one of four replicates). In the FSTRA at 

this low concentration, effects on female gonadal histopathology [decreased yolk synthesis, and 

occasional disruptions of follicular vitellogenin (VTG) concentration] were observed. It is noted 

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PHOSMET FINAL 

Page 2 of 55 

that there were no other effects in the low concentration observed including alterations in 

gonadal somatic index (GSI) and plasma VTG. Therefore, the available data do not suggest that 

phosmet has the potential to act as an estrogen agonist based on the in vitro and uterotrophic 

study. Additionally, while the in vitro aromatase assay was positive which could indicate a 

potential decrease in estradiol production, the in vitro steroidogenesis was negative and the 

results of both the female pubertal and FSTRA, do not support an anti-estrogenic effect. 

For phosmet, while the Tier 1 AR binding assay was positive, the Tier 1 Hershberger was 

negative and no androgen-related effects were observed in the Tier 1 male pubertal assay in the 

absence of overt toxicity. Additionally, the Tier 1 steroidogenesis assay was negative. In the 

FSTRA, based on the data available, there were no androgen-related effects observed in the 

absence of overt toxicity. The only androgen-related effect observed in the mammalian Part 158 

studies was an increase in the incidence of testicular atrophy in the mouse carcinogenicity study. 

Overall there is no convincing evidence for potential interaction with the androgen pathway. 

In the Tier 1 male pubertal assay, T4 levels were increased and changes in thyroid 

histopathology (decreased colloid area and increases in follicular cell height) were observed at 

both doses, although the effects at the high dose occurred in the presence of overt toxicity. 

Similarly, thyroid effects (increased T4 levels and histopathology) were observed in the Tier 1 

female pubertal assay; however, both test doses were considered to be overtly toxic in this assay. 

In the Part 158 mammalian studies, histopathological changes in the thyroid were observed at 

10.9 mg/kg/day in the chronic rat study. In the amphibian metamorphosis assay (AMA), while 

developmental alterations were observed (↓NF stage and HLL), there were no effects on thyroid 

histopathology. Therefore, the observed developmental delays are likely not thyroid-mediated. 

Overall, for phosmet there is no convincing evidence for a potential interaction with the estrogen 

or androgen pathway in mammals or wildlife. The available data indicate a potential interaction 

with the thyroid pathway in mammals, but not amphibians. 

In the EDSP Tier 1 male and female pubertal and OSRI, there was evidence for potential 

interaction with the thyroid pathway in studies conducted with adult animals, but no such data 

exists for potential thyroid toxicity in the young animals. 

For phosmet, the current Point of Departure (POD) of 3.56 mg/kg/day used for human health risk 

assessment is based on red blood cell (RBC) cholinesterase inhibition, which is the most 

sensitive endpoint for this class of chemicals, observed at 7.0 mg/kg/day in rats. This POD was 

used for deriving the chronic Reference Dose (RfD) used for assessing chronic dietary risk for 

the general population. A second POD of 0.16 mg/k/day used for deriving the chronic RfD for 

the young (the most sensitive population) is based on RBC cholinesterase inhibition in the 

offspring. This POD was also used for calculating the Margins of Exposure (MOEs) used for 

non-dietary risk assessments. 

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PHOSMET FINAL 

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In the case of phosmet, although no data exists for potential thyroid toxicity in the young, a 

comparative thyroid assay (CTA) is not recommended since: 1) the POD used for assessing risk 

for the young is approximately 63-fold lower than the low doses (10 mg/kg/day) that caused 

thyroid-related effects in the Tier 1 in vivo pubertal assays; 2) the POD for the adults and the 

young is based on the most sensitive endpoint (cholinesterase inhibition); 3) the CTA would be 

tested at higher doses and would not be expected to yield a POD lower than the level currently 

used for risk assessment; and 4) the chronic RfD and MOEs are protective of the thyroid related 

findings in the Tier 1 in vivo assays. Therefore, mammalian EDSP Tier 2 testing is not 

recommended for phosmet since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. 

As there was no evidence of potential interaction with the estrogen or androgen pathway in fish, 

or thyroid pathway in amphibians, additional testing is not recommended for these taxa. 

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard 

duck are available for phosmet. In bobwhite quail, effects on reproductive parameters were 

observed at concentrations where mortality and adult body weight decreases were observed; no 

reproductive effects were observed in the mallard duck study. The type of data obtained from 

Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating 

potential reproductive effects to birds from phosmet exposure. Relative to the EDSP, additional 

testing is not recommended. 

. 

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PHOSMET FINAL 

Page 4 of 55 

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 a pesticide chemical 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 April 9, 2014, the EDSP Tire 1 Assay Weight of Evidence Review Committee (T1WoERC) 

of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy 

(OSCP) conducted a weight-of-evidence analysis of the potential interaction of phosmet 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).

Phosmet is a synthetic organophosphate pesticide, and its mode of action is based on altered 

neurotransmission due to cholinesterase (ChE) inhibition. Phosmet has a low vapor pressure 

(3.7–4.9 × 10-7 mm Hg at 25°C), and therefore, volatilization is not expected to significantly 

contribute to the dissipation of phosmet in the environment. The compound has a water solubility 

of 20–25 mg/L at (20–25°C) and an octanol/water partition coefficient (log Kow) of 2.78 to 2.95. 

Phosmet is degraded by hydrolysis and atmospheric photolysis. Phosmet has a whole body 

bioconcentration factor value in fish ranging from 10 to 44. 

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

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

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

data are summarized in Sections III.A through III.C. An analysis of the data submitted to the 

agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in 

Section IV. The EDSP Tier 2 study recommendations are presented in Section V. 

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PHOSMET FINAL 

Page 5 of 55 

II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

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

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

in Appendix 1. 

B. Other Scientifically Relevant Information (OSRI) 

In response to the Agency’s Test Orders, data 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 phosmet (USEPA, 2010). Since then, the 

Agency has also conducted a more recent search of available scientific literature for any 

additional relevant information. Summaries of the available OSRI are presented in Appendix 2. 

Additionally, literature/studies 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-mediated processes (i.e., E, , 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. 

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 of elven assays 

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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 consistency 

of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple 

endpoints are measured in each assay. 

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

relative degree of diagnostic utility of a 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 to 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 whether the responses were 

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

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

endocrine-mediated effect from a non-endocrine toxic response. 

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This uncertainty in distinguishing whether the responses were endocrine-mediated was discussed 

at the FIFRA SAP meeting in 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. 

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 a substance has the potential to interact with the E, A or 

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T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for 

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

A. EDSP Tier 1 Screening Assays 

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

in Table 1. 

Table 1. Tier 1 Screening Assays for Phosmet.

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay 

(Rat uterine cytosol) 

OCSPP 

890.1250 

Requirement not satisfied1

(MRID 48618704) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 

890.1300; 

OECD 455 

Requirement satisfied 

(MRID 48618705) 

AR Binding Assay 

(Rat prostate cytosol) 

OCSPP 

890.1150 

Requirement satisfied 

(MRID 48618703) 

Steroidogenesis Assay 

(Human cell line H295R) 

OCSPP 

890.1550; 

OECD 456 

Requirement satisfied 

(MRID 48618701) 

Aromatase Assay 

(Human recombinant microsomes) 

OCSPP 

890.1200 

Requirement satisfied 

(MRID 48618702) 

Uterotrophic Assay (Rat) 

OCSPP 

890.1600; 

OECD 440 

Requirement satisfied 

(MRID 48618707) 

Hershberger Assay (Rat) 

OCSPP 

890.1400; 

OECD 441 

Requirement satisfied 

(MRID 48618706) 

Pubertal Female Assay (Rat) OCSPP 

890.1450 

Requirement not satisfied2 

 (MRID 48673003) 

Pubertal Male Assay (Rat) OCSPP 

890.1500 

Requirement satisfied 

(MRID 48673003) 

Fish Short-term Reproduction Assay 

OCSPP 

890.1350; 

OECD 229 

Requirement satisfied3

(MRID 48673002) 

Amphibian Metamorphosis Assay (Frog) 

OCSPP 

890.1100; 

OECD 231 

Requirement satisfied 

(MRID 48673001) 

 1 This assay is not satisfied due to issues with test chemical solubility. However a repeat of this assay is not 

required since negative results were seen in the Tier 1 ERTA and OSRI which evaluated potential interaction of 

phosmet with the ER. 

2 This assay is not satisfied due to overt toxicity seen at both doses tested. However, a repeat of the assay is not 

required since it would have no impact on the risk assessment. 

3 Although the mortality in the high treatment level, clinical signs of toxicity in the intermediate treatment level, 

and noteworthy rate of oocyte atresia in control females make the results difficult to interpret, when 

considered in context of other available Tier 1 EDSP data for phosmet, this study satisfies the EDSP Tier 1 

Test Order requirement for a Fish Short-Term Reproduction Assay (OCSPP Guideline 890.1350). 

Page 16 of 63
PHOSMET FINAL 

Page 9 of 55 

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

1. Effects on Estrogen Pathway 

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

various targets of the estrogen pathway across the relevant Tier 1 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. 

Phosmet was negative in the Tier 1 steroidogenesis assay and positive in the Tier 1 aromatase 

assay in which aromatase activity was decreased to 34% at 10−4 M. Although there was no 

evidence of an interaction of phosmet with the ER in the Tier 1 ER binding assay, issues with 

test chemical solubility were not adequately addressed which confounded the interpretation of 

the assay and lessened confidence in the results. However, phosmet was negative in the Tier 1 

ERTA and negative for ER agonism and antagonism in a published literature study by Kojima et 

al. (2004) which evaluated the potential to interact with two human estrogen receptor subtypes 

(hERα and hERβ) in transactivation assays in Chinese hamster ovary cells (CHO-K1 cells). 

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

assay, persistent tremors were seen at both doses (10 and 20 mg/kg/day) tested, and body weight 

losses (9-10%) were seen at the high dose (20 mg/kg/day) indicating overt toxicity. Although 

both doses were considered to be overtly toxic, it is noted that there were no treatment-related 

effects on vaginal opening, cycle status at necropsy, mean age at first estrus, mean cycle length, 

percent cycling, percent regularly cycling or histopathological changes in the ovaries or uterus. 

In the FSTRA, the mid-and high-treatment groups were considered to be overtly toxic as survival 

was 9% in the highest concentration tested and data from this treatment levelwere not 

statistically analyzed. In the mid-concentration, in addition to a 25% decrease in survival, there 

were generalized overt signs of toxicity in female fish, including lethargy in all surviving fish, 

and loss of equilibrium in females. At the low concentration, survival was reduced by 13%, 

whereas 3 out of 4 females in one replicate died, thereby reducing the ability to evaluate 

potential effects in this treatment. At the mid-concentration, there was a significant 81% 

decrease in fecundity, with a 7% decrease in fertilization. Additionally at this concentration, 

gonado-somatic index (GSI) was decreased (↓34%). Gonadal histopathology was affected as 

evidenced by decreased yolk synthesis in the low- and mid-concentration females, and 

occasional disruptions of follicular VTG concentration. There were no effects on plasma 

vitellogenin (VTG) concentrations at the low or mid-treatment. 

In the Part 158 mammalian toxicity studies, in the 2-generation reproduction study in the rat, 

absolute and relative ovarian weights were decreased in the high-dose P0 females at doses that 

caused significant decreases (≥ 10%) in body weight. The viability index was decreased at the 

Page 17 of 63
PHOSMET FINAL 

Page 10 of 55 

high dose in the rat reproduction study; however, maternal overt toxicity and substantial ChE 

inhibition was also present at this dose. An increased incidence of dilation and myometrial 

atrophy of the uterus were observed at 12 and 24 months, respectively, in the mouse 

carcinogenicity study at doses that caused significant decreases in brain ChE activity. 

In the Part 158 mallard duck reproduction study, there were no significant effects determined for 

any reproductive parameters assessed. In the bobwhite quail study, significant reductions were 

determined for eggs laid but these effects were observed at the highest treatment group (150 

mg/kg-diet) where increased mortality and decreased body weight relative to the control group 

were also observed. In the Part 158 fish early life stage study with rainbow trout, effects on 

growth were observed at 6.1 μg a.i/L. 

Page 18 of 63
PHOSMET FINAL 

Page 11 of 55 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Phosmet

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Phosmet1

Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

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 

(VTG) 

EDSP Tier 1 Assay

ER Binding (MRID 48618704) I ERTA (MRID 48618705) NAromatase (MRID 48618702) PSteroidogenesis (MRID 48618701) N Uterotrophic (MRID 48618707) N Female Pubertal Rat (MRID 48673003) 

--

4 

--

4

--

4

--

4

--

4

--

4

--

4

--

4 

FSTRA (MRID 48673002) NE N P5 (L) N N N 

OSRI

ER transactivation assay; (Kojima, et al., 2004) 

 N

Developmental toxicity (Rat; MRID 41962902) N N Developmental toxicity (Rat; MRID 00063192) NR N 
PHOSMET FINAL 

Page 12 of 55 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Phosmet

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Phosmet1

Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

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 

(VTG) 

Developmental toxicity (Rabbit; MRID 41962901) 

N N 

Developmental toxicity (Monkey; MRID 00043397) 

NR N 

Two-generation reproduction (Rat; MRID 41520001) 

NR N N N 

Chronic toxicity (Rat; MRID 41916401) NE N N NE Carcinogenicity (Mouse; MRID 00141659) 

P 

(H) NE N NE 

Chronic toxicity (Dog; MRID 00076436) 

NR NR NR NR 

Avian reproduction (Duck; MRID 00105999) 

 N 

Avian reproduction (Quail; MRID 00125786) 

 N 

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

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

Changes in weight are absolute unless otherwise indicated. CS=Clinical signs; BW=Body weight 
PHOSMET FINAL 

Page 13 of 55 

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

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 b

details see Section IV. A 

4. Both doses in the assay were considered to be overtly toxic, therefore no findings are reported in the table for these parameter

5. Gonadal histopathology was affected as shown by decreased yolk synthesis in the low-concentration females, and occasional 

VTG concentration. 

