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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

# &

Health Effects Divison

# ::~:E£'~:::e 

# f] g:ams d f 'v--

Environmental

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

THR O UG H: Je ss Rowland

## ~ S

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

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

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

.

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

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

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

CHEMICAL: ACEPHATE

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS 

### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY 

### U.S E NVIRONMENTAL 

### P ROTECTION 

### A GENCYACEPHATE FINAL

ii TABLE OF CONTENTS

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

I. Introduction.......................................................................................3

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

A. EDSP Tier 1 Screening Assays............................................................4

B. Other Scientifically Rele vant Information (OSRI)......................................4

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

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

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................8
1. Effects on Estrogen Pathway.....................................................8
2. Effects on Androgen Pathway..................................................12

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

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

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

1. Tier 1 in vivo Assays ............................................................19

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

B. Estrogen Pathway...........................................................................20

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

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

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

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

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

LIST OF TABLES: Table 1. Tier 1 Screening Assays for Acephate ..................................................7
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Acephate....................................9
Table 3. Androgenic/Anti-Andr ogenic Pathway for Acephate ................................13
Table 4. Thyroid Pathway for Acephate..........................................................17

APPENDIX 1. EDSP Tier 1 Screening Assays........................................................28
APPENDIX 2. Other Scientifically Relevant Information (OSRI)................................38

APPENDIX 3. References Not Utilized in the Acephate WoE Analysis..........................50ACEPHATE 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 enerationACEPHATE 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 Interagency Coordinating Committee on the Validation of Alternative Methods

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-dealkylaseACEPHATE FINAL

v Abbreviation Terminology

MTC Maximum Tolerated Concentration
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 TriiodothyronineACEPHATE FINAL

vi Abbreviation Terminology

T4 Thyroxine (tetraiodothyronine)

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-EvidenceACEPHATE FINAL

Page 1 of 51 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, 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 acephate interacts with E, A, or T hormone pathways, the number and type of effects induced, the magnitude and patt ern of responses observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whether or not endoc rine-related responses occurred at dose(s) that also resulted in general systemic toxicity.

On March 19, 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 we ight-of-evidence (WOE) analysis of the potential interaction of acephate with the E, A or T signaling pathways. The T1Wo ERC 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).

For acephate, there was no convincing evidence of potential interaction with the estrogen pathway in mammals or wildlife. All of the Tier 1 in vitro assays were negative. Furthermore, no estrogen-related effects were observed in th e Tier 1 uterotrophic and female pubertal rat assays. A minimal (3%) decrease on fertility wa s observed in the Fish Short Term Reproduction Assay (FSTRA) in the absence of any further endocrine-related endpoints. Effects on the HPG axis also are not supported across the battery of Tier 1 assays. The only estrogen-related effect
in the Part 158 mammalian studies was a reported decrease in fe rtility index in the 3-generation reproduction study. While effects on egg viability and chick survival were reported in the Part 158 avian reproduction studies, th ese endpoints are limited in their ability for diagnostic specificity for an endocrine-related interaction, especially given the relative lack of other estrogen-related effects in the Tier 1 battery. Additionally, decreases in brain ChE activity in adults were reported at all doses in both avian reproduction studies.

For the androgen pathway, there was no convincing evidence of potential interaction of acephate in mammals or wildlife. Acephate was negative for androgen receptor (AR) binding and negative in the steroidogenesis assay. Although acephate exposure resulted in decreased androgenACEPHATE FINAL

Page 2 of 51 sensitive tissues (AST) weights in the high dose in the anti-androgenic portion of the in vivo Hershberger assay, no endocrine-related effects (g onadal or AST weights/ histopathology, or age and weight at preputial separation) were noted in the male pubertal rat assay with an intact HPG
axis. It is noted that at the doses tested in th e Hershberger assay; usi ng a satellite group of noncastrated rats, both red blood cell (RBC) and br ain cholinesterase (C hE) activities were decreased 40 to 87% compared to controls. Wh ile there was a significant decrease in pituitary weight in the male pubertal assay there was no evidence of treatment-related pituitary histopathology in the OSRI Part 158 mammalian toxicity studies. There was a slight effect on fertility in reported in the FSTRA, but no othe r complementary responses were observed. While effects on offspring viability were observed in the avian reproduction studies, there are limited findings in the Tier 1 battery to suggest a potential androgen-r elated interaction.

In the female pubertal assay, no thyroid-related e ffects were noted. In the male pubertal assay, thyroid weights were decreased by 25 and 21% at the low and high dos e, respectively, and pituitary weight was decreased by 14% at the high dose, but no treatment-related effects were noted for thyroid histopathology. D ecreases in ChE activity were observed in the male pubertal assay at all doses tested. No ch anges in thyroid weight or histology were reported in the Part 158
mammalian studies.

In the amphibian metamorphosis assay (AMA), while there were signifi cant increases in the developmental endpoint of normalized hind-limb length (HLL) at Day 21, significant increases in this endpoint were not obser ved (p>0.05) at Day 7, which is a time point at which HLL has
been shown to be a sensitive endpoint for thyroid agonists [at Day 7, there was a non-significant (p>0.05) increase of 1.7% at the low and mid treat ment with a non-signifi cant 3.4% decrease at the high treatment]. Additionally, there were no significant differences in developmental stage,
nor treatment-related effects on t hyroid histopathology at Day 21. It is noted that tadpoles in this treatment group were larger than controls. Theref ore, overall, it appears that the st atistically significant increase in HLL at Day 21 in the acephate treatments compared to the control is not due to interaction with the thyroid pathway, but ma y be due to a generalized increase in growth in the tadpoles.

Based on weight of evidence considerations, ma mmalian and wildlife EDSP Tier 2 testing are not recommended for acephate since there was no convincing evidence of potential interaction with the estrogen, androgen or thyroid pathways.ACEPHATE FINAL

Page 3 of 51 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 acephate interacts with E, A, or T hormone pathways, the number and type of effects induced, the magnitude and patt ern of responses observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whether or not endoc rine-related responses o ccurred at does (s) that also resulted in general systemic toxicity or overt toxicity.

On March 19, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence analysis of the potential interaction of acephate with the estrogen (E), androgen (A) or thyroid (T) signaling pathways. The T1WoERC
conclusions from the WoE evaluation in this re port are presented by pathway (E, A and then T) beginning with the resu lts 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).

Acephate is an organophosphate insecticide and its mode of action is based on altered neurotransmission due to acetylcholinesterase (C hE) inhibition. The vapor pressure of acephate
is 1.7 × 10 -6 mm Hg at 25°C, and therefore, volatili zation is not expected to significantly contribute to the dissipation of acephate in the environment. The water solubility is 790 g/L at 20°C and 818 g/L at 25°C, and a low octanol/w ater partition coefficient (log Kow = -0.85 to 0.13). Acephate is photolytic ally stable, not subject to hy drolytic degradation, however quickly undergoes biotic degradatio n (aerobic soil metabolism half- lives <2 days). Acephate has a whole body bioconcentration factor va lue in fish of approximately 10.

The available information considered in determ ining the potential interaction of acephate with the E, A, or T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxi city studies and other pu blished articles. These data are summarized in Sections III.A through I II.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 recomme ndations are presented in Section V.ACEPHATE FINAL

Page 4 of 51 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 acephate (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 of the potential for a
substance to interact with endocrine-related processes ( i.e., E, A, or T hormonal pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

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

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

Page 5 of 51 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 concordance or consistency (complementarity) of the responses in the individual study is evaluated. For the
Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a 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 molecular initiating ev ent, 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.ACEPHATE FINAL

Page 6 of 51 This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting in July 2013 that evaluated scie ntific issues associ ated with the WoE
evaluation of the EDSP Tier 1 sc reening 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 effects in endocrine se nsitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013). For these WoE analyses, overt toxicity wa s generally defined in accordance with EPA’s current approach as used by OPP in reviewi ng 40 CFR Part 158 studies for both human and ecological risk assessments. Specifically, in these an alyses, the effects that EPA considered to be potential evidence of overt toxicity included, but were not limited to: mo rtality; clinical signs such as tremors, ataxia and abnormal swimmi ng (fish and aquatic-pha se amphibians); and body weight decreases of ≥ 10% in mammals. Additionally, ot her effects including morphological ( e.g., organ weights/histopat hology), biochemical ( e.g. , blood chemistry), and other clinical signs ( e.g., lethargy) were also considered when evaluating overt toxicity, especially if the effects were extreme. In some instances, one parameter ( i.e. , death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g. , changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addressing the three main components described above is fundamental to the WoE process and in determ ining whether additional data are needed. In addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting data; 4) overall strength of eviden ce supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing th e need for additional data is based on a caseby-case analysis which took all availa ble toxicity data into account.ACEPHATE FINAL

Page 7 of 51 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 biol ogically 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 acephate are shown in

Table 1.

Table 1: Tier 1 Screening Assays for Acephate.

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48614502)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48614503)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48669001)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48614506)

Aromatase Assay (Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48614501)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48614507) Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48614504) Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID No. 48614505) Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID No. 48614505)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48675101)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 48698501)ACEPHATE FINAL

Page 8 of 51 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for acephate 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.

Acephate was classified as negative (or not interactive) in the Tier 1 in vitro ER binding, ERTA, aromatase, and steroidogenesis assays. A cephate also was negative in the Tier 1 in vivo uterotrophic and female pubertal rat assays. Acephate was also negative in OSRI ERTAs that were conducted in CHO-K1 (Kojima et al ., 2004) and BG-1 cells (Kojima et al ., 2005).

In the FSTRA, there was a 3% decrease (p<0.05) in fertilization at the high concentration (91 mg a.i./L). No other effects (i.e. plasma VT G concentration, GSI, gonadal histopathology, body weight/length effects) were reported. ChE activity was not measured in the FSTRA.

In the Part 158 mammalian studies, there was a decrease in ovarian weight (absolute and relative) in high-dose female mice in the carcinogenicity study; however, there was no corresponding histopathol ogical effects and the decreased ovarian wei ghts occurred in the presence of overt toxicity (>10% body weight). In a 3-generati on reproductive toxicity study in the rat, fertility index was decreased for the th ird generation high dose gro up. Fertility was low in all groups including controls for the F1b and F2b litters, and the high dose (25 mg/kg/day) would be expected to inhibit brain and RBC ChE activiti es as observed with other mammalian toxicity studies at comparable dose levels. It is not ed that ChE activity was not measured in the reproduction study.

In the Part 158 mallard duck reproduction st udy, significant decreases in the number of embryo viability were reported at the mid-treatment group (20 mg/kg-diet); similar effects were reported at the high treatment, 80 mg/kg-di et, along with a decrease in th e number of 14-day old offspring ( ↓ 18%). No symptoms of toxicity or behavioral abnormalities or treatment-related mortalities were reported in the treatments (except for tw o incidences of prolapsed penis in the high treatment); however, slight, but significant, depressions in f ood consumption were reported in the low and high treatment. In the correspondi ng bobwhite quail repr oduction study, effects on egg viability and chick survival were reported at 80 mg/kg-diet. Concurrent studies of brain ChE activity in both the mallard duc k and bobwhite quail studies reported reduced activity in all treatments with decreases increasing with dosage.ACEPHATEFINALPage 9 of 51Table 2. Estrogenic/Anti-Estrogenic Pathway for AcephateLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Acephate1Study Type / LiteratureCitation E R Binding E R Activation Steroidogenesis Sex Steroid Hormone U terine Weigh t O varian Weigh t / GSI O varian/Gonad Staging and H istopathology P ituitary Weigh t E strous Cyclicity A ge & Weight at VO 2 ° Sex Characteristics F ertility(Frt) / F ecundity (Fcd) V itellogenin (VTG) Systemic Toxicity O bserved 2 O ver t Toxicity O bserved 3EDSP Tier 1 AssayER Binding(MRID 48614502)NERTA(MRID 48614503)NSteroidogenesis(MRID 48614506)NAromatase(MRID 48614501)NUterotrophic(MRID 48614507)NChE(M, H)NFemale Pubertal Rat(MRID 48614505)NENNNNNN↓KW↓AW(H)ChE(L, H)NFSTRA(MRID 48675101)NENNNFrt:↓3%(H)NNOSRIER Transactivation(Kojimaet al., 2004)NER Transactivation(Kojimaet al.,2005)NDevelopmental toxicity(Rat;MRID 41081602)NNNEX(H)ACEPHATEFINALPage 10 of 51Table 2. Estrogenic/Anti-Estrogenic Pathway for AcephateLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Acephate1Study Type / LiteratureCitation E R Binding E R Activation Steroidogenesis Sex Steroid Hormone U terine Weigh t O varian Weigh t / GSI O varian/Gonad Staging and H istopathology P ituitary Weigh t E strous Cyclicity A ge & Weight at VO 2 ° Sex Characteristics F ertility(Frt) / F ecundity (Fcd) V itellogenin (VTG) Systemic Toxicity O bserved 2 O ver t Toxicity O bserved 3Developmental toxicity(Rabbit; MRIDs00069684)NRNNRNDevelopmentalNeurotoxicity(Rat MRID 46151802)NENNNENThree-generationreproduction(Rat; MRIDs 40323401and 40605701)NR NR NRFrt:↓33%(H; F3gen)X4(H)NChronic toxicity /carcinogenicity(Rat; MRID 00101623)NNNNX(M, H)NCarcinogenicity (Mouse;MRID 00105197)NENNNX(H)X(M,H)Chronic toxicity(Dog; MRID 41812001)NENNNX(H)NAvian reproduction(Duck; MRIDs 00029691,00029689)P5X(L-H)NAvian reproduction(Quail; MRIDs 00029692,00029690)P5X(L-H)N1.Key to responses: L= Low treatment, ML=Medium-Low treatment, MH=Medium-high treatment, H=High treatment. Arrows (↓or↑) indicate thedirection of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight areabsolute unless otherwise indicated. LW= Liver weight; KW= Kidneyweight; AW= Adrenal weight; ChE= Cholinesterase activity; Frt=Fertility; F3gen=third generationACEPHATEFINALPage 11 of 512.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 anX in this column. For details see Section IV. A3.The overt toxicity in the Tier 1 assays are presented inthis column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV.4 ChE activity was not measured in this study. However, the high dose would be expected to inhibit ChE activity based on otherstudies with comparabledoses.5 In the mallard duck study, significantly decreased viable and live embryos≤20 mg/kg-diet, with decreased chick survival at 80 mg/kg-diet. In thebobwhite quail study, decreases in egg viability and chick survival at 80 mg/kg-diet.P Positive findingsN Negative findings (in vitro) / No effect (in vivo)NE Not examinedNR Not reportedACEPHATE FINAL

Page 12 of 51 2. Effects on Androgen Pathway

The potential for acephate 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 androgeni c, 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.

