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 82
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 82
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: CHLORPYRIFOS 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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CHLORPYRIFOS FINAL 

ii 

TABLE OF CONTENTS 

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

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

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

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

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

B. Other Scientifically 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..................................................15 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................21 

IV. Committee’s Assessment of Weight of Evidence..........................................25 

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................25 

1. Tier 1 in vivo Assays.............................................................26 

2. OSRI...............................................................................26 

B. Estrogen Pathway...........................................................................31 

C. Androgen Pathway.........................................................................31 

D. Thyroid Pathway...........................................................................32 

E. Conclusions.................................................................................32 

V. EDSP Tier 2 Testing Recommendations....................................................32 

VI. References.......................................................................................33 

LIST OF TABLES: 

 Table 1. Tier 1 Assays for Chlorpyrifos................................................................7 

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

 Table 3. Androgenic/Anti-Androgenic Pathway for Chlorpyrifos ..............................17 

 Table 4. Thyroid Pathway for Chlorpyrifos ........................................................23 

APPENDIX 1. EDSP Tier 1 Screening Assays.........................................................38 

APPENDIX 2. Other Scientifically Relevant Information (OSRI).................................49 

APPENDIX 3. References Not Utilized in the Chlorpyrifos WoE Analysis......................70 

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CHLORPYRIFOS FINAL 

iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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CHLORPYRIFOS FINAL 

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 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

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CHLORPYRIFOS FINAL 

v 

Abbreviation Terminology 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

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CHLORPYRIFOS FINAL 

vi 

Abbreviation Terminology 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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CHLORPYRIFOS FINAL 

Page 1 of 74 

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-pituitarygonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In addition to the available Tier 1 

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 chlorpyrifos interacts with E, A or T hormone pathways, the number and 

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

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

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

general systemic toxicity or overt toxicity. 

On May 21, 2014, the 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 (WoE) of the potential interaction of chlorpyrifos with 

the E, A or T hormone pathways. The T1WoERC conclusions from the WoE evaluation in this 

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

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

mammalian and wildlife studies (40 CFR Part 158 and literature) 

The Tier 1 Estrogen Receptor (ER) binding assay was negative. However, there were positive 

responses in the Tier 1 ER transactivation assay (ERTA) as well as multiple ERTA studies from 

the published literature. In the steroidogenesis assay, estradiol levels were increased at the two 

highest concentrations (10-5 to 10-4 M) of chlorpyrifos. However the in vitro effects were not 

supported by higher-tiered mammalian data such as the uterotrophic, the female pubertal and the 

fish short term reproduction assay (FSTRA). There were no estrogen-related effects observed in 

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

in the mammalian Part 158 studies in the absence of systemic toxicity. In the FSTRA, while 

there was a significant decrease (52-71%) on fecundity, there were no treatment-related changes 

in estrogen-related endpoints such as ovarian histopathology, GSI, plasma vitellogenin or plasma 

17β-estradiol; reductions in brain cholinesterase activity were observed at all test concentrations. 

In the Part 158 avian reproduction studies (n=4), all effects occurred in the presence of overt 

toxicity, except for a reduction in eggs laid for one of the studies where decreases in food 

consumption were observed. There were no effects on reproductive endpoints in the Part 158 

fish life-cycle study. Therefore, there is a lack of convincing evidence for potential interaction 

with the estrogen pathway in mammals and wildlife. 

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CHLORPYRIFOS FINAL 

Page 2 of 74 

For the androgen pathway, there was an equivocal finding (at 10-4 to 10-3 M) in the Tier 1 AR 

binding assay, and in the steroidogenesis assay, testosterone levels were decreased at the high 

test concentrations (10-5 to 10-4 M). However, these positive in vitro findings were not supported 

across the body of the higher-tiered in vivo data. The Tier 1 Hershberger assay was negative, and 

in the male pubertal assay, there were no treatment-related changes in age or body weight at 

preputial separation, weights of the testes, accessory sex tissue weights, pituitary weight, serum 

testosterone levels, or testicular and epididymal histopathology. In an OSRI study (Joshi et al. 

