UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: June 29, 2015 

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

List 1 Chemicals 

PC Code: See table, Attachment A 

Decision No.: NA 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

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

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

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

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

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

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

and report identifiers. 

Page I of2 

Page 1 of 59
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

Page 2 of 59
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: ACEPHATE 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S ENVIRONMENTAL PROTECTION AGENCY

 

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ii 

TABLE OF CONTENTS 

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

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

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

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

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

B. Other Scientifically Relevant Information (OSRI)......................................4 

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

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

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

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

2. Effects on Androgen Pathway..................................................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-Estrogenic Pathway for Acephate....................................9 

 Table 3. Androgenic/Anti-Androgenic 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..........................50 

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iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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iv 

Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Methods 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

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v 

Abbreviation Terminology 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

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Abbreviation Terminology 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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Page 1 of 51 

Executive Summary 

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

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

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

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

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

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

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

in this weight of evidence (WoE) assessment. 

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

type of effects induced, the magnitude and pattern of responses observed across studies, taxa, 

and sexes were considered. Additionally, the conditions under which effects occur were 

considered, in particular, whether or not endocrine-related responses occurred at dose(s) that 

also resulted in general systemic toxicity. 

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 (WOE) analysis of the potential interaction 

of acephate with the E, A or T signaling pathways. The T1WoERC conclusions from the WoE 

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

the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of 

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

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 the Tier 1 uterotrophic and female pubertal rat 

assays. A minimal (3%) decrease on fertility was 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 fertility index in the 3-generation 

reproduction study. While effects on egg viability and chick survival were reported in the Part 

158 avian reproduction studies, these 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 androgen 

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sensitive tissues (AST) weights in the high dose in the anti-androgenic portion of the in vivo

Hershberger assay, no endocrine-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 that at the doses tested in the Hershberger assay; using a satellite group of noncastrated rats, both red blood cell (RBC) and brain cholinesterase (ChE) activities were 

decreased 40 to 87% compared to controls. While 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 other 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-related interaction. 

In the female pubertal assay, no thyroid-related effects were noted. In the male pubertal assay, 

thyroid weights were decreased by 25 and 21% at the low and high dose, respectively, and 

pituitary weight was decreased by 14% at the high dose, but no treatment-related effects were 

noted for thyroid histopathology. Decreases in ChE activity were observed in the male pubertal 

assay at all doses tested. No changes in thyroid weight or histology were reported in the Part 158 

mammalian studies. 

In the amphibian metamorphosis assay (AMA), while there were significant increases in the 

developmental endpoint of normalized hind-limb length (HLL) at Day 21, significant increases 

in this endpoint were not observed (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 treatment with a non-significant 3.4% decrease at 

the high treatment]. Additionally, there were no significant differences in developmental stage, 

nor treatment-related effects on thyroid histopathology at Day 21. It is noted that tadpoles in this 

treatment group were larger than controls. Therefore, overall, it appears that the statistically 

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 may be due to a generalized increase in growth 

in the tadpoles. 

Based on weight of evidence considerations, mammalian 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. 

 

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I. Introduction 

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

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

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

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

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

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

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

in this weight of evidence (WoE) assessment. 

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

type of effects induced, the magnitude and pattern of responses observed across studies, taxa, 

and sexes were considered. Additionally, the conditions under which effects occur were 

considered, in particular, whether or not endocrine-related responses occurred at 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 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).

Acephate is an organophosphate insecticide and its mode of action is based on altered 

neurotransmission due to acetylcholinesterase (ChE) inhibition. The vapor pressure of acephate 

is 1.7 × 10-6 mm Hg at 25°C, and therefore, volatilization 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/water partition coefficient (log Kow = -0.85 

to 0.13). Acephate is photolytically stable, not subject to hydrolytic degradation, however 

quickly undergoes biotic degradation (aerobic soil metabolism half-lives <2 days). Acephate has 

a whole body bioconcentration factor value in fish of approximately 10. 

The available information considered in determining 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 toxicity studies and other published articles. These 

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

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

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

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II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

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

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

1. 

B. Other Scientifically Relevant Information (OSRI) 

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

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

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

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

Report of the Endocrine Disruptor Review Team on 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 considered but not utilized for the WoE analysis are listed in 

Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

The principles, criteria and approach used in the WoE determination 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 presented at the 2013 FIFRA 

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

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

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

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

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

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

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

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

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

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

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

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

above. 

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

EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, 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 were 

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

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

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

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

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

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

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

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

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

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

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

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

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

The second step in this WoE process is to formulate hypotheses and integrate the available data 

along different levels of biological organization. Two keys elements in the integration of data, as 

well as characterizing the extent to which the available data support a hypothesis that a chemical 

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

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

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

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

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

through potential adverse effects in apical endpoints such as sexual development and fecundity at 

the whole organism level. The extent of expression of responses at higher levels of biological 

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

After the data have been assembled and integrated, the third step is to characterize the main lines 

of evidence along with the conclusions; this characterization involves three components. The 

first component is whether the data provide relevant, robust and consistent evidence in terms of 

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

biological organization were the responses observed and whether organisms exhibit 

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

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

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

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

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

 

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

the FIFRA SAP meeting in July 2013 that evaluated scientific issues associated with the WoE 

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

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

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

evaluated in terms of primary interactions with the endocrine system vs. secondary effects 

related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 

2013). For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s 

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

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

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

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

weight decreases of ≥10% in mammals. Additionally, other effects including morphological 

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

(e.g., lethargy) were also considered when evaluating overt toxicity, especially if the effects were 

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

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

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

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

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

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

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

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

Section IV.A. 

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

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

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

the E, A or T pathway. Adequately addressing the three main components described above is 

fundamental to the WoE process and in determining whether additional data are needed. In 

addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their 

potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or 

conflicting data; 4) overall strength of evidence supporting a conclusion; and, 5) what, if any, 

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

 

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

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

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

which supports a conclusion of whether 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) 

ERα 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) 

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B. Effects on Hypothalamic-Pituitary-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 assays are delineated so as to 

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

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

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

considered further in the WOE assessment. 