In an evaluation of androgenic/an ti-androgenic activity from Tier 1 testing results, acephate was classified as negative (or a non-binder) in the in vitro AR binding assay and the steroidogenesis assay. Acephate was also negative in an AR tr anscriptional activation (A RTA) assay considered as OSRI (Kojima et al. 2004).

In the Hershberger assay, while glans penis weig hts were increased in bo th low- and high-dose groups by 25, and 19%, respectively, no other an drogen-sensitive tissue (AST) weights were affected. Therefore, the assay is considered negative in the agonist portion of the assay. However, in the antagonist portion of th e assay, absolute ventral prostate ( ↓ 24%) and Cowper’s gland ( ↓ 18%) weights were significantly decreased at the high test dose (30 mg/kg/day); ventral prostate weights were also signifi cantly decreased 18% at the mid- dose (10 mg/kg/day). It is also noted that while absolute seminal vesicle weight was not significantly decr eased, adjusted weight was significantly decreased 21% at the high-dose. Therefore, acephate was positive in the antiandrogen portion of the assay. At all acephate doses (3 to 30 mg/kg/day), ChE activities, measured using a satellite group of non-castrated rats, were decreased (p<0.01) compared to control in both the RBC (40 to 80% inhibition) and brain (42 to 87% inhibition).

In the male pubertal rat assay, no treatment-rela ted effects were observe d on age or weight at PPS, AST weights, or gross and microscopic pathology. In the high-dose group (30 mg/kg/day), pituitary weight was decreased ( ↓ 14%; p<0.05) and serum testosterone levels were decreased ( ↓ 53%; p<0.001). In the satellite animals at the intermediate and high doses, dose-dependent reductions (p<0.001) in both RBC ( ↓ 74 to 92%) and brain ( ↓ 72 to 87%) ChE activities were
observed.

In the FSTRA, a slight, yet signif icant (p<0.05) decrease of 3% fe rtility was reported at the high concentration; however, no other treatment-related effects were reported.

In the Part 158 mammalian studies, increases in re lative testes weights of 43% were observed at the high dose in the mouse carcinogeni city study. Body weight decreases ≥ 10% were also reported at this high dose. As reported in the es trogen pathway, effects on egg viability and chick survival were reported in both the mallard duck and bobwhite quail study at ≥ 20 and 80 mg/kgdiet; additionally, decreases in brain ChE activity were reported at all doses in both studies.ACEPHATEFINALPage 13 of 51Table 3. Androgenic/Anti-Androgenic Pathway for AcephateLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Acephate1Study 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 Obsereved 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48669001)NSteroidogenesis(MRID 48614506)NHershberger(MRID 48614504)NEVP:↓18/24%(M, H);CG:↓18%,(H)↓LW(H)ChE(H)NMale Pubertal Rat(MRID 48614505)↓53%(H)NNNN↓14%(H)NNChE(L,H)NFSTRA(MRID 48675101)NENNN↓3%(H)NNACEPHATEFINALPage 14 of 51Table 3. Androgenic/Anti-Androgenic Pathway for AcephateLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Acephate1Study 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 Obsereved 2 Overt Toxicity Observed 3OSRIARTA (Kojimaet al.,2004)NDevelopmental toxicity(Rat;MRID 41081602)NNE NDevelopmental toxicity(Rabbit; MRID 00069684)NNR NDevelopmentalNeurotoxicity(Rat MRID 46151802)NNNE NThree-generationreproduction(Rat; MRIDs 40323401and 40605701)NRNRNRNRNRNRFrt:↓33%(H; F3gen)X(H)4NChronic toxicity /carcinogenicity(Rat; MRID 00101623)N NNENNE NEX(M, H)NCarcinogenicity(Mouse; MRID 00105197)↑43%(H, Rel)N NE N NE NEX(H)NChronic toxicity(Dog; MRID 41812001)NNNENNNEX(H)NAvian reproduction(Duck; MRIDs 00029691,00029689)P5X(L-H)NACEPHATEFINALPage 15 of 51Table 3. Androgenic/Anti-Androgenic Pathway for AcephateLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Acephate1Study 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 Obsereved 2 Overt Toxicity Observed 3Avian reproduction(Quail; MRIDs 00029692,00029690)P5X(L-H)N1. Key to responses: L=Low treatment, ML= Medium-Low treatment, M=Medium treatment, MH=Medium High treatment, H=High treatment.Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay.Changes in weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV),Ventral prostate (VP),Cowper’s gland (CG), LW= Liver weight; ChE= Cholinesterase activity; Frt=Fertility; F3 gen=third generation2. 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. ChE activity was not measured in this study. However, the high dose would be expected to inhibit ChE activity based on otherstudies with comparabledoses5. In the mallard duck study, significantly decreased viable and live embryos≤20 mg/kg-diet, with decreased chick survival at 80 mg/kg-diet. In the bobwhitequail study, decreases in egg viability and chick survival at 80 mg/kg-diet.P Positive findingsN Negative findings (in vitro) / No effect (in vitro)NE Not examinedNR Not reportedACEPHATE FINAL

Page 16 of 51 C. Effects on Hypothalamic-Pit uitary-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 male and female (rat) assays, and the AMA (frog).

The potential for acephate to interact w ith 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 the female pubertal assay, no treatment-rela ted effects were noted for thyroid weight, histopathology, T 4 or TSH concentration. In the male pubertal assay, thyroid weights were decreased by 25 and 21% at the low and high do se, respectively, and pituitary weight was decreased by 14% at the high dose, but no treat ment-related effects were noted for thyroid histopathology. No changes in thyroid weight or histology were repor ted in the Part 158
mammalian studies.

In the AMA study, there were no gross or histol ogical effects on the thyroid; however, an increase of 12 and 27% in the Day 21 normalized hind-limb length (HLL) were observed in the low-(3.5 mg a.i./L) and high-dose (102 mg a. i./L) groups, respectively. A non-significant (p=0.08) increase of 6.8% in HLL was observed in the mid-dose group. An increase of 15% in body weight was reported on Day 7 in the hi gh-dose group. Additionally, snout-vent length (SVL) was increased by 9% in the high-dose group on Day 7 and an increase of 2.6% (p<0.05) was observed in the mid and high-dose groups on Day 21.ACEPHATE FINAL

Page 17 of 51 Table 4. Thyroid Pathway for Acephate

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

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

EDSP Tier 1 Assays

Female Pubertal Rat (MRID 48614505) N N N N N N ↓ KW; ↓ AW  ChE (L,H) (H) N

Male Pubertal Rat (MRID 48614505) ↓ 25, 22% (L, H) N N N ↓ 14% (H) N  ChE (L, H) N

AMA (Frog; MRID 48698501) N HLL: ↑ 12 and

27% Day 21 (L, H) BW: ↑ 15% Day 7 (H);
SVL: ↑ 9% Day 7 (H), ↑ 2.6% Day 21 (M, H) N

OSRI

Three-generation reproduction (Rat; MRIDs 40323401 and 40605701) N N N X (H) 4 N

Chronic toxicity / carcinogenicity (Rat; MRID 00101623) N N NE NE NE N N

Carcinogenicity (Mouse; MRID 00105197) NE N NE NE NE X (H) N

Chronic toxicity (Dog; MRID 41812001) N N NE NE N X (H) N
1. Key to responses: L=Low treatment, ML= Medium-Low treatment, M=Medium, 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 applicab le in this assay. Changes in weight are absolute unless otherwise indicated KW= Kidney weight; AW= Ad renal weight; ChE = Cholinesterase activity 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. ChE activity was not measured in this study. However, the high dose would be expected to inhibit ChE activity based on other studies with comparable doses N Negative findings NE Not examinedACEPHATE FINAL

Page 18 of 51 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 acephate to interact with 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 an alysis of those li nes of evidence.

The WoE evaluation includes consid erations of biological plausibility of the findings from the different lines of evidence by examining the co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substance may contribute to the WoE evalua tion in helping elucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively a sequelae of system ic effects. Endocrine modes of action may elicite 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 1assays 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).ACEPHATE FINAL

Page 19 of 51 1. Tier 1 in vivo Assays

In the Hershberger assay, at the high dose (30 mg/kg/day), body weight gain was decreased by 32% in the androgenic phase, and absolute liver weights were decrease d by 12 to 13% in both the androgenic and anti-androge nic phases of the assay. After the 10-day exposure, both RBC
and brain ChE activities were substantially decreased for all dose groups. At 3, 10 and 30 mg/kg/day, RBC activity was inhibited by 40 to 80% and brain ChE activity was inhibited by
42 to 87%.

No clinical signs of toxicity were noted in either the female or male pubertal assay at either dose (10 or 30 mg/kg/day). In the female pubertal assay, the final body weight and cumulative body weight gains were decreased by 5 and 8%, resp ectively, at the high dos e, and absolute and relative kidney and adrenal weights were decr eased by 7 and 13%, respectively. Compared to control, ChE activity at 10 and 30 mg/kg/day was significantly inhibi ted (p<0.001) in the RBC ( ↓ 69 and 89%) and brain ( ↓ 71% and 84%). In the male pubert al assay, pituitary weight was decreased by 14% at the high dos e, and thyroid weight was decr ease by 25 and 22% at the low and high dose. Compared to the control, ChE activity in males at 10 and 30 mg/kg/day was significantly inhibited (p<0.001) in the RBC ( ↓ 74 and 92%) and brain ( ↓ 72% and 87%).

In the uterotrophic assay, no c linical sign of toxicity were observed at any dose (3, 10 and 30 mg/kg/day). After the 5-day exposure period, final body weights and body weight gains at 3 and 10 mg/kg/day were comparable to control, but decreases were observed in body weight gain ( ↓ 93%, p<0.05) at the high dose. ChE activity was decreased (p<0.01) in RBC by 59% and 77% at 10 and 30 mg/kg/day, respectiv ely, and in brain by 35, 59 and 80% at 3, 10 and 30 mg/kg/day, respectively.

No overt signs of toxicity were observed in th e AMA or FSTRA at test concentrations up to 102 and 91 mg/L, respectively. In the AMA study, bent tail was observed (8 to 21%), but this finding
occurred in the cont rol and treated groups.

2. OSRI

In the Part 158 mammalian studies, the most se nsitive endpoint across studies was cholinesterase inhibition. In the developmental ra t study, clinical signs of toxicity (tremors and decreased motor activity) were observed in the dams at the hi gh dose (75 mg/mg/day). In the developmental neurotoxicity study in rats, no maternal effects were observed at ≤ 10 mg/kg/day, and no decreases in plasma, RBC or brain ChE activity were observed in pups on PND 4. By PND 21, brain ChE activity in pups (males and female s) was inhibited by 28, 30 and 60% at 0.5, 1.0 and 10 mg/kg/day, and plasma and RBC ChE activity was inhibited by 45 and 57%, respectively, in high-dose pups.ACEPHATE FINAL

Page 20 of 51 In the chronic rat study, decreased body weights ( ≥ 10%) and body weight gain (6-18%) were observed in high dose males (35 mg/kg/day), along with altere d behavior (hyperactivity and increased aggression). Plasma ChE activity was inhibited at all sampling times in the high-dose males (10 to 50%) and females (50 to 72%). In the mid-dose group (2.5 mg/kg/day), ChE inhibition was 0–29% (NS) in males and 0–38% (occasionally p<0.05 or 0.01) in females. Plasma ChE activity was generally not inhibited in the low-dose animals (0.25 mg/kg/day). RBC ChE activity was decreased at all sampling time s in the high-dose males (21–67%) and females (21–61%). In the mid-dose groups, RBC ChE activity was inhibited by ≤ 42% (generally NS) for males and females, and there was no effect in the low-dose animals. Brain ChE activity was inhibited by 0–13%, 34–43% and 69–77%, in the low- , mid-, and high-dose males, respectively, and by 1–13%, 33–45% and 66–83% in low-, mid- and high-dose females, respectively.