6. At the mid and treatment groups were considered to be overtly toxic as survival was 9% in the highest concentration tested an

were not statistically analyzed. In the mid-concentration, in addition to a 25% decrease in survival, there were generalized ov

female fish, including lethargy in all surviving fish, and loss of equilibrium in females 

7. Increased incidence of dilation and myometrial atrophy of the uterus were observed at 12 and 24 months, respectively. 

I Inadequate P Positive findings N Negative findings (in vitro) / No effect (in vivo) NE Not examined NR Not reported 
PHOSMET FINAL 

Page 14 of 55 

2. Effects on Androgen Pathway 

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

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

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

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

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

considered further in the WOE assessment. 

Phosmet was negative in the Tier 1 steroidogenesis assay. In the Tier 1 AR binding assay, 

phosmet showed >50% binding, with an IC50 value of 10-5 M. A published literature study that 

screened phosmet for its potential to interact with human AR in a transactivation assay in CHO-K1 

cells. In this, phosmet was reportedly negative for AR transactivation up to the highest 

concentration tested, 10-5 M (Kojima et al. 2004)

In the Hershberger assay, there were no increases in AST in the agonist phase of the assay. In the 

antagonist phase, decreases in all five AST weights were observed at the high dose (25 mg/kg/day) 

which was determined to be overtly toxic (12% decrease in body weight and tremors). 

In the Tier 1 male pubertal rat assay, there were no effects on age or weight at preputial separation 

(PPS), hormone concentrations, or gross and microscopic pathology. Slight decreases in ventral 

prostate weights (NS; ↓9 and 15%) were observed in the low (10 mg/kg/day) and high-dose (20 

mg/kg/day) males, respectively. tremors were seen at both doses (10 and 20 mg/kg/day) tested. At 

the 10 mg/kg/day, mild tremors were seen during one day only in 6/16 animals. At 20 mg/kg/day, 

mild to moderate tremors were seen on multiple days in 11/16 animals. The high dose in this assay 

was determined to be overtly toxic based on the frequency and severity of the tremors. 

In the FSTRA, as mentioned in the estrogen pathway section, the mid- and high treatments were 

overtly toxic, and a limited number of replicates in the low treatment available for evaluation. In 

this study, there was a 7% decrease in fertilization at mid dose, and histological examination 

indicated an increase in the proportion of spermatogonia in the testes at the high concentration. 

There were no other androgen-related effects observed in the study. 

In the mammalian Part 158 studies, testicular atrophy was observed in high-dose (14 mg/kg/day) 

males at termination (24 months) in the mouse carcinogenicity study. In the 2- generation 

reproduction study, absolute testis weights were decreased in high-dose F0 and F1 parents, with no 

treatment-related histopathological lesions seen in the testes in either parental group. In both 

studies, significant reductions in brain and/or RBC ChE activity were reported at the doses that 

showed effects on the testes. In a OSRI, phosmet had no effect on testis weight, sperm count or 

sperm morphology in mice dosed with phosmet (50 mg/kg/day) for 5 days (Quinto et al. 1989). 

There were no androgen-related effects identified for the available Part 158 wildlife studies.

Page 22 of 63
PHOSMET FINAL 

Page 15 of 55 

Table 3. Androgenic/Anti-Androgenic Pathway for Phosmet

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Phosmet1Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone 

Testes Weight / 

GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility (Frt) / 

Fecundity (Fcd) 

Age and Weight at 

EDSP Tier 1 

AR Binding (MRID 48618703) P Steroidogenesis (MRID 48618701) N Hershberger (MRID 48618706 NE N Male Pubertal Rat (MRID 48673003) N N N N N N N N N FSTRA (MRID 48673002) N N N N N 
PHOSMET FINAL 

Page 16 of 55 

Table 3. Androgenic/Anti-Androgenic Pathway for Phosmet

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Phosmet1Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone 

Testes Weight / 

GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility (Frt) / 

Fecundity (Fcd) 

Age and Weight at 

OSRI 

Male Reproductive toxicity (Mouse, Quinto et al., 1989) N N Two-generation reproduction (Rat; MRID 41520001) 

 N N N 

Chronic toxicity (Rat; MRID 41916401) N N NE N NE N Carcinogenicity (Mouse; MRID 00141659) 

N P7 N N NE NE 

Chronic toxicity (Dog; MRID 00076436) NR NR NR NR NR NR Avian reproduction (Duck; MRID 00105999) N Avian reproduction (Quail; MRID 00125786) N 

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

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

in weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventra

prostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), Glans penis (GPBW=Body weight 

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

Page 17 of 55 

4. At the high dose, mean body weight was decreased (p≤0.05) by 12% on Day 11 (with a NS trend to decrease noted on Days 4 

gains were decreased by 60-69%, compared to the control. Clinical signs of toxicity (tremors and uncoordinated movements) wdose. 

5. Clinical signs (tremors) were seen at both doses tested (10 and 20 mg/kg/day) 6. At the mid and treatment groups were considered to be overtly toxic as survival was 9% in the highest concentration tested and 

were not statistically analyzed. In the mid-concentration, in addition to a 25% decrease in survival, there were generalized ove

female fish, including lethargy in all surviving fish, and loss of equilibrium in females 

7. Testicular atrophy at 24 months at the high dose (18 mg/kg/day) P Positive findings N Negative findings (in vitro) / No effect (in vivo) 

NE Not examined NR Not reported 
PHOSMET FINAL 

Page 18 of 55 

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 phosmet 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 female pubertal assay, T4 concentrations were increased by 24 and 38%, 

respectively, in the low- and high-dose groups, but no treatment-related effects were noted for 

thyroid weight or TSH concentration. However, both doses in this assay were considered to be 

overtly toxic based on persistent tremors seen at both doses (10 and 20 mg/kg/day) and body 

weight losses (9-10%) seen at the high dose (20 mg/kg/day). In the male pubertal assay, T4

levels were increased by 48 and 43%, respectively, in the low (10 mg/kg/day)- and high-dose (20 

mg/kg/day) groups, with no treatment-related effects noted for thyroid weight or TSH 

concentration. The high dose in this assay was determined to be overtly toxic based on the 

frequency and severity of the tremors observed. Marginal decreases in thyroid colloid area and 

increases in follicular cell height were observed in all animals in the pubertal rat studies. No 

effects on pituitary weight were noted in either pubertal study. 

In the AMA, there were no histological effects on the thyroid. Phosmet decreased normalized 

hind-limb length (HLL) at the intermediate (↓12 and 15%) and high concentrations (↓10 and 

29%) on Days 7 and 21, respectively, with a 6.7% decrease in snout-vent length on Day 7 in the 

high-dose group. Median NF stage was unaffected on Day 7, although a delay was observed on 

Day 21 in the intermediate and high treatment groups (Stage 58 vs. 59); no asynchronous 

development was observed. 

In the mammalian Part 158 studies, no changes in thyroid weight or histology were seen except 

for mineralization of the thyroid in medium-high dose females (5/50 vs. 0/50) at study 

termination (24 months) in the mouse carcinogenicity study. 

Page 26 of 63
PHOSMET FINAL 

Page 19 of 55 

Table 4. Thyroid Pathway for Phosmet

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Phosmet1

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) Systemic Toxicity Observed2 

Overt Toxicity Observed3

EDSP Tier 1 Assays 

Female Pubertal Rat 

(MRID 48673003) --4 --4 --4 --4 --4 X 

(L, H) 

CS; BW 

(L, H)5

Male Pubertal Rat 

(MRID 48673003) N P6 ↑48% 

(L) N N X (L, H) 

CS 

(H) 

AMA 

(Frog; MRID 

48673001) 

N 

HLL: 

↓12, 9.6% 

Day 7, ↓15, 

27% Day 

21 (M, H) 

SVL: 

↓6.7% 

(Day 7, H) 

N 

OSRI

Chronic toxicity 

(Rat; MRID 

41916401) 

N P 

(MH) NE NE NE 

X 

(L,ML, 

MH,H) 

X 

(H) 

Chronic oncogenicity 

(Mouse; MRID 

00141659) 

N N NE NE N X 

(M, H) N 

Chronic toxicity 

(Dog; MRID 

00076436) 

NR NR NR NR NR X 

(H) N 

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

treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that 

is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unless 

otherwise indicated. CS= Clinical signs (tremors); BW= Body weight 

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

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

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

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

4. Both doses in the assay were considered to be overtly toxic, therefore no findings are reported in the table for 

these parameters. 

5. Persistent clinical signs (tremors) observed at both doses tested (10 and 20 mg/kg/day) and body weight loss (9-

10%) at 20 mg/kg/day. 

6. Marginal decreases in thyroid colloid area and increases in follicular cell height were observed in all animals 

 N Negative findings 

 P Positive findings 

NE Not examined 

NR Not reported 

Page 27 of 63
PHOSMET FINAL 

Page 20 of 55 

IV. Committee’s Assessment of Weight of Evidence 

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

potential of phosmet to interact with endocrine mediated processes (i.e., E, Aor 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 mediated through an endocrine pathway, therefore nonendocrine 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 Tier 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 assays 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 63
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1. Tier 1 in vivo Assays 

ChE activity was not measured in any of the mammalian in vivo Tier 1 assays. In the 

Hershberger assay, signs of clinical toxicity included tremors and/or uncoordinated movement up 

to four hours post-dose were seen in most of the high-dose (25 mg/kg/day) animals; no clinical 

signs were observed at doses ≤15 mg/kg/day. At the high dose, body weight gain was decreased 

(p<0.05) by 69% in the androgenic portion of the Hershberger assay. In the anti-androgenic 

portion of the assay, body weight and body weight gain were decreased (p<0.05) by 12% and 

60%, respectively, at Study Day 11. Organ weights, other than for the accessory sex organs, were 

not evaluated in the androgenic and anti-androgenic parts of the assay. 

In the uterotrophic assay, no clinical signs of toxicity were observed at either dose (5 or 

20 mg/kg/day). After the 5-day exposure period, final body weights and body weight gains at 

both doses were comparable to control. 

In the female pubertal assay, tremors were seen in 10/16 rats at the 10 mg/kg/day dose level 

(during 10 days) and all animals at the 20 mg/kg/day dose (during 18 days). The severity of the 

tremors ranged from “not characterized to moderate.” Decreased body weights (9 to 10%) were 

observed at the high dose during PND 26 to 36. Additionally, intermittent body weight gains in 

this group were decreased (p<0.05) by up to 30%, but overall body weight gain was decreased 

(NS) by only 4%. 

In the male pubertal assay, tremors were observed in one 10 mg/kg/day rat (time of occurrence not 

provided) and in 11 of 16 rats at 20 mg/kg/day. Body weight differences in treated animals versus 

control were <10%, and overall body weight gain was decreased (p<0.05) only 6 to 7%. 

In the FSTRA, survival was decreased in all treatment groups; 83, 75 and 9% in the low-, midand high-dose groups. However, 3 of the 4 females that died in the low treatment group were in 

the same replicate (replicate A). There were clinical signs of overt toxicity in the mid and high 

treatments (e.g., lethargy, loss of equilibrium). Body weights in the intermediate-concentration 

group were decreased (NS) by 12 to 15%. In addition, there was increased oocyte atresia in all 

treatment groups (69% in the control group and 36–83% in the treatment groups) due to 

microsporidia infections but fecundity in the control and low treatment groups, 16 and 19 

eggs/female/day/replicate, respectively, did not appear to be affected. 

In the AMA, no overt or systemic signs of toxicity were observed at test concentrations up to 

1.0 mg/L. Bent tail (spinal curvature) was observed (30 to 49%), but this finding occurred in the 

control and the two lower phosmet concentrations compared to the high concentration. 

Page 30 of 63
PHOSMET FINAL 

Page 23 of 55 

2. OSRI 

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

In the subchronic neurotoxicity study in rats, ChE activity was determined in whole blood, 

plasma, RBC, and six separate regions of the brain. In the low-dose group (1.5/1.6 mg/kg/day, 

m/f), mean RBC ChE activity was decreased (p<0.01) in males at Week 13 (↓19%) and females 

at Week 7 (↓42%). Decreases (p<0.05 or 0.01) were also observed at Week 13 in mean plasma 

ChE activity in females (↓29%), mean whole blood ChE activity in both sexes (↓16–19%), and 

ChE activity of the olfactory bulb (↓36%) and brain stem (↓21%) regions in females. In the middose group (2.7/3.1 mg/kg/day, m/f), decreased (p<0.05 or 0.01) mean ChE activities were 

observed in plasma ChE in the males at Week 3 (↓21%) and females at Weeks 3 and 13 

(↓27-46%); RBC ChE in the males at Weeks 3, 7, and 13 (↓26–39%) and females at Week 7 

(↓38%); whole blood ChE at Weeks 3, 7, and 13 in both sexes (↓24–36%); and whole brain ChE 

at Week 7 in the females (↓20%) and at Weeks 3 and 7 in the males (↓11–17%). In the high-dose 

group (9.4/11 mg/kg/day, m/f), decreased (p<0.05 or 0.01) mean ChE activities were observed in 

plasma ChE at Weeks 3, 7, and 13 in both sexes (↓23–71%); RBC ChE at Weeks 3, 7, and 13 in 

males (↓65–70%) and at Weeks 3 and 7 in females (↓66–89%); whole blood ChE in both sexes 

(↓59–74%) at Weeks 3, 7, and 13; whole brain ChE in both sexes (↓43–68%) at Weeks 3 and 7; 

and in all six brain regions in the females (↓36–67%) and in all brain regions (except for 

olfactory region) in the males (↓27–52%) at Week 13. 

