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 

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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 

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EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: TETRACHLORVINPHOS (TCVP) 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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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..................................................13 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................17 

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...........................................................................25 

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

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

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

LIST OF TABLES: 

 Table 1. Tier 1 Assay for Tetrachlorvinphos (TCVP)..............................................8 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Tetrachlorvinphos (TCVP).................10 

 Table 3. Androgenic/Anti-Androgenic Pathway for Tetrachlorvinphos (TCVP).............14 

 Table 4. Thyroid Pathway for Tetrachlorvinphos (TCVP).......................................19 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................31 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................44 

APPENDIX 3. References Not Utilized in the Tetrachlorvinphos (TCVP) WoE Analysis........57 

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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 

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Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

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Abbreviation Terminology 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

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vi 

Abbreviation Terminology 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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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 tetrachlorvinphos (TCVP) 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 30, 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 TCVP 

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

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

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

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

TCVP 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 

There is no convincing evidence or redundancy of responses to demonstrate potential for TCVP 

to interact with the estrogen pathway in mammals or wildlife. TCVP was classified as negative 

in the estrogen receptor (ER) binding and ER transactivation assay (ERTA). Although TCVP 

was positive in the aromatase assay, there were technical issues with the conduct of the 

steroidogenesis assay that lessen the confidence in the negative results for this assay and there 

were no redundant responses observed in the in vivo mammalian assays (Tier 1 or OSRI) to 

support an interaction with the estrogen pathway. The lack of responses to TCVP in female rats 

in the uterotrophic and female pubertal rat studies do not support a potential for ER interaction or 

altered steroidogenesis. Additionally, there was no redundancy of evidence across the other in 

vivo Part 158 studies. In the fish short-term reproduction assay (FSTRA), effects were observed 

at the highest concentration, 0.57 mg/L (i.e., ↓61% fecundity, ↓9% fertilization, and ↓30% 

female plasma VTG); however, there no effects on GSI or gonadal histopathology. 

Cholinesterase activity and sex steroids were not measured in this study. While there were no 

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clinical signs of toxicity observed in this study, the concentrations tested in the FSTRA were 

similar to concentrations that were shown to be overtly toxic in other fish assays [(FSTRA 

rangefinder (1.8 mg/L), Part 158 fish early-life stage study (≥0.15 mg/L), acute LC50 bluegill 

(0.53 mg/L)]. As such, the decreases observed in the FSTRA may be due to potential general 

stress on the fish from exposure to TCVP as oppose to potential endocrine-related interactions. 

A decrease in embryo viability (↓7.5%) and eggshell thinning (↓10%) was observed in the Part 

158 bobwhite quail study; however, there were no other reproductive effects including egg 

production. A decrease in offspring weight was observed in the mallard duck study, but adult 

female body weight was also affected, and there were no other reproductive effects. Therefore, 

while effects on reproduction were observed in the FSTRA, there were no effects on gonadal 

weight or histopathology, and there was limited redundancy in the other in vivo mammalian and 

wildlife studies. 

There is no convincing evidence or redundancy of responses to demonstrate potential for TCVP 

to interact with the androgen pathway in mammals or wildlife. TCVP was equivocal in the Tier 1 

in vitro AR binding assay and negative in an OSRI ARTA (Kojima et al., 2004). Although the in 

vitro steroidogenesis assay was considered inadequate due a number of technical issues in the 

conduct of the study, TCVP did not show a potential to interact with the androgen pathway in the 

male pubertal assay and was considered negative in the Hershberger assay. In the FSTRA, there 

was a 9% decrease (p<0.05) in fertilization in the high-dose group, and no other potentially 

androgen-related effects were observed. In the Part 158 mammalian studies, the only observed 

effect was a decrease in prostrate weight in the dog chronic study at the limit dose. While 

decreases in embryo viability was reported in the Part 158 quail reproduction study, no other 

androgen-related effects were observed in the Part 158 wildlife studies. 

In both the female and male pubertal assays, no treatment-related effects were noted for thyroid 

weights, but effects on thyroid histopathology were observed in both sexes. Serum T4 levels were 

decreased at the high dose in males and females (↓65% females; ↓68% males; decreases at low 

dose not significant). Thyroid stimulating hormone (TSH) was increased 31% in males at the 

high dose; however the change was not statistically significant. There was no treatment effect on 

TSH levels in females. In the Part 158 studies, alterations in thyroid histopathology were 

observed in the subchronic rat study and 28-day inhalation rat study. Cholinesterase activity was 

not measured in either Tier 1 pubertal assay, however, the effects observed in the Part 158 

studies occurred at doses that resulted in significant reductions in RBC ChE activity as well as 

increased incidences of histopathological effects in the liver and kidney or increased adrenal 

weights. 

In the amphibian metamorphosis assay (AMA), while effects on development and growth were 

observed in the highest treatment group, they occurred in the presence of overt toxicity. 

Additionally, in the highest treatment, which was considered to be overtly toxic, while 

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histopathological effects on the thyroid included apparent increases in gland hypertrophy, gland 

size, follicle size and follicle asymmetry compared to the negative control; however, when 

compared to the solvent control the only histopathological effect was an increase in follicle 

asymmetry. 

In the EDSP Tier 1 male and female pubertal assays as well as in the 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 TCVP, the current point of departure (POD) of 4.23 mg/kg/day used for human health risk 

assessment is based on histological liver and adrenal changes and reduced weight gain at 43.2 

mg/kg/day (LOAEL) in a chronic oral toxicity study in rats. This POD is used for deriving the 

chronic Reference Dose (RfD) for chronic dietary risk assessment. The second POD of 6.7 

mg/kg/day) is based on plasma/RBC ChE inhibition at 142 mg/kg/day (LOAEL) in a subchronic 

toxicity study in rats. This POD is used for deriving the Margins of Exposure (MOEs) for nondietary risk assessments. 

In general, since the POD and the toxicity endpoints of concern are based on data obtained from 

adult animals, there is a concern that the POD may not be protective of potential thyroid toxicity 

in the young. However in the case of TCVP, although no data exists for potential thyroid toxicity 

in the young, a comparative thyroid assay (CTA) is not recommended since: 1) the POD is 

approximately 83 and 52-fold lower, respectively, than the dose (350 mg/kg/day) that induced 

thyroid effects in the pubertal assays; 2) the POD is based on a more sensitive endpoint 

(cholinesterase inhibition) for this class of chemicals (organophosphate); and 3) the CTA would 

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

used for risk assessment. Consequently, the RfD and MOEs are expected to be protective of the 

thyroid-related effects seen in the male pubertal assay. 

Based on the weight of evidence considerations, mammalian or wildlife EDSP Tier 2 testing is 

not recommended for TCVP since additional testing will not impact current EPA established 

regulatory point of departure or endpoint for human health risk assessments. 

Given the lack of convincing evidence for a potential interaction with the estrogen, androgen and 

thyroid pathways in wildlife no additional testing is recommended. 

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

duck are available for TCVP. In both species, effects on reproductive parameters were observed. 

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 TCVP exposure. 

Relative to the EDSP, additional testing is not recommended. 

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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 tetrachlorvinphos (TCVP) 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 30, 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 TCVP 

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

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

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

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

Tetrachlorvinphos (TCVP) is an organophosphate insecticide with a vapor pressure of 4.2 × 10-8

mm Hg at 20°C. TCVP is slightly soluble in water (11 mg/L at 20°C) and has an octanol/water 

partition coefficient (log Kow) of 3.53. TCVP is susceptible to photodegradation in the vapor 

phase and hydrolyzes slowly in water, and it is not considered to be persistent in the 

environment. Volatilization is not expected to significantly contribute to the dissipation of TCVP 

in the environment. A bioconcentration factor of approximately 17 has been determined for 

TCVP. 

The available information considered in determining the potential interaction of 

tetrachlorvinphos 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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II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

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

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

1. 

B. Other Scientifically Relevant Information (OSRI) 

In response to the Agency’s Test Orders, data 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 TCVP (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-related processes (i.e., E, A or T hormone pathways) were 

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

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

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

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

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

foundation for WoE evaluations. The first step is to evaluate the individual studies for their 

scientific quality and relevance in evaluating potential endocrine interaction(s). The second step 

is to integrate the data along different levels of biological organization while examining the 

extent of complementarity (i.e., the concordance of endpoints within an assay that measures 

multiple endpoints) and redundancy (i.e., the concordance of endpoints/responses across assays) 

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

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

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

above. 

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

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

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

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

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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 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-related effect from a non-endocrine toxic response. 

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

the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of 

the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “In summary, the 

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

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

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

For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s current 

approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological 

risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential 

evidence of overt toxicity included, but were not limited to: mortality; clinical signs such as 

tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight 

decreases of ≥10% in mammals. Additionally, other effects including morphological (e.g., organ 

weights/histopathology), biochemical (e.g., blood chemistry), and other clinical signs (e.g.,

lethargy) were also considered when evaluating overt toxicity, especially if the effects were 

extreme. In some instances, one parameter (i.e., death or >10% decrease in mammalian body 

weight) was sufficient to consider a dose/concentration to be overtly toxic. However, in other 

instances, more than one parameter was needed to determine overt toxicity. For example, in the 

FSTRA, generally, body weight decreases were considered along with other responses when 

assessing potential overt toxicity. Additionally, effects which were considered to be signs of 

systemic toxicity were also captured and these effects were generally considered as less severe 

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

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

Section IV.A. 

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

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

known modes of action in its overall characterization of a chemical’s potential to interact with 

the E, A or T pathway. Adequately addressing these three main questions 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 case-by-case analysis 

which took all available toxicity data into account. 

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The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 

assays and OSRI which are depicted in Tables 2 - 4. These tables contain data that are 

considered scientifically and biologically relevant with regard to a treatment-related effect 

which supports a conclusion of whether a substance has the potential to interact with the E, A, 

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

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

A. EDSP Tier 1 Screening Assays 

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

Table 1. 

Table 1. Tier 1 Assays for Tetrachlorvinphos (TCVP)

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay 

(Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID 48765001) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID 48619501) 

AR Binding Assay 

(Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID 48703101) 

Steroidogenesis Assay 

(Human cell line H295R) 

OCSPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID 48805201) 

Aromatase Assay 

(Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID 48673801) 

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID 48880601) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID 48946701) 

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied 

(MRID 49186901) 

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied 

(MRID 49186901) 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID 48838201) 

Amphibian Metamorphosis Assay 

(Frog) 

OCSPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID 48688401) 

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Page 9 of 58 

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

1. Effects on Estrogen Pathway 

The potential for TCVP 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. 

TCVP was negative (not interactive) in the Tier 1 in vitro ER binding assay and ERTA. TCVP 

was also negative in an ERTA considered as OSRI (Kojima et al., 2004) Although the results of 

the Tier 1 steroidogenesis assay did not show an effect on estradiol production, there were 

numerous deficiencies noted in the study that preclude making a definitive conclusion with 

regards to the effects of TCVP on hormone production in this assay. TCVP was classified as 

positive in the aromatase assay with mean aromatase inhibition being 63, 33 and 17% of control 

activity at concentrations of 10-4, 10-3.5 and 10-3 M, respectively, with an IC50 of -3.76 Log M. 

There were no estrogen-related effects in the Tier 1uterotrophic assay or female pubertal assays. 

In the Part 158 mammalian toxicity studies, the only potential estrogen-related effects observed 

were increased resorptions, post-implantation loss and decreased live fetuses in the rabbit 

developmental toxicity study which occurred in conjunction with increased maternal toxicity, 

including mortality. 

In the FSTRA, there was a 61% decrease (p<0.05) in fecundity and a 9% decrease (p<0.05) in 

fertilization in the high-dose group (0.57 mg/L). Female vitellogenin (VTG) was decreased 30% 

(p<0.05) in the high-dose group. There were no effects on gonadal histopathology and sex 

steroids were not measured; additionally cholinesterase activity was not measured. While there 

were no clinical signs of toxicity observed in this study, effects on survival (50%) and clinical 

signs of toxicity (i.e., loss of equilibrium, lethargy and lying on bottom of tank) were observed at 

1.8 mg/L after 14-day in the rangefinder conducted for the FSTRA. Also, reduced growth and 

survival was observed at the top three concentrations (≥0.15 mg/L) in a fish early life-stage study 

with the fathead minnow. 

In the Part 158 avian reproduction studies and in the mallard duck reproduction study, effects on 

14-day offspring weight (↓6.8%) were observed at 979 mg/kg-diet; decreases in female body 

weight gain were also reported (290 & 979 mg/kg-diet; ↓24.5 and 25.7%, respectively). In the 

quail reproduction study at the highest concentration tested (979 mg/kg-diet), decreases in 

embryo viability (↓7.5% in 21-day live embryos) and eggshell thickness (↓10%) were reported in 

the absence of clinical signs of toxicity. 

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Table 2. Estrogenic/Anti-Estrogenic Pathway for TCVP

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for TCVP1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight / 

GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt) / 

Fecundity (Fcd) 

Vitellogenin 

(VTG) 

EDSP Tier 1 Assay 

ER Binding (MRID 48765001) N ERTA (MRID 48619501) N4 Steroidogenesis (MRID 48805201) I5Aromatase (MRID 48673801) PUterotrophic (MRID 48880601) N Female Pubertal Rat (MRID 49186901) NE N N N N N N FSTRA (MRID 48838201) NE N N N 

Frt: ↓9% 

/ Fcd: 

↓61% 

(H) 

F; 

↓30%

(H) 

OSRI 

ERTA (Kojima et al., 2004) N 
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Page 11 of 58 

Table 2. Estrogenic/Anti-Estrogenic Pathway for TCVP

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for TCVP1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight / 

GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt) / 

Fecundity (Fcd) 

Vitellogenin 

(VTG) 

Developmental toxicity (Rat; MRIDs 40152701, 41828001, 42520101) 

NR N 

Developmental toxicity (Rabbit; MRID 00127831) 

NR N 

Developmental Neurotoxicity (Rat; MRID 46660601) 

 N N 

Two-generation reproduction (Rat; MRID 42054301) 

 NR NR NR NR N 

Chronic toxicity / carcinogenicity (Rat; MRIDs 42980901, 43335101) 

N N N N 

Carcinogenicity (Mouse; MRID 00126039) 

NR NR N NR 

Chronic toxicity (Dog; MRID 42679401) 

N N N N 

Avian reproduction (Duck; MRID 48951301) 

P6 

(H) 

Avian reproduction (Quail; MRID 48952701) 

P8 

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

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

weight are absolute unless otherwise indicated. F = Female; LW= liver weight, AW= adrenal weight 

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

this column. For details see Section IV. A 

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

details see Section IV. A 

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

sensitivity of the assay to very weak agonists. Although the conditions of this assay were not optimal to detect very weak activity, similar to those of the negative control corticosterone and not comparable to the responses of 17α-methyltestosterone. TCVP is cothis assay. 