In the mouse carcinogenicity study, body weights were decreased (>10%) in high-dose males and females (146 or 167 mg/kg/day) from Week 2 until study termination, and in mid-dose males and females (36 or 42 mg/kg/day) from Week s 65 to 78 until study termination. High-dose females had a higher incidence of hepatocellular ca rcinomas and hyperplastic nodes in the liver,
and there were increased lesions in the liver, lung and nasal cav ity in the mid- and high-dose males and females. High-dose males had decrea ses in liver and kidne y weight and high-dose females had decreased kidney and brain weights comp ared to the control. ChE activities were not examined in the mouse study.

In the chronic dog study, no signs of clinical toxicity were observed at any dose (0.27, 3.1 and 20.2 mg/kg/day). However, brain ChE activity was inhibited (p<0.05) at study termination (Week 53) in males from all dose groups (17, 5 3, and 66%, respectively) and in the mid- and high-dose females (49 and 66%, respectively). RBC ChE activity was inhibited (p<0.05) in the mid- (42–55%) and high-dose (76–87%) groups of bot h sexes, with no change at the low dose.
Other treatment-related effects were increased (NS) absolute liver weights in high-dose males (29%) and females (17%), and perivascul ar infiltration and pigment in hepatic
reticuloendothelial cells of one mid-dose male and many high-dose males and females.

In the Part 158 mallard duck and bobwhite qua il reproductive studies brain ChE activity was reduced in all treatment groups with reductions increasing with dose.

B. Estrogen Pathway

There was no convincing evidence of potential interaction with the estrogen pathway in
mammals or wildlife. All of the Tier 1 and OSRI in vitro assays were negative. Furthermore, no estrogen-related effects were obser ved in the Tier 1 uterotrophic and female pubertal rat assays. A minimal (3%) decrease on fertility was observed in the FSTRA in the absence of any further endocrine-related endpoints. Effects on the HPG ax is also are not supported across the battery of Tier 1 assays. The only estrogen- related effect in the Part 158 mammalian studies was a reportedACEPHATE FINAL

Page 21 of 51 decrease in fertility index in the 3-generati on reproduction study. While effects on egg viability and chick survival were reported in the Part 158 avian reproduction studi es, these endpoints are limited in their ability for diagnos tic specificity for an endocrine-r elated interaction, especially given the relative lack of othe r estrogen-related effects in th e Tier 1 batter y. Additionally, decreases in brain ChE activity were reported at all doses in both avian reproduction studies.

C. Androgen Pathway

There was no convincing evidence of potential inte raction of acephate in mammals or wildlife. Acephate was negative for androgen receptor (A R) binding and negative in the steroidogenesis assay. Acephate was also negative in an AR TA considered as OSRI. Although acephate exposure resulted in decreased androgen sensitive tissues (AST) weights in the high dose in the anti-androgenic portion of the in vivo Hershberger assay, no endocri ne-related effects (gonadal or AST weights/ histopathology, or age and weight at preputial separation) were noted in the male pubertal rat assay with an intact HPG axis. It is noted th at at the doses tested in the Hershberger assay; using a satellite group of non-castrated rats, both red blood cell (RBC) and brain cholinesterase (ChE) activities were decr eased 40 to 87% compared to controls. While there was a significant d ecrease in pituitary weight in the male pubertal assay there was no evidence of treatment-related p ituitary histopathology in the OSRI Part 158 mammalian toxicity studies. There was a slight effect on fer tility in reported in the FSTRA, but no other complementary responses were observed. While e ffects on offspring viability were observed in the avian reproduction studies, there are limited findings in the Tier 1 battery to suggest a potential androgen-re lated interaction.

D. Thyroid Pathway

In the female pubertal assay, no thyroid-related e ffects were noted. In the male pubertal assay, thyroid weights were decreased by 25 and 21% at the low and high dos e, respectively, and pituitary weight was decreased by 14% at the high dose, but no treatment-related effects were noted for thyroid histopathology. D ecreases in ChE activity were observed in the male pubertal assay at all doses tested. No ch anges in thyroid weight or histology were reported in the Part 158
mammalian studies.

In the AMA, while there were significant increa ses in the developmental endpoint of normalized hind-limb length (HLL) at Day 21, significant in creases in this endpoi nt were not observed (p>0.05) at Day 7, which is a time point at whic h HLL has been shown to be a sensitive endpoint for thyroid agonists [at Day 7, there was a non-si gnificant (p>0.05) increase of 1.7% at the low and mid treatment with a non-significant 3.4% decrease at the high treatment]. Additionally, there were no significant differe nces in developmental stage, nor treatment-related effects on thyroid histopathology at Day 21. It is noted that tadpoles in th is treatment group were larger than controls. Therefore, overall, it appears that the statistically significant increase in HLL atACEPHATE FINAL

Page 22 of 51 Day 21 in the acephate treatments compared to th e control is not due to interaction with the thyroid pathway, but may be due to a genera lized increase in growth in the tadpoles.

E. Conclusions

Based on the data considered in the WoE analys is, acephate did not demonstrate a potential to interact with the estrogen, androgen or thyroid pathways.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations, ma mmalian and wildlife EDSP Tier 2 testing are not recommended since there was no convincing evidence of potential interaction with the estrogen, androgen or thyroid pathways.ACEPHATE FINAL

Page 23 of 51 VI. References

Akhurst, L.C. (2011). Acephate technical estrog en receptor transcriptio nal activation (Human cell line (HeLa-9903)). Unpublished prepar ed by Huntingdon Life Sciences, Ltd. Cambridgeshire, England. Laborator y Report No.: ADB0061, October 14 2011. (MRID 48614503).

Akhurst, L.C. (2011). Acephate technical H295R steroidogenesis assay. Unpublished study prepared by Huntingdon Life Sciences Ltd., Ca mbridgeshire, England. Laboratory Study No. ADB0064, October 25, 2011. (MRID 48614506).

Beavers, J.B., et al. (1979). [Companion to duck repr oductive study examining ChE inhibition in ducks. No citation available] (MRID 00029689).

Beavers, J.B., et al. (1979). [Companion to quail repr oductive study examining ChE inhibition in quails. No citation available] (MRID 00029690).

Beavers, J.B., Fink, R., Grimes, J., et al. (1979). Final Report: On e-Generation Reproduction Study--Mallard Duck: Project No. 162-107. Unpublished study prepared by Wildlife International, Ltd., submitted by Chevron Ch emical Co., Richmond, Calif.; CDL: 241824-C). (MRID 00029691).

Beavers, J.B.; Fink, R., Grimes, J., et al. (1979). Final Report: On e-Generation Reproduction Study—Bobwhite Quail: Project No. 162-106. Unpublished study prepared by Wildlife International, Ltd., submitted by Chevron Ch emical Co., Richmond, Calif.; CDL:241824-D). (MRID 00029692).

Brorby, G.; Rosenberg, D. (1987). The Cholines terase Inhibition Potential of Acephate Technical (SX-1102) Following 4-, 9-, or 13-Week Dietary Administration in Male and Female Rats: Laboratory Project ID S- 3068. Unpublished study prepared Ch evron Environmental Health Center, Inc. (MRID 40504819).

Dalgard, D

. (1991). One-Year Oral Toxicity Study in Dogs with Chevron Acephate Technical : Lab Project Number: HWA 2107-165

. Unpublished study prepared by Hazleton Washington

. (MRID 41812001).

Donington, S. (2011). Acephate Technical: Hershberger bioassa y in the castrated rat (oral administration). Unpublished prepared by Hunti ngdon Life Sciences, Cambridgeshire, England. Laboratory Project Study ID: ADB 0062, October 13, 2011. (MRID 48614504).ACEPHATE FINAL

Page 24 of 51 Foster J.R. (2011). Acephate aromatase (human recombinant) assay. Unpublished prepared by Huntingdon Life Sciences, Ltd., Cambridgeshire, England. Laboratory Report No.: ADB0059, October 14, 2011. (MRID 48614501).

Geil, R.G. (1981). Lifetime Oral Carcinogenicity Study in Mice: Histopathology Data: IRDC No. 415-006. Unpublished study received June 8, 1981 under 239-2418; prepared by International Research and Development Cor p., submitted by Chevron Chemical Co., Richmond, CA; CDL:245374-A. (MRID 00077209).

Glickman, A. (1983). Orthene Lifetime Study in Mice: Hepatocarcinoma in Female Mice. Unpublished study received June 27, 1983 unde r 239-2471; submitted by Chevron Chemical Co., Richmond, CA; CDL:250666-B. (MRID 00129156).

Hoberman, A. (1987). Two-Generation (Two-Litte r) Reproduction Study in Rats with Chevron Acephate Technical: Laboratory Project ID: Argus Research Laborat ories: 303005: Chevron Chemical Co. Study: S-2497. Unpublished study prepar ed by Argus Research Laboratories Inc. in cooperation with Chevron Chemical Co. (MRID 40323401).

Hoberman, A. (1988). Two-Generation (Two-Litte r) Reproduction Study in Rats with Chevron Acephate Technical: Laboratory Project ID 303005 and S-2497. Unpublished study prepared by Argus Research Laborator ies, Inc. (MRID 40605701).

Hoberman, A. (2003). Oral (gav age) developmental neurotoxicity study of Acephate technical in rats. Unpublished prepared by Argus Research, Horsham

, PA

. Laboratory Project No

. VP23747. December 4, 2003. (MRID 46151802).

Hoberman, A.M. (2003). Oral (gavage) dosage-ra nge study of acephate technical in adult rats. Argus Research, 905 Sheehy Drive, Buildi ng A, Horsham, PA 19044-1297. Argus 222-004; Laboratory Project Identification VP-25064. November 25, 2003. Unpublished. (MRID 46151803).

Hoberman, A.M. (2003). Oral (gavage) dosage- range study of acephate technical in neonatal rats. Argus Research, 905 Sheehy Drive, Build ing A, Horsham, PA 19044-1997. Argus 222-003; Laboratory Project Identification VP-25056. December 9, 2003. Unpublished. (MRID 46151804).

Hoberman, A.M. (2003). Oral (gavage) acute rela tive sensitivity study of acephate technical in neonatal and adult rats. Argus Research, 905 Sheehy Drive, Building A, Horsham, PA 19044- 1297. Argus 222-005; Laboratory Project Identification VP-25072. December 9, 2003. Unpublished. (MRID 46151801).ACEPHATE FINAL

Page 25 of 51 Hoberman, A.M. (2003). Oral (gavage) maternal and fetal exposure study of acephate technical in rats. Argus Research, 905 Sheehy Drive, Building A, Horsham, PA 19044-1297. Argus 222- 007; Laboratory Project Identificatio n VP-25267. December 1, 2003. Unpublished. (MRID 46151805).

Hoberman, A.M. (2003). Oral (gavage) repeat ed dose relative sensi tivity study of acephate
technical in neonatal and adult rats. Argus Research, 905 Sheehy Drive, Building A, Horsham, PA 19044-1297. Argus 222-006; Laboratory Project Identification VP-25081. November 25, 2003. Unpublished. (MRID 46151806).

Knezevich, A. and Hogan, G. (1982). A lifetime oral toxicity/carcinoge nicity study technical RE-12420 in rats: Ref.78-2135. (Unpublished study received May 18, 1982 under 239-2471; prepared by Bio/dynamics, Inc., submitted by Chevron Chemical Co., Richmond, CA; CDL:247508-A)

. (MRID 00101623).

Kojima, H., Katsura, E., Takeuchi, S., Niiyami, K., and Kobayashi, K. (2004) Screening for estrogen and androgen receptor activi ties in 200 pesticides by in vitr o reporter gene assays using Chinese hamster ovary cells. Environ. Health Perspect . 112 (5): 524-531.

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.

Leary, J. (1981). Addendum to Orthene (S X-1032) Mouse Lifetime Feeding Study. IRDC Project No. 415-006: Diet Analyses: File N o. 721.11/s-1338. Unpublished study received June 22, 1982 under 239-2471; submitted by Chevron Chemical Co., Richmond, CA; CDL:247718-A. (MRID 00105198).

Lochry, E. (1989). Oral Teratoge nicity and Developmental Toxici ty Study in Rats with Chevron Acephate

- Technical. Unpublished study prepared by Argus Research Labs, Inc. (MRID 41081602).

Matthews, A. (2011). Acephate: Estrogen Recep tor Binding Assay Using Rat Uterine Cytosol (ER-RUC). Unpublished prepared by Huntingdon Life Sciences, Huntingdon, Cambridgeshire, UK. Laboratory Study No.: ADB 0060, October 31, 2011. (MRID 48614502).