Acephate was classified as negative (or not interactive) in the Tier 1 in vitro ER binding, ERTA, 

aromatase, and steroidogenesis assays. Acephate 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 VTG 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 histopathological effects and the decreased ovarian weights occurred in the 

presence of overt toxicity (>10% body weight). In a 3-generation reproductive toxicity study in 

the rat, fertility index was decreased for the third generation high dose group. 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 activities as observed with other mammalian toxicity 

studies at comparable dose levels. It is noted that ChE activity was not measured in the 

reproduction study. 

In the Part 158 mallard duck reproduction study, 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-diet, along with a decrease in the 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 two incidences of prolapsed penis in the high 

treatment); however, slight, but significant, depressions in food consumption were reported in 

the low and high treatment. In the corresponding bobwhite quail reproduction 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 duck and bobwhite quail studies reported reduced activity in all 

treatments with decreases increasing with dosage. 

Page 16 of 59
ACEPHATE 

Page 9 of 51 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Acephate

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Acephate1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid Hormone 

Uterine Weight

Ovarian Weight / GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight

Estrous Cyclicity 

Age & Weight at VO 

2° Sex Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

EDSP Tier 1 Assay 

ER Binding (MRID 48614502) N ERTA (MRID 48614503) N Steroidogenesis (MRID 48614506) 

N

Aromatase (MRID 48614501) N Uterotrophic (MRID 48614507) N Female Pubertal Rat (MRID 48614505) NE N N N N N N FSTRA (MRID 48675101) NE N N N Frt: ↓3% (H) 

OSRI 

ER Transactivation (Kojima et al., 2004) N ER Transactivation  (Kojima et al., 2005) N 

Developmental toxicity (Rat; MRID 41081602) N N 
ACEPHATE 

Page 10 of 51 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Acephate

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Acephate1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid Hormone 

Uterine Weight

Ovarian Weight / GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight

Estrous Cyclicity 

Age & Weight at VO 

2° Sex Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Developmental toxicity (Rabbit; MRIDs 00069684) 

NR N 

Developmental Neurotoxicity (Rat MRID 46151802) 

 NE N N 

Three-generation reproduction (Rat; MRIDs 40323401 and 40605701) 

 NR NR NR 

Frt: ↓33%

(H; F3 

gen) 

Chronic toxicity / carcinogenicity (Rat; MRID 00101623) 

N N N N 

Carcinogenicity (Mouse; MRID 00105197) NE N N N Chronic toxicity (Dog; MRID 41812001) NE N N N Avian reproduction (Duck; MRIDs 00029691, 00029689) 

P5 

Avian reproduction (Quail; MRIDs 00029692, 00029690) 

P5 

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

direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. absolute unless otherwise indicated. LW= Liver weight; KW= Kidney weight; AW= Adrenal weight; ChE= Cholinesterase ac

gen=third generation 
ACEPHATE 

Page 11 of 51 

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

X in this column. For details see Section IV. A 

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

details see Section IV. 

 4 ChE activity was not measured in this study. However, the high dose would be expected to inhibit ChE activity based on other 

doses. 

 5 In the mallard duck study, significantly decreased viable and live embryos 

≤20 mg/kg-diet, with decreased chick survival at 80 

bobwhite quail study, decreases in egg viability and chick survival at 80 mg/kg-diet. 

P Positive findings 

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

 NE Not examined  NR Not reported 
ACEPHATE 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 androgenic, anti-androgenic, or HPG axis effects. This 

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

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

considered further in the WOE assessment. 

In an evaluation of androgenic/anti-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 transcriptional activation (ARTA) assay considered 

as OSRI (Kojima et al. 2004). 

In the Hershberger assay, while glans penis weights were increased in both low- and high-dose 

groups by 25, and 19%, respectively, no other androgen-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 the 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 significantly decreased 18% at the mid-dose (10 mg/kg/day). It is also 

noted that while absolute seminal vesicle weight was not significantly decreased, 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-related effects were observed 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 significant (p<0.05) decrease of 3% fertility was reported at the high 

concentration; however, no other treatment-related effects were reported. 

In the Part 158 mammalian studies, increases in relative testes weights of 43% were observed at 

the high dose in the mouse carcinogenicity study. Body weight decreases ≥10% were also 

reported at this high dose. As reported in the estrogen 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. 

Page 20 of 59
ACEPHATE 

Page 13 of 51 

Table 3. Androgenic/Anti-Androgenic Pathway for Acephate

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Acephate1Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Age and Weight at 

PPS 

EDSP Tier 1 Assay 

AR Binding (MRID 48669001) N Steroidogenesis (MRID 48614506) N Hershberger (MRID 48614504) NE 

VP: ↓18/24% 

(M, H); 

CG: ↓18%, 

(H) 

Male Pubertal Rat (MRID 48614505) 

↓53%

(H) N N N N ↓14% (H) N N 

FSTRA (MRID 48675101) NE N N N ↓3% (H) 
ACEPHATE 

Page 14 of 51 

Table 3. Androgenic/Anti-Androgenic Pathway for Acephate

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Acephate1Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Age and Weight at 

PPS 

OSRI 

ARTA (Kojima et al., 2004) N Developmental toxicity (Rat; MRID 41081602) N Developmental toxicity (Rabbit; MRID 00069684) N Developmental Neurotoxicity (Rat MRID 46151802) 

N N 

Three-generation reproduction (Rat; MRIDs 40323401 and 40605701) 

NR NR NR NR NR NR 

Frt: 

↓33% 

(H; F3 

gen) 

Chronic toxicity / carcinogenicity (Rat; MRID 00101623) 

N N NE N NE NE 

Carcinogenicity (Mouse; MRID 00105197) 

↑43% 

(H, Rel) N NE N NE NE 

Chronic toxicity (Dog; MRID 41812001) N N NE N N NE Avian reproduction (Duck; MRIDs 00029691, 00029689) 

P5 
ACEPHATE 

Page 15 of 51 

Table 3. Androgenic/Anti-Androgenic Pathway for Acephate

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Acephate1Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Avian reproduction (Quail; MRIDs 00029692, 00029690) 

P5 

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

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

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

Cowper’s gland (CG), LW= Liver weight; ChE= Cholinesterase activity; Frt=Fertility; F3 gen=third generation 

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

this column. For details see Section IV. A 

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

details see Section IV. A 

4. ChE activity was not measured in this study. However, the high dose would be expected to inhibit ChE activity based on other stud

doses 

5. In the mallard duck study, significantly decreased viable and live embryos 

≤20 mg/kg-diet, with decreased chick survival at 80 mg

quail study, decreases in egg viability and chick survival at 80 mg/kg-diet. 