5. Study was considered inadequate. Study did not adequately meet performance criteria. Only 2 of the required 3 experimental runs w

6. Decreases in 14-d offspring body weight at 979 mg/kg-diet. 7. Decreases in female body weight gain at 290 and 979 mg/kg-diet (↓24.5 and 25.7%, respectively). No other clinical signs of toxic8. Decreases in ratio of live 21-d embryos to eggs set (89.3% vs. 96.6% in control) and eggshell thickness at 979 mg/kg-diet. P Positive findings N Negative findings (in vitro) / No effect (in vivo) I Inadequate NE Not examined NR Not reported 
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2. Effects on Androgen Pathway 

The potential for TCVP 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. 

TCVP was equivocal in the Tier 1 in vitro AR binding assay. TCVP reduced the mean specific 

binding of [3

H]-R1881 to between 50- 75% at the highest concentration tested (10-3 M). 

However, TCVP did not show an AR agonist or antagonist response in an AR transcriptional 

activation (ARTA) assay considered as OSRI (Kojima et al., 2004). The Tier 1 steroidogenesis 

assay was considered to be inadequate. Although the results did not show an effect on 

testosterone production, there were numerous deficiencies noted in the study that preclude 

making a definitive conclusion with regard to the effects of TCVP on hormone production in this 

assay. In the Hershberger assay, no treatment-related changes were seen in the androgenic phase 

of the assay. In the anti-androgenic phase, however, atypical effects were observed in the 

weights of some accessory sex organs. Ventral prostate (VP) and Cowper’s gland (CG) weights 

were increased (not decreased) in the antagonist portion of the study, with minimal increases in 

levator ani-bulbocavernosus (LABC) weights in both the agonist and antagonist portions of the 

study. The etiology of these effects is unclear since the weight changes were not dose-dependent 

and the direction (increase) of the observed effect occurred in the phase of the assay designed to 

detect decreases in AST weights. Based on guideline criteria, TCVP is considered negative in the 

Hershberger assay. No androgen-related effects were observed in the Tier 1 male pubertal rat 

assay. In the mammalian Part 158 toxicity studies, the only androgen-related effect observed was 

a 44% decrease prostate weight in the dog chronic study at the limit dose. 

In the FSTRA, there was a 9% decrease (p<0.05) in fertilization in the high-dose group 

(0.57 mg/L). No other potentially androgen-related effects were observed. Slight decreases in 

embryo viability (↓7.5% in 21-day live embryos) was reported in the Part 158 quail reproduction 

study at 979 mg/kg-diet; offspring weight was also reported in the Part 158 mallard duck study. 

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Page 14 of 58 

Table 3. Androgenic/Anti-Androgenic Pathway for TCVP

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for TCVP1Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

hormones 

Testes Weight / 

GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility (Frt) / 

Fecundity (Fcd) 

Age and Weight at 

PPS 

EDSP Tier 1 Assay 

AR Binding (MRID 48703101) E4Steroidogenesis (MRID 48805201) I5 Hershberger (MRID 48946701) NE N Male Pubertal Rat (MRID 49186901) N N N N N N N N FSTRA (MRID 48838201) NE N N N

Frt: 

↓9% 

(H) 

N 
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Page 15 of 58 

Table 3. Androgenic/Anti-Androgenic Pathway for TCVP

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for TCVP1Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

hormones 

Testes Weight / 

GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility (Frt) / 

Fecundity (Fcd) 

Age and Weight at 

PPS 

OSRI 

ARTA (Kojima et al., 2004) N Developmental Neurotoxicity (Rat; MRID 46660601) 

N N 

Two-generation reproduction (Rat; MRID 42054301) 

NR NR NR NR NR NR N 

Chronic toxicity / Carcinogenicity (Rat; MRIDs 42980901, 43335101) 

N N N N N N 

Carcinogenicity (Mouse; MRID 00126039) NR N NR NR NR NR Chronic toxicity (Dog; MRID 42679401) N N N N N 

prostate 

weight 

↓44% 

(H)

Avian reproduction (Duck; MRID 48951301) 

P6 

(H) 

Avian reproduction (Quail; MRID 48952701) 

P8

(H) 

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

indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this are absolute unless otherwise indicated. LW = Liver weight; AW = Adrenal weight; KW = kidney weight. 
TCVP FINAL 

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

this column. For details see Section IV. A 

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

details see Section IV. A 

4. Equivocal in Runs 1 and 2, and “non-binder” in Run 3. 5. Study was considered inadequate. Study did not adequately meet performance criteria. Only 2 of the required 3 experimental runs w

6. Decreases in 14-d offspring body weight at 979 mg/kg-diet. 7. Decreases in female body weight gain at 290 and 979 mg/kg-diet (↓24.5 and 25.7%, respectively). No other clinical signs of toxic8. Decreases in ratio of live 21-d embryos to eggs set (89.3% vs. 96.6% in control) at 979 mg/kg-diet. P Positive findings N Negative findings (in vitro) / No effect (in vivo) E Equivocal NE Not examined NR Not reported 
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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 TCVP to interact with thyroid regulation are 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 both the Tier 1 female and male pubertal assays, no treatment-related effects were noted for 

thyroid weights, but thyroid histopathology indicated increased follicular cell height and 

decreased follicular cell area, with greater changes noted in males. The thyroid histopathology 

findings were statistically significant at the high dose (350 mg/kg/day) in both sexes. Serum T4

levels were decreased at both doses (50 and 350 mg/kg/day) in males and females, although the 

decreases were statistically significant (p<0.05) only at the high dose (↓65% females; 

↓68% males). TSH was increased 31% in males at the high dose; however the change was not 

statistically significant. There was no treatment effect on TSH levels in females. 

In the AMA, median NF stage was decreased one stage in the high-concentration group 

compared to the negative control at Day 7 and 21, but was only significant on Day 7; 

additionally, no asynchronous development was reported. Normalized hind limb length (HLL) 

was decreased at Day 7 (12%, NS) and 21 (38%, p<0.01) at the high concentration, as were 

snout-vent length (SVL; ↓7 and 22% Day 7 and 21) and body weight (↓47% Day 21; p<0.05). 

Frogs in the solvent control group displayed significantly increased growth and development 

compared to the negative control, so decreases in growth and developmental endpoints at the 

highest test concentration were more pronounced when compared to the solvent control. 

Histopathological effects on the thyroid in the highest treatment included apparent increases in 

gland hypertrophy, gland size, follicle size and follicle asymmetry compared to the negative 

control. However, compared to the solvent control, the only histopathological effect was an 

increase in follicle asymmetry. Increased follicle asymmetry as observed in the high treatment 

group is a finding that is associated with increased gland size, but by itself is not diagnostic of 

thyroid gland hypertrophy. Additionally, this high dose was also considered overtly toxic based 

on increased mortality (10%) and clinical signs of toxicity including loss of equilibrium (n=1) 

and lying on the bottom of the tank (n=1). 

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An increased incidence of thyroid follicular cell hypertrophy was observed in females at 142 and 

in both sexes at 375 mg/kg/day in the Part 158 rat subchronic oral study. These effects occurred 

at doses that resulted in significant reductions in RBC ChE activity (↓30 and 72%, respectively) 

as well as increased incidences of histopathological effects in the liver and kidney. In a 28-day 

inhalation study in rats, an increase in mild follicular cell hypertrophy was seen at the mid and 

high dose (0.5 and 1.0 mg/L). Systemic toxicity was also observed at these two doses, including 

significant RBC ChE inhibition (↓35 and 30%, respectively) and increased adrenal weights. 

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Table 4. Thyroid Pathway for TCVP

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for TCVP1

Study Type / Literature 

Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

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

Overt Toxicity Observed 3 

EDSP Tier 1 Assays 

Female Pubertal Rat 

(MRID 49186901) N P (H) 

↓65%

(H) N N 

LW: 

↑21% (H);

AW: 

↑31% (L),

↑37% (H) 

N

Male Pubertal Rat 

(MRID 49186901 ) N P (H) 

↓68%

(H) N N 

LW: 

↑23% (H);

KW: 

↑11% (H) 

N

AMA 

(Frog; MRID 48688401) N N N X4(H) 

OSRI

Subchronic Study 

(Rat; MRID 43371201) 

P 

(M, H)

X 

(M, H) 

X 

(H)

Subchronic Inhalation 

Study 

(Rat; MRID 488033501) 

N P 

(M, H)

X 

(L, M, H) N 

Chronic toxicity / 

carcinogenicity 

(Rat; MRIDs 42980901, 

43335101) 

NE N N X 

(M, H) 

X 

(H)

Carcinogenicity 

(Mouse; MRID 00126039) N N N X 

(H) 

X 

(H) 

Chronic toxicity 

(Dog; MRID 42679401) NE N N X 

(L, M, 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. LW = Liver weight; AW = Adrenal weight; KW = kidney 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. Clinical signs in the high-concentration group included loss of equilibrium (n=1) and lying on the bottom of the 

tank (n=1). Mortality was increased at the high concentration at Day 7 (2.5%) and Day 21 (10%); solvent control 

mortality also was increased at Day 7 (2.5%) and Day 21 (5.2%); no mortality in the negative control. 

P Positive findings 

N Negative findings 

NE Not examined 

 

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IV. Committee’s Assessment of Weight of Evidence 

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

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

pathways) as well as recommendations regarding Tier 2 testing. The results of the Tier 1 assays 

are considered, along with other scientifically relevant information (e.g., 40 CFR Part 158 test 

guidelines and published or publicly available peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence (i.e., specific Tier 1 assays and 

OSRI), then performing an integrated analysis of those lines of evidence. 

The WoE evaluation includes considerations of biological plausibility of the findings from the 

different lines of evidence by examining the consistency, coherence and interrelationships among 

the measured endpoints within and across studies. The available findings from standard 

toxicology studies on the substance may contribute to the WoE evaluation in helping elucidate if 

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

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

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

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

whether or not the responses are related through an endocrine pathway, therefore non-endocrine 

toxic responses (including but not limited to mortality or body weight changes) are also 

considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated 

with weight of evidence evaluation of the results of the 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). 

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1. Tier 1 in vivo Assays 

In the uterotrophic assay, no treatment-related effects were observed on mortality, clinical signs, 

body weights or body weight gains. In the Hershberger assay, body weights for each TCVPtreated group were comparable to control throughout the androgen and anti-androgen assays. At 

350 mg/kg/day, there was a mean daily body weight loss on Days 1 to 2 and body weight gain on 

Days 2 to 3 was decreased (NS) by 46%, resulting in an overall reduction (Days 1 to 10) of 23% 

(NS) in body weight gain. In the 150 mg/kg/day group, body weight gain was decreased on Days 

2 to 3, but increased on Days 6 to 7. Overall body weight gain (Days 1 to 10) for the 50 and 

150 mg/kg/day groups was similar to control. Liver weights in the agonist assay were increased 

by 6, 13 and 30% in the 50, 150 and 350 mg/kg/day groups, respectively, but the increase was 

only significant at the highest dose. Liver weight in the anti-androgen assay was increased by 

17% at 350 mg/kg/day, but was comparable to control at the two lower doses. 

There was no TCVP-related mortality, and no treatment-related clinical signs of toxicity were 

observed during the daily examinations in either the female and male pubertal rat studies. There 

were no effects of TCVP treatment on body weights, body weight gains or food consumption. In 

the female pubertal study, absolute and relative adrenal weights were increased by 29–31% at 

50 mg/kg/day. At 350 mg/kg/day, absolute and relative liver weights were increased by 21 to 

25%, relative kidney weights were increased by 10%, and absolute and relative adrenal weights 

were increased by 37 to 42%. In the male pubertal rat study, absolute and relative liver weights 

were increased by 23 to 27% and absolute and relative kidney weights were increased by 11 to 

15% at 350 mg/kg/day. 

In the FSTRA study, survival in males and females was 100% for all treatment concentrations, 

with the exception of 88% male survival at the 0.055 mg/L (medium) concentration. There were 

no notable observations with regards to behavior or changes in appearance, specifically 

color/banding, ovipositor appearance or size of dorsal nape pad in the control or treated groups, 

or clinical signs of toxicity for any treatment group compared to the control groups. 

In the AMA study, clinical signs of toxicity (i.e., behavioral and other sub-lethal effects) were 

noted at the 2.5 mg/L concentration (high dose) including loss of equilibrium (n=1) and lying on 

the bottom of the tank (n=1). Spinal curvature (crooked tail) was observed in the control groups 

and in every treatment except at the high concentration. Mortality was equivalent in the solvent 

control and the high-concentration group at Day 7 (2.5%), and mortality was increased in the 

high-concentration group at Day 21 (10 vs. 5%), but was greater in the solvent control group 

compared to the low- and medium-concentration groups (1.7 and 3.4%, respectively). No 

mortality was observed in the negative control. 

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2. OSRI 

In the developmental toxicity stud with rats, clinical signs of toxicity observed in the high-dose 

group included urine-stained abdominal fur in 2/25 dams, chromodacryorrhea in 6/25 dams, and 

tremors in one rat from Day 10 through termination. At the high dose (300 mg/kg/day), mean 

maternal body weight gains were decreased by 52% for GD 6 to 15 and increased by 8% (NS) 

for GD 16 to 20, with an overall decrease of 14% for GD 0 to 20. At the medium dose 

(100 mg/kg/day), mean maternal body weight gains were decreased (NS) by 11% for GD 6 to 15, 

with an overall decrease (NS) of 2% for GD 0 to 20. Food consumption in the high-dose group 

was decreased by 22% for GD 6 to 16, and overall consumption was decreased by 9% for GD 0 

to 20. Treatment-related clinical signs observed in the rabbit developmental study consisted of 

maternal toxicity that manifested as mortality (0/18, 1/18, 1/8, 2/18), abortions [one at the low 

dose (75 mg/kg/day) and three at the high dose], and red vaginal fluid passed by 8/11 does in the 

high-dose group and one each in the low- and medium dose groups. 

In a developmental neurotoxicity study in rats, there were no treatment-related effects on 

mortality, clinical signs, body weight, body weight gain, feed consumption, FOB or gross 

pathology in the dams. ChE activity was not determined in the study. In offspring, there were no 

treatment-related effects on viability, litter observations, clinical signs, body weight or food 

consumption. Body weight gains were decreased during pre-weaning at several intervals in 

males (↓12–28%) and females (↓13–26%). Decreases in body weight gains during post-weaning 

were observed in the males on PNDs 21 and 22 (↓36%) and in the females on PNDs 21 and 22 

and 22 to 29 (↓7–28%). 

In a two-generation reproduction study, no treatment-related clinical signs were reported during 

the study. Determination of ChE activity was not reported in this study. Overall mean body 

weight gains in all dose groups for F0 males were decreased to a similar extent (↓7–9%), with 

body weight decreases at Week 10 or 16 of only 5 to 7% (NS). Body weight and body weight 

gains for F0 females were not affected by treatment. Decreases in mean overall body weight gain 

of high-dose (130 mg/kg/day) F1 males (p≤0.05, ↓6), and body weight gain of high-dose F1

females (168 mg/kg/day) during the premating period (p≤0.001, ↓11), were observed, but no 

significant effects on weekly body weights were noted. Mean adrenal weights were increased in 

F0 females [↑9% at the low (7.3 mg/kg/day) and medium dose (40 mg/kg/day, and ↑28% at the 

high dose (155 mg/kg/day]. In the F1 females adrenal weights in the medium- and high-dose 

females were increased by 8 and 9%. No changes in adrenal weights were observed in males in 

either generation. 