Nemec, M. (1997). A Subchronic (13-Week) Neur otoxicity Study of Orthene Technical in Rats: Lab Project No. WIL-194014. U npublished study prepared by WIL Research Labs., Inc. (MRID 44203304).ACEPHATE FINAL

Page 26 of 51 Odum, J and Matthisen, P. (20012). Acephate: Wei ght of Evidence Evaluation of US EPA EDSP Tier 1 Studies (MRID No. 48983001)

Parker, J.A., Palmer, A., Barton, S., Offer, J., et al. (1983). Effect of Technical RE-12420 on Reproductive Function of Multiple Generations in the Rat: HRC Report No

. CHR 11/81957. (Unpublished study received July 13, 1983 under 239-2471; prepared by Huntingdon Research Centre, Eng., submitted by Chevron Chemical Co., Richmond

, CA ; CDL : 250752- A; 250753)

. (MRID 00129508).

Parker, R.M. (2011). Acephate technical: A ssessment of pubertal development, thyroid function, and cholinesterase inhibition in juvenile male and female rats. Unpublished prepared by Huntingdon Life Sciences, East Millstone, NJ. Laboratory Study No.: 10-4360, October 26, 2011. (MRID 48614505).

Rodwell, D.E., Janes, J

. M., Jessup, D.C., et al. (1980). Teratology Stud y in Rabbits: IRDC No. 415-024. (Unpublished study received May 19, 1981 unde r 239-2471; prepared by International Research and Development Corp., submitted by Chevron Chemical Co., Richmond, CA; CDL: 245262-A). (MRID 00069684).

Schneider, S.Z., Kendall, T.Z., and Krueger, H. O. (2012). Acephate: Amphibian Metamorphosis Assay. Unpublished study performed by Wild life International, Ltd., Easton, MD 21601. Laboratory report number 701A-103. Study sponsor ed by Acephate Task Force Steering Committee/Joint Venture, c/o Consumer Special ty Products Associati on, Washington, DC 2006. (MRID 48698501).

Schneider, S.Z., Martin, K.H., Kendall, T.Z., and Krueger, H.O. (2012). Acephate; Fish Shortterm Reproduction Assay with the Fathead Minnow ( Pimephales promelas ). Performed by Wildlife International, Ltd., Easton, Maryland, Report No.: 701A-102. Submitted by Acephate Task Force Steering Committee/Joint Venture, c/o Consumer Specialty Products Association, Washington, DC. (MRID 48675101).

Sharif, I. (2011). Acephate Technical: Uterotro phic Bioassay in Ovariectomised Rats (Oral Administration). Unpublished prepared by H untingdon Life Sciences Ltd, Cambridgeshire, England. Laboratory Proj ect Identity ADB0065, Nove mber 4, 2011. (MRID 48614507).

Spicer, E

. , Geil, R., and Phillips, L. (1982). Life time Oral Carcinogenicity Study in Mice: Orthene Technical: 415- 006. (Unpublished st udy received June 22, 1982 under 239-2471; prepared by International Research and Deve lopment Corp., submitted by Chevron Chemical

Co

. , Richmond, CA; CDL:247717- A). (MRID 00105197).ACEPHATE FINAL

Page 27 of 51 USEPA. (2001). Interim Reregistration Eligib ility Decision (IRED) for Acephate. CASE 0042. September, 2001. EPA 738-F-01-013, Office of Pollu tion Prevention and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC.

USEPA. (2010). Acephate: Report of the Endoc rine Disruptor Review Team — Test Order #: EDSP-103301-22. July 28, 2010. Office 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) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting held July 30 - August 1, 2013 on Weight-of-Ev idence: Evaluating Resu lts of EDSP Tier 1 Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

Willoughby, J.A. (2012). Acephate: Androgen R eceptor Binding (Rat Prostate Cytosol). Unpublished prepared by CeeTox, Inc., Kalam azoo, MI. Laboratory Study Number: 9146V100602ARB, February 13, 2012. (MRID 48669001).ACEPHATE FINAL

Page 28 of 51 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosi s Assay; (OCSPP 890.1100)

The 21-day assay (MRID No. 48698501) of acephate (97.7% purity) on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stag e 51 (80 tadpoles per gr oup) were exposed to
nominal concentrations of 0 (negative control), 4.0, 20, and 100 mg a.i./L; mean-measured concentrations were <3.00 (<L OQ), 3.5, 19, and 102 mg a.i./L. The test system was maintained at 21.4 to 22.2°C and a pH of 8.0 to 8.3.

One tadpole in the mid treatment group was killed on Day 3 during siphoning, and no other mortalities were observed during th e study. By test termination, curv ed tails were ob served in 21, 8, 14, and 18% of tadpoles in the negative control, low, mid, and high treatment groups, respectively.

Acephate did not significantly affect Day 7 norma lized (for snout-vent length) hind-limb length (HLL) at any treatment level. However, Day 21 normalized HLL was significantly increased in the low and high treatment groups by 12 and 27% respectively, relative to the negative control (p<0.05); a non-significant (p=0.08 ) increase of 7% was observe d in the mid treatment group. Minor increases (p<0.05) in snout-vent length (SVL ) were also observed on Day 7 (high treatment, 9%) and Day 21 (mid and high treatment, 3%), and body weights in the high treatment group showed a trend toward a significant increase on Days 7 and 21 (p=0.051 and 0.08, respectively). No significant acceleration or dela y of median NF developmental stage was observed at Day 7 or 21 at any treatment level. Further, no asynchr onous development was observed. No treatmentrelated effects on thyroid gland histopathology were observed in any treatment group. Mild to moderate thyroid hypertrophy, mild thyroid atrophy, and mild to seve re follicular ce ll hypertrophy
were observed in the control and treatm ent groups, though incidence was low.

Androgen Receptor (AR) Binding; (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48669001), ventral prostate cytosol from male Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment. The competitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations of acephate (98.8% purity; l ogarithmic increase from 10

− 11 to 10

− 3 M). Dimethyl sulfoxide (DMSO) was used as a vehicle at a final assay concentration of approxima tely 3.2%. The assay included dexamethasone as a weak positive contro l, and R1881 as the ligand reference standard.

The saturation binding experiment was conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and wa s functioning with appropriate affinity for theACEPHATE FINAL

Page 29 of 51 radiolabeled reference androgen (R1881) prior to conducting AR competitive binding experiment. Saturation binding da ta were not originally provid ed in the study report; however, summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency. The dissociation constant (K d ) for [ 3 H]-R1881
was 0.613±0.041 nM and the estimated B max was 0.817±0.049 fmol/100 μg protein for the single batch of prostate cytosol used for th is assay. The mean and individual K d and B max values were below the expected ranges repor ted in the EPA validation program (0.685 to 1.57 nM and 7 to 16 fmol/100 μg protein, respectively).

In the competitive binding experiment, precipitation of acephate was visually observed at several of the higher concentrations (10

− 3 , 10

− 4 , and/or 10

− 5 M) in the first two r uns, therefore the highest
concentrations used for data evaluation were 10

− 6 M in the first run and 10

− 5 M in the second and
third runs. It is unclear if the testing laboratory made additi onal efforts to address chemical insolubility issues at th e higher concentrations ( e.g., use of water or ethanol solvents, or testing half log concentrations). The estimated average log IC 50 s for R1881 and the weak positive control (dexamethasone) were

− 9.0 and

− 4.6 M, respectively, and the mean RBA for the weak positive control was 0.0041%.

Acephate is classified as “unt estable” in the first run because acephate was not soluble at >10

− 6 M and the binding curve did not cross 50%. In th e other two runs, acephate is classified as “nonbinding” as specific binding was >75% at all concentrations ( ≤ 10

− 5 M) tested.

Based on the results across all three runs, acephate is classified as a N on-Binder in the Androgen Receptor Binding Assay.

Aromatase; (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48614501) , Acephate (98.8% a.i. Lot # 09-750- 10) was incubated with human recombinant ar omatase and tritiated androstenedione (1- β [ 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) in ethanol at l og concentrations of 10 -10 to 10 -3 M for 15 minutes to assess the potential of acephate 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) with three repetitions per concentration.

Aromatase activity in the fu ll activity controls ranged from 0.332 to 0.364 nmol·mgprotein

− 1 ·min

− 1 for the three test runs, with a mean and standard deviation of 0.351 ± 0.016ACEPHATE FINAL

Page 30 of 51 nmol·mg-protein

− 1 ·min

− 1 . Activity in the background controls ranged from 0.60% to 0.83% and averaged 0.70% of the full activity controls. Th e response of each full acti vity control within a run was between 90 to 110% of the average full activity.

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.314-0.343 nmol·mg-protein

− 1 ·min

− 1 at the lowest tested concentration, 10

− 10 M, and 0.0025 nmol·mgprotein

− 1 ·min

− 1 at the highest tested concentration, 10

− 5 M. These results were generally within the recommended ranges for the top of the curve, bottom curve, Hill slope, log IC 50 , and %CV for replicates of each concentrati on within runs. The mean log IC 50 value was

− 7.64 M, which was just outside the guideline range of

− 7.3 to

− 7.0 M, and the Hill slope was

− 0.96. The variation in the positive control values was acceptable ( ≤ 6.6% CV).

For acephate, aromatase activity averaged 0.331-0.367 nmol·mg-protein

− 1 ·min

− 1 at the lowest tested concentration, 10

− 10 M, and 0.311-0.360 nmol·mg-protein

− 1 ·min

− 1 at the highest tested concentration, 10

− 3 M. The data for acephate were not modeled. The average dose-response
curve indicated that the aromatase activity of acephate at all concen trations tested was essentially equivalent to the activity observed in the full activity control.

Based on the data from the average response curve, acephate is classified to be a non-inhibitor of aromatase activity in this assay.

Estrogen Receptor (ER) Binding; (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48614502), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct saturation and competitive binding experiments. A saturation binding experiment wa s conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radiolabeled reference estrogen prior to conducting ER competitive binding experiments. A 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 of acephate (logarithmic increase from 10

− 10 to 10

− 3 M). Ethanol was used as the solvent vehicle at a final concentration of 2%. The assay included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17 β -estradiol as the na tural ligand reference
material.ACEPHATE FINAL

Page 31 of 51 For the saturation binding assay, the protein concentration was determined to be 0.1 mg/mL as it bound 27% of the [ 3 H]-17 β -estradiol (within the Guideline ra nge of 25 to 35% binding). The K d for [ 3 H]-17 β -estradiol was 0.125 nM, and the estimated B max was 20.8 fmol/100 μ g protein for the prepared rat uterine cytosol. The K d for each of the three runs was within the expected range
of 0.03 to 1.5 nM.

For the competitive binding assay, the protein concentration was de termined to be 0.3 mg/mL as it bound 13% of the [ 3 H]-17 β -estradiol (within the Guideline ra nge of 10 to 15% binding). The mean log IC 50 was

− 8.75 M for 17 β -estradiol (strong positive control and

− 5.44 M for 19- norethindrone (weak positive control). Less than 13% of the radiolabeled ligand was displaced by the negative control, octyltriethox ysilane at concentrations up to 10

− 3 M. Acephate did not displace more than 25% of the radiolabeled estradiol in any run. The estimated mean log IC 50 for the test material could not be determined as it did not result in 50% displacement of radiolabeled estradiol at any concentration tested.

Based on the results of the three runs, acephate is classified as Not Interactive in the Estrogen Receptor Binding Assay.

ER α Transcriptional Activat ion (ERTA); (OCSPP 890.1300)

In an estrogen receptor transcriptional activation assay (MRID 48614503), HeLa-9903 cells
cultured in vitro were exposed to acephate (98.8% a.i. ) at serial log c oncentrations of 10

− 9 to 10

− 3 M in DMSO (final concentration 0.1%) for 23 ± 1 hours. The experiments were performed using 96-well plates and each acephate concentration wa s tested in 6 wells/plate. The reference
chemicals 17 β -estradiol (strong estrogen), 17 α -estradiol (weak estr ogen), corticosterone (negative compound) and 17 α -methyltestosterone (very weak a gonist) were also tested. Cells were exposed to the test agents for 23 ±1 hours to induce reporter (lucifer ase) gene products. For each run of the assay, two test plates were establ ished: one plate for the luciferase assay and one for the cytotoxicity assay. Luciferase expres sion was measured upon addition of a luciferase
substrate and detection with a luminometer w ith acceptable sensitivity. Cytotoxicity was
measured after 2.5 hours incubation with MTS. Relative transcriptional activity was expressed as percent of the positive control (PC) 1 nM 17 β -estradiol) and compared to 10% of the PC maximal response (PC 10 ). Data demonstrating laborator y proficiency was not available.

Acephate was tested up to the limit concentration of 10

− 3 M. No precipitation or cytotoxicity was observed at any acephate concentration tested. The mean RPC Max was 4.1% for the first run,
6.4% for the second run and 12.3% for the third run. The associated PC Max values were 10

− 3

, 10

− 5 and 10

− 3 M, respectively. The assay performance criteria were generally met, although Hill slopes for 17 β -estradiol and 17 α -estradiol and the log PC 10 for 17 α -methyltestosterone were lower than the expected values which indicate s increased sensitivity of the cells to weakACEPHATE FINAL

Page 32 of 51 agonists. All other performance criteria were acceptable. Although the third test run gave a positive result (RPC Max > PC 10 ), comparison of the third run with the two earlier runs is complicated by the use of a new set of cells for th e third run, particularly as the proficiency test for either cell stock was not provided.