In the two-generation reproduction study, tremors were exhibited in the high-dose (23 mg/kg/day) 

F0 and F1 animals. Serum ChE activity was decreased in mid-dose (6.1 mg/kg/day) F0 females 

(p<0.01, ↓24%) and high-dose F0/F1 males (p<0.01, ↓30/41%) and females (p<0.01, ↓65/63%). 

RBC ChE activity was decreased in low-dose (1.5 mg/kg/day) F0 males (p<0.05, ↓8%), low-dose 

F0/F1 females (p<0.01, ↓11/16%), mid-dose F0/F1 males (p<0.01, ↓37/48%) and females (p<0.01, 

↓48/59%), and high-dose F0/F1 males (p<0.01, ↓74/85%) and females (p<0.01, ↓81/80%).

In the chronic oral toxicity/carcinogenicity study in rats, dose-related inhibition (p≤0.01 or 0.05) 

of serum ChE activity occurred in males in the high-dose (23/27 mg/kg/day, m/f) group at all 

sample times and in the high-medium group (9.4/10.9 mg/kg/day, m/f) at 18 months, and in 

females in the high-medium- and high-dose groups at all sample times and in the low-medium 

group at 6 months. RBC ChE was inhibited (p≤0.01 or 0.05) in males and females in the lowmedium (1.8/2.1 mg/kg/day, m/f), high-medium and high-dose groups at all sample times, except 

at 12 months in the low-medium animals. RBC ChE also was inhibited in the low-dose males 

(1.1 mg/kg/day) only at the 6-month sample time. Brain ChE was inhibited (p≤0.01) in males 

and females in the high-medium- and high-dose groups at all sample times, and in the 24-month 

low-dose samples in males (p<0.05). 

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In the mouse carcinogenicity study, a dose-related trend in the incidence of convulsions was 

attributed to phosmet administration. Inhibition (significance not reported) of plasma ChE 

activity occurred in males in the low (1.0/1.2 mg/kg/day, m/f) and mid- (4/5 mg/kg/day, m/f) 

dose groups and mid-dose females (↓11 to 13%) at the interim sacrifice, with lower inhibition 

(↓4 to 10%) observed at study termination. Greater inhibition (significance not reported) of 

plasma ChE activity was noted in high-dose (14/18 mg/kg/day, m/f) males and females (↓54 to 

56%) at the interim sacrifice, with a similar degree of inhibition (↓47 to 52%) observed at study 

termination. RBC ChE activities were not determined at the interim sacrifice, but were not 

different from control at study termination. Brain ChE was inhibited (significance not reported) 

in males and females in all dose groups to a similar degree (↓22 to 34) at the interim sacrifice; at 

study termination, brain ChE activity in males was not different from control, and was decreased 

only in mid- and high-dose females (↓14 and 22%, respectively). 

In the chronic dog study, brain and RBC ChE inhibition was observed in the high-dose 

(10 mg/kg/day) animals (significance and details were not reported); no additional treatmentrelated effects on ChE activity were observed at the mid (1.0 mg/kg/day) or low (0.5 mg/kg/day) 

doses. 

In the 21-day dermal toxicity study in rats, brain ChE activity was decreased (p≤0.01, ↓36%) at 

study termination in high-dose (60 mg/kg/day) males, but plasma and RBC ChE activities were 

unaffected by treatment. In high-dose females, ChE activity was decreased in plasma (p≤0.01, 

↓38%) on Day 22 and decreased in RBC (p≤0.05, ↓29%) on Day 8, and brain ChE activity was 

inhibited (p≤0.01) in a dose-dependent manner (↓13, 20, and 61%) in the low (15 mg/kg/day)-, 

mid (22.5 mg/kg/day)-, and high- (60 mg/kg/day) dose groups, respectively. 

In the non-Guideline developmental study (Staples et al., 1976), animals in the high-medium and 

high-dose gavage groups (25 and 30 mg/kg/day) died. Additionally, in the rabbit developmental 

toxicity study, deaths and signs of clinical toxicity occurred in the high-dose group (15 

mg/kg/day). 

Several wildlife Part 158 studies were also available. However, it should be noted that ChE was 

not measured in any of these studies. Observations were primarily based on clinical signs of 

toxicity. Two avian reproduction studies were conducted including the mallard and the bobwhite 

quail, both at 25, 60, and 150 ppm dose levels. There were no reported signs of clinical or overt 

toxicity in either study. 

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B. Estrogen Pathway 

While the Tier 1 ER binding assay was considered inadequate (due to solubility issues), the Tier 

1 ERTA assay as well as an additional ERTA study which evaluated both agonism and 

antagonism was negative. Furthermore, the Tier 1 uterotrophic assay was negative. While the 

Tier 1 in vitro aromatase assay was positive, the steroidogenesis assay was negative. There were 

no estrogen-related effects observed in the Part 158 mammalian and wildlife studies, except for 

an increased incidence of dilation and myometrial atrophy of the uterus observed at 12 and 24 

months, respectively, in the mouse carcinogenicity study. In the female pubertal assay, although 

all test doses were considered to be overtly toxic, no estrogen-related effects were observed in 

this study. Similarly, the mid and high treatment groups in the FSTRA were considered to be 

overtly toxic, and the information obtained from the low concentration was limited (due to 

essentially loss of one of four replicates). In the FSTRA at this low concentration, effects on 

female gonadal histopathology (decreased yolk synthesis, and occasional disruptions of follicular 

VTG concentration) were observed. It is noted that there were no other effects in the low 

concentration observed including alterations in GSI and plasma VTG. Therefore, the available 

data do not suggest that phosmet has the potential to act as an estrogen agonist based on the in 

vitro and uterotrophic studies. Additionally, while the in vitro aromatase assay was positive 

which could indicate a potential decrease in estradiol production, the in vitro steroidogenesis was 

negative and the results of both the female pubertal and FSTRA, do not support an antiestrogenic effect. 

C. Androgen Pathway 

While the Tier 1 AR binding assay was positive, the Tier 1 Hershberger was negative and no 

androgen-related effects were observed in the Tier 1 male pubertal assay in the absence of overt 

toxicity. Additionally the Tier 1 steroidogenesis assay was negative. In the FSTRA, based on 

the data available, there were no androgen-related effects observed in the absence of overt 

toxicity. The only androgen-related effect observed in the mammalian Part 158 studies was an 

increase in the incidence of testicular atrophy in the mouse carcinogenicity study. Overall there 

is no convincing evidence for potential interaction with the androgen pathway. 

D. Thyroid Pathway 

In the Tier 1 male pubertal assay, T4 levels were increased and changes in thyroid 

histopathology (decreased colloid area and increases in follicular cell height) were observed at 

both doses, although the effects at the high dose occurred in the presence of overt toxicity. 

Similarly, thyroid effects (increased T4 levels and histopathology) were observed in the Tier1 

female pubertal assay; however, both test doses were considered to be overtly toxic in this assay. 

In the Part 158 mammalian studies, histopathological changes in the thyroid were observed at 

10.9 mg/kg/day in the chronic rat study. In the AMA, while developmental alterations were 

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observed (↓NF stage and HLL), there were no effects on thyroid histopathology. Therefore, the 

observed developmental delays are likely not thyroid-mediated. 

E. Conclusions 

There is no convincing evidence for a potential interaction with the estrogen or androgen 

pathway in mammals or wildlife. The available data indicate a potential interaction with the 

thyroid pathway in mammals, but not amphibians. 

V. EDSP Tier 2 Testing Recommendations 

In the EDSP Tier 1 male and female pubertal and OSRI, there was evidence for potential 

interaction with the thyroid pathway in studies conducted with adult animals, but no such data 

exists for potential thyroid toxicity in the young animals. 

For phosmet, the current Point of Departure (POD) of 3.56 mg/kg/day used for human health risk 

assessment is based on red blood cell (RBC) cholinesterase inhibition, which is the most 

sensitive endpoint for this class of chemicals, observed at 7.0 mg/kg/day in rats. This POD was 

used for deriving the chronic Reference Dose (RfD) used for assessing chronic dietary risk for 

the general population. A second POD of 0.16 mg/k/day used for deriving the chronic RfD for 

the young (the most sensitive population) is based on RBC cholinesterase inhibition in the 

offspring. This POD was also used for calculating the Margins of Exposure (MOEs) used for 

non-dietary risk assessments. 

In the case of phosmet, although no data exists for potential thyroid toxicity in the young, a 

comparative thyroid assay (CTA) is not recommended since: 1) the POD for the young is 

approximately 63-fold lower than the low doses (10 mg/kg/day) that caused thyroid-related 

effects in the Tier 1 in vivo pubertal assays; 2) the POD for the adults and the young is based on 

the most sensitive endpoint (cholinesterase inhibition); 3) the CTA would be tested at higher 

doses and would not be expected to yield a POD lower than the level currently used for risk 

assessment; and 4) the chronic RfD and MOEs are protective of the thyroid related findings in 

the Tier 1 in vivo assays. Therefore, mammalian EDSP Tier 2 testing is not recommended for 

phosmet since additional testing is not expected to impact EPA’s current regulatory point of 

departures and endpoints for human health risk assessments. 

As there was no evidence of potential interaction with the estrogen or androgen pathway in fish, 

or thyroid pathway in amphibians, additional testing is not recommended for these taxa. 

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard 

duck are available for phosmet. In bobwhite quail, effects on reproductive parameters were 

observed at concentrations where mortality and adult body weight decreases were observed; no 

reproductive effects were observed in the mallard duck study. The type of data obtained from 

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Page 27 of 55 

Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating 

potential reproductive effects to birds from phosmet exposure. Relative to the EDSP, additional 

testing is not recommended. 

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VI. References 

Cappon, G.D. (1999). A Dietary Subchronic (90-Day) Neurotoxicity Study of Phosmet in Rats. 

Unpublished study performed by WIL Research Laboratories, Inc. Study No. WIL-331002. 

(MRID 44811801). 

Chang, J.C.F., Morrissey, R.L., and Wyand, S. (1991). 2-Year Chronic Toxicity/Oncogenicity 

Study with R-1504 in Rats. Unpublished study performed by Ciba-Geigy Corp. Laboratory 

Report No. T-13241. (MRID 41916401). 

Cohle, P. (1988). Early Life Stage Toxicity of 14C-Phosmet to Rainbow Trout (Salmo gairdneri) 

in a Flow-Through System. Unpublished study performed by Analytical Bio-Chemistry 

Laboratories, Inc. Laboratory Report No. 35777. (MRID 40938701).

Courtney, K.D. (1968). Teratological investigation of Captan, Imidan and Thalidomide in 

Macaca mulatta. Final report. Unpublished study performed by Bionetics Research Laboratories, 

Inc. (MRID 00043397).

Fletcher, D., Jenkins, D., and Leonard, M. (1982a). Report to Stauffer Chemical Co.: Toxicity 

and Reproduction Study with Imidan Technical in Bob-white Quail. Unpublished study 

performed by Bio-Life Assoc., Ltd Laboratory Report No. BLAL 81 QR2 (T-10817). 

(MRIDs 00129519, 00125786). 

Fletcher, D., Jenkins, D., and Leonard, M. (1982b). Report to Stauffer Chemical Company: 

Toxicity and Reproduction Study with Imidan Technical in Mallard Ducks. Unpublished study 

performed by Bio-Life Associates, LTD Laboratory Report No. BLAL 81DR2 (T-10816). 

(MRID 00105999). 

Hilaski, R.J. (1999). A 21-Day Dermal Toxicity Study of Imidan in Rats. Unpublished study 

performed by MPI Research. Laboratory Report No.: 815-002. (MRID 44795801). 

Hodge, M.C.E. (1991). Phosmet: Teratogenicity Study in the Rat. Unpublished study performed 

by ICI Central Toxicology Laboratory Report No.: CTL/P/3360: RR0544. (MRID 41962902). 

Katz, A.C., Sprague, G.L., Frank, D.W., Turnier, J.C., Zwicker, G.M., and Freundenthal, R.I. 

(1984). Two-year Dietary Oncogenicity Study in Mice with lmidan Technical: Final Report: 

T-10719. Unpublished study performed by Stauffer Chemical Company Laboratory Report No.: 

T-10719. (MRIDs 00141659, 0016114, and 40595501). 

Kojima, H., Katsura, E., Takeuchi, S., et al. (2004). Screening for Estrogen and Androgen 

Receptor Activities in 200 Pesticides by In Vitro Reporter Gene Assays Using Chinese Hamster 

Ovary Cells. Environ. Health Perspect. 112(5):524–531. (MRID 48033008).

Page 36 of 63
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Lee, M.R. (2012). Phosmet—Amphibian Metamorphosis Assay with African Clawed Frog 

(Xenopus laevis) Following OPPTS Test Guideline 890.1100 and OECD Test Guideline No. 