2007), effects on androgen-related endpoints were observed (decreased testosterone, decreased 

testes weight/altered histopathology, and altered fertility). However, signs of toxicity were 

reported at all doses tested. Furthermore, although ChE activity was not determined in this study, 

the dose levels administered (7.5, 12.5, 17.5 mg/kg/day) have been observed to cause significant 

inhibition of ChE activities in other studies (e.g. male pubertal assay at 2 mg/kg/day). In the 

FSTRA, there were no treatment-related effects in androgen-related endpoints. There were no 

androgen-related effects in the Part 158 mammalian studies in the absence of overt toxicity; there 

were also no androgen-related effects observed in the Part 158 avian or fish reproduction studies. 

Therefore, while in vitro studies indicate some potential androgen-related effects, no significant 

corroborating effects were observed in the in vivo studies in the absence of systemic (e.g., 

cholinesterase inhibition) or overt toxicity. 

There were no treatment-related changes in any of the thyroid parameters (i.e., thyroid weight, 

serum thyroid hormone levels or histopathology) in the male and female pubertal assays at doses 

that caused significant cholinesterase inhibition. While developmental delays were observed in 

the AMA, the lack of effects on thyroid histopathology along with a reduction in growth do not 

support a potential thyroid-related effect. Furthermore, no agonist or antagonist activity was 

seen in a receptor transactivation assay using thyroid receptor transfected Vero cells. No thyroidrelated effects were seen in the mammalian Part 158 studies. Although there were reported 

effects on thyroid hormone and histopathology in the De Angelis et al. (2009), there is less 

confidence in these findings due to uncertainty in the experimental design. Overall, based on the 

available information the evidence does not support a potential interaction with the thyroid 

pathway. 

Based on weight of evidence considerations, EDSP Tier 2 testing is not recommended for 

chlorpyrifos since there was no evidence of potential interaction with the estrogen, androgen and 

thyroid pathways. 

 

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CHLORPYRIFOS FINAL 

Page 3 of 74 

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-pituitarygonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In addition to the available Tier 1 

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 chlorpyrifos interacts with E, A or T hormone pathways, the number and 

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

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

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

general systemic toxicity or overt toxicity. 

On May 21, 2014, the 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 Chlorpyrifos with 

the E, A or T hormone pathways. 

The water solubility of chlorpyrifos is 1.4 mg/L at 25°C. The vapor pressure of chlorpyrifos is 

2.02 x 10−5 mm Hg at 25°C. In the environment, the major route of dissipation appears to be 

aerobic and anaerobic biodegradation. Abiotic hydrolysis under neutral and acidic conditions, 

photodegradation, and volatilization do not seem to play a significant role in the dissipation 

process. Under alkaline conditions (pH 9), hydrolysis of chlorpyrifos occurs more rapidly with a 

half-life of approximately 2 weeks. Based on available data, chlorpyrifos appears to degrade 

slowly in soil under both aerobic and anaerobic conditions. However, the persistence appears to 

vary over about two orders of magnitude (from a few days to well over 100 days and typically 

greater than 200 days for termiticidal uses) depending on soil type, environmental conditions and 

possibly previous use history at the treatment site. Information on leaching and 

adsorption/desorption indicate that parent chlorpyrifos is largely immobile. Volatilization from 

soil and water surfaces is expected to occur slowly; however, chlorpyrifos has been detected in air 

samples several kilometers from its application site suggesting that some volatilization and 

atmospheric transport may occur. 

The available information considered in determining the potential interaction of chlorpyrifos 

with the E, A and/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 

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submitted to the Agency, using the WoE approach outlined by the Agency (USEPA, 2011), is 

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

II. Sources of Scientific Data and Technical Information 

A. EDSP T1 Screening Assays 

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

agency’s test order for chlorpyrifos 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 

Screening 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 chlorpyrifos (USEPA, 2010). Since 

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

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

Additionally, literature/studies considered but not utilized for the WoE analysis are listed in 

Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

The principles, criteria, and approach used in the WoE determination on the potential of a 

substance to interact with endocrine-related processes (i.e., E, A or T hormone pathways) were 

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

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

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

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

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

foundation for WoE evaluations. The first step is to assemblethe 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 concordance 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. 