P Positive findings N Negative findings (in vitro) / No effect (in vitro) 

NE Not examined NR Not reported 
ACEPHATE FINAL 

Page 16 of 51 

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

The current EDSP Tier 1 battery does not have a specific in vitro assay to detect chemicals with 

the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it 

does include three in vivo assays that provide data to detect changes in the HPT axis, i.e., the 

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

The potential for acephate to interact with thyroid regulation are summarized in Table 4. The 

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

facilitate determination of potential for thyroid or HPT axis effects. This table also includes 

HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of 

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

further in the WOE assessment. 

In the female pubertal assay, no treatment-related effects were noted for thyroid weight, 

histopathology, T4 or TSH concentration. In the male pubertal assay, thyroid weights were 

decreased by 25 and 21% at the low and high dose, respectively, and pituitary weight was 

decreased by 14% at the high dose, but no treatment-related effects were noted for thyroid 

histopathology. No changes in thyroid weight or histology were reported in the Part 158 

mammalian studies. 

In the AMA study, there were no gross or histological 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 high-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. 

Page 24 of 59
ACEPHATE FINAL 

Page 17 of 51 

Table 4. Thyroid Pathway for Acephate

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Acephate1

Study Type / Literature 

Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

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

Overt Toxicity Observed3

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 applicable in this assay. Changes in weight are absolute unless 

otherwise indicated KW= Kidney weight; AW= Adrenal 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 examined 

Page 25 of 59
ACEPHATE FINAL 

Page 18 of 51 

IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight 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 recommendations regarding Tier 2 testing. The results of the Tier 1 assays 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

with weight of evidence evaluation of the results of the Tier 1 assays stated that “In summary, 

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

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

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

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

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

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

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

for the in vivo Tier 1 assays and OSRI, other effects observed which may be considered 

overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% 

decrease in mammalian body weight) was sufficient for a dose/concentration to be considered 

overtly toxic. However, in other instances, more than one parameter was needed to determine 

overt toxicity. Effects which were considered to be signs of systemic toxicity were generally less 

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

Page 26 of 59
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 decreased by 12 to 13% in both 

the androgenic and anti-androgenic 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%, respectively, at the high dose, and absolute and 

relative kidney and adrenal weights were decreased by 7 and 13%, respectively. Compared to 

control, ChE activity at 10 and 30 mg/kg/day was significantly inhibited (p<0.001) in the RBC 

(↓69 and 89%) and brain (↓71% and 84%). In the male pubertal assay, pituitary weight was 

decreased by 14% at the high dose, and thyroid weight was decrease 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 clinical 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, respectively, 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 the 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 control and treated groups. 

2. OSRI 

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

inhibition. In the developmental rat study, clinical signs of toxicity (tremors and decreased motor 

activity) were observed in the dams at the high 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 females) 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. 

Page 27 of 59
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 altered 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 times 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 Weeks 65 to 78 until study termination. High-dose 

females had a higher incidence of hepatocellular carcinomas and hyperplastic nodes in the liver, 

and there were increased lesions in the liver, lung and nasal cavity in the mid- and high-dose 

males and females. High-dose males had decreases in liver and kidney weight and high-dose 

females had decreased kidney and brain weights compared 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, 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- (42–55%) and high-dose (76–87%) groups of both 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 perivascular 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 quail 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 observed 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 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 

Page 28 of 59
ACEPHATE FINAL 

Page 21 of 51 

decrease in fertility index in the 3-generation reproduction study. While effects on egg viability 

and chick survival were reported in the Part 158 avian reproduction studies, these 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 were reported at all doses in both avian reproduction studies. 

C. 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. Acephate was also negative in an ARTA 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 endocrine-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 that 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 decreased 40 to 87% compared to controls. While 

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 other 

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-related interaction. 

D. Thyroid Pathway 

In the female pubertal assay, no thyroid-related effects were noted. In the male pubertal assay, 

thyroid weights were decreased by 25 and 21% at the low and high dose, respectively, and 

pituitary weight was decreased by 14% at the high dose, but no treatment-related effects were 

noted for thyroid histopathology. Decreases in ChE activity were observed in the male pubertal 

assay at all doses tested. No changes in thyroid weight or histology were reported in the Part 158 

mammalian studies. 

In the AMA, while there were significant increases in the developmental endpoint of normalized 

hind-limb length (HLL) at Day 21, significant increases in this endpoint were not observed 

(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 treatment with a non-significant 3.4% decrease at the high treatment]. Additionally, 

there were no significant differences in developmental stage, nor treatment-related effects on 

thyroid histopathology at Day 21. It is noted that tadpoles in this treatment group were larger 

than controls. Therefore, overall, it appears that the statistically significant increase in HLL at 

Page 29 of 59
ACEPHATE FINAL 

Page 22 of 51 

Day 21 in the acephate treatments compared to the control is not due to interaction with the 

thyroid pathway, but may be due to a generalized increase in growth in the tadpoles. 

E. Conclusions 

Based on the data considered in the WoE analysis, 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, mammalian 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. 

Page 30 of 59
ACEPHATE FINAL 

Page 23 of 51 

VI. References 

Akhurst, L.C. (2011). Acephate technical estrogen receptor transcriptional activation (Human 

cell line (HeLa-9903)). Unpublished prepared by Huntingdon Life Sciences, Ltd. 

Cambridgeshire, England. Laboratory Report No.: ADB0061, October 14 2011. 

(MRID 48614503). 