231. Unpublished study performed by Smithers Viscient, Wareham, Massachusetts 02571. 

Laboratory report number 12791.6142. Study completed February 27, 2012. (MRID 48673001). 

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

R-1504. Unpublished study performed by Ciba-Geigy Corporation Laboratory Report No. 

T-13260. (MRID 41520001). 

Moxon, M.E. (1991). Teratology Study in the Rabbit. Unpublished study performed by ICI 

Central Toxicology Laboratory Report No.: CTL/P/3373: RB05045. (MRID 41962901). 

Quinto, I., DeMarinis, E., Mallardo, M., Arcucci, A., Della Morte, R., and Staiano, N. (1989). 

Effect of DNOC, Ferbam and Imidan Exposure on Mouse Sperm Morphology. Mutat Res

224:405–408. (MRID 48084301). 

Staples, R.E., Kellam, R.G., and Haseman, J.K. (1976). Developmental toxicity in the rat after 

ingestion or gavage of organophosphate pesticides (Dipterex, Imidan) during pregnancy. 

Environ. Health Perspect. 13:133–140. (MRID 00063192). 

USEPA. (2010). Phosmet: Report of the Endocrine Disruptor Review Team — Test Order #: 

EDSP-057801-36. August 5, 2010. Office of Chemical Safety and Pollution Prevention. U.S. 

Environmental Protection Agency, Washington, DC. 

USEPA. (2011). Endocrine Disruptor 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. 

Wagner, H. (2011). Phosmet: H295R Steroidogenesis Assay. Unpublished study conducted by 

CeeTox Inc., Kalamazoo, MI. Laboratory Study No. 9142V-100348STER, November 3, 2011. 

(MRID 48618701). 

Wilga, P.C. and C. Toole. (2011). Phosmet: Human Recombinant Aromatase Assay. 

Unpublished study conducted by CeeTox Inc., Kalamazoo, MI. Laboratory Study No. 

9142V-100348AROM, November 3, 2011. (MRID 48618702). 

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Willoughby, J.A (2011). Phosmet: Androgen Receptor Binding (Rat Prostate Cytosol). 

Unpublished study conducted by CeeTox, Kalamazoo, MI. Laboratory Study #: 

9142V-100348ARB, November 2, 2011. (MRID 48618703). 

Willoughby, J.A. Sr. (2011). Phosmet: Estrogen Receptor Binding (Rat Uterine Cytosol). 

Unpublished study conducted by CeeTox, Kalamazoo, MI. Laboratory Study #: 

9142V-100348ERB, November 2, 2011. (MRID 48618704). 

Willoughby, J.A. Sr. (2011). Phosmet: Estrogen Receptor Transcriptional Activation (Human 

cell Line, HeLa-9903). Unpublished study conducted by CeeTox, Kalamazoo, MI. Laboratory 

Study #: 9142V-100348ERTA, November 2, 2011. (MRID 48618705). 

York, D.O. (2012). Phosmet — Short-term Reproduction Assay with the Fathead Minnow 

(Pimephales promelas) Following OPPTS 890.1350 and OECD 229 Guidelines. Unpublished 

study performed by Smithers Viscient, Wareham, Massachusetts. Lab Study No.: 12791.6143. 

(MRID 48673002). 

Zorrilla, L. (2011). The Hershberger assay with phosmet in castrated CRL:CD(SD) IGS Rats. 

Unpublished study conducted by Integrated Laboratory Systems, Inc., Durham, NC. Laboratory 

Project Study ID: C194-200, October 26, 2011. (MRID 48618706). 

Zorrilla, L., and Codrea, E. (2011). The Uterotrophic Assay with Phosmet in Ovariectomized 

CRL:CD(SD) IGS Rats. Unpublished study conducted by Integrated Laboratory Systems, Inc., 

Durham, NC. Laboratory Project Study ID: C194-100, October 19, 2011. (MRID 48618707). 

Zorilla, L. (2012). Pubertal developmental and thyroid function with phosmet in 

juvenile/peripubertal CRL:CD(SD) IGS female and male rats. Unpublished study conducted by 

Integrated Laboratory Systems, Durham, NC, Laboratory Study No.: C194-300, February 24, 

2012. (MRID 48673003). 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100) 

The 21-day assay (MRID 48673001) of phosmet on amphibian metamorphosis of African 

Clawed Frog (Xenopus laevis) was conducted under flow-through conditions. Amphibian larvae 

(80 tadpoles/treatment group, 20/replicate) at Nieuwkoop-Faber (NF) stage 51 were exposed to 

phosmet (96.8% purity) at nominal concentrations of 0 (negative control), 0.020, 0.20, and 2.0 

mg a.i./L. Mean-measured concentrations were <0.0022 (<LOQ; control), 0.0081, 0.096, and 1.0 

mg a.i./L The test system was maintained at 20 to 23°C and a pH of 7.4 to 8.2. 

Phosmet significantly decreased normalized hind-limb length (HLL) in non-late stage tadpoles 

(NF stage < 60) at the mean-measured 0.091 and 1.0 mg a.i./L treatment groups on Days 7 and 

21. Median Nieuwkoop-Faber (NF) stage was unaffected on Day 7, though a significant delay 

was observed at on Day 21 at the mean-measured 0.096 and 1.0 mg a.i./L treatment groups. No 

asynchronous development was observed. Mild thyroid gland hypertrophy, follicular cell 

hyperplasia, and follicular cell hypertrophy were observed at all treatment levels, including 

controls; there was no apparent treatment-related pattern in histopathological findings. Tadpoles 

with spinal curvature were observed in 30 to 49% of tadpoles in the control and all treatment 

groups. Unless otherwise reported, all analyses are based on comparison to the negative 

(FETAX) control, and analyses of SVL, normalized HLL, and body weight were performed 

excluding late stage (NF stage > 60) tadpoles. 

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

In an androgen receptor (AR) binding assay (MRID 48618703), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of the AR. The saturation binding experiment was 

conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonable 

numbers and was functioning with appropriate affinity for the radiolabeled reference androgen 

(R1881). 

Saturation binding data were not originally provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. The mean dissociation constant (Kd) for [3

H]-R1881 was 

1.080±0.690 nM, and the estimated Bmax was 1.071±0.167 fmol/100 μg protein for the single 

batch of prostate cytosol that was used in the assay. Only one of the three Kd values was within 

the range reported in the EPA validation program (0.685 to 1.57 nM), and all Bmax values were 

below the recommended range (7 to 16 fmol/100 μg protein). Although the goodness of fit for 

individual runs was acceptable (R2

 = 0.880-0.986), confidence in these numbers is low based on 

the large variation in Kd estimates among runs.

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

concentration of [3H]-R1881 (10 nM) in the presence of increasing concentrations of phosmet 

(96.8% purity; logarithmic increase from 10-10 to 10-3 M). Dimethyl sulfoxide (DMSO) was 

used as a vehicle at a final assay concentration of approximately 3.2%. The assay included 

dexamethasone as a weak positive control, and R1881 as the ligand reference standard. Three 

independent runs were conducted with 3 replicates per concentration per run. The estimated log 

IC50s for R1881 and the weak positive control (dexamethasone) were in the ranges of -9.0 to -9.9 

M and -4.3 to -4.6 M, respectively. Phosmet displaced more than 50% of the radiolabeled R1181 

from the AR in all three competitive binding runs. The estimated log IC50 was -5.0 M in each 

run and averaged -5.0 M; therefore, phosmet is classified as an AR “binder”. The mean relative 

binding affinity (RBA) for the positive control dexamethasone was 0.0013%. The RBA for 

phosmet was calculated to be 0.0050%, indicating a stronger affinity for the AR than the weak 

positive control. Confidence in these numbers is high due to the small variation. The solvent 

control responses indicated no drift in the study assay. All performance criteria were met, with 

the exception of the bottom (% binding) of dexamethasone in Assay #1, and the bottom plateau 

level could not be accurately calculated because of a deviation where the concentration range for 

dexamethasone was prepared slightly different than that specified in the guideline. This was 

considered a minor deviation. 

Based on the results of the three competitive binding runs, phosmet is classified as a Binder in 

the Androgen Receptor Binding Assay. 

Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48618702), phosmet (96.8% a.i., batch # 

GQH9118000) in dimethyl sulfoxide (DMSO) was incubated with human recombinant 

aromatase and tritiated androstenedione ([1β3

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

concentrations of 0, 10−3, 10−3.5,10−4, 10−5, 10−6, 10−7, 10−8, 10−9, or 10−10 M for 15 minutes at 

37°C to assess the effect on aromatase activity. Due to slight precipitation that was observed at 

the high concentration in the first two runs, the high concentration was decreased to 10-3.5 M for 

the third run. 

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

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

quantified using liquid scintillation counting (LSC). Three independent runs were conducted, 

and each run included a full activity control, a background activity control, a positive control 

series (10−10 to 10−5

 M) using a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), and 

the test chemical series (10−10 to 10−3) with 3 replicates per concentration. 

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Aromatase activity in the full activity controls ranged from 0.150 to 0.376 nmol·mgprotein−1

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

·min−1. Activity in the background controls ranged from 0.37% to 0.97% and averaged 

0.56% of the full control activity. The response of the full activity controls and background 

controls were acceptable for each run. However, each full activity control was not generally 

within 10% of the average for the full activity controls, as recommended by the EPA. Run 1 had 

roughly half the activity of the other runs and the 0.5 mL count for total activity was roughly half 

that of the other runs. This discrepancy was not explained in the methods of the study report, 

and applies to both the controls and phosmet in run 1 compared to runs 2 and 3. 

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

nmol·mg-protein−1·min−1 at the lowest tested concentration (10−10 M) and 0.002±0.001 nmol·mgprotein−1

·min−1 at the highest tested concentration (10−5 M). The results were generally within 

the recommended ranges for the top of the curve, bottom curve, Hill slope, log IC50, and 

coefficient of variation for replicates of each concentration. 

For phosmet, aromatase activity averaged 0.231±0.059 nmol·mg-protein−1·min−1 at the lowest 

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

 at the highest tested 

concentration of 10−3 M. The lowest portion of the response curve across runs for phosmet was 

less than 50% activity, indicating that phosmet is an inhibitor of aromatase activity at the highest 

soluble concentration. Phosmet decreased aromatase activity to 34% at 10−4

 M. 

The estimated log IC50 ranged from -4.37 to -4.89 M for phosmet and averaged -4.54 M 

compared to 4OH-ASDN, which averaged -7.21 M. The average slope of the concentration 

response curve was -1.20 for phosmet and -0.97 for 4OH-ASDN. Confidence in the mean log 

IC50 for both chemicals and the Hill slope for the positive control is high due to the small 

variation, but the variation was moderate (22.5% CV) for the slope value of phosmet. At 10-10 M 

in Run 2, aromatase activity was only 69-73% of control, as compared to 95-101% of control in 

the other runs; no explanation was provided. 

Based on the data from the average response curve, phosmet is classified as an Inhibitor of 

aromatase activity in this assay 

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

In an estrogen receptor (ER) binding assay (MRID 48618704), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct saturation and competitive binding 

experiments. A saturation binding experiment 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 

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binding experiments. The competitive binding experiment was conducted to measure the 

binding of a single concentration of [3H]-17β-estradiol (1 nM) in the presence of increasing 

concentrations of phosmet (96.8% purity; logarithmic increase from 10-10 to 10-3 M). DMSO 

was used as a vehicle at a final concentration of <4%. The assay included 19-norethindrone as a 

weak positive control, octyltriethoxysilane as a negative control, and 17β-estradiol as the natural 

ligand reference material. 

Saturation binding data were not provided in the study report; however, summarized saturation 

binding data (MRID 48843501) from the performing laboratory were submitted following a 

request by the Agency. 

For the Competitive Binding Experiment, a log IC50 could not be calculated for phosmet. The 

mean log IC50 for 17β-estradiol was −9.0 M and the average log IC50 for 19-norethindrone was 

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

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

Based on the top % binding for 19-norethindrone at 49%, Run 2 should not be considered for 

evaluation of phosmet for binding to the ER. The Hill slope for 17β-estradiol in Run 3 (−1.7) 

was outside of parameter limits. Precipitation was observed at 10-4M (but not 10-3 M) in Run 1 

and at the 10-3 M in Runs 2 and 3. In these instances, data for those concentrations were 

disregarded by the study author despite indications of potential interaction with the estrogen 

receptor. The investigators failed to follow assay guidelines that recommend testing solubility of 

test substances in increasing solvent concentrations (up to 10% DMSO) and including half log 

concentrations at the limit of solubility. Because of insufficient attempts to resolve issues with 

solubility of the test substance at concentrations that suggested an ER interaction, there is lack of 

confidence in the results of the three runs; therefore the assay is considered to be technically 

inadequate. 

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

In an estrogen receptor transcriptional activation assay (ERTA; MRID 48618705) conducted by 

CeeTox, hERα-HeLa-9903 cells cultured in vitro were exposed to phosmet (96.8% a.i., lot# 

GQH91800) at concentrations increasing logarithmically from 10-11 to 10-4 M mg/mL in DMSO 

(0.1%) for approximately 24 hours. The assay was performed twice using 96-well plates and 

each phosmet concentration was tested in 6 wells/plate in each assay. Cells were exposed to the 

phosmet for 24 ± 2 hours to induce reporter (luciferase) gene products. Luciferase expression in 

response to activation of the estrogen receptor by phosmet was measured using a proprietary 

CeeTox Standard Operating Protocol (SOP), which was not included in the report. 