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

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

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

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

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

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

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

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

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

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

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

endpoints are measured in each assay. 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

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

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

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

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

biological organization were the responses observed and whether organisms exhibit 

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

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

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

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Page 6 of 74 

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

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

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

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

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

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

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

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

related to toxicity in 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 components discussed 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 T1 Screening Assays 

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

below in Table 1. 

Table 1. Tire 1 Screening Assays for Chloropyrifos 

Tier 1 Assays Test Guideline Test Order Status 

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied (MRID 

No. 48615502) 

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

No. 48615504) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OSCPP 890.1300; 

OECD 455 

Requirement Satisfied (MRID 

No. 48615505) 

Steroidogenesis Assay (Human cell line 

H295R) 

OSCPP 890.1550; 

OECD 456 

Requirement Satisfied (MRID 

No. 48615510) 

Aromatase Assay (human recombinant 

microsomes) OSCPP 890.1200 Requirement Satisfied (MRID No. 48615503) 

Uterotrophic Assay (Rat) OSCPP 890.1600; 

OECD 440 

Requirement Satisfied (MRID 

No. 48615511) 

Hershberger Assay (Rat) OSCPP 890.1400; 

OECD 441 

Requirement Satisfied (MRID 

No. 48615507) 

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied (MRID 

No. 48615508) 

Pubertal Male Assay (Rat) OSCPP 890.1500 

Requirement Satisfied by OSRI 

(Joshi et al., 2007 and De 

Angelis et al., 2009; however, a 

Tier 1 assay was submitted 

(MRID No. 48615509) 

Fish Short-term Reproduction Assay OSCPP 890.1350; 

OECD 229 

Requirement Satisfied (MRID 

No. 48615506) 

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; 

OECD 231 

Requirement Satisfied (MRID 

No. 48615501) 

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

1. Effects on Estrogen Pathway 

The potential interactions of chlorpyrifos with the estrogen pathway are 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. 

Chlorpyrifos was classified as not interactive (negative) in the Tier 1 in vitro estrogen receptor 

(ER) binding assay, and as negative in the Tier 1 aromatase assay up to the limit concentration (1 

mM). As summarized below, chlorpyrifos was positive in the Tier 1 ERα transcriptional 

activation assay and positive in several ER transcriptional activation assays considered as OSRI. 

In the Tier 1 ERTA, the mean RPCmax was 25.4% in the first run, 10.2% in the second run and 

19.5% in the third run; the associated PCmax was 10−4 M for the first and third runs, and 10−5 M 

for the second run. Because the RPCmax ≥PC10 in all three runs, chlorpyrifos was considered 

positive for estrogen receptor transcriptional activation. 

In a published literature study, Kojima et al. (2004) screened chlorpyrifos for its potential to 

interact with two human estrogen receptor subtypes (hERα and hERβ) in transactivation assays 

in Chinese hamster ovary cells (CHO-K1 cells). In this study, chlorpyrifos was reported to be 

positive for ERα (agonist) transactivation with a REC20 value of 7.5 x 10-6 M and negative as an 

ERα antagonist. Chlorpyrifos was also negative for ERβ activity (agonist and antagonist). Howe 

In another study (Andersen et al., 2002), chlorpyrifos was positive in a non-guideline ERαtranscriptional activation assay in MCF7 cells. In this study, chlorpyrifos induced a weak 

estrogenic response in both the cell proliferation assay (36% of the maximum 17β-estradiol 

response at 50 μM), and the ERTA (25% of the maximum 17β-estradiol response at 50 μM), but 

had no antagonistic effects on 17β-estradiol-induced cell proliferation. However, in a published 

study by Soto et al. (1995), chlorpyrifos did not induce a proliferative response in MCF-7 cells. 

In a third non-guideline ERTA study (Sun et al., 2012), chlorpyrifos demonstrated antagonistic 

activity in an ER reporter gene assay. Chlorpyrifos had no estrogenic activity at 0.003-3.0 mg/L 

but did show significant suppression of luciferase at 0.3 and 3.0 mg/L compared to estradiol, 

suggesting antagonistic activity. The relative inhibitory concentration (RIC)20 for chlorpyrifos 

was 1.15×10-6 M. Finally, in a fourth non-guideline ERTA, chlorpyrifos did not activate an ER 

response in a reporter gene assay system in human ovarian carcinoma (BG1) cells (Kojima et al., 

2005). 

