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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

## ~ S

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

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

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

.

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

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

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

PATHWAYS

CHEMICAL: TETRACHL ORVINPHOS (TCVP)

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYTCVP FINAL

ii TABLE OF CONTENTS

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

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

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

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

B. Other Scientifically Rele vant 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 fo r Tetrachlorvinphos (TCVP)..............................................8
Table 2. Estrogenic/Anti-Estrogenic Pathway for Tetrachlorvinphos (TCVP).................10
Table 3. Androgenic/Anti-Androgenic Pa thway 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 th e Tetrachlorvinphos (T CVP) WoE Analysis........57TCVP FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

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

CG Cowper’s Gland

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

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

iv Abbreviation Terminology

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

K OW Octanol/Water Partition Coefficient

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

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

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

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated ConcentrationTCVP FINAL

v Abbreviation Terminology

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

OPP Office of Pesticide Programs

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

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

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

T4 Thyroxine (tetraiodothyronine)TCVP FINAL

vi Abbreviation Terminology

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

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

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

Page 1 of 58 Executive Summary

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

In determining whether tetrachlo rvinphos (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 part icular, whether or not endocrine-rela ted 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) anal ysis of the potential interaction of TCVP with the E, A or T signaling pathways. The T1 WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in

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

TCVP is a member of the organophosphate class of insecticides whose primary mode of toxic
action is cholinesterase inhibi tion 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 assa y (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 fo r 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 pube rtal rat studies do not support a potential for ER interaction or altered steroidogenesis. Additionally, there wa s 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 noTCVP FINAL

Page 2 of 58 clinical signs of toxici ty observed in this study, the concentr ations 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 LC 50 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 oppos e 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 ef fects including egg production. A decrease in offspr ing weight was observed in th e mallard duck study, but adult female body weight was also affect ed, and there were no other repr oductive effects. Therefore, while effects on reproduction were observed in the FSTRA, there were no effects on gonadal weight or histopathology, and ther e 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 tec hnical 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 re production study, no other androgen-related effects were observe d 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 histopathol ogy were observed in both sexes. Serum T 4 levels were decreased at the high dose in males and females ( ↓ 65% females; ↓ 68% males; decreases at low dose not significant). Thyroid stimulating horm one (TSH) was increased 31% in males at the high dose; however the change was not statistica lly significant. There was no treatment effect on
TSH levels in females. In the Part 158 studi es, alterations in thyr oid histopathology were observed in the subchronic rat study and 28-day inhalation rat study. Cholin esterase activity was not measured in either Tier 1 pubertal assa y, however, the effects observed in the Part 158
studies occurred at doses that re sulted in significant reductions in RBC ChE activity as well as increased incidences of histopat hological 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, whileTCVP FINAL

Page 3 of 58 histopathological effect s 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 hist opathological effect was an increase in follicle
asymmetry.

In the EDSP Tier 1 male and female pubertal assa ys 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 thyr oid toxicity in the young animals.

For TCVP, the current point of departure (POD) of 4.23 mg/kg/day used fo r human health risk assessment is based on histological liver and ad renal changes and reduced weight gain at 43.2 mg/kg/day (LOAEL) in a chronic oral toxicity st udy in rats. This POD is used for deriving the chronic Reference Dose (RfD) for chronic di etary 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 fo r deriving the Margins of Exposure (MOEs) for nondietary risk assessments.

In general, since the POD and the toxicity endpo ints of concern are base d on data obtained from adult animals, there is a concern that the POD may not be protectiv e of potential thyroid toxicity in the young. However in the case of TCVP, although no data exists for poten tial 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 ch emicals (organophosphate); and 3) the CTA would be tested at higher doses and w ould 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 departur e or endpoint for human h ealth risk assessments.

Given the lack of convincing evidence for a potential interaction with the estrogen, androgen and thyroid pathways in wildlife no a dditional testing is recommended.

For birds, Part 158 avian reproduction studies w ith 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 re productive effects to bird s from TCVP exposure. Relative to the EDSP, additional testing is not recommended.TCVP FINAL

Page 4 of 58 I. Introduction

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

In determining whether tetrachlo rvinphos (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 part icular, whether or not endocrine-rela ted 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) anal ysis of the potential interaction of TCVP with the E, A or T signaling pathways. The T1 WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in

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

Tetrachlorvinphos (TCVP) is an organophosphate insec ticide 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 sign ificantly 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 (OSR I) 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.TCVP FINAL

Page 5 of 58 II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

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

1.

B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Orde r recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review T eam on 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 c onsidered but not util ized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and approach used in the WoE determination on the potential of a
substance to interact with endocrine-related processes ( i.e., E, A or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the indivi dual studies for their scientific quality and relevance in evaluating potential endocrine interaction(s). The second step is to integrate the data along different levels of biological organization while examining the extent of complementarity ( i.e ., the concordance of endpoints within an assay that measures multiple endpoints) and redundancy ( i.e ., the concordance of endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to
evaluate whether additional te sting is needed based on the evidence and conclusions described
above.

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of tw o categories: 1) EDSP Ti er 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 wereTCVP FINAL

Page 6 of 58 designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

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

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biological organization? Finally, under what conditions did the responses occur including co nsideration of whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.TCVP FINAL

Page 7 of 58 This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addressing th ese three main questions is fundamental to the WoE process and in determining whether addi tional data are needed. In addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) discussion of ke y studies; 3) description of inc onsistent or conflicting data; 4) overall strength of evidence supporting a conclusi on; and, 5) what, if a ny, additional data are needed and why. Assessing the need for additiona l data is based on a case-by-case analysis
which took all available toxic ity data into account.TCVP FINAL

Page 8 of 58 The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

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

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID 48765001)

ER α 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)TCVP FINAL

Page 9 of 58 B. Effects on Hypothalamic-Pit uitary-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 a ssays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