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Phosmet was tested up to the limit of cytotoxicity, 10-4 M. The mean RPCMax for phosmet was 

1.6-3.9%, the PCMax was 10-7 M and the PC10 could not be calculated as the RPCMax was less 

than 10%. Cytotoxicity of 20.1% was observed in the highest concentration in the first run. A 

similar trend of cytotoxicity was observed in the second run, with the highest concentration 

having cell viability of only 81.7%. 

In the main assays, the responsiveness of 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. The PC10 for 17α-methyltestosterone should have been reached by 

10-6 M but its maximum RPC was 8.8%, at that concentration. A higher concentration (10-5 M) 

reached 47% RPC but in the presence of cytotoxicity. Cytotoxicity was also observed at the 

highest concentrations of 17α-estradiol and corticosterone. Although the conditions of this assay 

were not optimal to detect very weak activity, phosmet responses were similar to those of the 

negative control corticosterone and not comparable to the responses of 17α-methyltestosterone, 

which was able to reach a maximum of 6.8-8.8% PC. Phosmet was only able to reach a 

maximum of 1.6-3.9% PC when tested up to the highest concentration possible based on 

cytotoxicity. Because the RPCMax < PC10 in both assay runs, phosmet was considered negative 

for estrogen receptor transcriptional activation in this test system. 

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

The 21-day short-term reproduction assay (MRID 48673002) of phosmet with fathead minnows 

(Pimephales promelas) was conducted under flow-through conditions. Sixteen groups of 6-

month old fish (2 males and 4 females per group; 4 groups per treatment) were exposed to 

phosmet (96.8% purity) at nominal concentrations of 0 (control), 0.0015, 0.015, and 0.150 mg 

a.i./L; mean-measured concentrations were <LOQ (<0.00021), 0.001, 0.0093, and 0.105 mg 

a.i./L. The test system was maintained at 24 to 25oC and a pH of 6.9 to 7.7. 

Survival was low at the high treatment level (8%), with only one male and one female surviving 

to study termination; endpoints at this treatment level were not statistically analyzed or included 

in the results. At the intermediate concentration, there was a 25% decrease in survival, compared 

to control, and generalized overt signs of toxicity in both males and females, including lethargy 

in all surviving fish and loss of equilibrium in females. At the low concentration, survival was 

reduced by 13% compared to control. Body weight and length were not significantly different in 

male or female fish at any treatment level. 

In the control group, spawning occurred at least every 4 days in two of the four replicates and 

fecundity was 16 eggs/female/day/replicate; fecundity was 15 eggs/female/day for three 

replicates and 6.4 eggs/female/day for one replicate. Fertilization success in the control group 

averaged 97%. At the intermediate concentration, phosmet significantly reduced fertility 

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(Jonckheere-Terpstra; p<0.05) by 7% and fecundity (Jonckheere-Terpstra; p<0.05) by 81%, 

compared to the control. 

Female gonado-somatic index (GSI) was significantly reduced by 34% at the intermediate 

concentration (Jonckheere-Terpstra; p<0.05) relative to control; male GSI was unaffected by 

phosmet treatment (p>0.05). While not statistically significant (Mann-Whitney; p=0.07), median 

male nuptial tubercle scores were reduced by 42% at the intermediate concentration compared to 

the control; no tubercles were observed for females. Male and female vitellogenin (VTG) levels 

were not significantly different from the control, although there was an apparent 1175% increase 

in male VTG at the intermediate concentration due solely to high levels in two males in this 

group. Plasma sex steroids were not measured. 

Gonadal staging effects were indicated for female fish at the low treatment concentration, and for 

males and females at the intermediate concentration. Effects on gonadal histopathology in male 

fish included an increased percentage of spermatogonia (4/6) and mild diffuse thickening of the 

germinal epithelium lining by spermatogonia at the intermediate concentration; in female fish, 

incidences of decreased yolk synthesis were observed at both low (2/11) and intermediate (7/12) 

concentrations relative to the controls (0/16). Occasional disruptions of VTG concentration were 

noted in the follicles of some females, and minimal interstitial fibrosis was observed at the 

intermediate concentration. In addition, incidences of increased oocyte atresia were observed in 

the control and treatment groups, with 11/16, 4/11, and 9/12 of the fish exhibiting this finding in 

the control and low and mid treatments, respectively. In 44% of these cases, granulomatous 

inflammation was observed; evidence of microsporidia infection was also observed. 

In summary, at the mid-concentration, fecundity, fertilization, and female were GSI were 

decreased compared to controls. Gonadal histopathology was affected as shown by decreased 

yolk synthesis in the low- and mid- concentration females, and occasional disruptions of 

follicular VTG concentration. The proportion of spermatogonia in the testes was increased at the 

high concentration. High mortality and clinical signs of toxicity in this study may confound the 

interpretation of these parameters. 

Hershberger Assay; (OCSPP 890.1400) 

The Hershberger Assay consists of androgenic and anti-androgenic components. To screen for 

potential androgenic activity, phosmet (96.8% a.i., Batch # 6QH9118000) in 0.5% aqueous 

carboxymethyl cellulose was administered daily via oral gavage to 59- to 60-day old, castrated 

male Sprague Dawley rats (8/dose group) at dose levels of 0 (vehicle), 5 or 25 mg/kg/day. An 

androgenic positive control group consisted of 10 castrated rats dosed by subcutaneous injection 

with 0.4 mg/kg/day testosterone propionate (TP) prepared in corn oil. 

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To screen for potential anti-androgenic activity phosmet (96.8% a.i., Batch # 6QH9118000) in 

0.5% aqueous carboxymethyl cellulose was administered daily via oral gavage to 59 to 60-day 

old, castrated male Sprague Dawley rats (8/dose group) at dose levels of 0 (vehicle), 5, 15, or 25 

mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by 

subcutaneous injection. An anti-androgenic positive control group consisted of 10 castrated rats 

exposed to 0.4 mg/kg/day TP and 3.0 mg/kg/day flutamide (FT). Flutamide and TP were 

prepared in corn oil. 

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 

the five androgen-dependent tissues. 

All animals survived until scheduled termination. Cholinergic signs (tremors) and a 12% 

decrease in terminal bodyweight was seen at 25 mg/kg/day suggesting that the high dose may 

have been excessive. 

In the androgen agonist assay, clinical signs in the 25 mg/kg/day group included tremors, 

uncoordinated movements and porphyrin staining around the eyes. No clinical signs were 

observed in the 5 mg/kg/day group. Body weights in the 5 and 25 mg/kg/day groups were 

comparable to controls throughout the duration of the assay. At 25 mg/kg/day, mean overall 

(Days 1-11) body weights gains were decreased (p≤0.05) by 69%. Animals dosed with 

0.4 mg/kg/day TP had increased (p≤0.05) mean body weights of 12% on Day 11, and increased 

mean overall body weight gains (p≤0.05) of 105%. There were no significant increases in the 

accessory sex organ weights in animals dosed with the test compound. Animals in the 0.4 

mg/kg/day TP group had accessory sex organ weight increases (p≤0.05) as follows: 1926% in 

seminal vesicles; 1264% in ventral prostate; 251% in levator ani-bulbocavernosus (LABC); 

794% in Cowper’s gland; and 72% in glans penis. All tissue weight CVs were below the 

maximum values recommended by the test guideline. The performance criteria indicated that 

this assay was performing as expected. 

In the anti-androgen assay, clinical signs in the 25 mg/kg/day group included tremors, and 

uncoordinated movements. No clinical signs were observed in the 5 or 15 mg/kg/day groups. At 

25 mg/kg/day, mean body weights were decreased (p≤0.05) by 12% on Day 11, and overall body 

weight gains were decreased (p≤0.05) by 60%. There were no effects on body weights or body 

weight gains in the 5 and 15 mg/kg/day group, or the positive control group. At 15 mg/kg/day, 

the weights of seminal vesicles were decreased 25% (p≤0.05). At 25 mg/kg/day, accessory sex 

organ weight decreases (p≤0.05) were noted as follows: 50% in seminal vesicles; 40% in ventral 

prostate; 27% in LABC; 34% in Cowper’s gland; and 10% in glans penis. Animals dosed with 

0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p≤0.05) accessory sex organ weights as 

follows: 85% in seminal vesicles; 76% in ventral prostate; 50% in LABC; 66% in Cowper’s 

gland; and 21% in glans penis. All tissue weight CVs were below the maximum values 

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recommended by the test guideline. The performance criteria indicated that this assay was 

performing as expected. 

Phosmet was negative for androgenic activity as no statistically significant increases in organ 

weights were seen in two or more of the five androgen sensitive tissues in the androgen agonist 

assay. However, phosmet was positive for anti-androgenicity in the Hershberger assay as 

statistically significant decreases in organ weights were seen in all five androgen sensitive tissues 

in the androgen antagonist assay. However, the decreases in androgen-sensitive tissue weights 

were seen at a dose (25 mg/kg/day) that resulted in overt toxicity (12% decrease in body weight 

and clinical signs), which confounds the interpretation of the results. 

Female Pubertal Assay; (OCSPP 890.1450) 

In a female pubertal assay (MRID 48673003), 16 Sprague-Dawley (Crl:CD®(SD) IGS) rats/dose 

group were treated daily by oral gavage with phosmet (96.8% a.i., Batch #: GQH9118000) in 

aqueous 0.5% carboxymethylcellulose (CMC) at doses of 0, 10, or 20 mg/kg/day (5 mL/kg 

dosing volume) from post-natal day (PND) 22 to 42 or 43. The animals were examined for 

vaginal opening (VO) daily beginning on PND 22, and age and body weight at day of attainment 

was recorded. After sacrifice on PND 42/43, serum thyroxine (T4) and thyroid stimulating 

hormone (TSH) concentrations were analyzed using radioimmunoassays. Various clinical 

chemistry parameters were measured in the serum. Liver, kidney, pituitary, adrenals, thyroid, 

ovaries, and uterus were weighed and examined macroscopically; microscopic examination was 

also conducted on the thyroid, ovaries, uterus, and kidneys. 

No treatment-related effects were observed on mortality, organ weights, serum TSH 

concentrations, clinical chemistry, or gross pathology. Changes (NS) in ovary, uterus, thyroid, 

and pituitary weights were noted in the low- and high-dose animals. There were no treatmentrelated histopathological findings noted in the ovaries, uterus, thyroid, or kidneys. In addition, 

no treatment-related effects were noted on cycle status, mean age at first estrus, mean cycle 

length, percent cycling, or percent regularly cycling. 

One 10-mg/kg/day rat exhibited localized head tremors on a single day, and tremors were 

observed at various times post-dose in up to 10/16 10-mg/kg/day rats on 10 days and up to 

sixteen 20-mg/kg/day rats at 18 days. No other treatment-related clinical signs were observed in 

animals from either dose group. Decreased body weights (p<0.05; ↓9% to 10%) were observed at 

20-mg/kg/day during PND 26 to 36. No significant change in mean final body weight or body 

weight gain was observed in either treatment group compared to the vehicle control group. 

The day of VO was comparable among the groups. Unadjusted and adjusted body weights at VO 

were decreased (p<0.05) by 9% each in the 20 mg/kg/day group compared to control. No 

treatment-related effect was noted on cycle status, mean age at first estrus, mean cycle length, 

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percent cycling, or percent regularly cycling. At 20 mg/kg/day, the number of ovarian antral 

follicles was increased (↑93%), and the number of atretic follicles was decreased (↓29%). All 

other values in the treated groups were similar to control. 

Serum T4 concentrations were increased (↑24 to 38%) in the treated groups. A marginal decrease 

in the thyroid colloid area and increase in the follicular cell height was observed in animals 

administered phosmet (10 and 20 mg/kg) compared to control. 

There were no effects on liver, kidney, pituitary, adrenal, uterus, or thyroid organ weights at 

either dose level compared to the control. 

Thyroid effects (increased serum T4 levels and thyroid histopathology) were seen at both doses. 

However, these doses (10 and 20 mg/kg/day) were determined to be excessive based on 

persistent tremors seen at both doses and severe body weight loss at the high dose. Additionally, 

data from the existing toxicity database show that lower doses should have been tested in this 

assay. Following the agency’s determination, the registrant submitted additional information 

(MRID 49610501) in response to the agency’s review of this assay. The submitted data show 

that tremors were seen in 10/16 rats at the 10 mg/kg/day dose level (during 10 days) and all 

animals at the 20 mg/kg/day dose (during 18 days). The severity of the tremors ranged from “not 

characterized to moderate.” Upon review of these data, the agency reaffirms its previous 

conclusion that both doses tested in this assay were excessive. 

Male Pubertal Assay; (OCSPP 890.1500) 

In a male pubertal assay (MRID 48673003), 16 Sprague-Dawley (Crl:CD®(SD) IGS) rats/dose 

group were treated daily via oral gavage with phosmet (96.8% a.i., Batch #: 6QH9118000) in 

aqueous 0.5% carboxymethylcellulose (5 mL/kg) at doses of 0, 10, or 20 mg/kg/day from postnatal day (PND) 23 to 53 or 54. The animals were examined for preputial separation (PPS) daily 

beginning on PND 30, and age and body weight at day of attainment was recorded. Following 

sacrifice on PND 53/54, total serum testosterone, thyroxine (T4), and thyroid stimulating 

hormone (TSH) levels were analyzed by radioimmunoassay. Various clinical chemistry 

parameters were measured in the serum. Liver, kidney, pituitary, adrenals, seminal vesicles with 

coagulating glands, and ventral and dorsolateral prostate were weighed and examined 

macroscopically; in addition, microscopic examinations were conducted on the epididymides, 

testis, thyroid, and kidneys. 