Page 16 of 82
CHLORPYRIFOS FINAL 

Page 9 of 74 

Chlorpyrifos was classified as positive in the in vitro Tier 1 steroidogenesis assay, increasing 

estradiol production 2.3- to 2.4-fold at 10 and 100 μm in all three runs. 

No estrogen-related findings were reported in either the female pubertal assay or the uterotrophic 

assay. 

In the FSTRA, there were reductions (p<0.05) in fecundity of 52-71% at all treatment levels, 

compared to the negative control, but fertility was not affected. There were no changes in 

plasma vitellogenin (VTG) or estradiol concentrations in females nor treatment-related effects on 

gonadal histopathology or GSI. There was significant decreases in cholinesterase (40 to 92%) 

within brain tissue of females at all treatment levels. At the highest dose (3.02 μg/L), male body 

weights were significantly reduced (17%); survival was not affected at any treatment. Clinical 

signs of toxicity, including injury to eyes and snout (along with females reported as bloated or a 

side injury) were reported at this concentration, however, similar effects and incident rates were 

reported in the control fish and other treatment groups, therefore it is uncertain if these clinical 

signs are treatment-related. 

In a Part 158 developmental neurotoxicity in rats, a minor 1-day increase in age at VO was 

observed (PND 33.4 treated vs. PND 32.4 controls) at the high dose (5 mg/kg/day); however, 

this finding occurred after reduced body weight gains were noted (↓30%; PND 18-22). In a 

developmental toxicity study in rabbits, post-implantation loss was increased (p<0.001; 13.9% 

treated vs. 6.5% controls) at the high dose (140 mg/kg/day); however, these animals exhibited a 

mean body weight loss of 99 g during dosing (GD 7-19). There were no estrogenic/antiestrogenic effects noted in the other Part 158 studies. Additionally, no effects on the estrogen 

pathway were observed in a modified developmental toxicity study in mice (De Angelis et al., 

2009). 

In the Part 158 bobwhite quail study, no treatment-related reproductive effects were noted at 

dietary levels up to 125 ppm; however, in the second quail study, there was a significant 27% 

reduction in the number of eggs laid at the high dose (130 mg/kg-diet); a reduction in parental 

food consumption was reported at this dose, however, there were no effects on body weight, 

mortality or clinical signs of toxicity. In a mallard duck reproduction study, significant decreases 

in egg laid (84%), viable embryos/hatchlings, and 14-d old offspring (92%) were observed along 

with significant decreases in egg weight (14%) and eggshell thickness (9%) at 125 mg/kg-diet. 

However, at this dose, adults exhibited locomotor dysfunction, reduced food consumption and 

body weights, and 8 mortalities (out of 35 exposed) were observed. In the second mallard duck 

study, there was a significant reduction (46%) in the number of eggs laid at the high dose (90/60 

mg/kg-diet). However, at this dose, there was treatment-related mortality and body weight 

decreases. 

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CHLORPYRIFOS FINAL 

Page 10 of 74 

In a Part 158 fathead minnow fish life cycle toxicity study, there were no treatment-related 

effects on reproduction; survival of young fry were significantly reduced in the F0 and F1 

generation at the highest treatment (1.09 μg/L). Three Part 158 early life-cycle studies with 

saltwater fish were available, Menidia beryllina, M. menidia, M. peninsulae. Effect on larvae 

survival were reported at concentrations ≥0.48 μg/L; effects on body weight were reported at 

higher concentrations than for survival. 

Page 18 of 82
CHLORPYRIFOS FIN

Page 11 of 74 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Chlorpyrifos 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Chlorpyrifos1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

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 48615504) N ERTA (MRID 48615505) P Aromatase (MRID 48615503) N Steroidogenesis (MRID 48615510) 

P

Uterotrophic (MRID 48615511) N Female Pubertal Rat (MRID 48615508) N N N N N N FSTRA (MRID 48615506) N N N N 

Fcd 

↓52-71% 

(L, M, H)

OSRI 

ER Transactivation (Andersen et al., 2002) P ER Transactivation (Kojima et al., 2004) PER Transactivation (Kojima et al., 2005) N ER Transactivation (Sun et al., 2012) P 
CHLORPYRIFOS FIN