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 estradio l 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 a ssay. TCVP was classified as positive in the aromatase assay with mean aromat ase inhibition being 63, 33 and 17% of control activity at concentrations of 10 -4 , 10 -3.5 and 10 -3 M, respectively, with an IC 50 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, th e 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 cholin esterase activity was not measured. While there were no clinical signs of toxicity observed in this study, eff ects 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 ra ngefinder conducted for the FSTR A. 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 reprod uction study, effects on 14-day offspring weight ( ↓ 6.8%) were observed at 979 mg/kg-diet; decreases in female body weight gain were also re ported (290 & 979 mg/kg-diet; ↓ 24.5 and 25.7%, respectively). In the quail reproduction study at the hi ghest concentration tested (9 79 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.TCVPFINALPage 10 of 58Table 2. Estrogenic/Anti-Estrogenic Pathway for TCVPLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for TCVP1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt) / Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48765001)NERTA(MRID 48619501)N4Steroidogenesis(MRID 48805201)I5Aromatase(MRID 48673801)PUterotrophic(MRID 48880601)NN NFemale Pubertal Rat(MRID 49186901)NENNNN N NLW:↑21%(H);AW:↑31%,↑37%(L, H)NFSTRA(MRID 48838201)NENNNFrt:↓9%/ Fcd:↓61%(H)F;↓30%(H)NOSRIERTA(Kojimaet al., 2004)NTCVPFINALPage 11 of 58Table 2. Estrogenic/Anti-Estrogenic Pathway for TCVPLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for TCVP1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt) / Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Developmental toxicity(Rat;MRIDs 40152701,41828001, 42520101)NRNX(H)X(M,H)Developmental toxicity(Rabbit;MRID 00127831)NRNX(H)X(H)DevelopmentalNeurotoxicity(Rat; MRID 46660601)NN(M, H)NTwo-generationreproduction(Rat; MRID 42054301)NR NR NRNRNX(H)NChronic toxicity /carcinogenicity(Rat; MRIDs42980901, 43335101)NNNNX(M, H)X(H)Carcinogenicity(Mouse;MRID 00126039)NRNRNNRX(H)X(H)Chronic toxicity(Dog;MRID 42679401)NNNNX(M, H)NAvian reproduction(Duck; MRID48951301)P6(H)N N7Avian reproduction(Quail; MRID48952701)P8(H)N NTCVPFINALPage 12 of 581. 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 indicatesthat is parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated. F= Female; LW= liver weight, AW= adrenal weight2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1 assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. The responsiveness of the cells to the very weak positive control 17α-methyltestosterone was lower than theexpected values, indicating a decreasedsensitivity of the assay to very weak agonists. Although the conditions of this assay were not optimal to detect very weak activity, TCVP responses weresimilar to those of the negative control corticosterone and not comparable to the responses of 17α-methyltestosterone. TCVP is considered to be negative inthis assay.5. Study was considered inadequate. Study did not adequately meet performance criteria. Only 2 of the required 3 experimental runs were performed.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 toxicity were observed.8. 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 findingsN Negative findings (in vitro) / No effect (in vivo)I InadequateNE Not examinedNR Not reportedTCVP FINAL

Page 13 of 58 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-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

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 cons idered as OSRI (Kojima et al ., 2004). The Tier 1 steroidogenesis assay was considered to be inadequate. A lthough the results did not show an effect on testosterone production, there were numerous deficiencies note d in the study that preclude making a definitive conclusion with regard to th e effects of TCVP on hor mone 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 pr ostate (VP) and Cowpe r’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 bo th the agonist and anta gonist portions of the study. The etiology of these effects is unclear since the weight changes were not dose-dependent and the direction (increase) of th e 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 chroni c 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 repor ted in the Part 158 quail reproduction study at 979 mg/kg-diet; offspring weight was also reported in th e Part 158 mallard duck study.TCVPFINALPage 14 of 58Table 3. Androgenic/Anti-Androgenic Pathway for TCVPLines of Evidence Indicating Potential Interactionwith the Androgenic/Anti-Androgenic Pathway for TCVP1Study Type / LiteratureCitation 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 Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48703101)E4Steroidogenesis(MRID 48805201)I5Hershberger(MRID 48946701)NENLW:↑17–30%(H);AW:↑39% (L),↑47% (M),↑44% (H)NMale Pubertal Rat(MRID 49186901)NNNNNNNNLW:↑23%(H);KW:↑11%(H)NFSTRA(MRID 48838201)NENNNFrt:↓9%(H)NNTCVPFINALPage 15 of 58Table 3. Androgenic/Anti-Androgenic Pathway for TCVPLines of Evidence Indicating Potential Interactionwith the Androgenic/Anti-Androgenic Pathway for TCVP1Study Type / LiteratureCitation 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 Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRIARTA(Kojimaet al., 2004)NDevelopmentalNeurotoxicity(Rat; MRID 46660601)NNX(M, H)X(H)Two-generationreproduction(Rat; MRID 42054301)NRNRNRNRNRNR NX(H)NChronic toxicity /Carcinogenicity(Rat; MRIDs 42980901,43335101)N N NNN NX(M, H)NCarcinogenicity(Mouse; MRID 00126039)NR N NR NR NR NRX(H)X(H)Chronic toxicity(Dog; MRID 42679401)NNNNNprostateweight↓44%(H)X(L, M, H)NAvian reproduction(Duck; MRID 48951301)P6(H)N N7Avian reproduction(Quail; MRID 48952701)P8(H)N N1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑)indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weightare absolute unless otherwise indicated. LW = Liver weight; AW = Adrenal weight; KW = kidney weight.TCVPFINALPage 16 of 582. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. 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 were performed.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 toxicity were observed.8. 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 findingsN Negative findings (in vitro) / No effect (in vivo)E EquivocalNE Not examinedNR Not reportedTCVP FINAL

Page 17 of 58 C. Effects on Hypothalamic-Pitui tary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it
does include three in vivo assays that provide data to de tect changes in the HPT axis, i.e ., the pubertal female and male rat assays, and the AMA (frog).

The potential for TCVP to interact wi th thyroid regulation are summarized in Table 4. The various targets of the th yroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. This table also includes HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the ta ble and not considered further in the WOE assessment.

In both the Tier 1 female and male pubertal assa ys, no treatment-related effects were noted for thyroid weights, but thyroid histopathology i ndicated increased follicular cell height and decreased follicular cell area, with greater changes noted in males. The thyroid histopathology findings were statistically signi ficant at the high dose (350 mg/kg/ day) in both sexes. Serum T 4 levels were decreased at both doses (50 and 350 mg/kg/day) in males and females, although the
decreases were statisti cally 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 trea tment effect on TSH levels in females.

In the AMA, median NF stage was decrease d one stage in the hi gh-concentration group compared to the negative control at Day 7 and 21, but was only significant on Day 7; additionally, no asynchronous development wa s reported. Normalized hind limb length (HLL) was decreased at Day 7 (12%, NS) and 21 (38% , p<0.01) at the high c oncentration, 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 si gnificantly increased gr owth and development compared to the negative control, so decreases in growth and developm ental endpoints at the highest test concentration we re more pronounced when compared to the solvent control. Histopathological effects on the thyroid in the hi ghest treatment included apparent increases in gland hypertrophy, gland size, follicle size and follicle asymmetry compared to the negative control. However, compared to the solvent cont rol, the only histopathol ogical effect was an increase in follicle asymmetry. Increased follicl e 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 botto m of the tank (n=1).TCVP FINAL

Page 18 of 58 An increased incidence of thyr oid follicular cell hypert rophy was observed in females at 142 and in both sexes at 375 mg/kg/day in the Part 158 ra t subchronic oral study. These effects occurred at doses that resulted in signifi cant 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 incr ease in mild follicular cell hyper trophy was seen at the mid and high dose (0.5 and 1.0 mg/L). Systemic toxicity wa s also observed at these two doses, including significant RBC Ch E inhibition ( ↓ 35 and 30%, respectively) and increased adrenal weights.TCVP FINAL

Page 19 of 58 Table 4. Thyroid Pathway for TCVP

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

Study Type / Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmentalstage (±),asynchronous,normalized HLLGrowth(BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

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 X 4 (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 resp onse. A shaded cell indicates that is parameter is not routinely evaluated or is not applicab le 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 examinedTCVP FINAL

Page 20 of 58 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of TCVP to interact w ith endocrine related processes ( i.e., E, A or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated analysis of those lines of evidence.