In the chronic rat study, minimal (↓4–6%), decrements in body weight were observed 

occasionally in males. In females, treatment-related decreases in body weight were generally 

significant from Weeks 2 to 104 for the medium-dose group (↓≤10%) and the high-dose group 

(↓≤12%). 

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At each sampling interval in the chronic rat study, plasma ChE activity in females was decreased 

29–55% at medium-dose (63 mg/kg/day) and 34–68% at high-dose (125 mg/kg/day). Decreases 

in ChE activity in plasma were also noted in males at high dose (89 mg/kg/day; ↓24 to 41%) and 

at medium dose (43 mg/kg/day; ↓21%, 18 months). No treatment-related effect on RBC ChE 

activity was apparent in male rats in this study. RBC ChE activity was decreased in all treated 

females (with dose-dependent decreases at 18- and 24-months), but the decreases were only 

significant at the 18-month sample time in the medium- and high-dose animals (↓29 and 36%, 

respectively). Brain ChE activity was not altered in males at the interim and terminal sacrifices, 

and inhibition in the high-dose females (↓17 and 15%, respectively) was NS. Liver weights were 

decreased by 16–22% in males at Week 104 (all dose groups) and decreased by 23% in highdose females at Week 52. Kidney weights were not affected in male rats, but were decreased in 

low-dose females at Week 104 (↓16%) and in high-dose females at Weeks 52 and 104 (↓18 and 

15%, respectively). 

In the mouse carcinogenicity study, an increased incidence of hunched/thin appearance in highdose rats beginning at Week 54. ChE activity was not measured in the study. At study 

termination, mean body weights at the two highest doses (1200 and 2400 mg/kg/day) were 

decreased in males (↓18.6 and 32.4%, respectively) and females (↓24.9 and 34.5%). Treatmentrelated increases in absolute liver weights occurred at Week 26 (high dose, male, ↑33%) and 

Week 103 (high dose, male and female, ↑23 and ↓11%, respectively; and 8000 ppm, female, 

↓18%). Treatment-related increases in absolute liver weights occurred at Week 26 (male, ↑37%), 

Week 52 (male, ↑79%), Week 78 (male and female, ↑63 and 51%, respectively), and Week 103 

(male and female, ↑85 and ↑41%, respectively). Kidney weights were decreased in male mice in 

the 8000 ppm dose group (NS, ↓17%) and female mice in the two highest dose groups (↓17 and 

12%, respectively). 

In the chronic dog study, plasma ChE activity was inhibited in low-dose (6.25 mg/kg/day) males 

and all medium- (500 mg/kg/day) and high-dose (1000 mg/kg/day) animals at all sample times. 

Plasma ChE activity was decreased in males by 26–30% at the low dose, 46–48% at the medium 

dose, and 55–61% at the high dose, and in females by 41–47% at the medium dose and 44–49% 

at the high dose. Brain and RBC ChE activity was not affected by treatment. Absolute liver 

weights were increased in medium-and high-dose males [↑20 (NS) and 43%] and females (↑43 

and 43%), and absolute kidney weights were increased (NS) in mid-and high-dose males (↑33 

and 30%) and females (↑21 and 24%). Relative liver and kidney weights were also increased in 

the medium- and high-dose groups. Absolute adrenal weights were increased in medium- and 

high-dose females (↑35 and 39%), with no change in relative weights or weights in males. 

Reduced growth and survival was observed at the top three concentrations (≥0.15 mg/L) in a Part 

158 fish early life-stage study with the fathead minnow. 

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In the Part 158 reproduction study with mallard ducks, decreases in female body weight gain 

were reported (290 & 979 mg/kg-diet; ↓24.5 and 25.7%, respectively). No other clinical signs of 

toxicity were noted. 

B. Estrogen Pathway 

There is no convincing evidence or redundancy of responses to demonstrate potential for TCVP 

to interact with the estrogen pathway in mammals or wildlife. TCVP was classified as negative 

in the ER binding and ERTA assays. Although TCVP was positive in the aromatase assay, there 

were technical issues with the conduct of the steroidogenesis assay that lessen the confidence in 

the negative results for this assay, and there were no redundant responses observed in the in vivo

mammalian assays (Tier 1 or OSRI) to support an interaction with the estrogen pathway. The 

lack of responses to TCVP in female rats in the uterotrophic and female pubertal rat studies do 

not support a potential for ER interaction or altered steroidogenesis. Additionally, there was no 

redundancy of evidence across the other in vivo Part 158 studies. In the FSTRA, effects were 

observed at the highest concentration, 0.57 mg/L (i.e., ↓61% fecundity, ↓9% fertilization and 

↓30% female plasma VTG); however, there no effects on GSI or gonadal histopathology. 

Cholinesterase activity and sex steroids were not measured in this study. While there were no 

clinical signs of toxicity observed in this study, the concentrations tested in the FSTRA were 

similar to concentrations that were shown to be overtly toxic in other fish assays [FSTRA 

rangefinder (1.8 mg/L), Part 158 fish early-life stage study (≥0.15 mg/L), acute LC50 bluegill 

(0.53 mg/L)]. As such, the decreases observed in the FSTRA may be due to potential general 

stress on the fish from exposure to TCVP as oppose to potential endocrine-related interactions. 

A decrease in embryo viability (↓7.5%) and eggshell thinning (↓10%) was observed in the Part 

158 bobwhite quail study; however, there were no other reproductive effects including egg 

production. A decrease in offspring weight was observed in the mallard duck study, but adult 

female body weight was also affected, and there were no other reproductive effects. Therefore, 

while effects on reproduction were observed in the FSTRA, there were no effects on gonadal 

weight or histopathology, and there was limited redundancy in the other in vivo mammalian and 

wildlife studies. 

C. Androgen Pathway 

There is no convincing evidence or redundancy of responses to demonstrate potential for TCVP 

to interact with the androgen pathway in mammals or wildlife. TCVP was equivocal in the Tier 1 

in vitro AR binding assay and negative in an OSRI ARTA (Kojima et al., 2004). Although the in 

vitro steroidogenesis assay was considered inadequate due a number of technical issues in the 

conduct of the study, TCVP did not show a potential to interact with the androgen pathway in the 

male pubertal assay and was considered negative in the Hershberger assay. In the FSTRA, there 

was a 9% decrease (p<0.05) in fertilization in the high-dose group, and no other potentially 

androgen-related effects were observed. In the Part 158 mammalian studies, the only observed 

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effect was a decrease in prostrate weight in the dog chronic study at the limit dose. While 

decreases in embryo viability was reported in the Part 158 quail reproduction study, no other 

androgen-related effects were observed in the Part 158 wildlife studies. 

D. Thyroid Pathway 

In both the female and male pubertal assays, no treatment-related effects were noted for thyroid 

weights, but effects on thyroid histopathology were observed in both sexes. Serum T4 levels were 

decreased at the high dose in males and females (↓65% females; ↓68% males; decreases at low 

dose not significant). TSH was increased 31% in males at the high dose; however the change was 

not statistically significant. There was no treatment effect on TSH levels in females. In the Part 

158 studies, alterations in thyroid histopathology were observed in the subchronic rat study and 

28-day inhalation rat study. Cholinesterase activity was not measured in either Tier 1 pubertal 

assay, however, the effects observed in the Part 158 studies occurred at doses that resulted in 

significant reductions in RBC ChE activity as well as increased incidences of histopathological 

effects in the liver and kidney or increased adrenal weights. 

In the AMA, while effects on development and growth were observed in the highest treatment 

group, they occurred in the presence of overt toxicity. Additionally, in the highest treatment, 

which was considered to be overtly toxic, while histopathological effects on the thyroid included 

apparent increases in gland hypertrophy, gland size, follicle size and follicle asymmetry compared 

to the negative control; however when compared to the solvent control, the only 

histopathological effect was an increase in follicle asymmetry. 

E. Conclusions 

There is a lack of convincing evidence for potential interaction with the estrogen or androgen 

pathways in mammals and wildlife. TCVP appears to interact with the thyroid pathway only in 

mammals. 

V. EDSP Tier 2 Testing Recommendations 

In the EDSP Tier 1 male and female pubertal assays as well as in the 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 TCVP, the current point of departure (POD) 4.23 mg/kg/day used for human health risk 

assessment is based on histological liver and adrenal changes and reduced weight gain at 43.2 

mg/kg/day (LOAEL) in a chronic oral toxicity study in rats. This POD is used for deriving the 

chronic Reference Dose (RfD) for chronic dietary risk assessment. The second POD of 6.7 

mg/kg/day) is based on plasma/RBC ChE inhibition at 142 mg/kg/day (LOAEL) in a subchronic 

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toxicity study in rats. This POD is used for deriving the Margins of Exposure (MOEs) for nondietary risk assessments. 

In general, since the POD and the toxicity endpoints of concern are based on data obtained from 

adult animals, there is a concern that the POD may not be protective of potential thyroid toxicity 

in the young. However in the case of TCVP, although no data exists for potential thyroid toxicity 

in the young, a comparative thyroid assay (CTA) is not recommended since: 1) the POD is 

approximately 83 and 52-fold lower, respectively, than the dose (350 mg/kg/day) that induced 

thyroid effects in the pubertal assays; 2) the POD is based on a more sensitive endpoint 

(cholinesterase inhibition) for this class of chemicals (organophosphate); and 3) the CTA would 

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

used for risk assessment. Consequently, the RfD and MOEs are expected to be protective of the 

thyroid-related effects seen in the male pubertal assay. 

Based on the weight of evidence considerations, mammalian or wildlife EDSP Tier 2 testing is 

not recommended for TCVP since additional testing will not impact current EPA established 

regulatory point of departure or endpoint for human health risk assessments. 

Given the lack of convincing evidence for a potential interaction with the estrogen, androgen and 

thyroid pathways in wildlife no additional testing is recommended. 

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

duck are available for TCVP. In both species, effects on reproductive parameters were observed. 

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 TCVP exposure. 

Relative to the EDSP, additional testing is not recommended. 

 

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VI. References 

Barton, S. (1991). TCVP: Two Generation Reproduction Study in Rats: Lab Project Number: 

438712. Unpublished study prepared by Inveresk Research International. (MRID 42054301). 

Barnet, J.P. (2005). Oral (gavage) developmental neurotoxicity study of TCVP in Crl:CD®

(SD)IGS BR V AF/Plus® rats. Unpublished study prepared by CR-DDS Argus Division, 

Horsham, PA. Laboratory Project ID: CR-DDS Argus Protocol Number: 1608-003, September 9, 

2005. (MRID 46660601). 

Boulet, J. (2012). Hershberger Bioassay in Rats: A Short-Term Screening Assay for Androgenic 

and Anti-Androgenic Properties of TCVP. Unpublished study conducted by Product Safety 

Labs, Dayton, NJ. Laboratory Report No.: 33549, August 31, 2012. (MRID 48946701). 

Boulet, J. (2012). The Uterotrophic Bioassay in Rats: A Short-Term Screening Test for 

Estrogenic Properties of TCVP. Unpublished study conducted by Product Safety Labs, Dayton, 

NJ. Laboratory Report No.: 33548, June 26, 2012. (MRID 48880601). 

Boulet, J. (2013). Pubertal development and thyroid function in intact juvenile/peripubertal male 

and female rats exposed to TCVP. Unpublished study conducted by Product Safety Labs, 

Dayton, NJ. Laboratory Study No.: 33547, July 26, 2013. (MRID 49186901). 

Ford, W.; Killeen, J. (1987). A Teratology Study in Rats with Technical Rabon: Project ID: 

1019-003; 85-0074. Unpublished study prepared by Ricerca, Inc. in cooperation with Argus 

Research Laboratories, Inc. and Test Substance Analysis Laboratory. (MRID 40152701). 

Hazleton Laboratories America, Inc. (1980). 103-week Chronic Feeding Study in Mice: 

SD-8447 and Original SD-8447. Final rept. (Unpublished study received July 31, 1980 under 

unknown admin. no.; submitted by Shell Chemical Co., Washington, DC; CDL:242976-A; 

242977). (MRID 00126039). 

Hoberman, A. (1991). Response to EPA Review of a Rat Teratology Study in Rats with 

Technical Rabon: Lab Project Number: Argus No. 1019-003; Ricerca No. 85-0074; Du Pont 

Report No. 1123-85-0074-TX-003: Supplement No. 1. Unpublished study prepared by Argus 

Research Laboratories, Inc., in cooperation with Ricerca, Inc. (MRID 41828001). 

Hoberman, A. (1992). A Teratology Study in Rats with T-142-4: An Amendment: Lab Project 

Number: 1019-003. Unpublished study prepared by Argus Research Laboratories, Inc. 

(MRID 42520101). 

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Page 28 of 58 

Hofman-Hüther, H. (2011). Endocrine Disruptor Test: Aromatase Assay (Human Recombinant) 

with TCVP (TCVP). Unpublished study conducted by BSL Bioservice, Scientific Laboratories 

GmbH, Planegg, Germany. Laboratory Study No.: 114587, December 23, 2011. 

(MRID 48673801). 

Hofman-Hüther, H. (2012). Endocrine Disruptor Test: In vitro Steroidogenesis Assay with 

TCVP (TCVP). Unpublished study conducted by BSL Bioservice, Scientific Laboratories 

GmbH, Planegg, Germany. Laboratory Study No.: 114586, April 4, 2012. (MRID 48805201). 

Kojima, M., K Fukunaga, M. Sasaki, M. Nakamura, M. Tsuji, T. Nishiyama. (2005) Evaluation 

of estrogenic activities of pesticides using an in vitro reporter gene assay. Int. J. Environ. Health 

Res. 15:271-280. 

Laveglia, J.; Killeen, J.; lgnatoski, J. (1982). A Teratology Study in Rabbits with DS-36779: 

Document No. 572-STX-82-0007-003. (Unpublished study received Apr 13, 1983 under 

unknown admin. no.; submitted by Diamond Shamrock Agricultural Chemicals, Cleveland, OH; 

CDL:249988-A). (MRID 00127831). 

McDonald, P. (2012). A 28 Day Study of Tetrachlorvinphos (TCVP) by Inhalation in Rats with a 

Two Week Recovery Period: Final Report. Project Number: 671774, 32916, 

110826/01/TCVPTF. Unpublished study prepared by Charles River Laboratories. 425p.(MRID 

428803501). 

Mulhern, M.; Robb, D.; Perry, C.; et al. (1993). TCVP: 104 Week Dietary Combined Chronic 

Toxicity/Carcinogenicity Study in Rats: Lab Project Number: 7929. Unpublished study prepared 

by Inveresk Research International. (MRID 42980901). 

Perry, C.; Mulhern, M.; Henderson, W. et al. (1990) Tetrachlorvinphos: 13 Week Dietary 

Toxicity Study in Rats: Lab Project Number: 7258: IRI/438141. Unpublished study prepared by 

Inveresk Research International. 194 p. (MRID 43371201) 

Perry, J.; Millar, P.; Mulhern, M. (1994). TCVP: 104 Week Dietary Combined Chronic 

Toxicity/Carcinogenicity Study in Rats: Addendum: Lab Project Number: 438403: 7929. 