Page 12 of 74 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Chlorpyrifos 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Chlorpyrifos1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

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) 

Two-generation reproduction (Rat; MRID 41930301) 

NE NE N NE N N 

Developmental neurotoxicity (Rat; MRID 44556901) 

N N 

Developmental toxicity (Rat; MRID 00130400) 

NE N 

Developmental toxicity (Rat; MRID 40436407) 

 NE N 

Developmental toxicity (Rabbit; MRID 40436408) 

 NE N 

Chronic toxicity/ carcinogenicity (Rat; MRID 40952802) 

NE N N NE 

Oncogenicity (Mouse; MRID 00054352) NE NE N NE Carcinogenicity (Mouse; MRID 42534201) 

NE NE N NE 

Subchronic toxicity (Rat; MRID 40436406) 

NE NE N NE 

Subchronic toxicity (Rat; MRID 40952801) 

 NE N N NE 
CHLORPYRIFOS FIN

Page 13 of 74 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Chlorpyrifos 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Chlorpyrifos1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

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) 

Subchronic inhalation toxicity (Rat; MRID 40013901) 

NE NE N NE 

Avian reproduction (Quail; MRID 00046957) 

N 

Avian reproduction (Quail; MRID 42144902) 

P5 

(H) 

Avian reproduction (Duck; MRID 00046952) 

N 

Avian reproduction (Duck; MRID 42144901) 

N 

Fish Full Life Cycle Toxicity (Minnow, MRID 42834401) 

N 

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

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

weight are absolute unless otherwise indicated. ChE=Cholinesterase inhibition 

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. At the highest dose (3.02 μg/L), male body weights were significantly reduced (17%); survival was not affected at any treatment. C

including injury to eyes and snout (along with females reported as bloated or a side injury) were reported at this concentration, howincident rates were reported in the control fish and other treatment groups, therefore it is uncertain if these clinical signs are treatm

5. Significant reduction of 27% in the number of eggs laid at the high dose (130 ppm) 
CHLORPYRIFOS FIN

Page 14 of 74 

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

NE Not examined 
CHLORPYRIFOS FINAL 

Page 15 of 74 

2. Effects on Androgen Pathway 

The potential interactions of chlorpyrifos with the androgen pathway are 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. 

Chlorpyrifos was classified as equivocal in the in vitro androgen receptor (AR) binding assay, as 

average ligand binding was reduced by 42 and 25% at concentrations of 10-3 M and 10-4 M, 

respectively; no effect on AR binding was noted at concentrations ≤10−5 M. In the Tier 1 

steroidogenesis assay, significant inhibition of testosterone production (0.4- to 0.6-fold) was 

observed at 10 and 100 μM. Three published studies were also available examining effects of 

chlorpyrifos on AR transactivation and/or steroidogenesis. Although chlorpyrifos had no agonist 

activity in an AR transcriptional activation (ARTA) assay using Chinese hamster ovary (CHO) 

cells at 10-8 to 10-3 M (Andersen et al., 2002) , chlorpyrifos demonstrated significant antagonist 

activity in an ARTA assay (Sun et al., 2012) in AR transfected Vero cells using testosterone as 

the positive control. No effect was seen in the ARTA assay using CHO-K1 cells treated with up 

to 10-5 M chlorpyrifos (Kojima, 2004).

In contrast, chlorpyrifos was negative in vivo for both androgenic and anti-androgenic activity in 

the Hershberger assay, and no androgen-related effects were observed in the male pubertal assay 

up to doses that resulted in significant inhibition of both brain and RBC cholinesterase activity (≥

2 mg/kg/day). In the FSTRA, there were no treatment-related effects on male GSI, VTG, or 

fertility. There was significant decreases in cholinesterase in brain tissue of males (72% and 

90%) at the 0.000812 and 0.00302 mg ai/L treatment groups, respectively. While not significant, 

a 65% decrease in cholinesterase activity in males was observed at the lowest treatment group 

compared to the control. At the highest dose (3.02 μg/L), male body weights were significantly 

reduced (17%); survival was not affected at any treatment. Clinical signs of toxicity, including 

injury to eyes and snout (along with females reported as bloated or a side injury) were reported at 

this concentration, however, similar effects and incident rates were reported in the control fish 

and other treatment groups, therefore it is uncertain if these clinical signs are treatment-related. 