The WoE evaluation includes consid erations of biological plausibility of the findings from the different lines of evidence by examining the cons istency, coherence and in terrelationships among the measured endpoints within and across studi es. The available findings from standard toxicology studies on the substance may contribute to the WoE evalua tion in helping elucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively a sequelae of system ic effects. Endocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence eval uation of the results of the Tier 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues shou ld be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l disruptions in homeostasis” (USEPA, 2013).

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

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

Page 21 of 58 1. Tier 1 in vivo Assays

In the uterotrophic assay, no treatment-related eff ects were observed on mortality, clinical signs, body weights or body weight gains. In the He rshberger 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 bod y weight gain (Days 1 to 10) for the 50 and 150 mg/kg/day groups was similar to control. Live r 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 wa s comparable to control at the two lower doses.

There was no TCVP-related mortality, and no treatm ent-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 weight s, body weight gains or food consumption. In the female pubertal study, absolute and relativ e adrenal weights were increased by 29–31% at 50 mg/kg/day. At 350 mg/kg/day, ab solute and relative liver we ights 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 fema les was 100% for all treatment concentrations, with the exception of 88% male survival at th e 0.055 mg/L (medium) concentration. There were no notable observations with regards to behavi or or changes in app earance, 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 su b-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 observe d in the control groups and in every treatment except at the high concentr ation. Mortality was equi valent 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-concentra tion groups (1.7 and 3.4%, respectively). No mortality was observed in the negative control.TCVP FINAL

Page 22 of 58 2. OSRI

In the developmental toxicity stud with rats, clin ical 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 terminat ion. 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 gain s 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 de velopmental study consisted of maternal toxicity that manifest ed 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 ra ts, 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 viab ility, litter obse rvations, 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 F 0 males were decreased to a similar extent ( ↓ 7–9%), with body weight decreases at Week 10 or 16 of onl y 5 to 7% (NS). Body weight and body weight gains for F 0 females were not affected by treatment. Decreases in mean overall body weight gain of high-dose (130 mg/kg/day) F 1 males (p ≤ 0.05, ↓ 6), and body weight gain of high-dose F 1 females (168 mg/kg/day) dur ing the premating period (p ≤ 0.001, ↓ 11), were observed, but no significant effects on weekly body we ights were noted. Mean adrena l weights were increased in

F 0 females [ ↑ 9% at the low (7.3 mg/kg/day) a nd medium dose (40 mg/kg/day, and ↑ 28% at the high dose (155 mg/kg/day]. In the F 1 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%).TCVP FINAL

Page 23 of 58 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) wa s NS. Liver weights were decreased by 16–22% in males at Week 104 (a ll dose groups) and decreased by 23% in highdose females at Week 52. Kidney weights were not a ffected 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 in cidence 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 hi ghest 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 weight s 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 we re 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 fe males ( ↑ 21 and 24%). Relative liver and kidney weights were also increased in the medium- and high-dose groups. Absolute adre nal weights were increased in medium- and high-dose females ( ↑ 35 and 39%), with no change in relati ve weights or weights in males.

Reduced growth and survival was observ ed at the top three concentrations ( ≥ 0.15 mg/L) in a Part 158 fish early life-stage study with the fathead minnow.TCVP FINAL

Page 24 of 58 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 st eroidogenesis assay that lessen the confidence in the negative results for this assay, and ther e were no redundant res ponses observed in the in vivo mammalian assays (Tier 1 or OSRI) to support an interaction with th e 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 al tered steroidogenesis. A dditionally, 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 toxici ty observed in this study, the concentr ations 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 LC 50 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 oppos e 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 ef fects including egg production. A decrease in offspr ing weight was observed in th e mallard duck study, but adult female body weight was also affect ed, and there were no other repr oductive effects. Therefore, while effects on reproduction were observed in the FSTRA, there were no effects on gonadal weight or histopathology, and ther e 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 tec hnical 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 observedTCVP FINAL

Page 25 of 58 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 re production study, no other androgen-related effects were observe d 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 histopathol ogy were observed in both sexes. Serum T 4 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 thyr oid histopathology were observed in the subchronic rat study and 28-day inhalation rat study. Cholines terase activity was not measured in either Tier 1 pubertal assay, however, the effects observed in the Part 1 58 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 gr owth were observed in the highest treatment group, they occurred in the pres ence of overt toxicit y. Additionally, in the highest treatment, which was considered to be overtly toxic, while histopathological effect s on the thyroid included apparent increases in gland hypertrophy, gland size, follicle size and follicle asymmetry compared to the negative control; however when co mpared to the solvent control, the only histopathological effect was an in crease in follicle asymmetry.

E. Conclusions

There is a lack of convincing evidence for pot ential 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 assa ys 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 thyr oid 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 ad renal changes and reduced weight gain at 43.2 mg/kg/day (LOAEL) in a chronic oral toxicity st udy in rats. This POD is used for deriving the chronic Reference Dose (RfD) for chronic di etary 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 subchronicTCVP FINAL

Page 26 of 58 toxicity study in rats. This POD is used fo r deriving the Margins of Exposure (MOEs) for nondietary risk assessments.

In general, since the POD and the toxicity endpo ints of concern are base d on data obtained from adult animals, there is a concern that the POD may not be protectiv e of potential thyroid toxicity in the young. However in the case of TCVP, although no data exists for poten tial 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 ch emicals (organophosphate); and 3) the CTA would be tested at higher doses and w ould 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 departur e or endpoint for human h ealth risk assessments.

Given the lack of convincing evidence for a potential interaction with the estrogen, androgen and thyroid pathways in wildlife no a dditional testing is recommended.

For birds, Part 158 avian reproduction studies w ith 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 re productive effects to bird s from TCVP exposure. Relative to the EDSP, additional testing is not recommended.TCVP FINAL

Page 27 of 58 VI. References

Barton, S. (1991). TCVP: Two Generation Repr oduction 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 prep ared 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 Ra ts: A Short-Term Screening Assay for Androgenic and Anti-Androgenic Properties of TCVP. U npublished study conducted by Product Safety Labs, Dayton, NJ. Laboratory Report No.: 33549, August 31, 2012. (MRID 48946701).

Boulet, J. (2012). The Uterot rophic Bioassay in Rats: A Shor t-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 thyroi d function in intact juve nile/peripubertal male and female rats exposed to TCVP. Unpublis hed 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 stu dy prepared by Ricerca, Inc

. in cooperation with Argus Research Laboratories, Inc

. and Test Substance Analys is Laboratory. (MRID 40152701).

Hazleton Laboratories America, Inc. (1980). 103-week Chronic Feeding Study in Mice: SD-8447 and Original SD-8447

. Final rept. (Unpublished stu dy 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 coope ration 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).TCVP FINAL

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. Laborator y 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 St udy 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 us ing 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. (Unpublis hed 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 St udy of Tetrachlorvinphos (TCVP) by Inhalation in Rats with a Two Week Recovery Period: Fina l 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 Di etary Combined Chronic Toxicity/Carcinogeni city Study in Rats : Lab Project Number: 7929. Unpublished study prepared by Inveresk Research Inte rnational. (MRID 42980901).