Unpublished study prepared by lnveresk Research International. (MRID 43335101). 

Rödig, K. (2012). Endocrine Disruptor Test: Androgen Receptor Binding (Rat Prostate Cytosol) 

with TCVP (TCVP). Unpublished study conducted by BSL Bioservice, Scientific Laboratories 

GmbH, Planegg, Germany. Laboratory Study No: 114588, January 17, 2012. 

(MRID 48703101). 

Rödig, K. (2012). Endocrine Disruptor Test: Estrogen Receptor Binding (Rat Uterine Cytosol) 

with TCVP (TCVP). Unpublished study conducted by BSL Bioservice, Scientific Laboratories 

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Page 29 of 58 

GmbH, Planegg, Germany. Laboratory Study No: 114589, January 18, 2012. 

(MRID 48765001). 

Schneider, S.Z., Kendall, T.Z., and Krueger, H.O. (2011). Tetrachlorvinphos (TCVP): 

Amphibian Metamorphosis Assay for the Detection of Thyroid Active Substances. Unpublished 

study prepared by Wildlife International, Ltd., Easton, MD. Project No. 699A-102. Sponsors: 

KMG-Bernuth, Inc. and THMC. Study completion date: December 29, 2011. 

(MRID 48688401). 

Schneider, S.Z., Martin, K.H., Kendall, T.Z., and Krueger, H.O. (2012). Tetrachlorvinphos 

(TCVP): Fish Short-term Reproduction Assay with the Fathead Minnow (Pimephales promelas). 

Unpublished study prepared by Wildlife International, Ltd., Easton, Maryland, Report No.: 

699A-101. Submitted by Tetrachlorvinphos Task Force (TCVPTF), c/o SRA International, 

Cambridge, Maryland. Study completion date: May 17, 2012. (MRID 48838201). 

Tompkins, E. (1993) One Year Oral (Capsule) Toxicity Study in Dogs with Rabon (TCVP): 

Final Report: Lab Project Number: WIL-149009. Unpublished study prepared by WIL Research 

Labs., Inc. MRID 42679401. 

U.S. Department of Health, Education, and Welfare (1978). Bioassay of Tetrachlorvinphos for 

Possible Carcinogenicity. HEW. (CAS no. 961-11-5; NCI·CG-TR-33; National Cancer Institute 

carcinogenesis technical report series no. 33; DHEW publication no. (NIH) 78-833; Public 

Health Service, National Cancer Institute, Div. of Cancer Cause and Prevention, Carcinogenesis 

Program, Carcinogen Bioassay and Program Resources Branch; published study; CDL:233821-

A). (MRID 00063589). 

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

EDSP-083702-55. August 9, 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. 

U.S. National Institutes of Health (1978). Bioassay of tetrachlorvinphos for possible 

carcinogenicity. By National Cancer Institute, Div. of Cancer Cause and Prevention, 

Carcinogenesis Program, Carcinogen Bioassay and Program Resources Branch. Bethesda, MD: 

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USNlli. (NCI-CG·TR-33; CAS No. 961· 11-5; DHEW publication no. (Nni)78-833; published 

study; CDL:238926-A). (MRID 00117443). 

Willoughby, J.A. (2011). TCVP: Estrogen Receptor Transcriptional Activation Assay (Human 

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

Report No.: 9180V-100538ERTA, November 4, 2011. (MRID 48619501). 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA): (OCSPP 890.1100) 

The 21-day assay (MRID 48688401) of tetrachlorvinphos (TCVP) (98.7% a.i.) on amphibian 

metamorphosis of the African clawed frog (Xenopus laevis) was conducted under flowthrough conditions. Amphibian larvae (four replicates per treatment, 20 larvae per replicate) at 

Nieuwkoop-Faber (NF) Stage 51 were exposed to negative (dilution water) control, solvent 

control (0.020 mg/L dimethyl formamide, DMF), and TCVP nominal concentrations of 0.030, 

0.30, and 3.0 mg a.i./L. Measured concentrations of TCVP were <0.020 mg a.i./L (LOQ) in the 

negative and solvent controls and 0.029, 0.30, and 2.5 mg a.i./L in the test item treatments. The 

test system was maintained at 17.6 to 22.6 °C (measured continuously) with a pH of 8.1 to 8.4. 

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

water) control. Clinical signs of toxicity (i.e., behavioral and other sublethal effects) were noted 

at 2.5 mg a.i./L including including loss of equilibrium and lying on the bottom of the tank were 

observed in two tadpoles. Spinal curvature (crooked tail) was observed in controls and in every 

treatment except the high treatment (2.5 mg a.i./L). 

At Day 21, tadpoles had shorter normalized hind-limb length (HLL) at the highest test 

concentration (2.5 mg a.i./L) than in the negative control (p<0.01). A statistically significant 

delay (one stage) of median NF developmental stage was observed at Day 7 (p=0.01) days at 2.5 

mg a.i./L. No asynchronous development was observed. Effects on thyroid gland histopathology 

were observed at 2.5 mg a.i./L. Histopathological effects included increases in gland 

hypertrophy, gland size, follicle size, and follicle asymmetry, compared to the negative control. 

Statistically significant effects on growth parameters (p<0.05), including snout-vent length and 

body weight, were observed at both the 7-day and 21-day sampling points at 2.5 mg 

a.i./L. Increased growth was seen at the lowest treatment (0.029 mg a.i./L) compared to the 

negative controls and was consistent with observations in the solvent controls. 

In the solvent control group, there was a significant increase in 7-day body weight and SVL 

(p<0.05), and 21-day body weight, SVL, HLL, and normalized HLL (p<0.05), compared to the 

negative controls. Compared to the solvent control, Day 7 body weight, SVL, and NF stage (2 

stages) were significantly decreased (p<0.05) at the highest test concentration. Day 21 body 

weight, SVL, normalized HLL, and NF stage (3 stages) were significantly decreased (p<0.05) at 

the highest test concentration. Compared to the solvent control, the only noteworthy 

histopathological finding was an increase in follicle asymmetry. 

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

In an androgen receptor (AR) binding assay (MRID 48703101), ventral prostate cytosol from 

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

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

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

functioning with appropriate affinity for the radio-labeled reference androgen (R1881), and the 

competitive binding experiment measured the binding of a single concentration of [3

H]-R1881 (1 

nM) in the presence of increasing concentrations (logarithmic increase from 10−10 to 10−3

 M) of 

tetrachlorvinphos (TCVP; 100.05% purity, Batch #TX 100113). Ethanol was used as a vehicle 

at a final assay concentration of 3.3%. A total of three runs were performed for the competitive 

binding assay, and each run included dexamethasone as a weak positive control, and R1881 as 

the ligand reference standard. 

The saturation binding experiment did not clearly demonstrate that the AR in the rat prostate 

cytosol was present in reasonable numbers and was functioning with appropriate affinity for 

reference ligand (R1881). The maximum binding capacity (Bmax) was 0.4298-0.859 fmol/100 μg 

protein for the three runs and the dissociation constant (Kd) was 0.5615-1.21 nM. The Kd was 

within the range reported in the EPA validation program for Runs 2 and 3 only (the value for 

Run 1 was slightly low); however, Bmax values were significantly below the expected range of 10 

to 150 fmol/100 μg protein for all runs. In addition, specific binding did not reach a plateau in 

Run 3, and non-specific binding ranged from 29.2 to 61.7% of total binding for Run 1 and from 

13.2 to 32.9% of total binding for Runs 2 and 3 (excluding one outlier), which is greater than the 

suggested value of <20% reported in the EPA validation program. The Scatchard plot of the data 

was linear for Runs 2 and 3 only. 

In the competitive binding experiment, the estimated mean log IC50s for R1881 (−8.69 M) and 

the weak positive control, dexamethasone (−4.39 M), were within the expected ranges. The 

mean RBA for the weak positive control was 0.0055%. Confidence in these numbers is high due 

to the small variation. The solvent control responses indicated a slight drift in the study assay for 

Run 1 (15%). Performance criteria were not met for top % binding for TCVP (Run 1), top % 

binding for R1881 (Run 2), bottom % binding for R1881 (all runs), and top % binding for 

dexamethasone (Run 2). 

A log IC50 and RBA for TCVP could not be determined as exposure to TCVP did not result in 

50% reduction in [3

H]-R1881 at any concentration. No precipitation of the test compound was 

reported. 

Based on the results from the three runs, TCVP is classified as Equivocal in the Androgen 

Receptor Binding Assay. 

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Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48673801), tetrachlorvinphos (TCVP; 100.05% 

a.i., Batch # TX 100113) was incubated with human recombinant aromatase and tritiated 

androstenedione (1-β [3

H(N)]-androst-4-ene-3,17-dione; [3H]ASDN) in 1.0% dimethyl sulfoxide 

(DMSO) at final log concentrations of 10−10 to 10−3 M for 15 minutes to assess the potential of 

TCVP to inhibit aromatase activity. 

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

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

using liquid scintillation counting (LSC). Three 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

 M for Run 1; 10−7 to 10−3 M for Runs 2 and 3) with three repetitions per 

concentration. Due to inhibition of aromatase activity observed in Run 1, the concentrations of 

the test item used in Runs 2 and 3 were adjusted to include mid-log concentrations. 

Aromatase activity in the full activity controls for the three successful runs was 0.181 ± 0.024 

nmol·mg-protein−1·min−1. The response of the full activity controls and background controls was 

acceptable for each run. Activity in the background controls ranged 1.7-4.4% and averaged 3.03 

± 0.93% of the full activity controls. 

For the positive control substance (4-OH ASDN), aromatase activity results were within the 

recommended ranges for the performance criteria. The estimated log IC50 for 4-OH ASDN 

averaged −7.3 M and the Hill slope was −1.05. 

For TCVP, aromatase activity averaged 0.203 ± 0.002 nmol·mg-protein−1

·min−1 at the lowest 

concentration tested in Run 1 (10−10 M) and 0.155 ± 0.037 nmol·mg-protein−1·min−1 at the lowest 

concentration tested in Runs 2 and 3 (10−7 M; includes data for Run 1). Aromatase activity was 

0.028 ± 0.008 nmol·mg-protein−1

·min−1 at the highest tested concentration of TCVP (10−3 M). 

The lowest portion of the response curve for TCVP across runs was less than 50% activity. 

Aromatase activity for TCVP was 103.6-105.9% at concentrations from 10−10 to 10−8

 M (tested 

in Run 1 only) and 90.0-95.4% at concentrations from 10−7

 to 10−5 M; activity decreased to 

62.6% at 10−4

 M, 33.1% at 3.16 × 10−4 M, and 17.2% at 10−3 M. The average log IC50 of TCVP 

was −3.76 M and the Hill slope was −1.01. 

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

aromatase activity in this assay. 

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

In an estrogen receptor (ER) binding assay (MRID 48765001) for tetrachlorvinphos (TCVP), 

uterine cytosol from Sprague Dawley rats was used as the source of ER to conduct saturation and 

competitive binding experiments. The saturation binding experiment was conducted to 

demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was 

functioning with appropriate affinity for the radiolabeled reference estrogen prior to routinely 

conducting competitive 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 (logarithmic increase from 10−10 to 10−3

 M) of TCVP 

(100.05% purity, Batch #TX 100113). Ethanol was used as the solvent vehicle at a final 

concentration of 2%. A total of 3 runs were performed, and each run included 19-norethindrone 

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

natural ligand reference material. 

In the saturation binding experiment, the mean maximum binding capacity (Bmax) was 5.464 

fmol/100 μg protein and the mean dissociation constant (Kd) was 0.1297 nM. The Bmax value 

was lower than the recommended range of 10-150 fmol/100 μg protein. The Scatchard plots for 

the three Saturation assays were linear. The specific binding reached a plateau in all runs, and 

non-specific binding was generally less than 20% of total binding. 

In the competitive binding experiment, the estimated mean log IC50 was −9.188 M for 17βestradiol and −6.484 M for the weak positive control. The mean relative binding affinity (RBA) 

was 0.1990% for the weak positive control. Confidence in these numbers is high due to the 

small variation in the data. All performance criteria were generally met. 

TCVP had minimal effect on specific binding of the [3

H]-ligand; therefore, a log IC50 and RBA 

could not be calculated for TCVP. Specific [3H]-ligand binding to the ER was >75% at all 

soluble concentrations of TCVP tested in all runs. Based on the results from the three runs, 

TCVP is classified as Not Interactive in the Estrogen Receptor Binding Assay. 

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

In an estrogen receptor transcriptional activation assay (MRID 48619501), hERα-HeLa-9903 

cells cultured in vitro were exposed to tetrachlorvinphos (TCVP, 100.05% a.i., Lot # TX100113) 

at logarithmically increasing concentrations from 10−12 to 10−4

 M in 0.1% DMSO for 

approximately 24 hours in two independent runs. The experiments were performed using 96-

well plates and each TCVP concentration was tested in 6 wells/plate in each run. Cells were 

exposed to the test agent for 24±2 hours at 37±2°C to induce reporter (luciferase) gene products. 

Luciferase expression in response to activation of the estrogen receptor was measured using a 

proprietary luciferase assay. 

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TCVP was tested up to the limit of cytotoxicity, which was observed at 10−4

 M in the first run 

and ≥10−6 M in the second run. In the main assays, the responsiveness of the cells to the very 

weak positive control 17α-methyltestosterone was lower than the expected values, indicating a 

decreased sensitivity of the assay to very weak agonists. Although the conditions of this assay 

were not optimal to detect very weak activity, TCVP 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 36.5-46.3% PC. TCVP was only able to reach a 

maximum of 0.8-2.4% PC when tested up to the highest concentration possible based on 

cytotoxicity (10−7 M). Because the RPCMax < PC10 in both assay runs, TCVP 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 48838201) of tetrachlorvinphos (TCVP) with 

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

conditions. Adult fish (20 spawning groups, 2 males and 4 females in each group; 5 months old) 

were exposed to TCVP (98.7%) at nominal concentrations of 0 (negative control), 0 [solvent 

control; dimethylformamide (DMF) at a final concentration of 0.02 mL/L], 0.006, 0.060, and 

0.60 mg a.i./L; mean-measured concentrations were <0.0025 (<LOQ; negative and solvent 

control), 0.006, 0.055, and 0.57 mg a.i./L. The test system was maintained at 24.5 to 25.6oC and 

a pH of 8.0 to 8.3. 

Unless otherwise noted, all results are presented based on comparison to the negative control. 

No significant differences (p>0.05) were detected between the negative and solvent control 

groups for any of the endpoints. Survival in males and females was 100% for all test levels, with 

the exception of 88% male survival at the 0.055 mg a.i./L level. Adult male and female survival 

was visually determined to be unaffected by treatment. 

Fecundity in negative and solvent control groups was 26.6 and 24.5 eggs/female/day/replicate, 

respectively; fertilization success in the negative and solvent control groups averaged 98.6 and 

98.1%, respectively. 