As the RPC Max was less than the PC 10 in two of the three test runs, acephate is considered negative for interaction with the human estroge n receptor to cause transcriptional activation under the conditions of this assay.

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

The 21-day short-term reproduction assay (MRID No. 48675101) of acephate with fathead minnows ( Pimephales promelas) was conducted under continuous flow-through conditions. Adult fish, 16 spawning groups (2 males and 4 females in each group; 6 months old) were exposed to acephate (98.8% purity) at nominal concentrations of 0 (control), 4.0, 20, and 100 mg a.i./L; meanmeasured concentrations were <LOQ (<1.00), 3.6, 18, and 91 mg a.i./L. The test system was
maintained at 24.5 to 25.8 o C and a pH of 7.8 to 8.3.

Overall survival values were 100% for both males and females in all treatment groups. There were
no notable observations with re gards to behavior or change s in appearance, specifically color/banding, ovipositor appearance, or size of dorsa l nape pad in the control or treated groups or clinical signs of toxicity for any treatment group compared to the controls.

Spawning frequency in the negative control was at least every four days in two of the four replicates and mean fecundity in negative control was 29.1 eggs /female/day/rep licate (range: 20.3- 32.7); fertilization success in the ne gative control group averaged 98.6%.

There was a slight but significant reduction in fer tility of 3% at the high treatment concentration compared to the negative control (p<0.05; J onckheere-Terpstra test). There were no other statistically significant differences between the trea tment levels and negative controls for any other endpoint for either males or females, including fecundity, tubercle scor e, plasma vitellogenin (VTG), gonado-somatic index (GSI), body weight, and body length. There were no differences noted between histopathology severi ty scores in treated groups comp ared to the negative controls. Sex steroids (testosterone and 17 β -estradiol) were not measured.

Hershberger; (OCSPP 890.1400)

In a Hershberger assay (MRID 48614504) screeni ng for androgenic activity, acephate (98.8% a.i., Lot# 09-750-10) in deionize d water was administered daily vi a oral gavage to groups of 55- to 59-day old, castrated male Sprague Dawley rats (10/group) at dose levels of 0 (vehicle), 10, orACEPHATE FINAL

Page 33 of 51 30 mg/kg/day. An androgenic positi ve control group consisting of 10 castrated rats exposed to 0.4 mg/kg/day testosterone propionate (TP) by subcutaneous (s.c.) injection was also included as a positive control.

To screen for potential anti-andr ogenic activity, acephate (98.8%, lot # 09-750-10) in deionized water was administered daily vi a oral gavage to 55- to 59-da y old, castrated male Sprague Dawley rats (10/group) at dose levels of 3, 10, or 30 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s. c. injection. The ve hicle control groups
consisted of ten castrated male rats dosed dail y with TP (0.4 mg/kg/day) by s.c. injection in deionized water and corn oil, and the anti-andr ogenic positive control group consisted of ten castrated male rats dosed orally with flutamide (FT) in corn oi l 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 animal s were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration to determine weights of the five androgen-dependent tissues. An additional intact (not castrated) satellite group of males (6/group) were treated with the water vehicle control and acepha te at 3, 10, and 30 mg/kg/day for 10 consecutive days to determine cholinesterase (ChE) activity in the brain and red blood cells (RBC).

In the androgen agonist assa y, body weights for the 10 and 30 mg /kg/day treatment groups were comparable to controls throughout the durati on of the assay. At 30 mg/kg/day, overall body weight gains (Days 1-11) were decreased (p ≤ 0.05) by 32%. Animals dosed with TP had increased (p ≤ 0.05) overall body weight gains ( ↑ 41%) compared to controls. At 30 mg/kg/day, the liver weights were decreased by 12% (p ≤ 0.05). Although the glans penis weights were increased by 25% (p ≤ 0.05) at 10 mg/kg/day and 19% (p ≤ 0.05) at 30 mg/kg/day, no other significant changes in the remaining four accessory sex tissues of acephate treated animals. Animals in the positive control (TP) group re sponded appropriately showing significant increases (p ≤ 0.05) in the five accessory sex organ weight s. All %CV values were less than or equal to the maximum recommended values.

In the anti-androgen assay, body wei ghts in the 3 and 10 mg/kg/day groups were comparable to controls throughout the duration of the assay. At 30 mg/kg/day, body wei ghts on Days 2-8 were decreased (p ≤ 0.05) by 7-8%. For the positive control group (TP + FT), overall body weight gains were decreased (p ≤ 0.05) by 17%. Liver weights were decreased by 13% (p ≤ 0.05) at 30 mg/kg/day. There were significant (p<0.05) decreases in two of the five target sex accessory tissues at 30 mg/kg/day: vent ral prostate weights were decr eased 24% and Cowper’s gland weights were reduced 18%. Ventral prostate we ights were also significantly decreased at 10 mg/kg/day (18%; p<0.05). Animals dosed w ith TP + FT (positive control) responded appropriately with decreases (p<0.05) in all five accessory sex organ weights. All %CV values were less than or equal to the maximum recommended values.ACEPHATE FINAL

Page 34 of 51 At all acephate dose levels (3- 30 mg/kg/day), ChE levels were si gnificantly decreased compared to the control in both the RBC (40-80% inhibiti on) and brain (42-87% inhibition). The data indicate that high dose of 30 mg/kg/day may have been excessive based on the 80-87% decrease
in RBC and brain ChE levels and the 32% d ecrease in body weight gain (androgen assay).

Acephate was negative for androgenic activity in the Hershberger assay as statistically significant increases in organ we ights were not seen in two or more of the five androgensensitive tissues in the androgen agonist assay. However, acephate was positive for antiandrogenicity in the Hershberger assay as stat istically significant decr eases in organ weights were seen in two or more of the five androgen sensitive tissues in the androgen antagonist assay. The target organ weight reducti ons were observed at a dose th at showed significant reduction (  87%; p<0.05) in brain ChE activity.

Female Pubertal; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48614505), 15 Sprague Dawley rats/dose group were treated daily by oral gavage (5 mL/kg) with acephate (98.8% a.i., lot# 09-750-10 ) in deionized water at doses of 0 (vehicle), 10, or 30 mg/kg/day from post-natal day (PND) 22 to 42. Animals were examined for vaginal opening (VO) daily begi nning on PND 22, and age and weight at day of attainment was recorded. After sacrifice on PND 42, total serum thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels were determ ined by radioimmunoassay (RIA). Weights were recorded for liver, kidneys, urogenital organs , pituitary, thyroid, and adrenal glands and microscopic examination of the ovaries, uter us, thyroid, and kidneys was performed. An additional satellite group (6 females/dose) was si milarly treated with acephate from PND 22 to
43 and used for red blood cell (RBC) and brain acetylcholines terase (AChE) measurements.

There were no effects of treatment on mortality, food consumption, age and weight at attainment of VO, mean age at first vaginal estrus, mean cycle length, percent cycling, percent regularly cycling, serum hormone concentrations, organ weights (except kidneys and adrenal) or histopathology parameters.

In the 30 mg/kg/day group, final body weights an d overall body weight gain were decreased (p<0.05) compared to the control ( ↓ 5% and 8%, respectively). Abso lute and adjusted kidney and adrenal glands weights were also decr eased (p<0.05) in the 30 mg/kg/day group ( ↓ 7% and 13%, respectively). Aspartate aminot ransferase concentrations we re increased (p<0.01) by 23 and 22%; alanine aminotransferase concentrations we re increased (p<0.05) by 13 and 9%; and total bilirubin concentrations were increased (p< 0.05) by 43 and 57% in the mid- and high-dose groups. These clinical chemistry changes were not considered to be treatment related because they were small in magnitude and values were compatible with normal biological variability.ACEPHATE FINAL

Page 35 of 51 For animals in the satellite study, AChE activ ity in RBC was significan tly inhibited (p<0.001) compared to the control at 10 (69% inhibiti on) and 30 (89% inhibitio n) mg/kg/day. AChE activity in the brain was also si gnificantly inhibited (p<0.001) at 10 mg/kg/day (71% inhibition) and 30 mg/kg/day (84% inhibition) . The doses tested were cons idered adequate based on the observed RBC and brain acetylcholinesterase activity.

Male Pubertal; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48614505), 15 Sprague-Dawley (Crl:CD ® [SD] IGS BR) rats/dose group were treated daily via oral gavage (5 mL/kg) with acephate (98.8% a.i., lot #: 09- 750-10) in deionized water at doses of 0, 10, or 30 mg/kg/day from post-natal day (PND) 23 to 53. An additional satellite group (6 males/dose) was similarly trea ted with acephate and used for red blood cell (RBC) and brain acetylcholinesterase (AChE) evalua tions (sacrificed on PND 54). The main study animals were examined for prep utial separation (PPS) daily beginning on PND 30, and age and body weight at day of attainment was recorded. Following sacrifice on PND 53, total serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were analyzed by radioimmunoassay. Kidney, liver, adre nal, pituitary, thyroid, and urogenital organ weights were recorded, and microscopic examina tions of the testes, epididymides, thyroid, and kidneys were performed.

No adverse treatment-related eff ects were observed in mortality, clinical signs of toxicity, body weight, body weight gain, food consumption, age or weight at PPS, clinical chemistry, or gross and histopathology. Dose-dependent reductions (p<0.001) in both RBC ( ↓ 74-92%) and brain ( ↓ 72-87%) AChE levels were obs erved at 10 and 30 mg/kg/day.

The age and weight at attainment of PPS at all doses was within the acceptable range of the performance criteria. The unadjusted values for most organ weights and hormone levels in the control group were within the acceptable range of the performance criteria provided in the Guideline. Thyroid glands examined in all dose groups were considered within normal limits of
control tissues. There were no treatment-related histopathological findings noted in the testes, epididymides or kidneys at any dose.

At 30 mg/kg/day, decreases (p<0.05 ) were observed in pituitary ( ↓ 14%) and thyroid/parathyroid ( ↓ 22%) weights. Treatment-related effects on hormone levels were limited to decreased (p<0.001) testosterone ( ↓ 53%) at this dose.

At 10 mg/kg/day, treatment-r elated findings were limited to decreased (p<0.05) thyroid/parathyroid weight ( ↓ 25%).ACEPHATE FINAL

Page 36 of 51 In the satellite animals at 10 and 30 mg/kg/da y, dose-dependent reduc tions (p<0.001) in both RBC ( ↓ 74-92%) and brain ( ↓ 72-87%) AChE levels were obser ved. The doses tested were judged to be adequate based on the observ ed RBC and brain cholinesterase activity.

Effects on androgen- and thyroid -related endpoints included decrea sed serum testosterone levels at 30 mg/kg/day, decreased pituitary weights at 30 mg/kg/day, a nd decreased thyroid/parathyroid weights at 10 and 30 mg/kg/day.

Steroidogenesis; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48615606), H295R cells cultured in vitro in 24-well plates were incubated with acephate (98.8% purity, Lot # 30560-19-1) at log concentrations of 10

− 10 to
10

− 4 M for 48 hours in triplicate for three indepe ndent experiments. Dimethyl sulfoxide (DMSO) was used as a vehicle, at a final concentration of 0.1%.

Testosterone and estradiol levels were measured using specific EIA kits. A Quality Control (QC) plate was run concurrently with each i ndependent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels. The positive controls included a known inhibitor (prochloraz) and a known inducer (forskolin) of estradiol a nd testosterone production.

Guideline acceptability recommendations and requirements were generally met, including lack of cytotoxicity, adequate producti on of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with positive controls, w ith the following exception: (i) testosterone induction by 10 μ M forskolin averaged 1.88-fold which is less than
the Guideline recommendation of ≥ 2-fold.

Precipitation of the test com pound and/or cytotoxicity was not observed at any tested
concentration. No treatment-related effect was obser ved on testosterone. There were no statistically significant differences in testoster one concentration in the acephate treated cells
relative to the solv ent control (0.87- to 1.09-fold change). Although significant differences were observed in estradiol concentrations in the acepha te-treated cells relative to the solvent control (SC) in Runs 1 and 2, these differences were not dose-dependent; no stat istically significant responses were observed in Run 3.

Based on hormone responses in at least three in dependent runs, acephate treatment did not result in statistically significant and re producible alterations in testosterone or estradiol production.ACEPHATE FINAL

Page 37 of 51 Uterotrophic; (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48614507) conducted to screen for potential estrogenic activity, acephate (98.8% purity, lot #09-750-10) in deioni zed water was administered daily via oral gavage to groups of eight 61- to 68-day old ova riectomized female Sprague Dawley rats at dose levels of 0 (vehicle), 3, 10 or 30 mg/kg/day fo r four consecutive days. A positive control group was concurrently treated with 17 α -ethynyl estradiol (EE) in corn oil by daily subcutaneous (s.c.) injection at 0.3 mg/kg/day. Animals were necropsied on post-natal day (PND) 65-72 approximately 24 hours after the final dose administ ration to determine liver, and wet and blotted uterine weights.