Perry, C.; Mulhern, M.; Henderson, W. et al. (1990) Tetrachlorvinphos: 13 Week Dietary Toxicity Study in Rats: Lab Project Numb er: 7258: IRI/438141. Unpublished study prepared by
Inveresk Research Intern ational. 194 p. (MRID 43371201)

Perry, J.; Millar, P .; Mulhern, M. (1994). TCVP : 104 Week Dietary Combined Chronic Toxicity/Carcinogeni city Study in Rats : Addendum : Lab Project Number: 438403: 7929. Unpublished study prepared by lnveresk Re search International. (MRID 43335101).

Rödig, K. (2012). Endocrine Disruptor Test: Andr ogen Receptor Binding (Rat Prostate Cytosol) with TCVP (TCVP). Unpublished study conducted by BSL Bioservice, Scie ntific Laboratories GmbH, Planegg, Germany. Laboratory Study No: 114588, January 17, 2012. (MRID 48703101).

Rödig, K. (2012). Endocrine Disruptor Test: Es trogen Receptor Binding (Rat Uterine Cytosol) with TCVP (TCVP). Unpublished study conducted by BSL Bioservice, Scie ntific LaboratoriesTCVP FINAL

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., East on, 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, Lt d., Easton, Maryland, Report No.: 699A-101. Submitted by Tetrachlorvinphos Task Fo rce (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 Nu mber: WIL-149009. Unpublished study prepared by WIL Research Labs., Inc

. MRID 42679401.

U

. S. Department of Health, Educ ation, and Welfare ( 1978). Bioassay of Tetrachlorvinphos for Possible Carcinogenicity

. HEW. (CAS no. 961-11-5; NCI·CG-TR-33; National Cancer Institute carcinogenesis technica l 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 Endocri ne Disruptor Review Team; Test Order # EDSP-083702-55. August 9, 2010. Offi ce of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

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

USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
held July 30 - August 1, 2013 on Weight-of

- Evidence: Evaluati ng Results of EDS P Tier 1 Screening. Office of Chemical Safety and Pollution Pr evention. U.S. Environmental Protection Agency, Washington, DC.

U.S. National Institutes of Health (1978). Bioassay of tetrachlorvinphos for possible carcinogenicity. By National Ca ncer Institute, Div. of Ca ncer Cause and Prevention, Carcinogenesi s Program , Carcinogen Bioassay and Progr am Resources Branch. Bethesda, MD :TCVP FINAL

Page 30 of 58 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). Unpublis hed study conducted by CeeTox, Kalamazoo, MI. Laboratory Report No.: 9180V-100538ERTA, N ovember 4, 2011. (MRID 48619501).TCVP FINAL

Page 31 of 58 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA): (OCSPP 890.1100)

The 21-day assay (MRID 48688401) of tetrachlorv inphos (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 we re exposed to negative (dilution water) control, solvent control (0.020 mg/L dimethyl formamide, DM F), 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 e ffects are reported based on comparison to the negative (clean water) control. Clinical signs of toxicity (i.e., be havioral and other sublet hal effects) were noted at 2.5 mg a.i./L including incl uding loss of equilibrium and lyi ng on the bottom of the tank were observed in two tadpoles. Spinal cu rvature (crooked tail) was obser ved in controls and in every treatment except the high tr eatment (2.5 mg a.i./L).

At Day 21, tadpoles had shorter normalized hi nd-limb length (HLL) at the highest test concentration (2.5 mg a.i./L) than in the ne gative 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 ob served. 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 lowe st 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, a nd normalized HLL (p<0.05), compared to the negative controls. Compared to the solvent cont rol, Day 7 body weight, SVL, and NF stage (2 stages) were significantly decr eased (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 in crease in follicle asymmetry.TCVP FINAL

Page 32 of 58 Androgen Receptor (AR) Bindi ng 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 sa turation binding experiment was conducted to demonstrate that the AR in the rat prostate cy tosol was present in reasonable numbers and was functioning with appropriate affi nity for the radio-labeled re ference 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% pu rity, Batch #TX 100113). Etha nol 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 (B max ) was 0.4298-0.859 fmol/100 μg protein for the three runs a nd the dissociation constant (K d ) was 0.5615-1.21 nM. The K d 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, B max values were significantly be low the expected range of 10 to 150 fmol/100 μg protein for all runs. In a ddition, 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 (exc luding one outlier), which is greater than the suggested value of <20% reported in the EPA valida tion program. The Scatchard plot of the data was linear for Runs 2 and 3 only.

In the competitive binding experiment, the estimated mean log IC 50 s 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 solven t control responses indicated a sl ight 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 IC 50 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 pr ecipitation 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.TCVP FINAL

Page 33 of 58 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; [ 3 H]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. Tritia ted water was quantified using liquid scintillation counting (LSC). Th ree runs were conducted and each run included a full activity control, a bac kground activity control, a pos itive 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 act ivity observed in Run 1, the concentrations of
the test item used in Runs 2 and 3 were adjusted to include mi d-log concentrations.

Aromatase activity in the full activity controls for the thr ee 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 backgr ound 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 IC 50 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 c oncentration of TCVP (10

− 3 M). The lowest portion of the response curve for TC VP across runs was less than 50% activity. Aromatase activity for TCVP was 10 3.6-105.9% 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 IC 50 of TCVP

was

− 3.76 M and the Hill slope was

− 1.01.

Based on the data from the average response curv e, TCVP is classified as an Inhibitor of aromatase activity in this assay.TCVP FINAL

Page 34 of 58 Estrogen Receptor (ER) Bindi ng Assay; (OCSPP 890.1250)

In an estrogen receptor (ER) binding a ssay (MRID 48765001) for tetrachlorvinphos (TCVP), uterine cytosol from Sprague Dawley rats was us ed as the source of ER to conduct saturation and competitive binding experiments. The satura tion binding experiment was conducted to demonstrate that the ER in the rat uterine cyto sol was present in reasonable numbers and was functioning with appropriate affin ity for the radiolabeled referen ce estrogen prior to routinely conducting competitive binding experiments. The competitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of 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, octyltriet hoxysilane as a negative control, and 17- β -estradiol as the natural ligand reference material.

In the saturation binding experiment , the mean maximum binding capacity (B max ) was 5.464 fmol/100 μg protein and the m ean dissociation constant (K d ) was 0.1297 nM. The B max value was lower than the recommended range of 10-150 fm ol/100 μg protein. The Scatchard plots for the three Saturation assays were linear. The sp ecific binding reached a pl ateau in all runs, and non-specific binding was generally less than 20% of total binding.

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

− 9.188 M for 17 β

estradiol and

− 6.484 M for the weak positive control. The mean relative binding affinity (RBA) was 0.1990% for the weak positive control. Conf idence 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 IC 50 and RBA could not be calculated for TCVP. Specific [ 3 H]-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.