There was a significant reduction at the 0.57 mg a.i./L treatment level in fecundity and fertility of 

61.4 and 9.15%, respectively (p<0.05), compared to the negative control. There was also a 

significant decrease in female vitellogenin (VTG) at the 0.57 mg a.i./L treatment level of 30.0% 

compared to the negative control (p<0.05). There were no other significant differences between 

the treatment levels and the negative control for any other endpoint. There were no notable 

differences between histopathology severity scores or gonadal stages in treated groups compared 

to the negative control. There were no notable observations with regards to behavior or changes 

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

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control or treated groups or clinical signs of toxicity for any treatment group compared to the 

controls. 

 

Hershberger Assay; (OCSPP 890.1400) 

In a Hershberger assay (MRID 48880601) screening for androgenic activity, tetrachlorvinphos 

(TCVP, 100.05% a.i., Lot# TX 10113) in aqueous 0.1% (w/v) methylcellulose was administered 

daily by oral gavage to groups of 57-day old, castrated male Sprague Dawley (Crl:CD[SD]) rats 

(6/group) at dose levels of 0 (vehicle), 50, 150, or 350 mg/kg/day. The androgenic assay 

included a positive control group consisting of six castrated rats exposed to 0.4 mg/kg/day of 

testosterone propionate (TP) by subcutaneous (s.c.) injection. 

To screen for potential anti-androgenic activity, TCVP in aqueous 0.1% (w/v) methylcellulose 

was administered daily via oral gavage to 57-day old, castrated male Sprague Dawley rats 

(6/group) at dose levels of 50, 150, or 350 mg/kg/day in conjunction with a daily dose of 

reference androgen TP at 0.4 mg/kg/day by s.c. injection. The control group for the antiandrogenic assay consisted of six castrated male rats dosed daily with TP (0.4 mg/kg/day) by s.c. 

injection, and the positive control group consisted of six castrated male rats dosed orally with 

flutamide (FT) at 3 mg/kg/day in conjugation with a daily s.c. dose of TP (0.4 mg/kg/day). 

For both components of the assay, the animals were dosed for ten consecutive days and 

necropsied approximately 24 hours after the final dose administration to determine weights of 

the five androgen-dependent tissues. 

In the androgen agonist assay, body weights and body weight gains for each TCVP-treatment 

group were comparable to controls throughout the assay. Food consumption was initially 

decreased (p<0.05) by 8% for Days 1-3 in the 350 mg/kg/day group, but was then increased for 

Days 3-10. For Days 3-10, food consumption in the 150 mg/kg/day group was increased 

(p<0.001) by 10%. Overall food consumption on Days 1-10 was increased (p<0.001) by 27% in 

the 150 mg/kg/day group, and increased 3% in both the 50 and 350 mg/kg/day groups. No 

effects on food efficiency were observed for the TCVP treatment groups. For the positive 

control group (TP), food consumption was increased (p<0.01) by 11% and food efficiency was 

increased (p<0.01) by 50% for Days 1-10. 

Liver weights in the agonist assay were increased by 6, 13 and 30% in the 50, 150, and 350 

mg/kg/day group, respectively, but the increase was only significant (p≤0.01) at the highest dose. 

Adrenal gland weights were also increased (p≤0.01) by 39, 47, and 44% in the 50, 150, and 350 

mg/kg/day groups, respectively. Except for a 32% increase (p≤0.01) in the levator anibulbocavernosus (LABC) weight of the 50 mg/kg/day group, there were no significant changes 

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in accessory sex organ weights of the TCVP-treated groups. Animals in the TP group had 

increases (p≤0.01) in unadjusted accessory sex organ weights as follows: 370% in ventral 

prostate, 295% in seminal vesicles, 179% in LABC, 205% in Cowper’s gland, and 72% in glans 

penis. 

In the anti-androgen assay, body weights for each TCVP-treated group were comparable to 

controls throughout the assay. At 350 mg/kg/day, there was a mean daily body weight loss 

(p≤0.05) on Days 1-2 and body weight gains on Days 2-3 were decreased (NS) by 46%, resulting 

in an overall reduction (Days 1-10) of 23% (NS) in body weight gains. In the 150 mg/kg/day 

group, body weight gains were decreased (p<0.05) on Days 2-3 but increased (p<0.05) on Days 

6-7. Overall body weight gains (Days 1-10) for the 50 and 150 mg/kg/day groups were similar 

to controls. For the positive control group (TP + FT), overall mean daily body weight gains were 

decreased (p≤0.05) by 14%. Food consumption in the 150 and 350 mg/kg/day groups were 

decreased (p<0.01) by 14-16% for Days 1-3. For Days 3-10, food consumption in the 50 

mg/kg/day group was decreased (p<0.01) by 3%. Compared to the TP-control, overall food 

consumption (Days 1-10) was decreased (p<0.01) by 5% in the 350 mg/kg/day group, but there 

was no effect in the 50 and 150 mg/kg/day groups. For the positive control group (TP+FT), food 

consumption was increased (p<0.01) by 7-8%. There was an initial decrease (p<0.01) of 96% in 

food efficiency in the 350 mg/kg/day group, but otherwise food efficiency in the TCVP-treated 

and positive control group were similar to the TP control. 

Liver weights in the anti-androgen assay were increased by 17% (p≤0.05) at 350 mg/kg/day, but 

were comparable to the control at the two lower doses. There were no decreases in accessory sex 

organ weights of TCVP-treated animals. However, non-dose-dependent increases (p≤0.01) in 

the following accessory sex organ weights were observed in all TCVP-treated groups: 85-139% 

in ventral prostate, 143-203% in seminal vesicles, and 47-60% in the Cowper’s gland. In 

addition, there was a 24% increase (p≤0.01) in LABC weights in the 50 mg/kg/day group. 

Animals dosed with FT + TP (positive control) had the following decreases in accessory sex 

organ weights: 61% in ventral prostate; 67% in seminal vesicles; 44% in LABC; 56% in 

Cowper’s glands; and 27% in glans penis. 

The high dose of TCVP selected for this study (350 mg/kg/day) was considered adequate based 

on decreased overall body weight gains and increased liver weights observed in the current 

assay. 

TCVP was negative for androgenic activity in the Hershberger assay as statistically significant 

increases in organ weights were not seen in two or more of the five androgen sensitive tissues in 

the androgen agonist assay. TCVP was also negative for anti-androgenicity in the Hershberger 

assay as no statistically significant decreases in organ weights were seen in two or more of the 

five androgen sensitive tissues in the androgen antagonist assay. The biological significance of 

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the increases in accessory sex organ weights of TCVP-treated rats in the anti-androgen assay is 

unclear. The accessory sex organ weight increases were not dose dependent, and TCVP did not 

give a positive response in the agonist assay. However, the data suggest that TCVP may be 

affecting the metabolism of the testosterone, which would not have been evident in the agonist 

assay as the castrated rats were already testosterone deficient. Testosterone concentrations were 

not determined in this assay. 

Female Pubertal Assay; (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 49186901), 15 Sprague Dawley [SD® IGS] rats/dose group 

were treated daily by oral gavage with tetrachlorvinphos (TCVP) (99.52% ai; Lot # TX 100113) 

in aqueous 0.1% (w/v) methylcellulose at doses of 0 (vehicle), 50, or 350 mg/kg/day from postnatal day (PND) 22 to 42/43. In addition, a reference group of 15 females dosed with 17αethynylestradiol (EE), by subcutaneous injection, at 0.003 mg/kg/day was included in the study. 

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

weight at day of attainment were recorded. After sacrifice on PND 42 or 43, blood was collected 

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

levels were determined using a radioimmunoassay (RIA). Adrenal, liver, kidney, pituitary, 

thyroid, and urogenital organs were weighed, and microscopic examinations were performed on 

the ovaries, uterus, thyroid, liver, and kidneys. 

There were no adverse, treatment-related effects on mortality, clinical signs of toxicity, body 

weights, body weight gains, food consumption, clinical chemistry, or macroscopic pathology. 

Serum T4 levels were decreased by18% (not statistically significant [NS]) and 65% (p<0.05) at 

50 and 350 mg/kg/day, respectively, but serum TSH concentrations at 50 and 350 mg/kg/day 

were comparable to control. Follicular cell height was slightly increased and follicular colloid 

area was slightly decreased in a dose-dependent manner in the 50 and 350 mg/kg/day treatment 

groups; it was reported that the differences were only statistically significant in the 350 

mg/kg/day group. 

There were no effects of treatment on age and body weight at attainment of VO. All females 

achieved complete VO. There were no effects of treatment on mean age at first vaginal estrus, 

mean cycle length (a slight increase was considered non-adverse), % cycling or regularly 

cycling, or cycle status at necropsy. Corpora lutea were present in the ovary of all individuals, 

and no atretic follicles were noted in controls or TCVP-treated groups. 

Compared to the control group, relative (to body weight) liver weights were increased (p<0.001) 

by 8%, and absolute and relative adrenal weights were increased (p<0.001) by 30-31% at 

50 mg/kg/day. At 350 mg/kg/day, absolute and relative liver weights were increased (p<0.001) 

by 21-25%, and absolute and relative adrenal weights were increased (p<0.001) by 37-42%. 

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There were increased incidence and severity of chronic progressive nephropathy in the 350 

mg/kg/day group compared to the control. 

The high dose (350 mg/kg/day) was considered to be adequate, based on increased liver and 

adrenal weights and increased incidence and severity of chronic progressive nephropathy. Mean 

adrenal glands weight was 37.6 mg, but the acceptable range is 38.34-48.84 mg. 

For animals in the positive control group (EE group), food consumption over the course of the 

study was decreased (p<0.05, ↓9%), leading to decreased overall body weight gain (p<0.05, 

↓14%). The mean age at attainment of VO was decreased (p<0.01) by 8 days (PND 25.6 treated 

vs. PND 33.6 controls) and body weight at attainment of VO was decreased (p<0.01) by 39%. 

All EE-treated females were non-cycling in constant estrus from PND 26 through the entire 

observation period. Absolute ovaries weights were decreased (p<0.001, ↓48%) and relative 

pituitary weights were increased (p<0.05, ↑21%). The following clinical chemistry parameters 

were increased (p<0.05) compared to controls: bilirubin (↑18%), total protein (↑9%), albumin 

(↑10%), globulin (↑8%), and calcium (↑4%). Many of these changes were as expected for an 

estrogen agonist. 

Male Pubertal Assay; (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 49186901), 15 Sprague Dawley [SD® IGS] rats/dose group 

were treated daily via oral gavage with tetrachlorvinphos (TCVP) (99.52% ai; Lot # TX 100113) 

in aqueous 0.1% (w/v) methylcellulose at doses of 0 (vehicle), 50, or 350 mg/kg/day from postnatal day (PND) 23 to 53/54. In addition, two separate positive control reference groups of 15 

males were dosed with either testosterone propionate (TP), via subcutaneous injection, at 1.0 

mg/kg/day, or with the androgen antagonist, flutamide (FT) via oral gavage at 3.0 mg/kg/day. 

Animals from each group were examined for preputial separation (PPS) daily beginning on PND 

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

54, blood was collected for clinical chemistry analyses, and total serum testosterone, thyroxine 

(T4), and thyroid stimulating hormone (TSH) levels were determined using a radioimmunoassay 

(RIA). Adrenal, liver, pituitary, thyroid, and urogenital organs were weighed, and microscopic 

examinations were performed on the testes, epididymides, thyroid, liver, and kidneys. 

There was no TCVP-related mortality, clinical signs of toxicity, body weights, body weight 

gains, food consumption, or macroscopic pathology. There were no effects of TCVP treatment 

on age and body weight at attainment of PPS. In the control group and two TCVP dose groups, 

14/15 males achieved complete PPS. 

Thyroid weight was unaffected by TCVP treatment. Follicular cell height was increased and 

colloid area was decreased in a dose-dependent manner in the 50 and 350 mg/kg/day treatment 

groups, with the differences being significant in the 350 mg/kg/day group. Serum T4 levels were 

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decreased by 15% (not statistically significant [NS]) and 68% (p<0.05) at 50 and 350 mg/kg/day, 

respectively, and serum TSH levels were increased (NS) by 8% and 31% at 50 and 350 

mg/kg/day. 

There were no effects of TCVP treatment on the weights of pituitary, adrenals, seminal vesicle 

plus coagulating gland (with or without fluid), ventral prostate, dorsolateral prostate, levator anibulbocavernosus (LABC) muscle complex, epididymides, testes, or thyroid. There were no 

treatment-related microscopic findings in the male reproductive tissues. Serum testosterone 

levels were unaffected by treatment. 

At 350 mg/kg/day, absolute and relative liver weights were also increased (p<0.001) by 23-27% 

and absolute and relative kidney weights were increased (p<0.05) by 11-15%. There was 

increased incidence (9/15) of chronic progressive nephropathy in the 350 mg/kg/day group 

compared to the control (1/15). In the liver, slight to moderate hepatocellular hypertrophy was 

also observed in all males in the 350 mg/kg/day group. 

The high dose (350 mg/kg/day) was considered to be marginally adequate based on increased 

liver and kidney weights and increased incidence of chronic progressive nephropathy. 

Groups receiving either with the androgen agonist (TP) or the antagonist (FT) showed the 

expected effects on sexual development and reproductive tissues. For rats receiving TP, age at 

attainment of PPS was decreased (p<0.01) by 5.6 days (PND 43.4 treated vs. PND 49.0 controls) 

and body weight at attainment of PPS was decreased (p<0.01) by 20%. The weights of seminal 

vesicle plus coagulating gland (with fluid), ventral prostate, dorsolateral prostate, and LABC 

were increased (p<0.001, incr 77%, 50%, 29%, and 27%, respectively) and the weights of 

epididymides and testes were decreased (p<0.001, decr 15-16% and 48%, respectively) 

compared to the controls. Serum testosterone was increased (p<0.05) by 617% compared to 

controls. Microscopic findings attributed to TP administration included testicular degeneration 

and/or depletion of the round spermatids and spermatocytes, degeneration and/or depletion of the 

elongating spermatids, atrophy of the Leydig cells, reduction in testicular size, and effects in the 

epididymis (ductular epithelium cribiform change, sloughed germ cells, and reduced sperm) in 

13-14/14 males. 

For rats receiving FT, age at attainment of PPS was increased (p<0.01) by 4.7 days (PND 53.7 

vs. PND 49.0). The weights of the following organs were decreased (p<0.01) compared to 

controls: seminal vesicle plus coagulating gland with fluid (decr 52%), ventral prostate (decr 

27%), dorsolateral prostate (decr 35%), LABC (decr 29%), and epididymides (decr 23-25%). 

Microscopic findings attributed to FT administration included hyperplasia of the Leydig cells in 

the testis in 14/15 males. 

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Steroidogenesis Assay; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48805201), H295R cells cultured in vitro in 24-well plates 

were incubated with TCVP (100.05% purity, Batch # TX 100113) at serial log concentrations 

from 0.0000001 to 100 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as a 

vehicle, at a final assay concentration of 0.1%. Only two independent experiments were 

performed, rather than the Guideline recommended three runs. 