To assess effects on cholinesterase (ChE) activity, an additional six intact female SpragueDawley rats (approximately PND 61-68) were sim ilarly dosed with acephate at 0 (vehicle), 3, 10 or 30 mg/kg/day. The ChE activity in red blood cells (RBC) and brain of control and acephate treated rats were determined from samples co llected from 6 rats/group, approximately 3 hours
after the last dose.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in animals for any acephate treated groups. Body we ights and body weight gains (Days 1-5) in the acephate treated groups were comparable to the controls throughout the study, except for a lower increase (p<0.05) in the overall body weight gain (0.9%) in the high dose group as compared to the body weight gain of the control group (13.3%). Liver and uterine weights in the acephate treated groups were compar able to the controls.

Body weights in the positive co ntrol (EE) group were comparable to the control group throughout the duration of the a ssay, but overall body weight gain s decreased (p<0.01) by 4.1%. Absolute wet and blotted uterus weights for th e positive control group were increased (p<0.001) by 936% and 302%, respectively, as expected. Li ver weights were also increased (p<0.01) by 8% in the positive control group.

In the cholinesterase phase of the assay, RBC Ch E activity were significantly decreased (p<0.01) at 10 mg/kg/day (59%) and at 30 mg/kg/day (7 7%). Brain ChE activity was significantly decreased at all dose levels (p<0.01) 35%, 59% and 80% inhibition, at 3, 10 and 30 mg/kg/day respectively), compared to the controls. Th ese dose-dependent reductions in ChE activity
indicate that the dose levels tested were adequate.

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

Page 38 of 51 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

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

Receptor Transcriptional Activation Assays

In a published, non-Guideline study ( Kojima et al ., 2004) , the potential of 200 chemicals (including acephate; purity 95- 100%) to interact with two 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. Acephate did not show agonist or antagonist activity for ER α , ER β , or AR in this study.

In a published study by Kojima et al . (2005) , the response of an in vitro ER–related reporter gene assay was investigated af ter exposure to thirty two pest icides either alone or in combination. The source of the test compounds was not reported; however, acephate had a reported purity of 98%. The test com pounds were prepared in DMSO at 10

− 2 M.

The E-CALUX Assay (Xenobiotic Detection Systems, Inc., NC, USA) uses a human ovarian carcinoma cell line (BG1) transfected with a luciferase reporter gene under the control of a mouse mammary tumor virus (MMTV) promoter . Estrogen-responsive concatemers (repetitive DNA sequences), inserted directly above the pr omoter, confer estrogen responsiveness to the viral promoter and reporter gene. The transfected cells are made st able by an antibiotic resistance gene resulting in the cell lin e used in the assay (BG1Luc4E 2 ). Compounds that interact withACEPHATE FINAL

Page 39 of 51 estrogen-responsive DNA are identif ied by the luciferase gene re sponse and quantified in terms of Relative Light Units (RLU).

The BG1Luc4E 2 cells were grown in medium for f our days, then the assay medium was replaced, and cells were transferred (2 × 10 5 cells/mL) into 96-well pl ates and incubated at 37  ̊ C
with 5% CO 2 for 24 hours. The medium was removed, and the test compound or estrogen standard (E2) was added. After incubation at 37  ̊ C with 5% CO 2 for another 24-hour period, the medium was removed and the cells were lyse d. Luciferase activity was measured with a luminometer in order to compare treatment RLU to the E2 standard. For comparative purposes,
the EC 10 (Effective Concentration at which 10% of the maximum response was observed) was
used. The data were not sufficien t to allow calculation of an EC 50 . Although the article does not specifically identify the E 2 standard, the reviewers assumed that E2 referred to 17 β -estradiol. Acephate was reported ne gative in this study.

Prenatal Development in Rodents (Rat)

In a developmental toxicity study (MRID 41081602), female SD rats (25/dose) were administered 0, 5, 20, or 75 mg/kg/day of acetate (99.7% purity) in reverse osmosis deionized water by oral gavage from GD 6 through 15. The rats were observed daily and weighed on Days 0, 3, and 6 through 20 (prior to sacrifice). The following parameters were examined at study termination: gross necropsy on the dams; dete rmination of the uterin e weights, number of implantations, pre- and post-implantation losses, resorptions, corpora lutea/dam, living and dead fetuses, and sex and body weights of fetuses; and examination of fetuses for malformations,
variations and sk eletal defects.

No mortality was observed in the study. The matern al animals of the high-dose group presented with an increased incidence (p ≤ 0.01) of clinical signs (tremors and decreased motor activity). The mid- and high-dose animals had decreased (p ≤ 0.05 or 0.01) body weights ( ≤ 9%), and decreased (p ≤ 0.05 or 0.01) body weight gains during GD 6 to 16, 6 to 20, and 0 to 20. There was a rebound (p ≤ 0.01) in body weight gain in the hi gh-dose group during GD 16 to 20, and a decrease (p ≤ 0.05 or 0.01) in body weight gain during GDs 16 to 20, 6 to 20, and 0 to 20 when corrected for gravid uterine weight (mid- a nd high-dose for GD 6 to 20 and 0 to 20 periods). Decreased (p ≤ 0.01) food consumption also was observed du ring the study. The maternal toxicity NOAEL was 5 mg/kg/day and the LOAEL was 20 mg /kg/day based on reduced body weight and body weight gain, reduced food consum ption, and reduced food efficiency.

No treatment-related effects were observed on any cesarean section parame ters; pregnancy rate

was ≥ 92% across all groups. In addition, no treatme nt-related effects were observed in the
external or soft-tissue examina tions. Developmental toxicity was noted in the high-dose group as slight decreases in the mean number of ossified caudal vertebrae, sternal centers, metacarpals and the fore- and hindlimb phalanges, with the hindlimb phalanges significantly decreasedACEPHATE FINAL

Page 40 of 51 (p ≤ 0.05). The developmental toxicity NOAEL was 20 mg/kg/day and the LOAEL was 75 mg/kg/day based on decreases in mean nu mbers of ossification centers per litter.

Prenatal Development in Non-Rodents (Rabbit)

In a developmental toxicity study (MRID 00069684) , artificially-inseminated and chorionic gonadotropin-injected (to induce ov ulation) Dutch Belted rabbits, 16/ group, were administered 0, 1, 3 and 10 mg/kg/day of acephate (92.8% purity) by oral gavage from GD 6 through 27. Doses selected for this study were based on the result s of the pilot study in which doses of 3, 10, 30 and 100 mg/kg/day of acephate were tested; (40% de aths and 10% weight loss were observed on GD 24 in the 30 mg/kg group). In the main study, the rabbits were observed daily and weighed every 6 days, and also on Day 28 prior to sacrifice. The following parameters were examined at study termination: gross necropsy on the dams; dete rmination of the uterin e weights, number of implantations, post-implantation losses, resorptions , corpora lutea/dam, living and dead fetuses, and sex and body weights of fetuses; and examina tion of fetuses for malformations, variations
and skeletal defects.

Two rabbits in the high-dose group aborted a nd were sacrificed and discarded without examination, the first on GD 25 and the other on GD 27. A slight increase in nasal discharge, possibly treatment-related, was observed in the mid- and high-dose groups compared to control. Acephate had no other effects on any maternal and developmental (f etotoxic/teratogenic) parameters examined. Based on 2/16 (12.5%) abor tions in the high-dose group and none in the control group, the LOAEL and NOAEL for maternal toxicity are 10 mg/kg/ day and 3 mg/kg/day, respectively. The NOAEL for developmental toxi city is >10 mg/kg/day; a LOAEL is not
established.

Subchronic Neurotoxicity in Rodents (Rat)

In a subchronic neurotoxicit y study (MRID 44203304), SD rats (30/ sex/dose) were administered acephate (99% purity) in the di et at nominal doses of 0, 5, 50, and 700 ppm (mean analytical values of 0/0, 0.33/0.41, 3.31/3.95, and 48.63/58.27 mg /kg/day in males and females, respectively). Cholinesterase (ChE ; ChE or NS ChE) activities in brain (six brain regions), RBC, and plasma were determined for 6 rats/sex /dose at Weeks 3, 7, and 13. The remaining 12
rats/sex/dose were used for ne uropathology evaluations [Functiona l Observational Battery (FOB) and Locomotor Activity assessments]. In addition, other parameters determined included clinical toxicity/mortality, body weight, food consum ption, and a general necropsy examination.

No signs of clinical toxicity and no mortality were reported during the study. Decreases (p<0.01 or 0.05) in mean body weight were noted in the high-dose males at Weeks 1 to 3 ( ↓ 5 to 6%), with decreases in body weight gain in high-dos e males and females (Week 1). No effects on body weight/body weight gain were observed in the low- and mi d-dose animals, and no effects on food consumption were noted in any study groups. No treatment-related effects on FOBACEPHATE FINAL

Page 41 of 51 parameters were observed during the study, but some ambulatory a nd total motor activity counts were decreased in high-dose females. Brain ChE activity was generally inhibited (p<0.01) by >20% in male and female mid- and high-dose animals at Weeks 3, 7, and 13 in all six brain regions; decreases (p<0.01 or 0.05) were obser ved occasionally in low-dose animal brain regions. Inhibition (p<0.01 or 0.05) of RBC ChE activity was obs erved in high-dose males at Weeks 3, 7, and 13 ( ↓ 37 to 46%), and observed (p<0.01 or 0.05) in high-dose females at Weeks 3 and 7 ( ↓ 42 and 43%). Plasma ChE activity was in hibited (p<0.01) in high-dose males and females at Weeks 3, 7, and 13 ( ↓ 55 to 57% and 67 to 74%, respectively). There were no
treatment-related effects on ab solute brain and brain region weights due to acephate. The NOAEL for systemic toxicity and behavioral pa rameters is 50 ppm (3.31/3.95 mg/kg/day). The NOEL for RBC ChE inhibition is 50 ppm (3.31 mg/kg/day) in males and 5 ppm (0.41 mg/kg/day) in females. The NOEL for plas ma and brain region ChE inhibition is 5 ppm (0.33 mg/kg/day) in males and <5 ppm (<0.41 mg/kg/day) in females.

Developmental Neurotoxicity in Rodents (Rat)

In a developmental neurotoxicity study (MRID 46151802), acephate (99.2% purity) in deionized water was administered daily by or al gavage to pregnant SD rats (25/dose) at doses of 0, 0.5, 1, or 10 mg/kg/day from GD 6 through lactation day (LD) 6. Additionally, the F1 pups were similarly dosed on PND 7 to 21. Dams were allowe d to deliver naturally and were sacrificed on LD 6. On PND 4, litters were standardized to 10 pups/litter (5 males and 5 females when possible); the remaining offspring were sacrifi ced and examined grossly, as well as for ChE activity. Subsequently, 10 pups/sex/group were allocated to Subsets 1 to 4, and up to 10 pups/sex/group in Subset 5. Select ed subsets were examined for detailed clinical and functional observational battery (FOB), moto r activity, auditory startle hab ituation, passive avoidance, and water maze learning and memory tests, brai n weight, neuropathology, and/or brain and RBC ChE determinations. Pups were weaned on PND 21, and all offspring were sacrificed by PND

71.

No treatment-related effect was observed on mate rnal mortality, clinical signs, abbreviated functional observations, body weight, food cons umption, reproductive performance, and gross pathology. The maternal NOAEL is 10 mg/kg/day; a maternal LOAEL is not established.

Treatment had no adverse effect on offspring survival, body weight, body weight gain, food consumption, clinical signs, FOB, developmental landmarks, auditory star tle reflex, learning and memory, brain weights, brai n morphology or neuropathology. Assessment for motor activity revealed dose-related decreases (N S) in the number of movements ( ↓ 19% at 1 mg/kg/day to ↓ 30% at 10 mg/kg/day) that wa s accompanied by comparable dos e-related decreases (NS) in time spent in movement ( ↓ 19% at 1 mg/kg/day to ↓ 28% at 10 mg/kg/day) in females on PND 21. However, it was determined that no conclusions can be drawn regarding the effect of acephate on motor activity due to the high variability in the data.ACEPHATE FINAL

Page 42 of 51 No treatment-related inhibition wa s observed in brain, plasma or RBC ChE activity of male or female pups at PND 4. On Day 21, dose-dependent inhibition (p ≤ 0.01) of brain ChE activity was seen. Inhibition in the low-, mid- and high-dose groups was 28%, 30% and 60%, respectively (M + F combined). There were also decreases (p ≤ 0.01) in plasma (45%) and RBC (57%) ChE activity in high-dose pups on PND 21. The offspr ing LOAEL is 0.5 mg/kg/day (LDT), based on dose-dependent inhibition of brain ChE activ ity in male and female pups on Day 21. An offspring NOAEL is not established.