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

In an estrogen receptor transcript ional activation assay (MRID 48619501), hER α -HeLa-9903
cells cultured in vitro were exposed to tetrachlorvinphos (TCVP, 100.05% a.i., Lot # TX100113) at logarithmically increasi ng 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 concentr ation was tested in 6 wells/pl ate 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.TCVP FINAL

Page 35 of 58 TCVP was tested up to the limit of cy totoxicity, 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 th e cells to the very weak positive control 17 α -methyltestosterone was lower than the expected values, indicating a decreased sensitivity of the assay to very weak agonists. Alt hough 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 no t comparable to the responses of 17 α -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 concentra tion possible based on cytotoxicity (10

− 7 M). Because the RPC Max < PC 10 in both assay runs, TCVP was considered negative for estrogen receptor transcrip tional activation in this test system.

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

The 21-day short-term reproduction assay (MRI D 48838201) of tetrachlorvinphos (TCVP) with fathead minnows ( Pimephales promelas) was conducted under c ontinuous flow-through conditions. Adult fish (20 spawning groups, 2 male s and 4 females in each group; 5 months old) were exposed to TCVP (98.7%) at nominal concen trations of 0 (negative control), 0 [solvent control; dimethylformamide (DMF) at a fina l 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 te st system was maintained at 24.5 to 25.6 o C and a pH of 8.0 to 8.3.

Unless otherwise noted, all result s are presented based on comparis on to the negative control. No significant differences (p>0.05) were det ected between the negativ e and solvent control groups for any of the endpoints. Survival in male s 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 a nd 24.5 eggs/female/day/replicate, respectively; fertilization success in the negati ve 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 fec undity 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 ne gative control for any other en dpoint. There were no notable differences between histopathology severity scores or gonadal stag es in treated groups compared to the negative control. There we re no notable observations with regards to behavior or changes in appearance, specifically color/banding, ovipositor appearance, or size of dorsal nape pad in theTCVP FINAL

Page 36 of 58 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) screeni ng 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, castr ated 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 si x castrated rats exposed to 0.4 mg/kg/day of testosterone propionate (TP) by s ubcutaneous (s.c.) injection.

To screen for potential anti-a ndrogenic 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 conjunc tion with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The cont rol 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 castrat ed 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 assa y, body weights and body weight gain s 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 incr eased (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 th e increase was only significant (p ≤ 0.01) at the highest dose. Adrenal gland weights we re 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/k g/day group, there were no significant changesTCVP FINAL

Page 37 of 58 in accessory sex organ weights of the TCVP-t reated 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 LA BC, 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 decr eased (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) fo r 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-c ontrol, 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%. Ther e was an initial decrea se (p<0.01) of 96% in food efficiency in the 350 mg/kg/day group, but ot herwise food efficiency in the TCVP-treated and positive control group were si milar to the TP control.

Liver weights in the anti-androge n 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 vesicl es, 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 incr eased liver weights observed in the current

assay.

TCVP was negative for androgenic activity in the Hershberger assa y as statistically significant increases in organ weights were not seen in two or more of the fi ve androgen sensitive tissues in the androgen agonist assay. TCVP was also ne gative for anti-androgenicity in the Hershberger assay as no statistically significan t decreases in organ weights were seen in two or more of the five androgen sensitive tissues in the androgen antagonist assay. The bi ological significance ofTCVP FINAL

Page 38 of 58 the increases in accessory sex organ weights of TCVP-treated rats in the anti-androgen assay is unclear. The accessory sex organ weight increase s were not dose dependent, and TCVP did not give a positive response in the agonist assay. However, the da ta suggest that TCVP may be affecting the metabolism of the testosterone, whic h would not have been evident in the agonist assay as the castrated rats were already testosterone de ficient. 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 tetr achlorvinphos (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 17 α

ethynylestradiol (EE), by subcutaneous injectio n, 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 we re recorded. After sacrifice on PND 42 or 43, blood was collected for clinical chemistry analyses, and total thyroxine (T 4 ) and thyroid stimul ating hormone (TSH) levels were determined using a radioimmunoassa y (RIA). Adrenal, liver, kidney, pituitary, thyroid, and urogenital organs we re weighed, and microscopic ex aminations were performed on the ovaries, uterus, thyr oid, liver, and kidneys.

There were no adverse, treatment -related effects on mortality, c linical signs of toxicity, body weights, body weight gains, food consumption, c linical chemistry, or macroscopic pathology.

Serum T 4 levels were decreased by18% (not statis tically significant [NS] ) and 65% (p<0.05) at 50 and 350 mg/kg/day, respectively, but serum TS H concentrations at 50 and 350 mg/kg/day were comparable to control. Follicular cell he ight 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 we re only statistically si gnificant in the 350 mg/kg/day group.

There were no effects of treatment on age and bo dy 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 cons idered non-adverse), % cycling or regularly cycling, or cycle status at necr opsy. 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 we re increased (p<0.001) by 8%, and absolute and relative adrenal weig hts were increased (p<0.001) by 30-31% at 50 mg/kg/day. At 350 mg/kg/day, ab solute and relative liver we ights were increased (p<0.001) by 21-25%, and absolute and re lative adrenal weights were increased (p<0.001) by 37-42%.TCVP FINAL

Page 39 of 58 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 seve rity 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 gr oup), food consumption over the course of the study was decreased (p<0.05, ↓ 9%), leading to decreased ov erall body weight gain (p<0.05, ↓ 14%). The mean age at attainment of VO wa s decreased (p<0.01) by 8 days (PND 25.6 treated vs. PND 33.6 controls) and body weight at atta inment of VO was decr eased (p<0.01) by 39%. All EE-treated females were non-cycling in cons tant 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 A ssay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 49186901), 15 Sp rague Dawley [SD® IGS] rats/dose group were treated daily via oral gavage with te trachlorvinphos (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 sepa rate positive control reference groups of 15 males were dosed with either testosterone pr opionate (TP), via subcut aneous injection, at 1.0 mg/kg/day, or with the androgen antagonist, flutamide (FT) via or al gavage at 3.0 mg/kg/day. Animals from each group were examined for pr eputial 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 anal yses, and total serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) leve ls 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 pathol ogy. 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 heig ht 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 signifi cant in the 350 mg/kg/day group. Serum T 4 levels wereTCVP FINAL

Page 40 of 58 decreased by 15% (not statistically significan t [NS]) and 68% (p<0.05) at 50 and 350 mg/kg/day, respectively, and serum TSH levels were in creased (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, epidid ymides, 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 re lative liver weights were also increased (p<0.001) by 23-27% and absolute and relative kidney weights we re increased (p<0.05) by 11-15%. There was increased incidence (9/15) of chronic progressive nephropat hy in the 350 mg/kg/day group compared to the control (1/15). In the liver, slight to modera te 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 inci dence of chronic progressive nephropathy.

Groups receiving either with the androgen agoni st (TP) or the antagon ist (FT) showed the expected effects on sexual deve lopment 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 decr eased (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 testoster one 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 sper matids and spermatocytes, degene ration and/or depletion of the elongating spermatids, atrophy of the Leydig cells, re duction in testicular si ze, 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 or gans were decreased (p<0.01) compared to controls: seminal vesicle plus coagulating gla nd 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 hype rplasia of the Leydig cells in
the testis in 14/15 males.TCVP FINAL

Page 41 of 58 Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 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 con centration of 0.1%. Only two independent experiments were performed, rather than the Guid eline recommended three runs.