Testosterone and estradiol concentrations were measured using an enzyme-linked 

immunosorbent assay (ELISA) method. A Quality Control (QC) plate was run concurrently with 

each independent run of a test chemical plate to demonstrate that the assay responded properly to 

positive control agents at two concentration levels; positive controls included the known 

inhibitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production. The 

highest concentration of TCVP suitable for evaluation was 10 μM due to cytotoxicity observed 

at 100 μM TCVP in both runs; half log or intermediate concentrations were not investigated. 

Minimum basal hormone production for testosterone (≥500 pg/mL) was not met in any blank or 

solvent control (SC) wells (mean testosterone values were ≤180 pg/mL), but minimum basal 

production of estradiol (≥40 pg/mL) was achieved. The reviewers could not determine if basal 

hormone production met the fold-increase criteria in the SC wells of ≥5-fold for testosterone and 

≥2.5-fold for estradiol above the minimum determination limit (MDL) of the assay because the 

MDLs for the ELISA were not reported. 

On the QC plates, exposure to 10 μM forskolin induced testosterone production ≥2-fold and 

estradiol production ≥7.5-fold over SC. Exposure to 1 μM prochloraz inhibited synthesis of 

testosterone by 2.22% and 84.62%, and inhibited estradiol synthesis by 47.96% and 56.69%, 

relative to the SC (Guideline recommendation is ≥50% inhibition). The mean fold-change data 

did not meet the performance criteria recommended by the Guideline, and there was a high 

degree of variability in testosterone induction (forskolin) and inhibition (prochloraz) responses 

between the two assay runs. In Run 1, the induction of testosterone in the presence of 1 μM 

forskolin was approximately 7-fold greater than the induction achieved at 10 μM forskolin. The 

testosterone induction at 1 μM forskolin in Run 1 was also approximately 7-fold greater than the 

response to 1 μM forskolin in Run 2. Inhibition of testosterone synthesis in the presence of 

0.1 μM prochloraz varied approximately 18-fold between the two assay runs (fold differences of 

7.27 versus 0.43). This variability may be associated with poor analytical performance of the 

ELISA or poor cell response; however, the reviewer’s assessment was hampered by the lack of 

methodological details and proficiency testing data. 

Only a single hormone concentration value for testosterone and estradiol, respectively, was 

reported for the SC replicates for each assay run; no distinction between SC values for the test or 

QC plates was provided. The % CVs for absolute testosterone concentrations for the SC 

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replicates within the test plates were 50% and 26%; therefore, only the second run met the ≤30% 

performance criteria recommended in the Test Guideline. The % CVs for absolute estradiol 

concentrations for the SC replicates within the test plates were 17% and 9%, with both values 

meeting the recommended performance criteria. The between-plate % CVs for the absolute 

hormone concentrations in the SC, which were 58% for testosterone and 38% for estradiol, were 

greater than the recommended performance criteria of ≤30%. All % CV determinations were 

calculated by the reviewers. 

With regards to the effects of TCVP, the results from the two runs were variable for testosterone. 

In Run 1, there was a 22.4-fold induction in testosterone production at 10 μM TCVP, but no 

induction (≤0.70-fold) was observed at the other reported TCVP concentrations, and there was a 

marked inhibition in testosterone production (0.26-fold) at the lowest concentration of TCVP 

(0.000001 μM). In Run 2, no induction or inhibition of testosterone production (0.87- to 1.24-

fold) was observed at 0.000001 to 10 μM TCVP, but there was again a marked reduction in 

testosterone (0.32-fold) at the lowest TCVP concentration tested (0.0000001 μM). For estradiol 

production, there was no consistent induction or inhibition in either run at 0.000001 to 10 μM 

TCVP, with fold-change values ranging from 0.80- to 1.70-fold in Run 1 and 0.83- to 1.14-fold 

in Run 2. Although the results of the assay do not indicate a role for TCVP as an inhibitor or 

inducer of testosterone or estradiol production, the numerous deficiencies noted in the study 

preclude making a definitive conclusion with regards to the effects of TCVP on hormone 

production in this assay. 

Uterotrophic Assay; (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48880601) conducted to screen for potential estrogenic activity, 

tetrachlorvinphos (100.05% a.i., Lot #TX 100113) in 0.1% aqueous methylcellulose was 

administered daily via oral gavage to groups of 6 ovariectomized female Sprague Dawley rats at 

dose levels of 0 (vehicle), 50, 150, or 350 mg/kg/day on post-natal days (PND) 56-58. A 

positive control group of 6 ovariectomized rats was treated with a daily dose of 17α-ethynyl 

estradiol (EE) in 0.1% aqueous methylcellulose at 0.3 μg/kg/day by subcutaneous (sc.) injection. 

Body weights and food consumption were determined daily. All animals were terminated and 

necropsied on PND 59, approximately 24 hours after the final dose administration to determine 

wet and blotted uterine weights. 

No treatment-related effects were observed on mortality, clinical signs, body weights, or body 

weight gains. Food consumption on Days 1-4 was increased (p<0.001) by 5-6% in all 

tetrachlorvinphos treatment groups, but there was no adverse effect on food efficiency. Uterine 

weights in the tetrachlorvinphos treated groups were comparable to the vehicle control. Absolute 

wet and blotted uterus weights for the positive control (EE) group were increased (p<0.001) by 

75% and 71%, respectively, as expected. 

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Although no evidence of systemic toxicity was observed in this study, the high dose 

(350 mg/kg/day) was considered adequate based on increases in liver and kidney weights seen at 

350 mg/kg/day in the female pubertal assay (MRID 49186901). 

No statistically significant changes were seen in uterine weight in this assay. Tetrachlorvinphos 

is negative in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Published literature and Part 158 general toxicity studies considered relevant for determining the 

potential interaction of TCVP with the endocrine system are summarized below. Given the 

breadth of toxicity data available on TCVP, 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.). 

ER/AR Transcriptional Activation 

In a published, non-Guideline study (Kojima et al., 2004), the potential of 200 chemicals 

(including TCVP; purity 95-100%) to interact with two human ER subtypes (hERα and hERβ) 

and one human androgen receptor (hAR) in transactivation assays with Chinese hamster ovary 

cells (CHO-K1 cells) was investigated. For detection of hERα or hERβ activity, cells were 

transfected with pcDNAERα or pcDNAERβ, pGL3-tkERE, and pRL-SV40. For detection of 

hAR activity, cells were transfected with pZeoSV2AR, pIND-ARE, and pRL-SV40. Flutolanil 

was tested over a concentration range from 10−8

 to 10−5 M, and the responses were compared to 

the activity of 17β-estradiol (E2; purity >97%) or 5α-dihydrotestosterone (DHT; purity 95%) for 

the ER or AR assays, respectively. For measurement of antagonistic activity to hERα, hERβ and 

hAR, either 10−11 or 10−10 M E2 or 10−10 M DHT was added to the cell cultures along with the 

test compound, respectively. Agonistic activities were evaluated by the relative activity 

expressed as 20% relative effective concentration (REC20), the concentration of the compound 

demonstrating 20% of the activity of 10−10 M E2, 10−9 M E2, or 10−9 M DHT for ERα, ERβ, and 

AR, respectively. Antagonistic activities were evaluated similarly by the relative activity 

expressed as 20% relative inhibitory concentration (RIC20), the concentration of the compound 

showing 20% inhibition of the activity of E2 or DHT against ERα, ERβ, and AR, respectively. 

TCVP did not show agonist or antagonist activity for ERα, ERβ, or AR in this study. 

Prenatal Development in Rodents (Rat) 

In a developmental toxicity study (MRID 40152701, 41828001, 42520101), TCVP (98.6% 

purity) was administered to presumed-pregnant female SD rats (25/dose) by oral gavage in 0.5% 

methyl cellulose at concentrations of 0, 75, 150, and 300 mg/kg/day from gestation days (GD) 6 

to 15. On GD 20, the dams were euthanized and subjected to gross necropsy. All fetuses were 

weighed, sexed and examined for gross external alterations. One-half of the fetuses in each litter 

were examined for visceral soft tissue alterations using a variation of Wilson’s sectioning 

technique. The remaining one-half of the fetuses in each litter were examined for skeletal 

alterations. An initial study was terminated at C-section due to poor pregnancy rates; a second 

study was conducted with an identical study design. 

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There were no mortalities; clinical signs of toxicity (p<0.01) observed in the high-dose only 

included urine-stained abdominal fur in 2/25 dams and chromodacryorrhea in 6/25 dams. 

Tremors were observed in one rat from Day 10 through termination. At the high dose, mean 

maternal body weight gains were decreased (p<0.01) by 44% for GD 6 to 16 (52% for GD 6 to 

15) and increased by 8% (NS) for GD 16 to 20, with an overall decrease (p<0.05) of 14% for 

GD 0 to 20. At the medium dose, mean maternal body weight gains were decreased (NS) by 9% 

for GD 6 to 16 (11% for GD 6 to 15), with an overall decrease (NS) of 2% for GD 0 to 20. 

Gravid uterine weights were not presented in the report. Food consumption in the high-dose 

group was decreased (p<0.05) by 22% for GD 6 to 16, and overall consumption was decreased 

(p<0.01) by 9% for GD 0 to 20. No compound-related lesions were observed in any females at 

necropsy. 

There were no treatment-related effects of treatment on corpora lutea, implantations, litter size, 

live/dead fetuses, resorptions, or live fetal body weights. There were also no treatment-related 

external or visceral alterations. Initial assessment of was hampered due to a laboratory error 

during staining that resulted in the complete damage of 64 fetuses and partial damage of an 

additional 11 fetuses from the control, low-, and high-dose groups. Additional information 

provided by the Registrant in report amendments provided sufficient data to complete the 

skeletal evaluations. The data do not indicate a treatment-related effect on skeletal malformations 

or variations. 

The maternal NOAEL is 75 mg/kg/day (LDT), and the maternal LOAEL is 150 mg/kg/day based 

on decreased body weight gain of 11%. The developmental NOAEL is 300 mg/kg/day (HDT), 

and a developmental LOAEL is not determined. 

Prenatal Development in Non-Rodents (Rabbit) 

In a prenatal developmental toxicity study (MRID 00127831) in female New Zealand White 

rabbits (18/dose), TCVP (98%) was administered by gavage in 5 mL/kg carboxymethylcellulose 

(CMC) at doses of 0 (CMC), 150, 375, or 750 mg/kg/day on GD 6 to 19. Rabbits were 

artificially inseminated, followed by i.v. injection of HCG (100 USP units). On GD 29, the does 

were euthanized and subjected to complete gross necropsy. All fetuses were weighed, sexed and 

examined for external abnormalities, visceral soft tissue abnormalities and skeletal abnormalities 

by standard procedures. 

Maternal toxicity was manifested as mortality (0/18, 1/18, 1/8, 2/18), abortions (one at the low 

dose and three at the high dose), and red vaginal fluid passed by 8/11 does in the high-dose 

group and one each in the low- and medium-dose groups. 

Developmental toxicity in the high-dose group was manifested as an increase in early 

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implantation loss (10.6%, 5.6%, 10.5%, and 21.9%, respectively) and a decrease in live 

fetuses/doe (7.7, 7.8, 6.9, and 5.8) for the control, low, medium, and high-dose groups. No 

treatment-related skeletal retardations were noted during the fetal examinations, and there were 

no treatment-related external, visceral, or skeletal variations or malformations. 

The maternal NOAEL is 375 mg/kg/day and the maternal LOAEL is 750 mg/kg/day based on 

mortality, abortions, and the red vaginal discharge in the high-dose does. The developmental 

NOAEL is 375 mg/kg/day and the developmental LOAEL is 750 mg/kg/day based on the 

number of viable fetuses. 

Developmental Neurotoxicity in Rodents (Rat) 

In a developmental neurotoxicity study (MRID 46660601), TCVP (99.6% purity) in aqueous 1% 

(w/v) methylcellulose was administered by gavage (10 mL/kg) to pregnant SD rats (25/dose) 

from GD 6 to lactation day (LD) 6 at doses of 0, 10, 50, or 200 mg/kg/day. Additionally, the F1

pups were similarly dosed on postnatal days (PND) 7 to 21. Dams were allowed to deliver 

naturally and were sacrificed on LD 21. On PND 4, litters were standardized to 10 pups/litter; the 

remaining offspring and dams were sacrificed and subjected to a gross necropsy. Subsequently, 1 

pup/sex/litter/group (at least 10 pups/sex/dose when available) were allocated to the following 

subsets: Subset 1, PND 21 brain weights and neurohistological evaluations; Subset 2, water maze 

and passive avoidance test; Subset 3, motor activity and auditory startle habituation; Subset 4, 

terminal brain weights and neurohistological evaluations; and Subset 5, standardize litter size to 

ten pups (5 male and 5 female) per litter on PND 4 to 21. 

In dams, there were no treatment-related effects on mortality, clinical signs, body weight, body 

weight gain, feed consumption, FOB, or gross pathology. No treatment-related effects on 

reproductive parameters were observed. The maternal LOAEL was not observed. The maternal 

NOAEL was 200 mg/kg/day (HDT). 

In offspring, there were no treatment-related effects on viability, litter observations, clinical 

signs, body weight, food consumption, sexual maturation, FOB, motor activity, acoustic startle 

habituation, learning and memory (passive avoidance and water maze), gross pathology, or 

histopathology parameters. In the high-dose group, decreases (p≤0.05) in body weight were 

noted in males (↓5 to 8%) on PNDs 15 and 16 (pre-weaning) and PND 22 (post-weaning) and in 

females (↓6%) on PND 29 (post-weaning). Body weight gains were decreased (p≤0.05) during 

pre-weaning at several intervals in males (↓12 to 28%) and females (↓13 to 26%). Decreases 

(p≤0.05) in body weight gains during post-weaning were observed in the males on PNDs 21 and 

22 (↓36%) and in the females on PNDs 21 and 22 and 22 to 29 (↓7 to 28%). Absolute brain 

weight was decreased (p≤0.01) by 8% in the males. This finding was judged to correlate with the 

treatment-related decreases (p≤0.05) in several microscopic linear brain measurements. It is 

noted that on PND 21, a decrease in single morphometric parameter was seen in both sexes with 

the small magnitude (4 to 5%) whereas on PND 70, decreases in multiple morphometric 

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measurements were seen in both sexes with greater magnitude (7 to 21%) compared to PND 21. 

No treatment related effects were noted in the ≤50 mg/kg/day F1 offspring. 

The offspring LOAEL was 200 mg/kg/day, based on decreases in body weight, body weight 

gain, and several morphometric linear brain measurements in both sexes, and decreased absolute 

brain weight in the males on PND 70. The offspring NOAEL was 50 mg/kg/day. 