Multi-Generation Reproduc tion in Rodents (Rat)

In this multi-generation reproduction study (MRIDs 40323401; and 40605701), SD rats, 30/sex/group, were fed diets cont aining acephate (98.7% purity) fo r 75 days before they were bred to produce the F1a, and la ter the F1b, litters. Pups from th e F1b litters were selected to produce the F2a and F2b litters. B ecause of low fertility in all groups, including control, in the F1b and F2b litters, a third generation (F3) was produced from the F2b li tters. All rats were continuously exposed to the test material or the control diet either dire ctly in their feed or through the mothers' milk during lactation. The nominal doses used were 0, 25, 50 and 500 ppm,
and were based on the results of an earlier re production study in rats (MRID 00129508) that did not determine a reproductive NOEL. These doses we re considered equivalent to 0, 1.25, 2.5 and 25 mg/kg/day, respectively, by using the FDA/HE W conversion factor (1 ppm in food ~ 0.05 mg/kg/day). Parameters examined were those routinely examined in a multi-generation rat reproduction study.

Treatment-related effects were observed only in the high-dose group and in cluded: (1) Decreased (significance not reported) body weights (<10%) a nd/or weight gains for adult males (in each generation) and females (in some generations), and for pups in the F2a and F3a generations; (2) Increased food consumption for males and fema les during the premating period and decreased food consumption for females during the gestation and lactation periods (h ighly variable); (3) Minimal clinical signs in males [increased inci dence of alopecia in the first generation and
increased incidence of soft or liquid stools in the second and th ird generations]; (4) Decreased mating performance (60%, p<0.01) for the F2b pare ntal generation; (5) Decreased mean litter size (25 to 30%, p<0.05 or 0.01) for the F1b, F2a, F2b and F3a generations; and (6) Decreased (p<0.01) pup survival to Day 4 for the F1a (3.2%) and the F2a (6.3%) generations. No gross pathology or histopathologi cal lesions were noted for any pare ntal animals. Based on decreased body weight and/or weight gain for adult males (each generation), and for adult females and pups (some generations), decreased food consumption during gestation and l actation, and decreased litter size (some generations), the parental LOAEL and NOAEL are 500 ppm (25 mg/kg/ day) and 50 ppm (2.5 mg/kg/day), respectively. Base d on decreases in viability index (two generations) and in mating performance (one generation), the reproductive LOAEL and NOAEL are also 500 ppm (25 mg/ kg/day) and 50 ppm (2.5 mg /kg/day), respectively.ACEPHATE FINAL

Page 43 of 51 In a previous 2-generation reproduction study (MRID 00129508), SD rats (12 males and 24 females/dose) were fed acephate (93% purity ) in the diet at 0, 50, 150, or 500 ppm for 15 weeks prior to mating. Feeding of acephate was continued through pregnancy and lactation. After weaning the F1 pups (LD 21), similar numbers of males and females were selected as the F1 parents, fed acephate for 15 to 18 weeks, w ith treatment continued as above through mating, pregnancy, and lactation. F2 pups were weaned after Week 3, and the study was terminated. Calculated values for acephate intake are 3.3, 9.8, and 34.53 mg/kg/day (F0 males); 4.04, 12.13, and 41.82 mg/kg/day (F0 females); 3.43, 10.21, and 36.82 mg/kg/day (F1 males); and 4.12, 12.35, and 45.12 mg/kg/day (F1 females) in the lo w-, mid-, and high-dose groups, respectively. Parameters examined were those routinely examined in a multi-generation rat reproduction study. Organ weights and histopathology were determ ined in the F0 and F1 animals and included testes, seminal vesicles, prostate, uterus, ova ries, pituitary, liver, ki dneys, and thyroid and adrenal glands.

Mortality was observed in three F0 (low- and hi gh-dose) and one F1 (mid-dose) females, but
these deaths were not treatment related. Clinical signs were minimal. Weight differences in males and females (prior to pregnanc y and post-lactation) were generally ≤ 10%; weights in pregnant F0 and F1 high-dose fe males (GD 20) were decreased by approximately 25 to 42%, but these decreases were not dose-dependent (low- and mid-dose female weight decreases ranged from 0 to 21%). Organ weight effects were minima l, but a decrease (p<0.05) in prostate weight was observed in high-dose F0 males ( ↓ 25.3%). Decreased pregnancy rates were observed in midand high-dose F0 females and low- ( ↓ 42%) and high-dose F1 females. Five and one total litters
were lost in the F0 and F1 low- dose groups, respectively, and three a nd five total litters were lost in the high-dose groups, respectively. Overall, the highest fetal losses occurred in the F1 lowdose group. In addition, there were decreases in the size and wei ght of total litters and the number of young born alive in the F0 (12 to 16%) and F1 (2 to 5%) low-dose groups. Due to the many treatment-related effects observed w ith low-dose dams and pups, a NOAEL was not determined in this study; the LAOEL is the lo w dose, 50 ppm (approximately 3 to 4 mg/kg/day).

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat)

In a chronic feeding/carcinoge nicity study (MRID 00101623), SD ra ts (75/sex/group) received acephate (92.5% purity) in the diet for 28 m onths at nominal doses of 0, 5, 50 and 700 ppm. These doses were estimated to be equivale nt to 0, 0.25, 2.5 and 35.0 mg/kg/day, respectively (FDA/HEW conversion factor). No justificati on was presented for the selection of doses. Contamination of the control diet with acepha te was noted for a 69-day period during the study with acephate concentrations of approximately 0.4 to 4.0 ppm. Parameters examined in all rats were those routinely examined in a chronic or al toxicity/carcinogenici ty study. Absolute and relative (% of body weight) weight s of organs were determine d, including adrenals, kidneys, liver, testes/ovaries, and thyr oid gland, for all scheduled sacrifices; histopathology was conducted on these tissues as well as epididym ides, mammary gland, pituitary, prostate, andACEPHATE FINAL

Page 44 of 51 uterus with cervix. Plasma and RBC ChE activiti es were determined during Weeks 6, 7, 19 and 28, and at 12, 18, 22, 24 and 28 months, from 4, 5 or 10 rats/sex/group (randomly selected). Brain ChE activity was determined at W eeks 7 and 19, and at 12, 22 and 28 months.

No treatment-related effect on mortality was obs erved during the study. The following treatmentrelated findings were observed in the high-dose male rats: (1) Hyperactivity in some of the males (8%) during the initial 5 months of the study; (2) Increased incidence of aggressive behavior (31% vs 5%), also during th e initial 5 months of the study; and (3) Decreased body weights ( ≥ 10%, p<0.01) from Weeks 25 to 106 and Week 118 (NS) and body weight gains ( ↓ 6–18%; p<0.01) during Weeks 8 to 106. Aggressive behavior was also observed in 13% of the low- and mid-dose male rats. No weight or aggression effects were observed in female rats.

Plasma ChE activity was inhibited at all samp ling times in the high-dose males (10 to 50%, p<0.01 or NS) and females (50 to 72%; p<0.01). In the mid-dose group, the inhibitions ranged from 0 to 29% (NS) in males and 0 to 38% (o ccasionally p<0.05 or 0.01) in females. Plasma ChE activity was generally not inhibited in the low-dose animals. RBC ChE activity was decreased (p<0.05 or 0.01, or NS) at all samp ling times in the high-dose males (21–67%) and females (21–61%). In the mid-dose groups, RBC Ch E was inhibited (generally NS; occasionally p<0.05 or 0.01) up to 42% for males and females, respectively, and there was no effect in the low-dose animals. Brain ChE activity in the low- , mid-, and high-dose males was inhibited 0 to 13%, 34 to 43% and 69 to 77%, respectively. The co rresponding values for the female rats were 1 to 13%, 33 to 45% and 66 to 83%, respectivel y. These decreases were generally significant (p<0.01) for the mid- and high-dose groups, but mixed (p<0.05 or 0.01, or NS) at the low dose.

There was a higher incidence of adrenal medu llary tumors (possible, but unconfirmed, pheochromocytomas) in the treated male rats than in the concurrent control males. However, the reported incidences for the low-, mid- and high-dose groups (9.7, 15.5 and 12.2%, respectively) were within the historical control range. All of the tumors, but two, in the current study were reported to be benign. No other treatment-rela ted effects were reported for the study. Based on the above findings, the systemic LOAEL and NOAEL for the male rats are 700 ppm (35 mg/kg/day) and 50 ppm (2.5 mg/kg/day), respectively; the systemic NOAEL for the female rats is > 700 ppm. The LOAEL and NOAEL for the inhibition of plasma, RBC, and brain ChE activities in males and females are 50 ppm (2.5 mg/kg/day) and 5 ppm (0.25 mg/kg/day; borderline value), respectively. Acephate was not carcinogenic in this study.

Carcinogenicity in Rodents (Mouse)

In a carcinogenicity study (MRIDs 00105197; 00077209; 00105198; and 00129156), CD-1 mice (75/sex/group) were administered acephate (92.7% purity) in the diet at nominal doses of 0, 50, 250 and 1000 ppm. The mean analytical doses (determined from diet analysis and food consumption) were 0, 7, 36 and 146 mg/kg/day, respectively, for males and 0, 8, 42 and 167 mg/kg/day, respectively, for females. The possib ility of feeding the wrong diets was discussedACEPHATE FINAL

Page 45 of 51 due to the high variability in analytical measurements. No explanation was given for the selection of dosages. Ten mice/ sex/group were sacrificed afte r 12 months, and the remaining mice were sacrificed after 24 months. Parameters examined in all mice were those routinely examined in a carcinogenicity study, including absolute and relative (% of body weight) weights of brain with stem, heart, liver, gonads, and kidneys. Histopathology was conducted on these tissues as well as epididymides, mammary gland, pituitary, prostate, seminal vesicles, thyroid,
and uterus with cervix. Tissues from mice wh ich died during the study or were sacrificed moribund were also examined microscopically.

Mortality was increased in low-dose males (p ≤ 0.06) and low- and mid-dose females (NS) compared to control. Mean body we ights were decreased (>10%; p ≤ 0.01 or NS) in high-dose males and females from Week 2 to study te rmination (Week 104). Significant body weight decreases (p ≤ 0.05 or 0.01) were generally present by Week 52 in the mid-dose animals, with

≥ 10% decreases present by Weeks 65 and 78 until study termination in males and females, respectively. Female mice in the high-dose group had a higher incidence of hepatocellular carcinomas and hyperplastic nodules compared to control. The incidence of hepatocellular carcinomas in the control and treatment groups for female mice was 1.3, 1.3, 0, and 15.8%, respectively. The corresponding values for the males were 5.3, 2.7, 4.0, and 4.0%, respectively.
All of these carcinomas were obser ved at the terminal sacrifice. The incidence of hyperplastic nodules in the control and treatment gr oups for female mice was 2.7, 1.3, 0 and 19.7%, respectively, and the corresponding values for the males were 13.3, 9.3, 5.3, and 17.3%, respectively. Most of the nodules were observed at the terminal sacrifice. The incidence of hepatocellular carcinoma in the historical cont rols (22 studies; 1630 CD-1 mice) ranged from 0
to 6%.

Other treatment-related findings were: (1) liver lesions in the mid- and high-dose males and females; (2) lung lesions and lesions in nasal ca vity (acute rhinitis) in the mid- and high-dose males and females; and (3) organ we ight effects in high-dose males ( ↓ liver and kidney weights) and females ( ↓ kidneys, brains and ovaries) relative to control. Based on decreased body weight, liver weight effects, decreased kidney weight, and non-neoplastic lesions in liver and lungs, the systemic LOAEL is 250 ppm (36 and 42 mg/kg/day; males and females, respectively) and the systemic NOAEL is 50 ppm (7 and 8 mg/kg/day; males and female s, respectively). Based on the increased incidence of hepatocellular carcinomas in the high-dose females, acephate was carcinogenic to female mice in this study.

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

In a chronic feeding study (MRID 41812001), beag le dogs (5/sex/group) were administered acephate (99.9% purity) in th e diet for one year at nominal doses of 0, 10, 120, and 800 ppm (mean analytical values of 0, 0.27, 3.11, and 20.16 mg/kg/day, respectively). Dosages used in this study were based on the results of a 4- week preliminary study (No. HWA 2107-164) withACEPHATE FINAL

Page 46 of 51 nominal dietary acephate doses of 8, 20, 250, and 500 ppm. Parameters examined in all dogs were those routinely examined in a chronic oral toxicity study, and in cluded ChE activity in plasma, RBC, and brain. Absolute and relative (% of terminal body weight and brain weight) weights of organs were determined, including adrenals, kidneys, liver , testes/ovaries, and pituitary and thyroid glands ; histopathology was conducted on these tissues as well as epididymides, mammary gland, prostate, and uter us with cervix. Plasma and RBC ChE activities were determined for all dogs dur ing Weeks -3, -2, -1, 4, 13, 26, and 52; brain ChE activities were assayed at study termination.