Testosterone and estradiol concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) method. A Quality C ontrol (QC) plate was run concurrently with each independent run of a test chemical plate to demonstrate that the as say responded properly to positive control agents at two concentration le vels; positive controls included the known inhibitor (prochloraz) and indu cer (forskolin) of es tradiol and te stosterone production. The highest concentration of TCVP suitable for evaluation was 10 μM due to cytotoxicity observed

at 100 μ M 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 reviewer s could not determine if basal hormone production met the fold-incr ease criteria in th e SC wells of ≥ 5-fold for testosterone and

≥ 2.5-fold for estradiol above the minimum determ ination limit (MDL) of the assay because the MDLs for the ELISA were not reported.

On the QC plates, exposure to 10 μM fo rskolin 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 sy nthesis 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 recomm ended by the Guideline, and there was a high degree of variability in testoste rone induction (forskolin) and i nhibition (prochloraz) responses between the two assay runs. In Run 1, the induction of testoster one 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 μ M forskolin in Run 1 was also approximately 7-fold greater than the response to 1 μM forskolin in Run 2. Inhibition of testosterone synthesi s in the presence of 0.1 μM prochloraz varied approximately 18-fold be tween 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; howev er, the reviewer’s assessment was hampered by the lack of methodological details and pr oficiency testing data.

Only a single hormone concentration value for te stosterone 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 absolu te testosterone concentrations for the SCTCVP FINAL

Page 42 of 58 replicates within the test pl ates were 50% and 26%; theref ore, only the second run met the ≤ 30% performance criteria recommended in the Test Gu ideline. 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 recommende d performance criteria of ≤ 30%. All % CV determinations were calculated by the reviewers.

With regards to the effects of TCVP, the results fr om the two runs were vari able for testosterone. In Run 1, there was a 22.4-fold induction in te stosterone production at 10 μM TCVP, but no induction ( ≤ 0.70-fold) was observed at the other reporte d TCVP concentrations, and there was a
marked inhibition in testosterone production (0.26-fold) at the lowest concentration of TCVP (0.000001 μ M). In Run 2, no induction or inhibition of testosterone pr oduction (0.87- to 1.24- fold) was observed at 0.000001 to 10 μ M TCVP, but there was again a marked reduction in testosterone (0.32-fold) at the lowest TCVP concentration tested (0.0000001 μ M). For estradiol production, there was no consiste nt induction or inhibition in either run at 0.000001 to 10 μ M TCVP, with fold-change values ranging from 0.8 0- 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 producti on, the numerous deficiencies noted in the study preclude making a definitive conclusion with re gards 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 17 α -ethynyl estradiol (EE) in 0.1% aqueous methylcellulose at 0.3 μ g/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 afte r 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 tr eated groups were comparable to the vehicle control. Absolute wet and blotted uterus weights for the positiv e control (EE) group were increased (p<0.001) by 75% and 71%, respectively, as expected.TCVP FINAL

Page 43 of 58 Although no evidence of systemic toxicity was observed in this study, the high dose (350 mg/kg/day) was considered ad equate based on increases in liv er and kidney weights seen at 350 mg/kg/day in the female pubertal assay (MRID 49186901).

No statistically sign ificant changes were seen in uterine weight in this assay. Tetrachlorvinphos is negative in the ut erotrophic assay.TCVP FINAL

Page 44 of 58 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

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

ER/AR Transcript ional Activation

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

− 8 to 10

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

− 11 or 10

− 10 M E 2 or 10

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

− 10 M E 2 , 10

− 9 M E 2 , or 10

− 9 M DHT for ER α , ER β , and AR, respectively. Antagonistic activities were evaluated similarly by the relative activity expressed as 20% relative i nhibitory concentration (RIC 20 ), the concentrat ion of the compound showing 20% inhibition of the activity of E 2 or DHT against ER α , ER β , and AR, respectively. TCVP did not show agonist or antagonist activity for ER α , ER β , 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 fema le 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 s ubjected to gross necropsy. All fetuses were weighed, sexed and examined for gross external alte rations. One-half of the fetuses in each litter
were examined for visceral soft tissue altera tions using a variation of Wilson’s sectioning technique. The remaining one-half of the fetuse s in each litter were ex amined for skeletal alterations. An initial study was terminated at C-section due to poor pr egnancy rates; a second study was conducted with an identical study design.TCVP FINAL

Page 45 of 58 There were no mortalities; clini cal 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, w ith 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-relate d lesions were observed in any females at

necropsy.

There were no treatment-related effects of treatm ent 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 assessm ent 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, lo w-, 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 e ffect on skeletal malformations
or variations.

The maternal NOAEL is 75 mg/kg/day (LDT), a nd the maternal LOAEL is 150 mg/kg/day based on decreased body weight gain of 11%. The de velopmental NOAEL is 300 mg/kg/day (HDT), and a developmental LOAEL is not determined.

Prenatal Development in Non-Rodents (Rabbit)

In a prenatal developm ental toxicity study ( MRID 00127831 ) in female New Zealand White rabbits (18/dose), TCVP (98%) was administer ed 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. inject ion of HCG (100 USP units). On GD 29, the does were euthanized and subjected to complete gr oss necropsy. All fetuses were weighed, sexed and
examined for external abnormalitie s, visceral soft tissue abnorma lities 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 vagi nal fluid passed by 8/11 does in the high-dose group and one each in the low- and medium-dose groups.

Developmental toxicity in th e high-dose group was manifested as an increase in early resorptions/doe (0.4, 0.5, 0.3, a nd 1.4, respectively), with a co rresponding increase in postTCVP FINAL

Page 46 of 58 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 contro l, 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 developmen tal 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 F 1 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 an d subjected to a gross necropsy. Subsequently, 1 pup/sex/litter/group (at least 10 pups /sex/dose when available) we re allocated to the following subsets: Subset 1, PND 21 brain weights and neur ohistological evaluations; Subset 2, water maze and passive avoidance test; Subset 3, motor activit y and auditory startle habituation; Subset 4, terminal brain weights and neurohi stological 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 effect s on mortality, clinical signs, body weight, body weight gain, feed consumption, FOB, or gr oss pathology. No treatment-related effects on reproductive parameters were observed. The mate rnal LOAEL was not observed. The maternal NOAEL was 200 mg/kg/day (HDT).

In offspring, there were no treatment-related e ffects on viability, litter observations, clinical signs, body weight, food consumption, sexual matu ration, FOB, motor activ ity, acoustic startle habituation, learning and memory (passive avoi dance and water maze), gross pathology, or histopathology parameters. In th e 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 durin g 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 findi ng was judged to correlate with the treatment-related decreases (p ≤ 0.05) in several microscopic linea r brain measurements. It is noted that on PND 21, a decrease in single morpho metric parameter was seen in both sexes with the small magnitude (4 to 5%) whereas on P ND 70, decreases in multiple morphometricTCVP FINAL

Page 47 of 58 measurements were seen in both sexes with gr eater magnitude (7 to 21%) compared to PND 21.
No treatment related effects were noted in the ≤ 50 mg/kg/day F 1 offspring.