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Beavers, J.B.; Fink, R., Grimes, J., et al. (1979). Final Report: One-Generation Reproduction 

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Brorby, G.; Rosenberg, D. (1987). The Cholinesterase Inhibition Potential of Acephate 

Technical (SX-1102) Following 4-, 9-, or 13-Week Dietary Administration in Male and Female 

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Laboratory Project Study ID: ADB0062, October 13, 2011. (MRID 48614504). 

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October 14, 2011. (MRID 48614501). 

Geil, R.G. (1981). Lifetime Oral Carcinogenicity Study in Mice: Histopathology Data: IRDC 

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Glickman, A. (1983). Orthene Lifetime Study in Mice: Hepatocarcinoma in Female Mice. 

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Hoberman, A. (1987). Two-Generation (Two-Litter) Reproduction Study in Rats with Chevron 

Acephate Technical: Laboratory Project ID: Argus Research Laboratories: 303005: Chevron 

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in cooperation with Chevron Chemical Co. (MRID 40323401). 

Hoberman, A. (1988). Two-Generation (Two-Litter) Reproduction Study in Rats with Chevron 

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Argus Research Laboratories, Inc. (MRID 40605701). 

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Hoberman, A.M. (2003). Oral (gavage) dosage-range study of acephate technical in adult rats. 

Argus Research, 905 Sheehy Drive, Building 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, Building 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 relative 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). 

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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 Identification VP-25267. December 1, 2003. Unpublished. 

(MRID 46151805). 

Hoberman, A.M. (2003). Oral (gavage) repeated dose relative sensitivity 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/carcinogenicity study technical 

RE-12420 in rats: Ref.78-2135. (Unpublished study received May 18, 1982 under 239-2471; 

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

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

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

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

Res. 15:271-280. 

Leary, J. (1981). Addendum to Orthene (SX-1032) Mouse Lifetime Feeding Study. IRDC 

Project No. 415-006: Diet Analyses: File No. 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 Teratogenicity and Developmental Toxicity Study in Rats with Chevron 

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

41081602). 

Matthews, A. (2011). Acephate: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol 

(ER-RUC). Unpublished prepared by Huntingdon Life Sciences, Huntingdon, Cambridgeshire, 

UK. Laboratory Study No.: ADB0060, October 31, 2011. (MRID 48614502). 

Nemec, M. (1997). A Subchronic (13-Week) Neurotoxicity Study of Orthene Technical in Rats: 

Lab Project No. WIL-194014. Unpublished study prepared by WIL Research Labs., Inc. 

(MRID 44203304). 

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Odum, J and Matthisen, P. (20012). Acephate: Weight 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: Assessment 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 Study in Rabbits: IRDC No. 

415-024. (Unpublished study received May 19, 1981 under 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 Wildlife International, Ltd., Easton, MD 21601. 

Laboratory report number 701A-103. Study sponsored by Acephate Task Force Steering 

Committee/Joint Venture, c/o Consumer Specialty Products Association, 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: Uterotrophic Bioassay in Ovariectomised Rats (Oral 

Administration). Unpublished prepared by Huntingdon Life Sciences Ltd, Cambridgeshire, 

England. Laboratory Project Identity ADB0065, November 4, 2011. (MRID 48614507). 

Spicer, E., Geil, R., and Phillips, L. (1982). Lifetime Oral Carcinogenicity Study in Mice: 

Orthene Technical: 415- 006. (Unpublished study received June 22, 1982 under 239-2471; 

prepared by International Research and Development Corp., submitted by Chevron Chemical 

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

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USEPA. (2001). Interim Reregistration Eligibility Decision (IRED) for Acephate. CASE 0042. 

September, 2001. EPA 738-F-01-013, Office of Pollution Prevention and Toxic Substances, U.S. 

Environmental Protection Agency, Washington, DC. 

USEPA. (2010). Acephate: Report of the Endocrine 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 Screening Program — Weight of Evidence: Evaluating 

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

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

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

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

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

Agency, Washington, DC. 

Willoughby, J.A. (2012). Acephate: Androgen Receptor Binding (Rat Prostate Cytosol). 

Unpublished prepared by CeeTox, Inc., Kalamazoo, MI. Laboratory Study Number: 9146V100602ARB, February 13, 2012. (MRID 48669001). 

 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis 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) stage 51 (80 tadpoles per group) were exposed to 

nominal concentrations of 0 (negative control), 4.0, 20, and 100 mg a.i./L; mean-measured 

concentrations were <3.00 (<LOQ), 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 the study. By test termination, curved tails were observed 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 normalized (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 observed 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 delay of median NF developmental stage was observed at Day 7 or 

21 at any treatment level. Further, no asynchronous 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 severe follicular cell hypertrophy 

were observed in the control and treatment 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; logarithmic increase from 10−11 to 10−3

 M). Dimethyl sulfoxide 

(DMSO) was used as a vehicle at a final assay concentration of approximately 3.2%. The assay 

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

The saturation binding experiment was conducted to demonstrate that the AR in the rat prostate 

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

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radiolabeled reference androgen (R1881) prior to conducting AR competitive binding 

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

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

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

H]-R1881 

was 0.613±0.041 nM and the estimated Bmax was 0.817±0.049 fmol/100 μg protein for the single 

batch of prostate cytosol used for this assay. The mean and individual Kd and Bmax values were 

below the expected ranges reported 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 runs, 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 additional efforts to address chemical 

insolubility issues at the higher concentrations (e.g., use of water or ethanol solvents, or testing 

half log concentrations). The estimated average log IC50s 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 “untestable” in the first run because acephate was not soluble at >10−6 

M and the binding curve did not cross 50%. In the 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 Non-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 aromatase and tritiated androstenedione (1-β

[

3

H(N)]-androst-4-ene-3,17-dione; [3H]ASDN) in ethanol at log 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. Tritiated water was quantified 

using liquid scintillation counting (LSC). Three runs were conducted and each run included a 

full activity control, a background activity control, a positive control series (10−10 to 10−5

 M) 

using a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), and the test chemical series 

(10−10 to 10−3

 M) with three repetitions per concentration. 