All rats survived until study termination. Tremors were observed periodically in the majority of 

the rats at 20 mg/kg/day. No other clinical signs of toxicity were observed in the study at either 

dose. 

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The decreases in body weight gains and final body weights were minimal (<10%) and not 

biologically significant. The small variations in clinical chemistry parameters observed at 20 

mg/kg/day were not considered to be indicative of toxic effects on the kidney or liver. 

There was no treatment-related effect on preputial separation at either dose level. 

The adjusted ventral prostate weights showed a linear trend of decreased weight with increasing 

dose; however, no statistically significant effect was detected between the treated groups and 

control. Both means were lower than historical control mean ventral weight for Sprague-Dawley 

male rats but were within the acceptable range. There were no effects on liver, kidney, pituitary, 

adrenal, testis, or other sex organ weights at either dose level compared to the control. 

Testosterone levels were lower in both dose groups compared to the control, but statistical 

significance was not attained and the means were within the expected range for Sprague-Dawley 

rats. It is to be noted that the standard deviation for testosterone in the 20 mg/kg/day dose group 

(1.62) was similar to the mean value (1.80). 

Serum T4 levels were increased in both treatment groups compared to the concurrent control and 

the mean value for Sprague-Dawley male rats, but they were within the acceptable range in the 

performance criteria. Additionally, these increases were within the range of control values 

observed at the analytical laboratory (3-7 μg/dL). TSH levels were slightly higher at both dose 

levels compared to the control, but the control value was below the acceptable range and the 

Guideline criteria for this parameter were not met. 

During the agency’s initial review, the doses (10 and 20 mg/kg/day) used in this study were 

considered excessive, based on the dose-selection rationale and the periodic occurrence of 

tremors in the majority of the males at 20 mg/kg/day. Subsequently, the registrant submitted 

additional data (MRID 4961502) in response to the agency’s review of this assay. The submitted 

data show at the 10 mg/kg/day dose level, mild tremors were seen during one day only in 6/16 

animals. In contrast, the severity of tremors at the 20 mg/kg/day dose level ranged from mild to 

moderate and were seen on multiple days in 11/16 animals. Based on this additional 

information, the low dose was determined to be adequate with only the high dose showing 

evidence of overt toxicity. 

Steroidogenesis Assay; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48618701), H295R cells cultured in vitro in 24-well plates 

were incubated with phosmet (96.8% purity, Batch # GQH9118000) at concentrations of 100, 

10, 1, 0.1, 0.01, 0.001, and 0.0001 μM for 48 hours in triplicate for four independent 

experiments. Run 1 was not analyzed because one of the forskolin concentrations on the quality 

control plate was different than the proposed concentration outlined in the test guideline (3.33 

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μM was used instead of 1 μM). Dimethyl sulfoxide (DMSO) was used as a vehicle, at a final 

concentration of 0.05% v/v. 

Testosterone and estradiol levels were measured using HPLC-MS/MS. A Quality Control (QC) 

plate was run concurrently with 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. 

All guideline acceptability recommendations and requirements were met, including lack of 

cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low 

%CV), and appropriate induction and inhibition with positive controls. 

Neither testosterone nor estradiol concentrations were affected by phosmet. Decreases (p<0.05) 

of 10-11% in estradiol concentration were observed at 0.0001, 0.001, and 0.01 μM of phosmet in 

Run 2, but not at higher concentrations nor at any concentration in the other runs. An increase 

(p≤0.05) of 10% in estradiol concentration was observed at 100 μM of phosmet in only one of 

the three runs. 

Based on hormone responses in each of three independent runs, phosmet treatment did not result 

in statistically significant and reproducible alterations in testosterone or estradiol production. 

Uterotrophic Assay; (OCSPP 890.1600) 

In an uterotrophic assay (MRID 48618707) conducted to screen for potential estrogenic activity, 

phosmet (96.8% a.i., Batch # 6QH9118000) in 0.5% carboxymethyl cellulose was administered 

daily via oral gavage to groups of eight ovariectomized female Sprague-Dawley rats at dose 

levels of 0 (vehicle), 5, or 20 mg/kg/day on post-natal days (PND) 63-65. A positive control 

group was treated with 17α-ethynyl estradiol (EE) by daily oral gavage at a dose level of 0.1 

mg/kg/day. All animals were terminated and necropsied on PND 66 approximately 24 hours 

after the final dose to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. Two 20 mg/kg/day rats had decreased 

movements at 1-4 h post-dosing on Days 1 and 2. Body weights in the dose groups were 

comparable to the controls throughout the duration of the assay. Uterine weights in the dose 

groups were comparable to the controls. Absolute wet and blotted uterus weights for the 0.1 

mg/kg/day EE group were increased (p<0.05) by 407% and 231%, respectively. 

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The dose levels selected for this study are adequate based on previously conducted studies which 

showed no mortality at doses up to 27 mg/kg/day and significant cholinesterase inhibition at 10 

mg/kg/day. 

No statistically significant changes were seen in uterine weight in this assay. Phosmet was 

negative in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Published literature and Part 158 studies considered relevant for determining the potential 

interaction of phosmet with the endocrine system are summarized below. Given the breadth of 

toxicity data available on phosmet, the only Part 158 studies discussed in this appendix are those 

that fulfill current guideline requirements and are directly relevant to assessing potential 

endocrine effects (i.e., multiple-generation rodent reproductive studies, development studies, 

chronic toxicity studies, etc.). 

In vitro transactivation assays 

In a published literature study, Kojima et al. (MRID 48033008) screened 200 pesticides, 

covering nine chemical classes, for their potential to interact with two human estrogen receptor 

subtypes (hERα and hERβ) and the human androgen receptor (hAR) in transactivation assays in 

Chinese hamster ovary cells (CHO-K1 cells). In this study phosmet was reportedly negative for 

ER and AR transactivation up to the highest concentration tested was 10-5 M (range 10-8 to 10-5

M).

21-Day Dermal Toxicity in Rodents (Rats) 

In a subchronic dermal toxicity study (MRID 44795801), 10 SD (Crl:CD VAF/Plus) 

rats/sex/dose were administered phosmet (71.2% purity as Imidan 70-WSB) in deionized 

distilled water by dermal application (once a day for 6 hours, 5 days/week) for 15 days at 

concentrations of 0, 15, 22.5, or 60 mg/kg/day. Hair was clipped from 20 to 25% of the body to 

allow an application area of approximately 30 cm2

. 

There were no deaths, and clinical signs, body weight, food consumption, hematology, clinical 

chemistry, organ weights, macro- and microscopic lesions were comparable among the groups 

(both sexes), except as noted. Dermal irritation was not observed in either sex at any dose. The 

only effects observed were on inhibition of ChE activity. In high-dose males, brain ChE activity 

was decreased (p≤0.01, ↓36%) at study termination. Plasma and RBC ChE activities were 

unaffected by treatment in males. In high-dose females, ChE activity was decreased in plasma 

(p≤0.01, ↓38%) on Day 22 and decreased in RBC (p≤0.05, ↓29%) on Day 8, and brain ChE 

activity was inhibited (p≤0.01) in a dose-dependent manner (↓13, 20, and 61%) in the low-, mid-, 

and high-dose groups, respectively. 

Because historical control data were unavailable, an expanded control study was initiated 

immediately after completion of the 21-day dermal study. The expanded control group consisted 

of 20 rats/sex of the same strain and age under identical test conditions. Decreases (p≤0.01, 

↓33%) were observed in plasma ChE activity in males in the mid- and high-dose groups at 

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Day 22 compared to the expanded control group. Brain ChE activity was decreased (p≤0.01) by 

25% and 53% in high-dose males and females, respectively, compared to the expanded control 

group. 

The NOAEL for systemic effects is 60 mg/kg/day, the highest dose tested. The NOAEL for ChE 

inhibition is 15 mg/kg/day, and the LOAEL is 22.5 mg/kg/day based on decreased plasma ChE 

activity in males and decreased brain ChE activity in females. 

Prenatal Development in Rodents (Rat)- Guideline 

In a rat teratology study (MRID 41962902), randomized groups of 24 assumed pregnant 

Alpk:APfSD Wistar rats were administered phosmet (96.4% purity) in corn oil at 0 (vehicle 

only), 5, 10, and l5 mg/kg/day by gavage during GD 7 to 16. All dams survived until sacrifice on 

GD 22, except for one control animal killed in extremis on GD 14. Food-consumption was 

decreased (p<0.01) GD 7 to 16 (↓16) and GD 16 to 22 (↓14) in the high-dose dams. Body weight 

gain was decreased (p<0.01 and 0.05) for the dosing period (GD 7 to 16) in the mid- and highdose females (↓12 and 47%, respectively); decreases were also noted for the entire gestation 

period (p<0.01, ↓20%) and corrected body weight gain (p<0.05, ↓56%) at the high dose. No 

treatment-related gross pathology findings were noted. Clinical signs of toxicity were seen in the 

high dose group including one or more of the following symptoms: piloerection, tremors, 

salivation, urinary incontinence, and subdued mood. 

Comparable incidences between control and treated groups were observed for corpora lutea/dam, 

implantations/dam, and the number of live fetuses. In addition, no differences were noted in the 

incidences of intrauterine death, pre- and post-implantation loss, and fetal sex ratio. Mean fetal 

weight was decreased (p<0.05) for the high-dose group, but the value was within the historical 

control range. No treatment-related findings or malformations were noted in the fetal external 

examinations, and visceral examination findings were incidental. There were no apparent 

treatment-related fetal skeletal defects due to phosmet treatment; the total percentage of fetuses 

displaying major and minor defects in the treatment groups were similar to or less than control. 

The maternal toxicity LOAEL is 15 mg/kg based on clinical signs, and decreased food 

consumption and body weight gain. The NOAEL is 10 mg/kg/day. Developmental toxicity was 

not observed in this study, so the NOAEL is 15 mg/kg/day, and the LOAEL is not determined. 

Prenatal Development in Rodents (Rat) — Non-guideline 

In a non-Guideline developmental toxicity study (MRID 00063192; Staples et al.), Imidan 

(phosmet; 95.8% purity) administered to assumed-pregnant female CD rats in the diet or by 

gavage. Nominal dietary concentrations were 0, 10, 25, 40, and 50 mg/kg/day (prepared in 23 g 

of ground Wayne Lab Blox; 25 g of treated feed were provided daily/dam on GD 6 through 15). 

Mean phosmet doses consumed were 0, 10, 22, 27, and 29 mg/kg/day. Gavage dosing of 

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phosmet was conducted at doses of 0 (0.5% methylcellulose), 5, 10, 20, 25, and 30 mg/kg/day on 

GD 6 through 15. All dams were weighted and sacrificed on GD 21; reproductive status was 

determined and general fetal examinations were conducted (live fetuses weighed, determination 

of external and visceral malformations, and skeletal alterations). 

Selection of dietary and gavage phosmet doses to achieve a MTD acknowledged the tendency of 

phosmet effects to accumulate and a lessened susceptibility of pregnant females compared to 

non-pregnant females. Food intake was decreased at higher doses of phosmet; therefore, 

achieved doses at the low-medium, high-medium, and high concentrations were similar (22, 27, 

and 29 mg/kg/day, respectively). No parental mortality occurred in the dietary portion of the 

study, and the percentage pregnant was not affected (89 to 100%). Food intake and body weight 

gain were decreased (p<0.05) in a dose-dependent manner (up to ↓46 and 102%, respectively) in 

all but the low-dose dietary group, with no fetal effects noted after dietary administration of 

phosmet. In the gavage administration portion, five dams died at the high-medium dose (16%) 

and both dams died in the high-dose group (100%). The number of pregnant dams in the highmedium group was decreased (p<0.05, ↓25%), but dams at the lower doses were not affected (83 

to 100%). Food intake was decreased (p<0.05) in all but the low-dose group in a dose-dependent 

manner (↓8 to 38%) and body weight gain was decreased (p<0.05) in the medium and highmedium dose groups (↓88 to 73%, respectively). Fetal weights were decreased (p<0.05) in the 

low- and low-medium-dose groups (↓8%) and the medium- and high-medium-dose groups 

(↓12%). No increases in stunted or malformed fetuses were observed. 

Phosmet administration (dietary or gavage) was not teratogenic even up to demonstrating 

toxicity/lethality to dams (≥25 mg/kg/day by gavage). Decreased fetal weights (↓12%) at gavage 

doses ≥20 mg/kg/day further demonstrate systemic toxicity. 

Prenatal Development in Non-Rodents (Rabbit) 

In a rabbit teratology study (MRID 41962901), randomized groups of 20 assumed pregnant New 

Zealand White rabbits were administered phosmet (96.4% purity) in corn oil at 0 (vehicle only), 

2, 5, and l5 mg/kg/day by gavage during GD 7 to 19. All does were sacrificed on GD 30. One 

high-dose doe died on GD 10, one low- and one high-dose animal were killed due to 

moribundity, and three animals in the mid-dose group were sacrificed on GD 27 due to signs of 

abortion. Two high-dose does exhibited multiple signs of clinical toxicity (e.g., salivation, 

shaking, irregular breathing, or unsteady gait) and two additional animals exhibited salivation 

only. Body weight and food-consumption were not affected by phosmet treatment. Body weight 

gain was decreased (p<0.0001) for the dosing period (GD 7 to 19) in the high-dose females 

(↓74%); decreases were also noted for the entire gestation period (p<0.05, ↓23%). No treatmentrelated gross pathology findings were noted. 