The offspring LOAEL was 200 mg/kg/day, base d on decreases in body weight, body weight gain, and several morphometric linear brain measur ements in both sexes, and decreased absolute brain weight in the males on PND 70. The offspring NOAEL was 50 mg/kg/day.

Multi-Generation Reproduc tion 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 F 0 and F 1 parental generation at dosages of 0, 100, 500, or 2000 ppm. Mean-measured intake during the premating period in the F 0 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 th e premating period (15 weeks old) in the F 1 parents was 6.7, 34, and 130 mg/kg/day in males and 8.3, 43, and 168 mg/kg/day in females. The F 0 generation was treated for 10 w eeks prior to mating. Treatment continued throughout the mating, gestation and lactation periods , until termination after weaning of these litters. The F 1 generation was treated for 11 weeks prio r to mating to produce the F 2 generation, with treatment continued throughout the mating, gestation and lactati on periods, until termination for all F 1 and F 2 animals at the weaning for these litters.

Study measurements included body weight/body we ight gain, food consumption, determination of reproductive parameters, organ weight s, and gross and microscopic pathology.

There was no evidence of a treatment-related eff ect on survival; no deaths occurred in the F 0 animals and the only deaths in the F 1 animals included two control animals (male and female) and two females in the medium-dose group. No tr eatment-related clinical signs were reported during the study. Overall mean body wei ght gains in all dose groups for F 0 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 F 0 females were not affected by
treatment. Decreases in mean ove rall body weight gain of high-dose F 1 males (p ≤ 0.05, ↓ 6), and body weight gain of high-dose F 1 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 dur ing the study. No consistent ev idence of treatment-related effects on fertility, gestation or lactation indices were observed in litters produced by the F 0 or F 1 generations. In the F 2 generation, pups had a greater mean increase in weight compared to F 1 pups over the 21-day period across all groups (including control).

Determination of ChE activity was not reporte d in this study. Mean adrenal weights were
increased in F 0 females (significance NR; ↑ 9% at the low and medium dose, and ↑ 28% at the high dose). A similar effect was not repeated in the F 1 females, although adrenal weights in the medium- and high-dose females were increased (significance NR) by 8 and 9%. No changes inTCVP FINAL

Page 48 of 58 adrenal weights were observed in males in either generation. No treatment-related histopathological lesi ons were reported in any examined tissues.

The NOAEL for systemic toxicity is 500 ppm (26 mg/kg/day in F 0 males, 40 mg/kg/day in

F 0 females, 34 mg/kg/day in F 1 males, and 43 mg/kg/day in F 1 females) based on decreased body weight gains and the increased mean adrenal weight in F 0 females. The LOAEL is 2000 ppm (102 mg/kg/day in F 0 males, 155 mg/kg/day in F 0 females, 130 mg/kg/day in F 1 males, and 168 mg/kg/day in F 1 females). The NOAEL for reproducti ve 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 re duced (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 signifi cantly reduced mean plasma ChE (-74% and -83%) and RBC ChE (-79%, -91% ). 5000 ppm females had reduced (-24%, not statistically significant) brain ChE. A notew orthy finding was that 2/10 male s and 2/10 females at 5000 ppm had no measurable RBC ChE activity at 13 weeks. The incidence and se verity of bilateral basophilic tubules of the kidneys was signifi cantly increased in males at 2000 and 5000 ppm.
The incidence of increased fat de position in the adrenal cortex wa s significantly elevated in 2000 ppm (7/10 animals) and 5000 ppm (9/10) fema les. 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 enlargemen t was noted in 2/10 2000 ppm and 7/10 5000 ppm males, but not in any females. Thyroid fo llicular 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 female s. Mean absolute liver weights were significantly elev ated in 5000 ppm females; mean covariantly adjusted (by body weight) liver weights were si gnificantly elevated in both sexes at 2000 and 5000 ppm. Mean absolute and covariantly (body weig ht) adjusted adrenal weights we re 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 inhalati on 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 hour s/day, 5 days/week for 3 weeks at exposure concentrations of 0,TCVP FINAL

Page 49 of 58 0.05, 0.5, or 1.0 mg/L. During the final week of expos ure (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 af ter completion of the 28-day e xposure period to determine the reversibility of any effects obser ved. Actual exposure concentrati ons as measured by HPLC were 100%, 100%, and101% of target values. The mass median aerodynamic diameters (MMADs) ± geometric standard deviations (GSD) were 2.57 μ m ±3.785, 4.51 μ m ±3.541, and 4.27 μ m ±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 bod y 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%) re lative 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 consum ption 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 examinati ons. 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 elevat ed 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 inhibiti on (p<0.001) was observed in females at the mid- and high-exposure concentrations (35% an d 30%, respectively). Stat istically significantAChE and RBC ChE activity inhibition (p<0.001) was observed in males at the mid- and highexposure concentrations (38% and 31% for AC hE, respectively; 24% and 31% for RBC ChE, respectively). Plasma acetylcholinestera se activity (58% to 70%) and plasma butyrylcholinesterase activity ( 66% to 87%) were decreased relati ve to controls at all exposure
concentrations in females. All cholinesterase activ ities were lower relative to controls in the male and female recovery groups, but the differe nces were not statis tically significant.

Treatment-related gross findings were limited to discoloration and enlarg ement of the adrenal glands at the mid- and high-expos ure concentrations in males, and in females at all exposure
concentrations. Diffuse adrenal co rtical 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 thyroi d gland was observed with an exposure-related incidence in both sexes at the mid- and high-expo sure concentrations. Minimal to mild ovarian
interstitial cell vacuolation was observed with an exposure-relate d incidence in females at theTCVP FINAL

Page 50 of 58 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 vacuol ation of the ovaries in female s, 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 cholin esterase inhibition was 0.05 mg/L, based on an
increase in RBC cholinesterase inhib ition 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 tw o years at nominal doses of 0, 100, 1000, or 2000 ppm, e quivalent 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 week s, all surviving rats in all groups were sacrificed for gross and histopathologi cal evaluation. Select or gans were weighed at each sacrifice interval (includi ng liver, kidneys, testes with ep ididymides, prostate, ovaries, uterus, adrenals, and pituitary). Gross and micr oscopic pathological examination included the liver, kidney, pituitary, thyr oid/parathyroids, adrenals, mammary gland, testes with epididymides, prostate, seminal vesi cles, uterus, vagina, and ovaries.

No clinical signs were observed th at were not considered normal fo r 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 we ight 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 acro ss all treatment groups. No changes in hematology parameters were noted in treated animals; changes in clinical chemistry parameters (primarily liver-related enzymes orTCVP FINAL

Page 51 of 58 compounds) included increased c holesterol in high-dose female s 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 male s 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 eff ect on RBC ChE activity was apparent in male rats in this study. RBC ChE activity was decreased in all treated females (with dos e-dependent decreases at 18- and 24-months), but the decrease s 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 weig hts of the testes with epididymides, prostate, ovaries, uterus, adrenals, or pituitary. Inconsis tent, 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 decr eased (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 hi gh 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 di d not demonstrate any consistent effect on
endocrine-related e ndpoints or tissues.