In a developmental neurotoxicity study in rats, there was a minor delay (2.8 d) in preputial 

separation at the high dose (5 mg/kg/day); however, the delay was also accompanied by a 

reduction in body weights for male pups (↓11-17%; PND 22-66). There were no androgenrelated effects observed in the other Part 158 mammalian studies. 

In addition to the Part 158 toxicity studies, three published studies were reviewed that examined 

effects of chlorpyrifos on testes and reproductive parameters in rodents. In a testicular toxicity 

study (Joshi et al., 2007), male rats dosed for 30 days by oral gavage with chlorpyrifos (purity 

Page 23 of 82
CHLORPYRIFOS FINAL 

Page 16 of 74 

not reported) at 12.5 or 17.5 mg/kg/day (mid and high doses) dose-dependently decreased serum 

testosterone levels, testes weights, sperm counts in the testes and epididymis, and sperm motility, 

resulting in reduced fertility in males. Additionally, histopathology of the testes showed severe 

degenerative changes marked with damaged epithelium, irregular and incomplete 

spermatogenesis and lumen filled with cellular debris and devoid of sperm. No mortality or 

changes in body weights were observed in this study; however, it was reported that all treated 

animals exhibited toxicity symptoms (types of symptoms not reported). ChE inhibition was not 

evaluated in the study. In another study examining reproductive toxicity in male mice following 

oral dosing with chlorpyrifos at 5, 15 or 25 mg/kg/day for 4 weeks (Farag et al., 2010), serum 

testosterone levels were decreased at the mid and high doses and the weights of the testes and 

several secondary accessory sex tissues were decreased at the high dose. In addition, male 

fertility was reduced at the mid and high doses, and histopathological changes were noted in all 

treatment groups. However, clinical signs of toxicity (tremors) were observed at the mid and 

high dose and decrease body weight (21%) was observed at the high dose. 

In the Part 158 avian reproduction toxicity studies (two bobwhite quail and two mallard duck), 

there were no androgen-related effects observed. In a Part 158 fathead minnow fish life cycle 

toxicity study, there were no treatment-related effects on reproduction; survival of young fry 

were significantly reduced in the F0 and F1 generation at the highest treatment (1.09 μg/L). 

Page 24 of 82
CHLORPYRIFOS FINA

Page 17 of 74 

Table 3: Androgenic/Anti-Androgenic Pathway for Chlorpyrifos Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Chlorpyrifos1Study 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 

Vitellogenin 

EDSP Tier 1 Assay 

AR Binding (MRID 48615502) E Steroidogenesis (MRID 48615510) P Hershberger (MRID 48615507) NE N Male Pubertal Rat (MRID 48615509) N N N N N N N N FSTRA (MRID 48615506) N N N N N N 

OSRI 

AR Transactivation (Andersen et al., 2002) 

N

AR Transactivation (Kojima et al., 

2004) 

N

AR Transactivation (Sun et al., 2012) P Two-Generation Reproduction (Rat; MRID 41930301)

N NE N NE N NE N 
CHLORPYRIFOS FINA

Page 18 of 74 

Table 3: Androgenic/Anti-Androgenic Pathway for Chlorpyrifos Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Chlorpyrifos1Study 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 

Vitellogenin 

Testicular Toxicity (Rat; Joshi et al., 

2007) 

↓

(M. H) 

↓18, 

28% 

(M, H)

P 5

(M, H) NE P 5 (M, H) NE NE Frt: 

↓80, 85% 

(M, H) 5

Male Reproductive Toxicity (Mouse; Farag et al., 2010) 

N N 

P7

(L) N N NE N N 

Developmental Neurotoxicity (Rat; MRID 44556901) 

NE NE NE NE NE NE 

N 

Chronic Toxicity / Carcinogenicity (Rat; MRID 40952802) 

N N NE N NE NE 

Carcinogenicity (Mouse; MRID 00054352) 

N N NE N NE NE 

Carcinogenicity (Mouse; MRID 42534201) 

N NE N NE NE NE 

Subchronic Toxicity (Rat; MRID 40436406) 

N N NE NE NE NE 

Subchronic Toxicity (Rat; MRID 40952801) 