Aromatase activity in the full 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 ± 0.016 

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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. The response of each full activity 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 IC50, and %CV 

for replicates of each concentration within runs. The mean log IC50 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 concentrations 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 assay (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 was conducted to demonstrate that the ER in the 

rat uterine cytosol was present in reasonable numbers and was functioning with appropriate 

affinity for the radiolabeled reference estrogen prior to conducting ER competitive binding 

experiments. A competitive binding experiment was conducted to measure the binding of a 

single concentration of [3

H]-17β-estradiol (1 nM) in the presence of increasing concentrations 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 17β-estradiol as the natural ligand reference 

material. 

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For the saturation binding assay, the protein concentration was determined to be 0.1 mg/mL as it 

bound 27% of the [3

H]-17β-estradiol (within the Guideline range of 25 to 35% binding). The Kd

for [3H]-17β-estradiol was 0.125 nM, and the estimated Bmax was 20.8 fmol/100 μg protein for 

the prepared rat uterine cytosol. The Kd 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 determined to be 0.3 mg/mL as 

it bound 13% of the [3

H]-17β-estradiol (within the Guideline range of 10 to 15% binding). The 

mean log IC50 was −8.75 M for 17β-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, octyltriethoxysilane 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 IC50

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. 

ERα Transcriptional Activation (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 concentrations 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 was tested in 6 wells/plate. The reference 

chemicals 17β-estradiol (strong estrogen), 17α-estradiol (weak estrogen), corticosterone 

(negative compound) and 17α-methyltestosterone (very weak agonist) were also tested. Cells 

were exposed to the test agents for 23 ±1 hours to induce reporter (luciferase) gene products. For 

each run of the assay, two test plates were established: one plate for the luciferase assay and one 

for the cytotoxicity assay. Luciferase expression was measured upon addition of a luciferase 

substrate and detection with a luminometer with 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 17β-estradiol) and compared to 10% of the PC 

maximal response (PC10). Data demonstrating laboratory 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 RPCMax was 4.1% for the first run, 

6.4% for the second run and 12.3% for the third run. The associated PCMax values were 10−3

, 

10−5

 and 10−3 M, respectively. The assay performance criteria were generally met, although Hill 

slopes for 17β-estradiol and 17α-estradiol and the log PC10 for 17α-methyltestosterone were 

lower than the expected values which indicates increased sensitivity of the cells to weak 

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agonists. All other performance criteria were acceptable. Although the third test run gave a 

positive result (RPCMax > PC10), comparison of the third run with the two earlier runs is 

complicated by the use of a new set of cells for the third run, particularly as the proficiency test 

for either cell stock was not provided. 

As the RPCMax was less than the PC10 in two of the three test runs, acephate is considered 

negative for interaction with the human estrogen 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 25.8o

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 regards to behavior or changes in appearance, specifically 

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

clinical signs of toxicity for any treatment group compared to the 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/replicate (range: 20.3-

32.7); fertilization success in the negative control group averaged 98.6%. 

There was a slight but significant reduction in fertility of 3% at the high treatment concentration 

compared to the negative control (p<0.05; Jonckheere-Terpstra test). There were no other 

statistically significant differences between the treatment levels and negative controls for any other 

endpoint for either males or females, including fecundity, tubercle score, plasma vitellogenin 

(VTG), gonado-somatic index (GSI), body weight, and body length. There were no differences 

noted between histopathology severity scores in treated groups compared to the negative controls. 

Sex steroids (testosterone and 17β-estradiol) were not measured. 

Hershberger; (OCSPP 890.1400) 

In a Hershberger assay (MRID 48614504) screening for androgenic activity, acephate (98.8% 

a.i., Lot# 09-750-10) in deionized water was administered daily via oral gavage to groups of 55- 

to 59-day old, castrated male Sprague Dawley rats (10/group) at dose levels of 0 (vehicle), 10, or 

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30 mg/kg/day. An androgenic positive 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-androgenic activity, acephate (98.8%, lot # 09-750-10) in deionized 

water was administered daily via oral gavage to 55- to 59-day 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 vehicle control groups 

consisted of ten castrated male rats dosed daily with TP (0.4 mg/kg/day) by s.c. injection in 

deionized water and corn oil, and the anti-androgenic positive control group consisted of ten 

castrated male rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg/day in conjugation 

with a daily s.c. dose of TP (0.4 mg/kg/day). 

For both components of the assay, the animals were dosed for 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 acephate 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 assay, body weights for the 10 and 30 mg/kg/day treatment groups were 

comparable to controls throughout the duration 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 responded appropriately showing significant 

increases (p≤0.05) in the five accessory sex organ weights. All %CV values were less than or 

equal to the maximum recommended values. 

In the anti-androgen assay, body weights 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 weights 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: ventral prostate weights were decreased 24% and Cowper’s gland 

weights were reduced 18%. Ventral prostate weights were also significantly decreased at 10 

mg/kg/day (18%; p<0.05). Animals dosed with 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. 

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At all acephate dose levels (3-30 mg/kg/day), ChE levels were significantly decreased compared 

to the control in both the RBC (40-80% inhibition) 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% decrease in body weight gain (androgen assay). 

Acephate was negative for androgenic activity in the Hershberger assay as statistically 

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

sensitive tissues in the androgen agonist assay. However, acephate was positive for antiandrogenicity in the Hershberger assay as statistically significant decreases in organ weights 

were seen in two or more of the five androgen sensitive tissues in the androgen antagonist assay. 

The target organ weight reductions were observed at a dose that 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 beginning on PND 22, and age and weight at day of 

attainment was recorded. After sacrifice on PND 42, total serum thyroxine (T4) and thyroid 

stimulating hormone (TSH) levels were determined by radioimmunoassay (RIA). Weights were 

recorded for liver, kidneys, urogenital organs, pituitary, thyroid, and adrenal glands and 

microscopic examination of the ovaries, uterus, thyroid, and kidneys was performed. An 

additional satellite group (6 females/dose) was similarly treated with acephate from PND 22 to 

43 and used for red blood cell (RBC) and brain acetylcholinesterase (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 and overall body weight gain were decreased 

(p<0.05) compared to the control (↓5% and 8%, respectively). Absolute and adjusted kidney and 

adrenal glands weights were also decreased (p<0.05) in the 30 mg/kg/day group (↓7% and 13%, 

respectively). Aspartate aminotransferase concentrations were increased (p<0.01) by 23 and 

22%; alanine aminotransferase concentrations were 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. 