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Comparable incidences between control and treated groups were observed for corpora lutea/doe, 

implantations/doe, and the number of live fetuses, and mean litter and fetal weights. In addition, 

no differences were noted in the incidences of intrauterine death, pre- and post-implantation loss, 

and fetal sex ratio. No treatment-related findings or malformations were noted in the fetal 

external examinations, and visceral examination findings were incidental. Several treatmentrelated increases (p<0.05) in skeletal variations and minor defects were observed in the highdose group, along with an increased (p<0.05) pes score. Overall, the total percentage of fetuses 

displaying major and minor defects in the treatment groups was similar to or less than control. 

The maternal toxicity LOAEL is 15 mg/kg based on clinical signs and decreased body weight 

gain. The NOAEL is 5 mg/kg/day. Developmental toxicity was present in the increased 

incidence of fetal skeletal variations at the high dose; the LOAEL is 15 mg/kg/day, and the 

NOAEL is 5 mg/kg/day. 

Prenatal Development in Non-Rodents (Monkey) 

In a developmental toxicity study (MRID 00043397), randomized groups of 7 pregnant rhesus 

monkeys (Macaca mulatta) were administered phosmet (purity not reported) in a 50% honey 

solution at doses of 2.0, 4.0 or 8.0 mg/kg/day on GD 22 to 32. Pregnancies were terminated by 

cesarean section on GD 84. Also investigated in this study were various doses or regimens of a 

positive control (thalidomide) and another pesticide (captan); the results from these compounds 

are not discussed further. 

Six of seven monkeys at in the high-dose group produced anatomically normal fetuses, and one 

monkey aborted. All seven monkeys at the intermediate dose produced anatomically normal 

fetuses. Finally, five of seven monkeys in the low-dose group produced anatomically normal 

fetuses; one monkey in the low-dose group aborted and one monkey resorbed the fetus. Fetal 

mortalities present in this study were not dose-related and were within the historical range for 

fetal mortality. The body weights, x-rays, organ weights and organ to body weight ratios, as well 

as crown-rump lengths of the fetuses were within normal limits after exposure to phosmet. 

Maternal body weights and hematology results indicated no compound-related effects. The 

LOAELs for maternal and developmental toxicity are not determined. The NOAELs for maternal 

and developmental toxicity are equal to or greater than 8.0 mg/kg/day. 

Subchronic Neurotoxicity in Rodents (Rat) 

In this subchronic oral neurotoxicity study (MRID 44811801), phosmet (97.3% purity) was 

administered for 90 days to 32 Crl:CD(SD)IGS BR rats/sex/dose at nominal dietary 

concentrations of 0, 25, 50, or 150 ppm. Actual mean dietary concentrations of phosmet were 

0, 21.6, 39.7, and 136 ppm [equivalent to achieved doses of 0/0, 1.5/1.6, 2.7/3.1, or 9.4/11.0 

mg/kg/day (M/F)]. Cholinesterase activity was determined with a modified Ellman method in the 

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blood and brains of 2 rats/sex/dose during the pretest and 6 rats/sex/dose during Study Weeks 3, 

7, and 13. The remaining 12 rats/sex/dose were subjected to the functional observational battery 

(FOB) and motor activity measurements during the pretest period and Study Weeks 3, 7, and 12, 

and were perfused in situ at study termination. Five animals/sex from the control and high-dose 

groups were used for neurohistological examination. No animals died during the study. No 

changes in appearance or behavior, body weights or body weight gains, and food consumption 

were observed in any of the treated groups compared to control. Results of the FOB indicated no 

treatment-related findings, and there were no gross neurological findings. No treatment-related 

effects were observed on brain weight in non-perfused and perfused animals. An increase in the 

incidence of neuropathological changes (characterized by digestion chambers in the sciatic and 

peroneal nerves) was observed in the high-dose perfused males. 

In general, whole blood and RBC ChE activities were decreased in all treated animals, and 

plasma and regional brain ChE activities were decreased in all treated females. The details are 

summarized below: 

In the low-dose group, mean RBC ChE activity was decreased (p<0.01) in males at Week 13 

(↓19%) and females at Week 7 (↓42%). Decreases (p<0.05 or 0.01) were also observed at 

Week 13 in mean plasma ChE activity in females (↓29%), mean whole blood ChE activity in 

both sexes (↓16–19%), and ChE activity of the olfactory bulb (↓36%) and brain stem (↓21%) 

regions in females. 

In the mid-dose group, the following decreased (p<0.05 or 0.01) mean ChE activities were 

observed: (i) plasma ChE in the males at Week 3 (↓21%) and females at Weeks 3 and 13 (↓27–

46%); (ii) RBC ChE in the males at Weeks 3, 7, and 13 (↓26–39%) and females at Week 7 

(↓38%); (iii) whole blood ChE at Weeks 3, 7, and 13 in both sexes (↓24–36%); and (iv) whole 

brain ChE at Week 7 in the females (↓20%) and at Weeks 3 and 7 in the males (↓11–17%). 

In the high-dose group, the following decreased (p<0.05 or 0.01) mean ChE activities were 

observed: (i) plasma ChE at Weeks 3, 7, and 13 in both sexes (↓23–71%); (ii) RBC ChE at 

Weeks 3, 7, and 13 in males (↓65–70%) and at Weeks 3 and 7 in females (↓66-89%); (iii) whole 

blood ChE in both sexes (↓59–74%) at Weeks 3, 7, and 13; (iv) whole brain ChE in both sexes 

(↓43–68%) at Weeks 3 and 7; and (v) in all six brain regions in the females (↓36–67%) and in all 

brain regions (except for olfactory region) in the males (↓27–52%) at Week 13. 

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

established. 

Two-Generation Reproduction in Rodents (Rat)

In a Two-Generation Reproduction study in the rat (MRID 41520001), phosmet (95.2% purity) 

was administered at target doses of 0, 20, 80, and 300 ppm in the diet to SD rats (Crl:CD(SD)Br; 

25/sex/dose) for two generations. Compound intake values were determined for males 

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(premating) and females (premating, gestation, and lactation) in the F0 and F1 generations: the 

overall intake values for both sexes during the entire study period were 0, 1.5, 6.1, and 

23.4 mg/kg/day. The F0 rats were fed the diet for 8 weeks prior to mating, through gestation and 

lactation of the F1a litter, and three weeks after weaning prior to re-mating to produce the F1b

litters. After weaning, F1b rats were selected as F1 parents and treated for 11 weeks prior to 

mating. As previously, the F1 parents were allowed three weeks after weaning the F2a litters prior 

to re-mating to produce the F2b litters. Blood samples were collected at terminal sacrifice for the 

determination of serum and RBC ChE activities. 

No treatment-related mortality was observed; three and two females, respectively, in the F0 and 

F1 high-dose groups had tremors. Body weights during the premating periods for F0 and F1 highdose animals were occasionally decreased (p<0.01 or 0.05) by 3 to 8%, with a decrease (p<0.01) 

of 10% observed on Day 21 in high-dose F0 females. Effects on premating body weight gain 

were observed in the F0 animals, but observed decreases (↓8 to 10%) were NS. Maternal body 

weights during gestation were slightly decreased (NS) in the high-dose animals (↓≤8%), with one 

decrease (p<0.01) of 8% in F1 dams on GD 20. Mean body weight gains were generally 

decreased (NS) only in the high-dose animals. Maternal body weights during lactation were 

decreased (p<0.01 or 0.05) in the high-dose dams (↓9 to 13%; LD 7 to 21) with the F1a and F2a

litters; decreased body weights (p<0.05 or NS) with the F1b and F2b litters were ≤8%. Mean body 

weight gains (losses) were generally greater (NS) in the mid- and high-dose animals. Food 

consumption was decreased (NS; ↓7 to 21%) or (p<0.01; ↓18 to 40%) in high-dose dams during 

lactation. 

Mean absolute and relative ovarian weights were decreased (↓15 and 21%, respectively; p<0.05) 

in high-dose F0 parents, but no histopathological lesions were seen in the ovaries in either 

parental group. Absolute testis weights were decreased (↓10%; p<0.05) in high-dose F0 and F1

parents, but relative testis weights were comparable to control, and no treatment-related 

histopathological lesions were seen in the testes in either parental group. 

Serum ChE activity was decreased in mid-dose F0 females (p<0.01, ↓24%) and high-dose F0/F1

males (p<0.01, ↓30/41%) and females (p<0.01, ↓65/63%). RBC ChE activity was decreased in 

low-dose F0 males (p<0.05, ↓8%), low-dose F0/F1 females (p<0.01, ↓11/16%), mid-dose F0/F1

males (p<0.01, ↓37/48%) and females (p<0.01, ↓48/59%), and high-dose F0/F1 males (p<0.01, 

↓74/85%) and females (p<0.01, ↓81/80%). 

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

decreased (p<0.01) for litters from all high-dose parents. 

The mean number of live pups per litter generally were decreased (NS, or p<0.05 or 0.01), and 

mean pup weight per litter was decreased (p<0.05 or 0.01) for all litters from high-dose parents 

after Day 0. Pup sex ratio was not affected by treatment. No definitive treatment-related gross or 

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histopathological findings were observed in pups culled on PND 4 or any F1-generation 

weanlings. 

The NOAEL for parental toxicity is not determined and the LOAEL is 20 ppm (1.5 mg/kg/day), 

based on inhibition of RBC ChE activity. The NOAEL for reproductive toxicity is 20 ppm, and 

the LOAEL is 80 ppm (6.1 mg/kg/day) based on decreased fertility and decreased fertility and 

gestation indexes. The NOAEL for offspring toxicity is 80 ppm and the LOAEL is 300 ppm 

(23.4 mg/kg/day), based on decreased pup body weight gain and survival. 

Reproductive Toxicity in Male Rodents (Mouse) 

In a published non-Guideline study, Quinto et al. (MRID 48084301) examined the effects of 

selected pesticides on male mouse sperm morphology. Imidan (phosmet; 99.8% purity) was 

dissolved in distilled water; male C3H×C57BL/6 F1 mice were administered phosmet by p.o. or 

i.p. at doses of 0, 12.5, 25, and 50 mg/kg/day for 5 days. On Study Day 35, animals were killed 

by cervical dislocation and testes weight, sperm count, and sperm abnormalities were 

determined. 

No treatment-related effects on testes weight, sperm count, and sperm abnormalities were 

observed after phosmet treatment by either the p.o. or i.p. routes of administration. 

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat) 

In a chronic two-year oral toxicity study in rats (MRID 41916401), phosmet (R-1504 Technical, 

94.3/95.2% purity) was administered as 0 (control), 20, 40, 200, or 400 ppm in the diet to Crl:CD 

(SD) Br rats (70/sex in the control group, 60/sex in all other dose groups except for the high-dose 

group) for 104 weeks. An interim-sacrifice at Week 53 included 20/sex in the control group, 

10/sex in the 20-, 40-, and 200-ppm dose groups; the high-dose group (400 ppm; 20/sex) was 

sacrificed in toto after Week 52. For the first six weeks of the study, the low-medium-dose group 

(40 ppm) was administered a 100-ppm diet. Excluding this error, the mean concentrations of 

phosmet in the treatment diets were calculated as 19.3±1.1, 39.4±2.9, 194.1±5.6, and 389.5±17.9 

ppm. Calculated mean intake values for males and females were 1.1, 1.8, 9.4, and 23 mg/kg/day 

and 1.1, 2.1, 10.9, and 27 mg/kg/day, respectively. 

Parameters examined in all rats were those routinely examined in a chronic oral 

toxicity/carcinogenicity study. Absolute and relative (% of body and brain) weights of organs 

were determined for adrenals, kidneys, liver, testes/ovaries, and brain for both scheduled 

sacrifices; histopathology was conducted on these tissues as well as epididymides, mammary 

gland, pituitary, prostate, seminal vesicles, thyroid/parathyroid, vagina, and uterus with cervix. 

Serum and RBC ChE activities were determined at 6, 12, 18, and 24 month. Brain ChE activity 

was determined at Weeks 52 (10 rats/sex/dose group) and 104 (animals designated for laboratory 

tests). 