N N NE N NE NE 

Subchronic Inhalation Toxicity (Rat; MRID 40013901) 

N N NE NE NE NE 

Avian reproduction (Quail; MRID 00046957) 

 N 
CHLORPYRIFOS FINA

Page 19 of 74 

Table 3: Androgenic/Anti-Androgenic Pathway for Chlorpyrifos Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Chlorpyrifos1Study 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 

Vitellogenin 

Avian reproduction (Quail; MRID 42144902) 

 N 

Avian reproduction (Duck; MRID 00046952) 

 N 

Avian reproduction (Duck; MRID 42144901) 

 N 

Fish Full Life Cycle Toxicity (Minnow, MRID 42834401) 

 N 

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

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

Changes in weight are absolute unless otherwise indicated. ChE=Cholinesterase inhibition 

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 

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 th

Section IV. A 

4. At the highest dose (3.02 μg/L), male body weights were significantly reduced (17%); survival was not affected at any treatment. Clinica

including injury to eyes and snout (along with females reported as bloated or a side injury) were reported at this concentration, however, incident rates were reported in the control fish and other treatment groups, therefore it is uncertain if these clinical signs are treatment-re

5. At the mid and high doses (12.5 and 17.5 mg/kg/day), histopathology of the testes showed severe degenerative changes marked with dam

irregular and incomplete spermatogenesis, and lumen filled with cellular debris and devoid of sperm. Sperm motility in the cauda epidid

(p<0.05), and testes and epididymal sperm counts were decreased (p<0.05; ↓71-80% and 41-67%, respectively), resulting in reduced fert

6. No mortality or changes in body weights were observed in this study, however it was reported that all treated animals exhibited toxicity 

symptoms not reported). 

7. At the low dose (5 mg/kg/day), mild changes in spermatogenic cells in the testis were noted in the form of focal areas of irregular semin

and wide separation of seminiferous tubules were also noted. Mild to severe vacuolation of the epithelium lining and congestion of blood 

tubules were noted in a dose-dependent manner. 
CHLORPYRIFOS FINA

Page 20 of 74 

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

E Equivocal assay response NE Not examined 
CHLORPYRIFOS FINAL 

Page 21 of 74 

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 interactions of chlorpyrifos 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. 

There were no effects of chlorpyrifos noted on serum T4 or TSH levels, pituitary or thyroid 

weights, or thyroid histopathology in either the male pubertal or female pubertal assays up to a 

dose of 2.0 mg/kg/day that resulted in significant reductions (≥22%) in brain cholinesterase 

activity. 

In the AMA, the highest test concentration of chlorpyrifos (13.6 μg a.i./L) was associated with a 

significant stage delay in 21-day NF developmental stage and a 17% reduction in normalized 

HLL. Body wet weight at day 21 was significantly reduced by 23 and 39% at the mid-high (3.68 

μg a.i./L) and high test concentrations. The 21-day SVL was also significantly reduced at the 

mid-low (0.881 μg a.i./L), mid-high, and high concentrations (3.7, 7.5, and 15.3%, respectively). 

Reductions in ChE activity from hind limb tissue was observed in all treatment groups on Day 21 

compared to the negative control. ChE activity was also reduced in tail tissue at the mid-high 

and high concentrations on Day 21. Several tadpoles were observed swimming erratically on 

Day 8 in the high concentration group. 

There were no thyroid-related effects observed in the mammalian Part 158 studies. In a journal 

article considered as OSRI, chlorpyrifos had no agonist or antagonist activity in a receptor 

transactivation assay (Sun et al., 2012) using thyroid receptor transfected Vero cells. In a 

modified developmental toxicity study (De Angelis et al., 2009), dams were dosed via oral 

gavage at 0, 3 and 6 mg/kg/day during gestation days 15-18. After delivery the pups were dosed 

via subcutaneous injection (sc) during postnatal days 11-14. T4 levels were decreased in dams at 

an oral gavage dose of 6 mg/kg, and microscopic pathology showed increased colloid foaminess 

(NS) and increased (p<0.05) follicular cell height in the thyroid. In PND 2 pups from dams 

dosed at 6 mg/kg/day, higher follicular density in the thyroid was noted (pups were not directly 

dosed until PND 11). Also at this dose, follicular size and in particular colloid area were 

reduced. In the PND 150 offspring, T3 levels in the 3 mg/kg/day (sc) dose were reduced 

(p<0.05) by 17% in males and increased (p<0.05) by 12% in females. Also, T4 levels in the 3 

mg/kg/day (sc) male were reduced (p<0.05) by 11%. Histopathology showed a dose-dependent 

Page 29 of 82
CHLORPYRIFOS FINAL 

Page 22 of 74 

higher number of necrotic follicular epithelial cells exfoliated into the lumen and colloidal fluid. 