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For animals in the satellite study, AChE activity in RBC was significantly inhibited (p<0.001) 

compared to the control at 10 (69% inhibition) and 30 (89% inhibition) mg/kg/day. AChE 

activity in the brain was also significantly inhibited (p<0.001) at 10 mg/kg/day (71% inhibition) 

and 30 mg/kg/day (84% inhibition). The doses tested were considered 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 treated with acephate and used for 

red blood cell (RBC) and brain acetylcholinesterase (AChE) evaluations (sacrificed on PND 54). 

The main study animals were examined for preputial separation (PPS) daily beginning on PND 

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

total serum testosterone, thyroxine (T4), and thyroid stimulating hormone (TSH) levels were 

analyzed by radioimmunoassay. Kidney, liver, adrenal, pituitary, thyroid, and urogenital organ 

weights were recorded, and microscopic examinations of the testes, epididymides, thyroid, and 

kidneys were performed. 

No adverse treatment-related effects 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 observed 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-related findings were limited to decreased (p<0.05) 

thyroid/parathyroid weight (↓25%). 

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In the satellite animals at 10 and 30 mg/kg/day, dose-dependent reductions (p<0.001) in both 

RBC (↓74-92%) and brain (↓72-87%) AChE levels were observed. The doses tested were 

judged to be adequate based on the observed RBC and brain cholinesterase activity. 

Effects on androgen- and thyroid-related endpoints included decreased serum testosterone levels 

at 30 mg/kg/day, decreased pituitary weights at 30 mg/kg/day, and decreased thyroid/parathyroid 

weights at 10 and 30 mg/kg/day. 

Steroidogenesis; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 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 independent 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 independent 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 and testosterone production. 

Guideline acceptability recommendations and requirements were generally met, including lack 

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

%CV), and appropriate induction and inhibition with positive controls, with the following 

exception: (i) testosterone induction by 10 μM forskolin averaged 1.88-fold which is less than 

the Guideline recommendation of ≥2-fold. 

Precipitation of the test compound and/or cytotoxicity was not observed at any tested 

concentration. No treatment-related effect was observed on testosterone. There were no 

statistically significant differences in testosterone concentration in the acephate treated cells 

relative to the solvent control (0.87- to 1.09-fold change). Although significant differences were 

observed in estradiol concentrations in the acephate-treated cells relative to the solvent control 

(SC) in Runs 1 and 2, these differences were not dose-dependent; no statistically significant 

responses were observed in Run 3. 

Based on hormone responses in at least three independent runs, acephate treatment did not result 

in statistically significant and reproducible alterations in testosterone or estradiol production. 

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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 deionized water was administered daily via oral 

gavage to groups of eight 61- to 68-day old ovariectomized female Sprague Dawley rats at dose 

levels of 0 (vehicle), 3, 10 or 30 mg/kg/day for four consecutive days. A positive control group 

was concurrently treated with 17α-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 administration 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 similarly 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 collected 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 weights 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 comparable to the controls. 

Body weights in the positive control (EE) group were comparable to the control group 

throughout the duration of the assay, but overall body weight gains decreased (p<0.01) by 4.1%. 

Absolute wet and blotted uterus weights for the positive control group were increased (p<0.001) 

by 936% and 302%, respectively, as expected. Liver weights were also increased (p<0.01) by 

8% in the positive control group. 

In the cholinesterase phase of the assay, RBC ChE activity were significantly decreased (p<0.01) 

at 10 mg/kg/day (59%) and at 30 mg/kg/day (77%). 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. These 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. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Published literature and Part 158 general toxicity studies considered relevant for determining the 

potential interaction of acephate with the endocrine system are summarized below. Given the 

breadth of toxicity data available 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, chronic 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 (hERα and hERβ) 

and one human androgen receptor (hAR) in transactivation assays with Chinese hamster ovary 

cells (CHO-K1 cells) was investigated. For detection of hERα or hERβ activity, cells were 

transfected with pcDNAERα or pcDNAERβ, pGL3-tkERE, and pRL-SV40. For detection of 

hAR activity, cells were transfected with pZeoSV2AR, pIND-ARE, and pRL-SV40. Flutolanil 

was tested over a concentration range from 10−8

 to 10−5 M, and the responses were compared to 

the activity of 17β-estradiol (E2; purity >97%) or 5α-dihydrotestosterone (DHT; purity 95%) for 

the ER or AR assays, respectively. For measurement of antagonistic activity to hERα, hERβ and 

hAR, either 10−11 or 10−10 M E2 or 10−10 M DHT was added to the cell cultures along with the 

test compound, respectively. Agonistic activities were evaluated by the relative activity 

expressed as 20% relative effective concentration (REC20), the concentration of the compound 

demonstrating 20% of the activity of 10−10 M E2, 10−9 M E2, or 10−9 M DHT for ERα, ERβ, and 

AR, respectively. Antagonistic activities were evaluated similarly by the relative activity 

expressed as 20% relative inhibitory concentration (RIC20), the concentration of the compound 

showing 20% inhibition of the activity of E2 or DHT against ERα, ERβ, and AR, respectively. 

Acephate did not show agonist or antagonist activity for ERα, ERβ, 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 after exposure to thirty two pesticides either alone or in 

combination. The source of the test compounds was not reported; however, acephate had a 

reported purity of 98%. The test compounds 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 promoter, confer estrogen responsiveness to the 

viral promoter and reporter gene. The transfected cells are made stable by an antibiotic resistance 

gene resulting in the cell line used in the assay (BG1Luc4E2). Compounds that interact with 

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estrogen-responsive DNA are identified by the luciferase gene response and quantified in terms 

of Relative Light Units (RLU). 