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No clinical signs of toxicity could be attributed to administration of the test material. Survival 

was comparable among the control and the treated groups (84 to 100% at Week 52, and 20 to 

42% at termination). Although decreases (p<0.05 or 0.01; up to 10%) in mean body weight were 

observed in the mid-high (Week 1) and high-dose (Weeks 1 to 13) females, no other treatmentrelated weight effects were noted. Mean compound intake was not affected by treatment and, in 

general, no relevant, treatment-related effects were noted in any hematology, clinical chemistry, 

or urinalysis parameters (except for ChE activity). Dose-related inhibition (p≤0.01 or 0.05) of 

serum ChE activity occurred in males in the high-dose group at all sample times and in the highmedium group at 18 months, and in females in the high-medium- and high-dose groups at all 

sample times and in the low-medium group at 6 months. RBC ChE was inhibited (p≤0.01 or 

0.05) in males and females in the low-medium-, high-medium-, and high-dose groups at all 

sample times, except at 12 months in the low-medium animals. RBC ChE also was inhibited in 

the low-dose males only at the 6-month sample time. Brain ChE was inhibited (p≤0.01) in males 

and females in the high-medium- and high-dose groups at all sample times, and in the 24-month 

low-dose samples in males (p<0.05). Organ weights, gross pathology, and neoplastic/nonneoplastic microscopic pathology were generally unremarkable. Absolute kidney weight was 

decreased (p<0.05, ↓12%) in the high-dose females at Week 53. An increased (p<0.05) incidence 

of fatty liver was observed in the high-medium- and high-dose males at the interim sacrifice, and 

males in all remaining treatment groups at study termination. Females in the high-medium-dose 

group had increased (p<0.05) incidences of fatty liver and mineralization of the thyroid at study 

termination. No evidence of treatment-related carcinogenicity was observed in the present study 

at phosmet exposures of ≤400 ppm. A NOAEL of <20 ppm is determined in this chronic study, 

with a LOAEL of 20 ppm based on decreased RBC and brain ChE activity. The systemic 

NOAEL and LOAEL are also <20 ppm and 20 ppm, respectively, based on the increased 

incidence of fatty liver. 

Carcinogenicity in Rodents (Mouse) 

In a two-year oral oncogenicity study in mice (MRID 00141659), phosmet (R-1504 Technical, 

94.7% purity) was administered as 0 (control), 5, 25, or 100 ppm in the diet to B6C3F1-Crl-BR 

(60/sex/dose) for 104 weeks. At 12 months, 10 mice/sex/dose were selected for an interim 

sacrifice. Calculated mean intake values for males and females were 1.0, 4, and 14 mg/kg/day 

and 1.2, 5, and 18 mg/kg/day, respectively. 

Parameters examined in all mice were those routinely examined in a chronic carcinogenicity 

study. Absolute and relative (% of body) weights of organs were determined for heart, kidneys, 

liver, testes with epididymides, and brain; histopathology was conducted on these tissues as well 

as adrenals, mammary gland, ovaries, pituitary, prostate, seminal vesicles, thyroids, vagina, and 

uterus. Plasma, RBC, and brain ChE activities were determined at 12 (interim sacrifice animals 

only) and 24 months (surviving animals at study termination). Due to reported reagent problems, 

RBC ChE activity was not determined at the interim sacrifice. 

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A dose-related trend in the incidence of convulsions was attributed to administration of phosmet 

in male mice only. Survival was comparable among the control and the treated groups with 

unscheduled deaths ranging from 4 to 10 males and 11 to 15 females, respectively. No treatmentrelated effects on body weight were observed; food consumption was decreased in all treated 

males and females in the mid- and high-dose groups (data not provided). No relevant, treatmentrelated effects were noted in hematology parameters; clinical chemistry determinations were 

limited to ChE activity. Inhibition (significance not reported) of plasma ChE activity occurred in 

males in the low- and mid-dose groups and mid-dose females (↓11 to 13%) at the interim 

sacrifice, with lower inhibition (↓4 to 10%) observed at study termination. Greater inhibition 

(significance not reported) of plasma ChE activity was noted in high-dose males and females 

(↓54 to 56%) at the interim sacrifice, with similar inhibition (↓47 to 52%) observed at study 

termination. RBC ChE activities could not be determined at the interim sacrifice due to a 

technical issue, but were not different from control at study termination. Brain ChE was inhibited 

(significance not reported) in males and females in all dose groups to a similar degree (↓22 to 

34%) at the interim sacrifice; at study termination, brain ChE activity in males was not different 

from control and was decreased only in mid- and high-dose females (↓14 and 22%, respectively). 

At the interim sacrifice, there were increases (significance not reported) in absolute kidney and 

liver weights in high-dose females; at termination, liver weights were increased (significance not 

reported) in mid- and high-dose females and high-dose males. Gross pathology findings in the 

livers of high-dose males included increased incidences of masses, nodules, or cysts and areas of 

focal discoloration/depression. Microscopic examinations at study termination noted increases in 

high-dose animals of testicular atrophy (19/50 vs. 11/49) and myometrial/uterine atrophy (8/50 

vs. 2/49). Hepatocellular adenomas and carcinomas were occasionally present in treated male 

mice at the interim sacrifice, and in all control and treated mice at study termination. Increased 

(p=0.05 and 0.06, respectively) hepatocellular adenomas and carcinomas were observed in highdose males and females 35/60 vs. 23/60 and 18/60 vs. 10/60, respectively). Further analyses of 

the tumor data indicated significant increases (p<0.05) for high-dose males and females. A 

NOAEL of <5 ppm is determined in this oncogenicity study, with a LOAEL of 5 ppm based on 

inhibition of brain ChE activity in males and females. The systemic NOAEL is 5 ppm and the 

LOAEL is 25 ppm based on convulsions in male mice. 

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

In a chronic two-year oral toxicity study in dogs (MRID 00076436), phosmet (purity unknown) 

was administered as 0, 20, 40, and 400 ppm in the diet to Beagle dogs (3/sex) for 104 weeks. 

Phosmet doses were calculated as 0, 0.5, 1.0, and 10 mg/kg/day with the conversion factor of 

0.025 mg/kg/day per ppm. 

Organs weighed at study termination included adrenals, liver, kidneys, ovaries, pituitary, 

prostate, testes, thyroid, and uterus, and organs examined microscopically included adrenals, 

liver, kidneys, mammary gland, ovaries, pituitary, prostate, testes, thyroid, and uterus. Gross and 

microscopic pathology were unremarkable (no treatment-related findings). Brain and RBC ChE 

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inhibition was observed in the high-dose animals; no additional treatment-related effects on ChE 

activity were observed at the low and intermediate doses. No other systemic toxicity effects were 

observed due to phosmet treatment in this study. 

Avian Reproduction (OCSPP 850.2300) 

In an avian reproduction study (MRID 00105999), 14 to 16-week old Mallard ducks 

(Anas platyrhynchos) were administered phosmet (94.7% purity) in the feed at concentrations of 

0 (acetone; vehicle control), 25, 60, and 150 ppm for up to 27 weeks (includes 13-week 

administration prior to the initiation of egg laying). Mean measured phosmet concentrations in 

the control and treated diets were <2.0, 22.1 to 27.3, 62.0 to 95.0, and 144.0 to 183.0 ppm. Each 

dose group consisted of 12 ducks/sex, with a single drake and hen being paired in 12 pens/dose 

group. It was noted that photoperiod was gradually lengthened according to 1978 Guidelines. No 

treatment-related effect on body weight was observed. Weekly egg production data was 

provided. Eggs were candled during fourteen 7-day periods and eggshell thickness was 

determined. Feed consumption was measured every two weeks. Surviving ducklings were 

weighed on Day 14, and viability was reported on Day 21. ChE determinations were not 

conducted during this study. 

No adult mortalities occurred in any test group, and no overt signs of toxicity or clinical signs 

were observed. No effects were seen at any test dose on female body weight or average feed 

consumption in males and females. Male body weight was increased (significance not reported) 

in the low-dose group for Week 27; no other differences were noted. In general, no reproductive 

impairment (p>0.05) was seen at any dose. There were no apparent treatment-related effects on 

the total number of eggs laid, shell thickness, or egg quality. There were also no treatmentrelated effects on live 21-day old embryos or percentage viability. Occasional differences in 

duckling body weight (Day 1 or 14) and Day 14 viability were not considered to be treatment 

related. Overall, fewer total eggs were laid in the high-dose group, but this may be in part due to 

the failure of a single pen/mating pair (No. 8) to produce (only 1 egg laid over the 13-week 

period). 

In another avian reproduction study (MRID 00125786), 16 to 20-week old Bobwhite Quail 

(species not identified) were administered phosmet (97.4% purity) in the feed at concentrations 

of 0 (acetone; vehicle control), 25, 60, or 150 ppm during the laying season (includes 14-week 

administration prior to the initiation of egg laying). Each dose group consisted of 24 quails, with 

a single cock and hen paired in 12 pens/dose group. It was noted that photoperiod was gradually 

lengthened according to 1978 Guidelines. Eggs were collected during 7-day collection periods, 

stored at 16°C and 65% relative humidity, and turned twice daily. On Day 21, embryo survival 

was determined and eggs were placed in a hatcher (37.2 to 37.8°C). Hatchling were weighed on 

Day 14. 

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Mortality occurred in all treatment groups (low dose: one female; intermediate dose: one male 

and five females; high dose: three females). These deaths were considered to be due to open 

sores attributed to increased activity and stress during the 16-hour light period for egg 

promotion. Adult body weights and feed consumption in the treated groups generally were 

comparable to control, although significant decreases were observed at Week 26 in the low- and 

high-dose groups. High incidences of reduced fluid in the gall bladder with diffuse discoloration 

of bile were noted in males and females (females>males) in all treatment groups; this response 

was dose dependent in females. There was no treatment-related effect on the prevalence of 

cracked eggs; the number of eggs cracked in the vehicle-control group was approximately 2-fold 

greater compared to treatment groups. Decreases in eggs laid (p<0.05) were observed in the 

high-dose group (NS). Reproductive parameters in the low- and mid-dose groups generally were 

similar to, or better, compared to control values. 

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

In a freshwater fish early life-stage test (MRID 40938701), Rainbow trout (Oncorhynchus 

mykiss; previously Salmo gairdneri) embryos were exposed to [14C]-phosmet (97 to 99% purity) 

at nominal concentrations of 0 [negative control and dimethylformamide (DMF), vehicle 

control], 3.0, 6.0, 12, 25, and 50 μg/L. Achieved exposure concentrations were 3.2, 6.1, 12, 24, 

and 51 μg/L for the treated groups. Eggs were exposed from Day 1 (fertilization) until hatch 

(≥95% by Day 36); exposure was continued for 60 days post-hatch until termination on Day 96. 

Four replicates of 30 eggs/replicate were included in each treatment group on Day 1. On Day 39, 

unhatched eggs were removed, and the number of fry was reduced to 15/replicate. Fry survival, 

and length and weight, were determined on Day 35 and 60 post-hatch. 

No solvent effects on study parameters were observed with DMF. Egg viability determined on 

Day 11 ranged from 86 to 96%, with a mean value of 92.5%. The percent hatch was 99% 

(negative control), 98% (vehicle control), 99% (low, low-medium, and medium concentrations), 

and 97% (high-medium and high concentrations); no treatment-related effect on hatching was 

noted. Fry survival on post-hatch Day 35 was decreased (p<0.05) in the intermediate 

concentration group by 7%, but no significant decrease was noted at the higher concentrations. 

Mean length on post-hatch Day 35 was decreased (p<0.05) in the medium, high-medium, and 

high concentrations in a treatment-related manner (↓4, 7, and 15%, respectively). Fry survival on 

post-hatch Day 60 was decreased (p<0.05) at the high-medium and high concentrations by 16 

and 57%, respectively. From the low-medium to the high concentration, mean length was 

decreased (p<0.05) by 5, 13, 20, and 28%, respectively, compared to the negative control. Mean 

weight on post-hatch Day 60 also was decreased (p<0.05) in the medium, high-medium, and 

high concentrations in a treatment-related manner (↓35, 50, and 62%, respectively). 

 

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APPENDIX 3: References Not Utilized in the Phosmet 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 Phosmet 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. 

Burgess, D. (1988) Chronic toxicity of 14C-Imidan® to Daphnia magna under flow-through test 

conditions. Analytical Bio-Chemistry Laboratories, Inc. Laboratory report no. 35778. (MRID 

40652801) 

Cappon, G. (1999) A Dietary Subchronic (90-Day) Neurotoxicity Study of Phosmet in Rats. WIl 

Research Laboratories, Inc. Report No.: WIL-331002. (MRID 44811801) 

Fernandez-Perea, M. T., Prados, E. A., Villajos, A. N., Prados, J. L., and Baudin, J. M. (2009) 

Influence of avian reproduction ecotoxicological endpoints in the assessment of plant protection 

products. J Environ Sci Health B. 44(2):106-112. 

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Gajdova, M., Vargova, M., Jakubovsky, J., Grunt, J., Valky, J., and Galbavy, S. (1988) 

Estrogenic effects of Phosmet on the Uterus of Neonatal Rats. Bratisl. Lek. Listy. 89: 843-847. 

Guiñazú, N., Rena, V., Genti-Raimondi, S., Rivero, V., and Magnarelli, G. (2012) Effects of the 

organophosphate insecticides phosmet and chlorpyrifos on trophoblast JEG-3 cell death, 

proliferation and inflammatory molecule production. Toxicol In Vitro. 26(3):406-413. 

Hollingsworth, R., Johnston, C.D., Woodard, G., and. Cronin, M.T.I (1965) Three-Generation 

Reproduction Study in Rats. Woodard Research Corporation. (MRID 00081432) 

Jablonika, A. and Kobzova, D. (1988) Morphological Changes in Sperm in Experimental Mice 

after Administration of Phosmet and Mancozeb. Bratisl. Lek. Listy. 89: 611-614. 

Johnston, C.D. and Cronin, M.T.I. (1962) Imidan: An Evaluation of Safety of Imidan in the Rat 

and Dog. Woodard Research Corporation. (MRIDs 00081426, 00081428, and 00081429) 

Kenwood, S.M., (1988) 21-Day Dermal Toxicity Study with lmidan Technical in Rabbits. 

Hazleton Laboratories America, Inc. Report No.: HLA 6142-103. (MRID 40538101) 

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