Multi-Generation Reproduction in Rodents (Rat) 

In a two-generation reproduction study (MRID 42054301), male and female SD rats 

(28/sex/dose) were administered TCVP (99% purity) in the diet to the F0 and F1 parental 

generation at dosages of 0, 100, 500, or 2000 ppm. Mean-measured intake during the premating 

period in the F0 parents was 5.2, 26, and 102 mg/kg/day in males and 7.3, 40, and 155 mg/kg/day 

in females. Mean-measured intake during the premating period (15 weeks old) in the F1 parents 

was 6.7, 34, and 130 mg/kg/day in males and 8.3, 43, and 168 mg/kg/day in females. The F0

generation was treated for 10 weeks prior to mating. Treatment continued throughout the mating, 

gestation and lactation periods, until termination after weaning of these litters. The F1 generation 

was treated for 11 weeks prior to mating to produce the F2 generation, with treatment continued 

throughout the mating, gestation and lactation periods, until termination for all F1 and F2 animals 

at the weaning for these litters. 

Study measurements included body weight/body weight gain, food consumption, determination 

of reproductive parameters, organ weights, and gross and microscopic pathology. 

There was no evidence of a treatment-related effect on survival; no deaths occurred in the F0

animals and the only deaths in the F1 animals included two control animals (male and female) 

and two females in the medium-dose group. No treatment-related clinical signs were reported 

during the study. Overall mean body weight gains in all dose groups for F0 males were decreased 

to a similar extent (p≤0.05 or 0.01, 7 to 9%), with body weight decreases at Week 10 or 16 of 

only 5 to 7% (NS). Body weight and body weight gains for F0 females were not affected by 

treatment. Decreases in mean overall body weight gain of high-dose F1 males (p≤0.05, ↓6), and 

body weight gain of high-dose F1 females during the premating period (p≤0.001, ↓11), were 

observed, but no significant effects on weekly body weights were noted. No differences in mean 

food consumption were noted during the study. No consistent evidence of treatment-related 

effects on fertility, gestation or lactation indices were observed in litters produced by the F0 or F1

generations. In the F2 generation, pups had a greater mean increase in weight compared to F1

pups over the 21-day period across all groups (including control). 

Determination of ChE activity was not reported in this study. Mean adrenal weights were 

increased in F0 females (significance NR; ↑9% at the low and medium dose, and ↑28% at the 

high dose). A similar effect was not repeated in the F1 females, although adrenal weights in the 

medium- and high-dose females were increased (significance NR) by 8 and 9%. No changes in 

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adrenal weights were observed in males in either generation. No treatment-related 

histopathological lesions were reported in any examined tissues. 

The NOAEL for systemic toxicity is 500 ppm (26 mg/kg/day in F0 males, 40 mg/kg/day in 

F0 females, 34 mg/kg/day in F1 males, and 43 mg/kg/day in F1 females) based on decreased body 

weight gains and the increased mean adrenal weight in F0 females. The LOAEL is 2000 ppm 

(102 mg/kg/day in F0 males, 155 mg/kg/day in F0 females, 130 mg/kg/day in F1 males, and 

168 mg/kg/day in F1 females). The NOAEL for reproductive effects is 2000 ppm (HDT), 

because no reproductive toxicity was observed in this study. 

Subchronic Oral Toxicity (Rat) 

In a subchronic study (MRID 43371201), groups of 10 male and 10 female Sprague-Dawley rats 

received dietary exposure to 0, 100, 2000, or 5000 ppm tetrachlorvinphos (TCVP) over a 13-

week period. At 13 weeks, 5000 ppm males had significantly reduced mean body weights; males 

at 2000 and females at 2000 and 5000 ppm had reduced (non-significant) mean body weights. 

Mean 13-week body weight gains for 2000 and 5000 ppm males were 92% and 80% of their 

control value; for 2000 and 5000 ppm females the values were 84 and 88%, respectively. At 13 

weeks, 2000 and 5000 ppm males had significantly reduced mean plasma ChE activity (-25% 

and -41% respectively relative to controls) and significantly reduced mean RBC ChE activity (-

30%, -72%), 2000 and 5000 ppm females had significantly reduced mean plasma ChE (-74% and 

-83%) and RBC ChE (-79%, -91%). 5000 ppm females had reduced (-24%, not statistically 

significant) brain ChE. A noteworthy finding was that 2/10 males and 2/10 females at 5000 ppm 

had no measurable RBC ChE activity at 13 weeks. The incidence and severity of bilateral 

basophilic tubules of the kidneys was significantly increased in males at 2000 and 5000 ppm. 

The incidence of increased fat deposition in the adrenal cortex was significantly elevated in 2000 

ppm (7/10 animals) and 5000 ppm (9/10) females. All 2000 and 5000 ppm females exhibited 

centrilobular hepatocellular hypertrophy, also present in 8/10 2000 ppm and 1/10 5000 ppm 

males. General hepatocellular enlargement was noted in 2/10 2000 ppm and 7/10 5000 ppm 

males, but not in any females. Thyroid follicular cell hypertrophy was noted in 2/10 2000 ppm 

and 8/10 5000 ppm males and 5/10 2000 ppm and 8/10 5000 ppm females. Mean absolute liver 

weights were significantly elevated in 5000 ppm females; mean covariantly adjusted (by body 

weight) liver weights were significantly elevated in both sexes at 2000 and 5000 ppm. Mean 

absolute and covariantly (body weight) adjusted adrenal weights were significantly elevated in 

2000 and 5000 ppm females. The NOAEL (both sexes) was 100 ppm (6.7 mg/kg/day); the 

LOAEL (both sexes) was 2000 ppm (142 mg/kg/day). 

Subchronic Inhalation Toxicity (Rat) 

In a nose-only inhalation toxicity study (MRID 48803501), tetrachlorvinphos (TCVP; Lot No. 

TX 100113; 100% purity) aerosol was administered to male and female Sprague-Dawley rats 

(10/sex/concentration) for 6 hours/day, 5 days/week for 3 weeks at exposure concentrations of 0, 

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0.05, 0.5, or 1.0 mg/L. During the final week of exposure (week 4), the animals were exposed for 

7 days. An additional 10 rats/sex/concentration were included in the control and high-dose 

groups and sacrificed 14 days after completion of the 28-day exposure period to determine the 

reversibility of any effects observed. Actual exposure concentrations as measured by HPLC were 

100%, 100%, and101% of target values. The mass median aerodynamic diameters (MMADs) ± 

geometric standard deviations (GSD) were 2.57 μm ±3.785, 4.51 μm ±3.541, and 4.27 μm 

±3.343 for the low-, mid- and high concentrations, respectively. 

There were no treatment-related deaths. Treatment-related reduced body weights (↓7%* and 

↓8%**) and body weight gains (↓27%** and ↓34%***) were observed in males from the midand high-exposure groups, respectively. The body weight difference persisted through the 

recovery period in the high-exposure (↓12%) male recovery group, but the weight gain during 

those 2 weeks was slightly higher (5%) relative to the control group. Bodyweight was 

comparable among the female groups throughout the study. Body weight gain was reduced in 

mid- (↓23%) and high-exposure (↓32%) females, but body weight gain was comparable to the 

control during the recovery period. Food consumption was decreased throughout the study in the 

mid and high male groups and during the first two weeks in the low dose male group, whereas 

food consumption was comparable among the female groups. There were no treatment-related 

findings during the ophthalmoscopic examinations. Decreased hemoglobin concentration and 

increased platelets were noted in high exposure concentration females, but these findings were 

no longer evident at the end of the 2-week recovery period. Calcium was slightly, but 

significantly, elevated in males at all three exposure levels, but there was no dose-response. 

Cholesterol was significantly elevated in females from the mid- (↑22%) and high-exposure 

(↑27%) groups. These findings were not observed in the recovery group. 

Statistically significant RBC ChE activity inhibition (p<0.001) was observed in females at the 

mid- and high-exposure concentrations (35% and 30%, respectively). Statistically significant 

AChE and RBC ChE activity inhibition (p<0.001) was observed in males at the mid- and highexposure concentrations (38% and 31% for AChE, respectively; 24% and 31% for RBC ChE, 

respectively). Plasma acetylcholinesterase activity (58% to 70%) and plasma 

butyrylcholinesterase activity (66% to 87%) were decreased relative to controls at all exposure 

concentrations in females. All cholinesterase activities were lower relative to controls in the male 

and female recovery groups, but the differences were not statistically significant. 

Treatment-related gross findings were limited to discoloration and enlargement of the adrenal 

glands at the mid- and high-exposure concentrations in males, and in females at all exposure 

concentrations. Diffuse adrenal cortical cell vacuolation (minimal to moderate with an exposurerelated incidence) was observed in both males and females at all exposure concentrations. 

Minimal follicular cell hypertrophy of the thyroid gland was observed with an exposure-related 

incidence in both sexes at the mid- and high-exposure concentrations. Minimal to mild ovarian 

interstitial cell vacuolation was observed with an exposure-related incidence in females at the 

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mid- and high-exposure concentrations. The findings in the thyroid and ovary were referred to as 

“commonly observed in this strain and age of rat,” but no historical control data or literature 

citations were provided. In the recovery group, 2/5 males and 2/5 females at the high dose had 

minimal diffuse adrenal cortical cell vacuolation (0/5 incidence in recovery controls). No other 

treatment-related microscopic findings were observed in the recovery group. 

Based on the effects seen in this study, a NOAEL in male and female Sprague-Dawley rats was 

not identified. The LOAEL in male and female rats was 0.05 mg/L, based on diffuse adrenal 

cortical cell vacuolation in both sexes, enlarged adrenals in females, and increased adrenal 

weights in females. At 0.5 mg/L and 1.0 mg/L, in addition to the adrenal findings, there was a 

dose-related increase in vacuolation of the ovaries in females, an increase in squamous 

metaplasia of the larynx in both sexes, and an increase in follicular cell hyperplasia of the 

thyroids in both sexes. The NOAEL for cholinesterase inhibition was 0.05 mg/L, based on an 

increase in RBC cholinesterase inhibition in both sexes at 0.5 mg/L/day. 

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat) 

In a combined chronic toxicity/oncogenicity study (MRIDs 42980901 and 43335101), TCVP 

(99% purity) was administered to SD rats (50/sex/dose) in the diet for two years at nominal doses 

of 0, 100, 1000, or 2000 ppm, equivalent to respective mean values of 0, 4.23, 43.2 and 

88.5 mg/kg/day for males and 0, 5.93, 62.7 and 125.3 mg/kg/day for females. An interim 

sacrifice was conducted at 12 months (15/lsex/dose). After 104 weeks, all surviving rats in all 

groups were sacrificed for gross and histopathological evaluation. Select organs were weighed at 

each sacrifice interval (including liver, kidneys, testes with epididymides, prostate, ovaries, 

uterus, adrenals, and pituitary). Gross and microscopic pathological examination included the 

liver, kidney, pituitary, thyroid/parathyroids, adrenals, mammary gland, testes with 

epididymides, prostate, seminal vesicles, uterus, vagina, and ovaries. 

No clinical signs were observed that were not considered normal for the age and strain of rat. No 

increased mortality was seen in study animals at any treatment level. Possible treatment-related, 

but minimal, decrements in body weight were observed in males (p≤0.05, ↓4%) at Weeks 8 

and 40 (low- and high-dose groups, respectively) and (p≤0.01, ↓6%) at Week 8 in the high dose. 

Treatment-related decreases in body weight in females in the medium-dose group were generally 

significant from Weeks 2 through 80 (p≤0.05, 0.01, or 0.001) but were ≤9% except for a 10% 

decrease on Weeks 52 (p≤0.001) and104 (NS). Decreased body weight in females in the highdose group were significant from Weeks 2 through 80 (p≤0.05, 0.01, or 0.001) but were ≤9% 

through Week 28, with decreases (p≤0.001) of 11 and 12% at Weeks 40 and 52, respectively, 

and decreases of 9 and 7% at Weeks 80 (p≤0.05) and 104 (NS). Food consumption was generally 

similar to control across all treatment groups. No changes in hematology parameters were noted 

in treated animals; changes in clinical chemistry parameters (primarily liver-related enzymes or 

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compounds) included increased cholesterol in high-dose females during the second year of 

treatment and decreases in alkaline phosphatase in males and females. 

Plasma ChE activity was decreased (p<0.01 or 0.001) in medium- and high-dose females at each 

of the six-month sampling intervals (↓29/34% at 6 months; ↓55/66% at 12 months; ↓51/68% at 

18 months; and ↓41/54% at 24 months). Decreases in males were noted at the 12-, 18-, and 24-

month intervals in high-dose males (↓24 to 41%) and at 18-months in the medium-dose males 

(↓21%). No treatment-related effect on RBC ChE activity was apparent in male rats in this study. 

RBC ChE activity was decreased in all treated females (with dose-dependent decreases at 18- 

and 24-months), but the decreases were only significant (p<0.05 or 0.01) at the 18-month sample 

time in the medium- and high-dose animals (↓29 and 36%, respectively). Brain ChE activity was 

not altered in males at the interim and terminal sacrifices, and inhibition in the high-dose females 

(↓17 and 15%, respectively) was NS. 

No treatment related changes were noted in weights of the testes with epididymides, prostate, 

ovaries, uterus, adrenals, or pituitary. Inconsistent, generally non-dose-dependent decreases in 

liver and kidney weights were noted occasionally in males and females at the interim and/or 

terminal sacrifices. Liver weights were decreased (p<0.05 or 0.01) 16–22% in male rats at 

Week 104 (all dose groups) and decreased (p<0.01) 23% in high-dose females at Week 52. 

Kidney weights were not affected in male rats, but were decreased (p<0.01) in low-dose females 

at Week 104 (↓16%) and in high-dose females at Weeks 52 and 104 (↓18 and 15%, respectively). 

Histopathological effects at the medium and high doses in both sexes included an increased 

incidence and tendency to greater severity of diffuse lipidosis of the adrenal zona fasciculata, 

hypertrophy of periacinar hepatocytes in liver, and, in males only, centriacinar degenerative 

changes of the liver. Microscopic findings did not demonstrate any consistent effect on 

endocrine-related endpoints or tissues. 

Although low-dose females had decreased RBC ChE activities (NS), decreased alkaline 

phosphatase (NS), and increased serum cholesterol (at 18 months, but not dose dependent), these 

occurrences do not suggest a treatment-related pattern. The NOAEL for systemic toxicity is 

considered 100 ppm (4.23 and 5.93 mg/kg/day, respectively) in males and females. The LOAEL 

is 1000 ppm, based on decreased body weight in females, liver and adrenal changes in both 

sexes, and decreased plasma ChE activity in females. 

In the present study, the incidence of thyroid C-cell carcinomas and adenomas in males 

(sacrificed at Week 104 or dying during the study) were 8/48 for control and 13/28 at the high 

dose (p=0.1228 by Fisher’s Exact Test), but outside of the historical control data range. The 

incidences of thyroid C-cell carcinomas and adenomas in males in the low- and medium-dose 

groups were more similar to control and within the historical control data range. The incidence of 

thyroid c-cell adenoma in 2000 ppm females surviving to week 104 was 4/26, and in their 

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controls was 4/27. There was no indication of a tumorigenic effect in the adrenals of females in 

the present study. 

In a previous carcinogenicity study in rats (MRID 00117443), technical-grade TCVP (purity not 

reported) was administered in the diet to groups of Osborne-Mendel rats (50/sex/dose) at 

nominal doses of 0, 4250, or 8500 ppm (calculated equivalency of 0, 212.5, or 435 mg/kg/day). 