The BG1Luc4E2 cells were grown in medium for four days, then the assay medium was 

replaced, and cells were transferred (2 × 105

 cells/mL) into 96-well plates and incubated at 37 ̊C 

with 5% CO2 for 24 hours. The medium was removed, and the test compound or estrogen 

standard (E2) was added. After incubation at 37 ̊C with 5% CO2 for another 24-hour period, the 

medium was removed and the cells were lysed. Luciferase activity was measured with a 

luminometer in order to compare treatment RLU to the E2 standard. For comparative purposes, 

the EC10 (Effective Concentration at which 10% of the maximum response was observed) was 

used. The data were not sufficient to allow calculation of an EC50. Although the article does not 

specifically identify the E2 standard, the reviewers assumed that E2 referred to 17β-estradiol. 

Acephate was reported negative 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; determination of the uterine 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 skeletal defects. 

No mortality was observed in the study. The maternal 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 high-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- and high-dose for GD 6 to 20 and 0 to 20 periods). 

Decreased (p≤0.01) food consumption also was observed during 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 consumption, and reduced food efficiency. 

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

was ≥92% across all groups. In addition, no treatment-related effects were observed in the 

external or soft-tissue examinations. 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 decreased 

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(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 numbers 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 ovulation) 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 results of the pilot study in which doses of 3, 10, 30 and 

100 mg/kg/day of acephate were tested; (40% deaths 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; determination of the uterine weights, number of 

implantations, 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 skeletal defects. 

Two rabbits in the high-dose group aborted and 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 (fetotoxic/teratogenic) 

parameters examined. Based on 2/16 (12.5%) abortions 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 toxicity is >10 mg/kg/day; a LOAEL is not 

established. 

Subchronic Neurotoxicity in Rodents (Rat) 

In a subchronic neurotoxicity study (MRID 44203304), SD rats (30/sex/dose) were administered 

acephate (99% purity) in the diet 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 neuropathology evaluations [Functional Observational Battery (FOB) 

and Locomotor Activity assessments]. In addition, other parameters determined included clinical 

toxicity/mortality, body weight, food consumption, 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-dose males and females (Week 1). No effects on 

body weight/body weight gain were observed in the low- and mid-dose animals, and no effects 

on food consumption were noted in any study groups. No treatment-related effects on FOB 

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parameters were observed during the study, but some ambulatory and 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 observed occasionally in low-dose animal brain 

regions. Inhibition (p<0.01 or 0.05) of RBC ChE activity was observed 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 inhibited (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 absolute brain and brain region weights due to acephate. The 

NOAEL for systemic toxicity and behavioral parameters 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 plasma 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 oral 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 allowed 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 sacrificed 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. Selected subsets were examined for detailed clinical and functional 

observational battery (FOB), motor activity, auditory startle habituation, passive avoidance, and 

water maze learning and memory tests, brain 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 maternal mortality, clinical signs, abbreviated 

functional observations, body weight, food consumption, 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 startle reflex, learning and 

memory, brain weights, brain morphology or neuropathology. Assessment for motor activity 

revealed dose-related decreases (NS) in the number of movements (↓19% at 1 mg/kg/day to 

↓30% at 10 mg/kg/day) that was accompanied by comparable dose-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. 

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No treatment-related inhibition was 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 offspring LOAEL is 0.5 mg/kg/day (LDT), based on 

dose-dependent inhibition of brain ChE activity in male and female pups on Day 21. An 

offspring NOAEL is not established. 

Multi-Generation Reproduction in Rodents (Rat) 

In this multi-generation reproduction study (MRIDs 40323401; and 40605701), SD rats, 

30/sex/group, were fed diets containing acephate (98.7% purity) for 75 days before they were 

bred to produce the F1a, and later the F1b, litters. Pups from the F1b litters were selected to 

produce the F2a and F2b litters. Because of low fertility in all groups, including control, in the 

F1b and F2b litters, a third generation (F3) was produced from the F2b litters. All rats were 

continuously exposed to the test material or the control diet either directly 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 reproduction study in rats (MRID 00129508) that did 

not determine a reproductive NOEL. These doses were considered equivalent to 0, 1.25, 2.5 and 

25 mg/kg/day, respectively, by using the FDA/HEW 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 included: (1) Decreased 

(significance not reported) body weights (<10%) and/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 females during the premating period and decreased 

food consumption for females during the gestation and lactation periods (highly variable); (3) 

Minimal clinical signs in males [increased incidence of alopecia in the first generation and 

increased incidence of soft or liquid stools in the second and third generations]; (4) Decreased 

mating performance (60%, p<0.01) for the F2b parental 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 histopathological lesions were noted for any parental 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 lactation, 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. Based 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. 

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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, with 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 low-, mid-, and high-dose groups, respectively. 

Parameters examined were those routinely examined in a multi-generation rat reproduction 

study. Organ weights and histopathology were determined in the F0 and F1 animals and included 

testes, seminal vesicles, prostate, uterus, ovaries, pituitary, liver, kidneys, and thyroid and 

adrenal glands. 

Mortality was observed in three F0 (low- and high-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 pregnancy and post-lactation) were generally ≤10%; weights in 

pregnant F0 and F1 high-dose females (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 minimal, 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 and 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 weight 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 with low-dose dams and pups, a NOAEL was not 

determined in this study; the LAOEL is the low dose, 50 ppm (approximately 3 to 4 mg/kg/day). 

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat) 

In a chronic feeding/carcinogenicity study (MRID 00101623), SD rats (75/sex/group) received 

acephate (92.5% purity) in the diet for 28 months at nominal doses of 0, 5, 50 and 700 ppm. 

These doses were estimated to be equivalent to 0, 0.25, 2.5 and 35.0 mg/kg/day, respectively 

(FDA/HEW conversion factor). No justification was presented for the selection of doses. 

Contamination of the control diet with acephate 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 oral toxicity/carcinogenicity study. Absolute and 

relative (% of body weight) weights of organs were determined, including adrenals, kidneys, 

liver, testes/ovaries, and thyroid gland, for all scheduled sacrifices; histopathology was 

conducted on these tissues as well as epididymides, mammary gland, pituitary, prostate, and 

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uterus with cervix. Plasma and RBC ChE activities 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 Weeks 7 and 19, and at 12, 22 and 28 months. 