The primary treatment-related effect observed in this study was the inhi bition of brain and RBC ChE activities. Brain ChE activity was inhibited (p<0.05) in males in all dose groups (17, 53, and 66%, respectively) and in the mid- and high- dose females (49 and 66%, respectively). RBC ChE activity was inhibited (p<0.05) in the mid- (42 to 55%) and high-dose (76 to 87%) groups of both sexes, with no change at th e low dose. Plasma ChE activity was inhibited (NS) in the midand high-dose males and all female dose groups, but the decreases were not dose-related. Despite
severe brain ChE inhibition in the mid-dose a nd high-dose groups of both sexes, symptoms usually associated with ChE i nhibition were not observed. Other treatment-related effects were increased (NS) absolute liver weights (29 and 17%) in the high-dose males and females, respectively, and perivascular in filtration and pigment in hepatic reticuloendothelial cells of one mid-dose male and many high-dose males and females.

Based on hematological effects, increased absolute liver weights, and hist ological changes in the liver, the LOAEL and NOAEL for systemic ef fects are 800 ppm (20.16 mg/kg/day; HDT) and 120 ppm (3.11 mg/kg/day), respectively (b oth sexes). The LOAEL and NOAEL for the inhibition of brain ChE activity in females ar e 3.11 and 0.27 mg/kg/day, respectively; in males the LOAEL is 0.27 mg/kg/day and a NOAEL is not determined. The LOAEL and NOAEL for the inhibition of RBC ChE activity in bot h sexes are 3.11 and 0.27 mg/kg/day. The NOAEL for the inhibition of plasma ChE activities in both sexes is 20.16 mg/.kg/da y, and a LOAEL is not determined (>20.16 mg/kg/day).

Mechanistic Study: Special Cholinesterase Inhibition (Rat)

In a cholinesterase inhibition oral toxi city study (MRID 40504819), 45-day old SD rats (30/sex/dose) were administered acephate (98.2% purity) in the di et at nominal doses of 0, 2, 5, 10, and 150 ppm (mean analytical va lues of 0/0, 0.12/0.15, 0.21/0.36, 0.58/0.76, and 8.90/11.48 mg/kg/day in males and females, respec tively). Cholinesterase (ChE; ChE or NS ChE) activities in brain, RBC, and plasma were determined for 10 rats/sex/dose at Weeks 4, 9, and 13. In addition, other parameters determin ed included clinical toxicity/mortality, body weight, and a general necropsy examination.ACEPHATE FINAL

Page 47 of 51 No signs of clinical toxicity and no mortality were reported during the study, and body weights
were not affected. Brain ChE was inhibited (p<0.01; 7 and 9%, respectively) at Week 13 in lowdose males and females, and in low-dose females at Week 9 (p<0.01; 9%). It was concluded that this dose essentially represented a threshold ( i.e. , the dose response curve in this area was very flat becoming essentially parallel to the x-axis). Brain ChE ac tivity was inhibited (generally p<0.01) in all higher dose groups at each samp ling time. Inhibition (p<0.01 or 0.05) of RBC ChE was observed in high-dose males (Weeks 4 and 9) and females (Weeks 9 and 13). Plasma ChE activity was inhibited (p<0.01) only in high -dose females at Week 13. High variability was noted in the RBC ChE and plasma ChE determin ations, and may have impacted statistical comparisons. The NOEL for RBC and plasma Ch E inhibition is 10 ppm (0.58/0.76 mg/kg/day) due to inhibition observed at the high dose (LOAEL; 8.90/11.48 mg/kg/day). A NOEL for brain
ChE inhibition is not determined in this st udy (<2 ppm), due to i nhibition observed at 0.12/0.15 mg/kg/day (LOAEL).

Comparative Cholinesterase Assay

Aa series of special comparative cholinestera se inhibition (ChEI) assays were conducted for acephate to evaluate sensitivity in life stages. Acephate technical (99.2% a.i., LOT No. AS 40s, Batch No. VLD-622-37a) was administered by gava ge to groups of Crl:CD(SD)IGS BR rats. For time-course evaluation, groups of 14 adult rats/sex were given single oral doses of 0, 2.5, or 10 mg/kg (MRID 46151803) and groups of 14 PND 11 and 14 PND 21 pups/sex were given single oral doses of 0, 5, or 10 mg/kg (MRID 46151804); tw o rats/sex/dose were sacrificed 0, 1, 2, 3, 4, 8, and 24 hours later. This study (MRID 46151803) also included a range-finding study for repeat exposures in adult rats. In (MRID 46151804), groups of 8 PND 11 and groups of 8 PND 21 pups/sex were given single or al doses of 0, 0.5, 2.5, 5, or 10 mg/kg and observed for 24 hours for clinical signs and mortality. In th e same study (MRID 46151804), groups of 4 PND 11 pups/sex were given 11 repeat doses, PND 11 th rough 21, and observed for clinical signs and mortality. In the definitive acute study (MRID 46151801), groups of adult, PND 11, and PND 21 rats, 10/sex/age group, were give n single oral doses of 0, 0.5, 1, 2.5, or 10 mg/kg and sacrificed 3 hours later (time of peak eff ect). In the definitive rep eat oral dosing study (MRID 46151806) groups of 10 adult and 10 PND 11 rats/sex were given 11 daily oral doses of 0, 0.5, 1, 2.5, or 10 mg/kg/day. In a study of comparative maternal and fetal sensitivity (MRID 46151805), groups of 8 pregnant dams were given doses of 0, 0.5, 1, 2.5, or 10 mg/kg/day on gestation days (GD) 6 through 21. Plasma, red blood cell (RBC), and brain Ch E activities were measured in all animals in each study except the clinical observation part of study (MRID 46151804 )

.
All adult male and female rats and all pups surviv ed to scheduled sacrifice. No treatment-related clinical signs of toxici ty were observed in any animal duri ng the studies. No treatment-related
lesions were observed at necr opsy. Body weight was comparable among the control and treated groups for both male and female adults and for bot h male and female pups in all studies (both the single and repeat-dose studies). Fetuses from treated dams had body weight comparable to thoseACEPHATE FINAL

Page 48 of 51 in the control group. Reproduction performance, ge station, and implantation were not affected by treatment in dams, nor were litter size, viab ility, sex ratio, or post implantation affected.

The acute ChEI data were often variable and did not follow clear dose-response patterns (MRID 46151801). Acute exposure at doses  1 mg/kg resulted in biologically significant (  20%) inhibition of enzyme activity in the brain co mpartment of most gr oups. At 1 mg/kg, brain enzyme activity was biologically or statistica lly significantly inhibited in adult males and females, PND 11 males, and PND 21 females. PND 11 female pups and PND 21 male pups were affected at 2.5 and 10 mg/kg, respectively. RBC activities were genera lly unaffected by acute treatment up to and including 10 mg/kg, the ex ception being PND 21 male pups which were affected at 10 mg/kg. Plasma ChE activity showed a clearer dose-response pattern than the other compartments. Plasma ChEI occurred at 2.5 mg/kg (adult males and PND 11 male pups) and 10 mg/kg (adult females, PND 21 male pups, a nd PND 11 and PND 21 female pups). For all compartments, pups were no more sensitive to acute exposures than were adults except for RBC in PDN 21 male pups. Where adults and pups were a ffected at the same dos e, e.g., brain ChEI at 1 mg/kg, the inhibition in pups (23-25%) was similar to that in adults (30-35%).

Data for brain ChEI were also variable after 11- day repeated dosing in bo th adults and neonates (MRID 46151806). LOAELs were 1 mg/kg for adult males and females and PND 21 male pups and 2.5 mg/kg for PND 21 female pups.

The LOAELs for acute and repeated exposures in a dults are fairly similar. The brain ChEI data for pups were variable, with acute and repeat dose LOAELs for PND 21 male pups of 10 and 1 mg/kg, respectively, and for PND 21 females of 1 and 2.5 mg/kg, respectively. Treatment had no effect on RBC cholinesterase activity, with the exception of PND 21 male pups, affected at the highest dose (30% ChEI at 10 mg/kg/day). Plasma ChEI LOAELs ranged from 1 to 10 mg/kg/day with no clear rela tionship to sex or age.

In the repeat-exposure gestation study, lack of a dose-response relations hip between 0.5 and 1 mg/kg/day for ChEI in several compartments fo r both dams and fetuses precluded establishing a LOAEL (of 0.5 mg/kg/day).

For acute exposure, the overall adult LOAEL for c holinesterase inhibition in rats is 1 mg/kg based on enzyme inhibition in brai n; the adult NOAEL is 0.5 mg/kg.

For repeated exposure, the overall adult LOAEL for cholinesterase inhibition in rats is 1 mg/kg/day based on enzyme inhibition in brain and plasma; the adult NOAEL is 0.5 mg/kg.

The cholinesterase activity measurements follo wing an acute oral dose of acephate technical demonstrate approximately equal susceptibility between juvenile and adult rats. Following an acute exposure, brain enzyme inhibition in male and female adults and male and female pups was similar (LOAELs at 1 mg/kg, 23–35%). Repeated exposures show that sensitivity is stillACEPHATE FINAL

Page 49 of 51 similar between adults and PND 21 rats (LOAELs at 1 mg/kg, 21–29%). In both adults and pups the brain ChE activity appeared to be more sensitive than RBC or plasma enzyme activity, although the brain data were vari able and some groups were not affected at 1 mg/kg, e.g., acute exposure of PND 11 females and PND 21 males. This compartment susceptibility was observed
in terms of the dose level at which an effect was observed (i.e., the LOAEL for cholinesterase inhibition was generally lower for brain than for RBC or plasma). RBC enzyme activity was largely unaffected in adults and pre-weaning rats of both sexes and following acute and repeated exposures. Following repeated exposures, LOAELs for brain enzyme activity were variable among sexes and age groups, but showed no greater se nsitivity in pups than in adults. The ChEI
data for both dams and GD 21 fetuses were vari able, but showed no grea ter susceptibility in brain enzyme activity of GD 21 fetuses when co mpared to dams, following maternal exposure
from GD 6–21.

Avian Reproduction

In an avian reproduction study (MRID 00029691), mallard ducks ( Anas platyrhynchos ) were exposed to treated diet at 5, 20 and 80 mg/kg-diet. No symptoms of toxicity or behavioral
abnormalities or treatment-related mo rtalities were reported in the treatments (except for two incidences of prolapsed penis in the high treatment); however, sli ght, but significant, depressions in food consumption were reported in the low an d high treatment. Significa nt decreases in the number of viable embryos ( ↓ 16%) and live three-week embryos ( ↓ 16%) were reported at the mid-treatment group (20 mg/kg-diet); similar e ffects were reported at the high treatment, 80 mg/kg-diet, along with a decrease in the number of 14-day old offspring ( ↓ 18%). In another avian reproduction study (MRID 00029692, 1979), bobwhite quail ( Colinus virginianus ) were exposed to treated diet at 5, 20 and 80 mg/kg-diet. Effects on e gg viability and chick survival were reported at 80 mg/kg-diet.

Concurrent studies of brain ChE activity in both the mallard duck and bobwhite quail studies (MRID 00029689 and 00029690) reported reduced activity in all treatments with impairment increasing with dosage.ACEPHATE FINAL

Page 50 of 51 APPENDIX 3: References Not Utilized in the Acephate 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 Acephate 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 adequately 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.

Chakraborty, T., Katsu, Y., Zhou, L. Y., Miyagawa , S., Nagahama, Y., and Iguchi, T. (2011). Estrogen receptors in medaka ( Oryzias latipes ) and estrogenic environmental contaminants: an in
vitro-in vivo correlation. J Steroid Biochem Mol Biol. 123(3–5):115–121.

Geen, GH, McKeown B, Watson T, and Parker D. (1984) Effects of Acephate (Orthene®) on Development and Survival of the Salamander, Ambystoma Gracile (Baird). J. Environ. Sci. Health Part B 19(2): 157-170.

Ghodageri, M.G. and Katti, P. (2013). In vitro induction/inhibition of germinal vesicle breakdown (GVBD) in frog ( Euphlyctis cyanophlyctis ) oocytes by endocrine active compounds. Drug and Chem. Toxicol. : 36(2), 217–223.ACEPHATE FINAL

Page 51 of 51 Liu, X.J., Luo, Z., Zheng, J.L., and Xiong, B.X. ( 2013) Effects of waterborne acephate exposure on antioxidant responses and acetylcholinesterase activitie s in Synechogobius hasta. Environ Toxicol . 28(1):42-50.

Lu, X.T., Ma, Y., Wang, C., Zhang, X.F., Jin, d.Q ., and Huang, C. J. (2012) Cytotoxicity and DNA damage of five organophosphorus pesticides related by oxidative stress in PC12 cells and protection by vitamin E. J Environ Sci Health B . 47(5):445-454.

Sankhala, L.N., Tripathi, S.M., Bhavsar, S. K., Thaker, A.M., and Sharma, P. (2012) Hematological and Immunological Changes Due to Short-term Oral Administration of Acephate. Toxicol Int . 19(2):162-166.

Yang, J., Cao, J., Sun, X., Feng, Z., Hao, D., Zhao, X., and Sun, C. (2012) Effects of long-term exposure to low levels of organophosphorous pesticides and their mixture on altered antioxidative defense mechanisms and lipid peroxidation in rat liver. Cell Biochem Funct

. 30(2):122-128.