Matched controls consisted of 10 rats/sex; pooled controls consisted of the matched controls 

combined with 45 rats/sex from similar bioassays. TCVP-treated animals were dosed for 80 

weeks, and observed for an additional 31 weeks; all surviving animals were sacrificed at Week 

111. 

Apparent body weight decreases were reported for treated rats, but the data or details were not 

available for review. Mortality was affected in male rats (data not provided), and no other study 

results were available, except for a summary of the incidence of carcinogenicity. A significant, 

dose-related trend (significance not reported) in C-cell adenomas of the thyroid was noted in 

female rats, with an increased incidence (p=0.027) in the high-dose group. An increased 

incidence of C-cell hyperplasia was observed in male and female rats. A significant, dose-related 

trend (significance not reported) in cortical adenomas of the adrenal also was noted in female 

rats, with an increased incidence (p=0.022) in the high-dose group. 

Carcinogenicity in Rodents (Mouse) 

In a 103-week carcinogenicity study (MRID 00126039), TCVP (98.7% and 98.4% purity) was 

administered in the diet to groups of B6C3F1 BR strain mice (80/sex/dose) at nominal doses of 0 

(acetone/corn oil suspension; n=160/sex), 17.5, 64, 320, 1600, 8000, or 16000 ppm (calculated 

equivalency of 0, 2.6, 9.6, 48, 240, 1200, or 2400 mg/kg/day). Two groups of mice were 

administered the highest dose with either a new batch of TCVP (used for all the lower doses) or a 

previous (original) batch of TCVP. Unless indicated, data from the new batch of TCVP are 

reported. Interim sacrifices were conducted at Weeks 26, 53, and 78 with 10 mice/sex/treatment 

group and 20 mice/sex from the control group, and the remaining survivors were sacrificed at 

Week 103. Mortality, clinical signs of toxicity, body weights and food consumption were 

monitored at appropriate times during the study. Necropsy examinations were performed on all 

animals and organ weight determinations were conducted at the interim and terminal sacrifices. 

Histopathological examinations were made on a complete set of organs/tissues from all animals 

that died or were sacrificed in extremis during the study, and on all animals at the interim and 

terminal sacrifices. 

No treatment-related mortality was observed in the present study, with survival rates higher in 

the two highest treatment groups. No treatment-related clinical signs, including no signs 

associated with ChE inhibition, were noted during the study, although there was an increased 

incidence of hunched/thin appearance in high-dose rats beginning at Week 54. Mean body 

weights were decreased ≥10% by Week 2 and throughout the remainder of the study at the two 

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highest doses (8000 and 16000 ppm) in females; data were not available for males. At study 

termination, mean body weights were decreased (p<0.05) in males (↓18.6 and 32.4%, 

respectively) and females (↓24.9 and 34.5%, respectively) at the two highest doses. Similar 

decreased weights were noted with the original batch of TCVP. No effects on food consumption 

were observed after correction for food spilled. 

Treatment-related changes (p<0.05) in absolute liver weights occurred with the new batch of 

TCVP at Week 26 (high-dose, male, ↑33%) and Week 103 (high-dose, male and female, ↑23 and 

↓11%, respectively; and 8000 ppm, female, ↓18%). Treatment-related increases (p<0.05) in 

absolute liver weights occurred with the original batch of TCVP at Week 26 (male, ↑37%), 

Week 52 (male, ↑79%), Week 78 (male and female, ↑63 and 51%, respectively), and Week 103 

(male and female, ↑85 and ↑41%, respectively). Kidney weights were decreased in male mice in 

the 8000 ppm dose group (NS, ↓17%) and female mice in the two highest dose groups (p<0.05, 

↓17 and 12%, respectively). An increase (p<0.05) in adrenal weight was also noted for the 

original batch of TCVP in male mice (↑83%). Gross pathology findings were generally minimal, 

but increased incidences of liver and kidney alterations were noted at terminal sacrifice in 

animals in the two highest dose groups. 

Non-neoplastic lesions in the liver, kidneys, and adrenals were noted at the two highest doses. 

Available summary data is generally illegible, but included enlarged cells in the zona fasciculata 

and increased pigment in the zona reticularis (adrenals); activated Kupffer cells, enlarged 

hepatocytes, necrosis, and bile duct proliferation (liver); and increased basophilia and tubular 

hyperplasia (kidneys). There was an increased incidence of hepatocellular adenomas/carcinomas 

and kidney adenomas/carcinomas in male mice of the high dose group (new and/or original 

batch) and hepatocellular adenomas/carcinomas in female mice of the 8000 and 16000 ppm (new 

and original batch) groups. No treatment-related histopathological lesions were seen in the testes, 

epididymides, seminal vesicle, prostate, ovaries, uterus, mammary, adrenal, thyroid or pituitary 

glands. 

The NOAEL for systemic effects is 1600 ppm (240 mg/kg/day), and the LOAEL is 8000 ppm 

(1200 mg/kg/day) based on decreased body weights in males and females. 

In a previous carcinogenicity study in mice (MRID 00063589), technical-grade TCVP (purity 

not reported) was administered in the diet to groups of B6C3F1 mice (50/sex/dose) at nominal 

doses of 0, 8000, or 16000 ppm (calculated equivalency of 0, 1200, or 2400 mg/kg/day). 

Matched controls consisted of 10 mice/sex; pooled controls consisted of the matched controls 

combined with 40 mice/sex from similar bioassays. TCVP-treated animals were dosed for 80 

weeks, and observed for an additional 12 weeks; all surviving animals were sacrificed at 

Weeks 90 to 92. 

Apparent body weight decreases were reported for treated mice, but the data and study details 

were not available for review. No other study results were available, except for a summary of the 

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incidence of carcinogenicity. A significant, dose-related trend (p<0.001) in hepatocellular 

carcinomas was noted in male mice, with an increased incidence (p<0.001) in both groups. 

Female mice had an increased incidence of neoplastic nodules in the liver, with the incidence in 

the low-dose group (p<0.001) greater than the incidence in the high-dose group (p=0.007). 

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

In a chronic one-year oral toxicity study in dogs (MRID 42679401), TCVP (99% purity) was 

administered by capsule at 0, 6.25, 500, or 1000 mg/kg/day to Beagle dogs (4/sex/dose) for 12 

months. Mortality, clinical signs of toxicity, body weights and food consumption were monitored 

at appropriate times during the study. ChE activity was determined in plasma and RBC at Weeks 

12, 26, and 51, and in the brain at study termination. Organ weights determined included 

adrenals, kidneys, liver, ovaries, pituitary, prostate, testes with epididymides, and uterus; 

histopathological examinations were conducted on these tissues as well as the mammary glands, 

seminal vesicle, thyroid/parathyroid, and vagina. 

All animals survived until terminal sacrifice, with no treatment-related clinical signs and no 

effects on body weight or food consumption. Several clinical chemistry parameters were affected 

in the high-dose males and/or females. Alkaline phosphatase was increased at all sampling times 

in males and females (p<0.05 or 0.01; ↑2.05- and 2.13-fold at Week 12, ↑2.86- and 2.15-fold at 

Week 26, and ↑4.67- and 4.82-fold at Week 51, respectively). Cholesterol was decreased in highdose males at all sampling times (p<0.05 or 0.01; ↓27% at Week 12, ↓21% at Week 26, and 

↓23% at Week 51, respectively). 

Plasma ChE activity was inhibited (p<0.05 or 0.01) in low-dose males and all medium- and highdose animals at all sample times. Plasma ChE activity in males was decreased by 26–30% at the 

low dose, 46–48% at the medium dose, and 55–61% at the high dose; plasma ChE activity in 

females was decreased by 41–47% at the medium dose and 44–49% at the high dose. Brain and 

RBC ChE activity was not affected by treatment. 

No treatment related changes were noted in weights of ovaries, pituitary, prostate, testes with 

epididymides, or uterus. Absolute liver weights were increased (p<0.05) in medium-and highdose males (↑20 [NS] and 43%) and females (↑43 and 43%), and absolute kidney weights were 

increased (NS) in mid- and high-dose males (↑33 and 30%) and females (↑21 and 24% 

[p<0.05]). Relative liver and kidney weights were also increased (NS, or p<0.05 or 0.01) in the 

medium- and high-dose groups. Absolute adrenal weights were increased (p<0.05) in mediumand high-dose females (↑35 and 39%), with no change in relative weights or weights in males. 

Finally, prostate weights were decreased (NS) in low- and medium-dose males (↓22 and 30%, 

respectively), with a significant decrease (p<0.05) in high-dose males (↓44%). Gross and 

microscopic pathology were generally unremarkable, with no treatment-related findings in 

endocrine-related organs. 

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For ChE inhibition, the NOAEL is 6.25 mg/kg/day and the LOAEL is 500 mg/kg/day based on 

decreased plasma ChE activity. For systemic toxicity, the NOAEL is 6.25 mg/kg/day and the 

LOAEL is 500 mg/kg/day based on various hematology effects, decreased plasma ChE activity, 

and increases in alkaline phosphatase and liver, kidney, and adrenal weights. 

Avian Reproduction Toxicity (OCSPP 850.2300) 

The one-generation reproductive toxicity (MRID 48952701) of technical grade 

tetrachlorvinphos (TCVP; 100.05% a.i.) to 18 pairs per dietary treatment level of ca. 30-week 

old Northern bobwhite quail (Colinus virginianus) was assessed over ca. 25 weeks. 

Tetrachlorvinphos was administered to the birds in the diet at nominal concentrations of 0 

(negative control), 100, 300, and 1000 mg a.i./kg diet. Mean-measured concentrations were <0.4 

(LOD, control), 96, 290, and 979 mg a.i./kg diet. 

There were no treatment-related mortalities or overt signs of toxicity in the control or dietary 

treatment groups. No significant differences in overall average food consumption per bird per 

day or mean body weight were observed. Slight but significant (p<0.05) reductions were 

observed for eggshell thickness (10%) and in the ratio of live 21-day embryos to eggs set for 

birds in the 979 mg a.i./kg diet group, as compared to the negative control. Based on these 

reductions, the NOAEC and LOAEC were determined to be 290 and 979 mg a.i./kg diet. 

The one-generation reproductive toxicity (MRID 48951301) of technical grade 

tetrachlorvinphos (TCVP; 100.05% a.i.) to 16 pairs per dietary treatment level of ca. 25-week 

old mallard duck (Anas platyrhynchos) was assessed over ca. 24 weeks. Tetrachlorvinphos was 

administered to the birds in the diet at nominal concentrations of 0 (negative control), 100, 300, 

and 1000 mg a.i./kg diet. Mean-measured concentrations were <0.4 (LOD, control), 96, 290, and 

979 mg a.i./kg diet. 

There were no treatment-related mortalities or overt signs of toxicity in the control and treatment 

groups. Adult female body weight gain was significantly (p<0.05) lower in the 290 and 979 mg 

a.i./kg-diet treatment groups, compared to the negative control. Significant reductions (p<0.05) 

were also observed for 14-day survivor weight in the 979 mg a.i./kg diet treatment group, 

compared to the negative control. Based on the reductions in adult female body weight gain, the 

NOAEC and LOAEC in this study were 96 and 290 mg a.i./kg diet. 

Fish Early Life-cycle Toxicity (OCSPP 850.1400) 

The 33-day chronic toxicity (MRID 48732301) of technical grade tetrachlorvinphos (TCVP; 

98.7% active ingredient) to the early life stage of fathead minnow (Pimephales promelas) was 

studied under flow-through conditions. Fertilized embryos were exposed to a geometric series of 

five test concentrations of TCVP technical (nominal test concentrations were 0.014, 0.046, 0.15, 

0.47 and 1.5 mg./L), a negative control or a solvent control (dimethylformamide; DMF; 0.1 

mL/L); mean-measured TCVP concentrations were 0.014, 0.047, 0.15, 0.47 and 1.4 mg a.i./L. 

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The NOAEC forhatching success is 0.15 mg a.i./L based on statistically significant different 

(p<0.05) hatching success at Day 5 in fish treated with 0.47 mg a.i./L compared to the negative 

control. On Day 33 (28 days post-hatch), juvenile survival averaged 99 and 90% in the negative 

and solvent control, respectively, compared to 92, 94, 83, 90 and 80% for the 0.014, 0.047, 0.15, 

0.47, and 1.4 mg a.i./L (mean-measured) test groups, respectively. The NOAEC for juvenile 

survival is 0.047 mg a.i./L based on statistically significant (p<0.05) difference in larval survival 

at treatment concentrations greater than or equal to 0.15 mg a.i./L, compared to either the 

negative or solvent control. The NOAEC and LOAEC values for growth (mean dry weight and 

mean length at 28 days post-hatch) are 0.047 and 0.15 mg a.i./L respectively, compared to the 

negative control. At the LOAEC for growth, there was a 3.5% decrease in mean length and a 

9.2% decrease in mean dry weight. The NOAEC and LOAEC for time to hatch are 0.15 and 0.47 

mg a.i./L, respectively, compared to the negative control. 

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APPENDIX 3: References Not Utilized in the TCVP 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 TCVP 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. 

Doyle, R.L., Teske, R.H., and Elsea, J.R. (1968) Two-year Dietary Administration of SD 8447 to 

Dogs: P-99A. Unpublished study received Aug 17, 1968 under 9F0739; prepared by Hill Top 

Research, Inc., submitted by Shell Chemical Co., Washington, D.C.; COL: 091273. (MRID 

00077819) 

The Effect of Tetrachlorvinphos (TCVP) on Blood and Brain Cholinesterase over 21 Days of 

Dosing Rats. WIL 404001. WIL Research Laboratories. Hartz Mountain Corp. Nov. 2000. 

(MRID 45570601) 

Eisenlord, G., Loquvam, G.S., Nemenzo, J., et al. (1966) Results of Reproduction Study of Rats 

Fed Diets Containing SD 8447 Insecticide over Three Generations: Report No. 28. Unpublished 

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study received May 23, 1967 under 800665; prepared by Hine Laboratories, Inc., submitted by 

Shell Chemical Co., Washington, D.C.; CDL:091166-M. (MRID 00077802) 

Lamb, I. (1994) A Subchronic (13-Week) Neurotoxicity Study of Rabon in Rats: Final Report: 

Lab Project Number: WIU149019. Unpublished study prepared by WIL Research Lab., Inc. 

1295 p. (MRID 43294101) 

Seidel, G. (1973) Evaluation of the Effects of Rabon on Reproductive Characteristics of Beef 

Bulls. Final report. Unpublished study received Oct 17, 1973 under 201-359; prepared by 

Colorado State Univ., submitted by Shell Chemical Co., Washington, DC; CDL:050006-I. 

(MRID 00117300) 

Walker, A. and Hunter, C. (1967) The Oral Toxicity of the Halophenyl Vinyl Phosphate 

Insecticide Gardona (SD 8447): Two Year Oral Experiment in Rats: Research Report R(T)-9-67. 

Unpublished study received Aug 17, 1968 under 9F0739; prepared by Shell Research Ltd., Eng., 

submitted by Shell Chemical Co., Washington, DC; CDL:091274-0. (MRID 00112525) 

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