No treatment-related effect on mortality was observed 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 the 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 sampling 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% (occasionally 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 sampling times in the high-dose males (21–67%) and 

females (21–61%). In the mid-dose groups, RBC ChE 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 corresponding values for the female rats were 

1 to 13%, 33 to 45% and 66 to 83%, respectively. 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 medullary 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-related 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 possibility of feeding the wrong diets was discussed 

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due to the high variability in analytical measurements. No explanation was given for the 

selection of dosages. Ten mice/sex/group were sacrificed after 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 which 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 weights were decreased (>10%; p≤0.01 or NS) in high-dose 

males and females from Week 2 to study termination (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 observed at the terminal sacrifice. The incidence of hyperplastic 

nodules in the control and treatment groups 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 controls (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 cavity (acute rhinitis) in the mid- and high-dose 

males and females; and (3) organ weight 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 females, 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), beagle dogs (5/sex/group) were administered 

acephate (99.9% purity) in the 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) with 

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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 included 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 uterus with cervix. Plasma and RBC ChE activities 

were determined for all dogs during 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 inhibition 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 the 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 and high-dose groups of both sexes, symptoms 

usually associated with ChE inhibition 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 infiltration 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 histological changes in the 

liver, the LOAEL and NOAEL for systemic effects are 800 ppm (20.16 mg/kg/day; HDT) and 

120 ppm (3.11 mg/kg/day), respectively (both sexes). The LOAEL and NOAEL for the 

inhibition of brain ChE activity in females are 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 both 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/day, and a LOAEL is not 

determined (>20.16 mg/kg/day). 

Mechanistic Study: Special Cholinesterase Inhibition (Rat) 

In a cholinesterase inhibition oral toxicity study (MRID 40504819), 45-day old SD rats 

(30/sex/dose) were administered acephate (98.2% purity) in the diet at nominal doses of 0, 2, 5, 

10, and 150 ppm (mean analytical values 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, respectively). 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 determined included clinical toxicity/mortality, body 

weight, and a general necropsy examination. 

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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 activity was inhibited (generally 

p<0.01) in all higher dose groups at each sampling 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 determinations, and may have impacted statistical 

comparisons. The NOEL for RBC and plasma ChE 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 study (<2 ppm), due to inhibition observed at 

0.12/0.15 mg/kg/day (LOAEL). 

Comparative Cholinesterase Assay 

Aa series of special comparative cholinesterase 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 gavage 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); two 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 oral doses of 0, 0.5, 2.5, 5, or 10 mg/kg and observed for 24 hours 

for clinical signs and mortality. In the same study (MRID 46151804), groups of 4 PND 11 

pups/sex were given 11 repeat doses, PND 11 through 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 given single oral doses of 0, 0.5, 1, 2.5, or 10 mg/kg and sacrificed 3 

hours later (time of peak effect). In the definitive repeat 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 ChE 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 survived to scheduled sacrifice. No treatment-related 

clinical signs of toxicity were observed in any animal during the studies. No treatment-related 

lesions were observed at necropsy. Body weight was comparable among the control and treated 

groups for both male and female adults and for both male and female pups in all studies (both the 

single and repeat-dose studies). Fetuses from treated dams had body weight comparable to those 

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in the control group. Reproduction performance, gestation, and implantation were not affected by 

treatment in dams, nor were litter size, viability, 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 compartment of most groups. At 1 mg/kg, brain 

enzyme activity was biologically or statistically 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 generally unaffected by acute 

treatment up to and including 10 mg/kg, the exception 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, and 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 affected at the same dose, 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 both 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 adults 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 relationship to sex or age. 

In the repeat-exposure gestation study, lack of a dose-response relationship between 0.5 and 1 

mg/kg/day for ChEI in several compartments for both dams and fetuses precluded establishing a 

LOAEL (of 0.5 mg/kg/day). 

For acute exposure, the overall adult LOAEL for cholinesterase inhibition in rats is 1 mg/kg 

based on enzyme inhibition in brain; 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 following 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 still 

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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 variable 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 sensitivity in pups than in adults. The ChEI 

data for both dams and GD 21 fetuses were variable, but showed no greater susceptibility in 

brain enzyme activity of GD 21 fetuses when compared 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 mortalities were reported in the treatments (except for two 

incidences of prolapsed penis in the high treatment); however, slight, but significant, depressions 

in food consumption were reported in the low and high treatment. Significant 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 effects 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 egg 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. 

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APPENDIX 3: References Not Utilized in the Acephate WoE Analysis 

In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing 

Test Orders for the EDSP Tier 1 screening battery was made available in a Federal Register 

Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted 

as “other scientifically relevant information (OSRI) was provided in the Report of the Endocrine 

Disruptor Review Team (USEPA, 2010). 

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies 

previously submitted to the agency for registration/reregistration, published literature articles 

and/or Tier 1 assays. The agency also conducted a more recent search (2009 to 2014) of 

available scientific literature for any additional relevant information for their weight of evidence 

(WoE) evaluations. These articles were evaluated in accordance with the agencies Evaluation 

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

(http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species

_reregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance 

for considering and Using Open Literature Toxicity Studies to Support Human Health Risk 

Assessment (http://www.epa.gov/pesticides/science/lit-studies.pdf). 

The following published and unpublished references were considered for use in the WoE 

analysis for Acephate but were not utilized due to one or more of the following reasons: 1) the 

article was not available in English; 2) the compound of interest was not used in the study; 3) the 

test material was not adequately described; 4) a formulated end-use product or mixture of 

chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 

6)the experimental conditions were not adequately described; 7) only an abstract of the study 

was available; 8) the reference is a review article or book chapter and does not contain primary 

study data; 9) insufficient information was available to adequately assess the validity of the study 

results; 10) the 40 CFR Part 158 guideline study was classified as unacceptable/inadequate; 11) 

the study dealt only with non-EDSP assay development; 12) no specific endocrine-related 

endpoints were assessed in the study; and 13) the study contained only data on invertebrates. 

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. 

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Liu, X.J., Luo, Z., Zheng, J.L., and Xiong, B.X. (2013) Effects of waterborne acephate exposure 

on antioxidant responses and acetylcholinesterase activities 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. 

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