Although low-dose females had decreased RBC Ch E activities (NS), decreased alkaline phosphatase (NS), and increased serum cholestero l (at 18 months, but not dose dependent), these occurrences do not suggest a tr eatment-related pattern. The NOAEL for systemic toxicity is considered 100 ppm (4.23 and 5.93 mg/kg/day, resp ectively) in males and females. The LOAEL is 1000 ppm, based on decreased bo dy weight in females, liver and adrenal changes in both sexes, and decreased plasma ChE activity in females.

In the present study, the incidence of thyroi d 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 car cinomas and adenomas in males in the low- and medium-dose groups were more similar to control and within th e historical control data range. The incidence of thyroid c-cell adenoma in 2000 ppm females su rviving to week 104 was 4/26, and in theirTCVP FINAL

Page 52 of 58 controls was 4/27. There was no indi cation of a tumorigenic effect in the adrenals of females in the present study.

In a previous carcinogeni city study in rats ( MRID 00117443 ), technical-grade TCVP (purity not reported) was administered in the diet to gr oups of Osborne-Mendel ra ts (50/sex/dose) at nominal doses of 0, 4250, or 8500 ppm (calculate d equivalency of 0, 212.5, or 435 mg/kg/day).
Matched controls consisted of 10 rats/sex; pooled controls cons isted of the matched controls
combined with 45 rats/sex from similar bioass ays. 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 pr ovided), and no other study results were available, except for a summary of the incidence of carcinoge nicity. 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 wa s observed in male and female rats. A significant, dose-related trend (significance not reported) in cortical aden omas of the adrenal also was noted in female rats, with an increased incide nce (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 ba tch of TCVP (used for a ll 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 toxi city, 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 co nducted 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 dur ing the study, and on all animals at the interim and
terminal sacrifices.

No treatment-related mortality was observed in the present study, with su rvival rates higher in the two highest treatment groups . No treatment-related clin ical signs, including no signs associated with ChE inhibition, were noted during the study, although there was an increased incidence of hunched/thin appearance in hi gh-dose rats beginning at Week 54. Mean body weights were decreased ≥ 10% by Week 2 and throughout the re mainder of the study at the twoTCVP FINAL

Page 53 of 58 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 th e two highest doses. Similar decreased weights were noted w ith 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 orig inal 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 we re 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 TC VP in male mice ( ↑ 83%). Gross pathology findings were generally minimal,
but increased incidences of liver and kidney alterations were not ed at terminal sacrifice in animals in the two highest dose groups.

Non-neoplastic lesions in the liver, kidneys, and adrenals we re 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 K upffer cells, enlarged hepatocytes, necrosis, and bile duct proliferation (liv er); and increased ba sophilia and tubular hyperplasia (kidneys). There was an increased in cidence 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 tr eatment-related histopat hological 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 e quivalency of 0, 1200, or 2400 mg/kg/day).
Matched controls consisted of 10 mice/sex; pooled controls cons isted of the matched controls
combined with 40 mice/sex from similar bioass ays. 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 fo r treated m ice, but the data and study details were not available for review. No other study results were available, except for a summary of theTCVP FINAL

Page 54 of 58 incidence of carcinogenicity. A significant, dos e-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 neopl astic 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, clinic al 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 termin ation. Organ weights determined included adrenals, kidneys, liver, ovaries , pituitary, prostate, testes wi th 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 d ecreased 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 weig hts of ovaries, pituitary, prostate, testes with epididymides, or uterus. Absolute liver wei ghts 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 kidne y 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 relati ve weights or weights in males. Finally, prostate weights were decreased (NS) in low- and medium-dose males ( ↓ 22 and 30%, respectively), with a significant decr ease (p<0.05) in high-dose males ( ↓ 44%). Gross and microscopic pathology were gene rally unremarkable, with no tr eatment-related findings in endocrine-related organs.TCVP FINAL

Page 55 of 58 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 hemato logy effects, decreased plasma ChE activity, and increases in alkaline phosphatase a nd 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 di etary 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 di et. Mean-measured concentrations were <0.4 (LOD, control), 96, 290, and 979 mg a.i./kg diet.

There were no treatment-related mortalities or over t 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. Sli ght 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 comp ared to the negative control. Based on these reductions, the NOAEC and LOAEC were determ ined 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 di etary 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 nomin al concentrations of 0 (negative control), 100, 300, and 1000 mg a.i./kg diet. Mean-measured concen trations were <0.4 (LOD, control), 96, 290, and 979 mg a.i./kg diet.

There were no treatment-related mortalities or over t 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 wei ght 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 earl y life stage of fathead minnow ( Pimephales promelas ) was studied under flow-through condition s. Fertilized embryos were expos ed 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 so lvent 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.TCVP FINAL

Page 56 of 58 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 aver aged 99 and 90% in the negative and solvent control, respectively, compared to 9 2, 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 gr oups, respectively. The NOAEC for juvenile survival is 0.047 mg a.i./L based on statistically significant (p<0.05) differen ce in larval survival at treatment concentrations great er 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% d ecrease 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.TCVP FINAL

Page 57 of 58 APPENDIX 3: References Not Utiliz ed in the TCVP WoE Analysis

In 2009, after public review and comment, a fina l list of 67 chemicals and schedule for issuing
Test Orders for the EDSP Tier 1 screening battery was made av ailable in a Federal Register Notice issued October 21, 2009 (74 FR 54422). The ag ency’s review of the initial data submitted as “other scientifically releva nt information (OSRI) was provided in the Report of the Endocrine Disruptor Review Team (USEPA, 2010).

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies previously submitted to the agency for registra tion/reregistration, published literature articles and/or Tier 1 assays. The agency also c onducted a more recent search (2009 to 2014) of
available scientific literature fo r any additional relevant informa tion for their weight of evidence (WoE) evaluations. These articles were evalua ted in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/s cience/efed/policy_guidance/team _authors/endangered_species _reregistration_workgroup/PDF_ro t/esa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature To xicity Studies to Suppor t Human Health Risk Assessment ( http://www.epa.gov/pesticides /science/lit-studies.pdf).

The following published and unpublished references were considered for use in the WoE analysis for TCVP but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the com pound of interest was not us ed in the study; 3) the test material was not adequately described; 4) a formulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only da ta on invertebrates.

Doyle, R.L., Teske, R.H., and Elsea, J.R. (1968) Two-year Dietary Admi nistration of SD 8447 to Dogs: P-99A. Unpublished study received A ug 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 Bl ood and Brain Cholinesterase over 21 Days of Dosing Rats. WIL 404001. WIL Research Labo ratories. 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. UnpublishedTCVP FINAL

Page 58 of 58 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 Char acteristics of Beef Bulls. Final report. Unpublis hed 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 E xperiment 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., Wash ington, DC; CDL:091274-0. (MRID 00112525)