Histomorphometry showed a dose-dependent decreased follicular density at 1 and 3 mg/kg/day 

dose in males. However, it is unclear how the dosing regimen were conducted (i.e. 3 and 6 

mg/kg/day orally for dams during gestation days 15-18 versus 1 and 3 mg/kg/day subcutaneously 

for pups on days 11-14). Therefore there is uncertainty in the experimental dosing design, 

particularly for the pups and consequently the reported results. Although only the findings in 

dams and PND 2 pups were considered of this evaluation, there is reduced confidence in the 

study. While there were no clinical signs of cholinergic inhibition in this study, ChE activity was 

not assessed. 

Several open literature studies on amphibian development for chlorpyrifos were available. While 

decreases in growth or development delays were reported in these some of these studies, as there 

were no measurements on thyroid or thyroid hormones, these studies are limited in their ability 

to inform about potential thyroid-interactions (e.g., Sparling and Fellers, 2009; Reylea 2009, 

Bonfanti et al. 2004; Richards and Kendall 2003). 

Page 30 of 82
CHLORPYRIFOS FINAL 

Page 23 of 74 

Table 4. Thyroid Pathway for Chlorpyrifos.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Chlorpyrifos1

Study Type/ 

Literature Citation 

Thyroid receptors Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, SVL) Systemic Toxicity2 

Overt Toxicity Observed3

EDSP Tier 1 Assays 

Male Pubertal Rat 

(MRID 

48615509) 

 N N N N N ↓ChE 

(L, M, 

H) 

N 

Female Pubertal 

Rat (MRID 

48615508) 

 N N N N N ↓ChE 

(L, M, 

H) 

N 

AMA 

(MRID 

48615501) 

N 

Day 21: 

↓NF stage 

(1 stage); 

HLL 

↓17% (H) 

Day 21: 

SVL: 

↓4-15% 

(ML, 

MH, H); 

BW: 

↓23-39% 

(MH,H) 

↓ChE 

(L, ML, 

MH, H) 

N4 

OSRI

Thyroid Receptor 

Transactivation 

(Sun et al., 2012) 

N 

Chronic Toxicity/ 

Carcinogenicity 

(Rat; MRID 

40952802) 

 NE N NE X 

(MH, H) N 

Carcinogenicity 

(Mouse; MRID 

00054352) 

 NE N NE N N 

Carcinogenicity 

(Mouse; MRID 

42534201) 

 NE N NE (L, M, H) N 

Subchronic 

Toxicity (Rat; 

MRID 40436406) 

 NE N NE 

X 

 (L, M, 

H) 

N 

Subchronic 

Toxicity (Rat; 

MRID 40952801) 

 NE N NE 

X 

(L, ML, 

MH, H) 

N 

Subchronic 

Inhalation 

Toxicity (Rat; 

MRID 40013901) 

 NE N NE N N 

Page 31 of 82
CHLORPYRIFOS FINAL 

Page 24 of 74 

Table 4. Thyroid Pathway for Chlorpyrifos.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Chlorpyrifos1

Study Type/ 

Literature Citation 

Thyroid receptors Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, SVL) Systemic Toxicity2 

Overt Toxicity Observed3

Modified 

Developmental 

Toxicity (Mouse; 

De Angelis et al., 

2009) 

 NE P5

(H) 

↓34% 

(H) NE NE N N 

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

MH=Medium-high treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the response. A 

shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. ChE= 

Cholinesterase inhibition. Medium-Low treatment (ML) Medium high treatment (MH) 

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. Several tadpoles were observed swimming erratically on Day 8 in the high concentration group 

5. In the high dose (6 mg/kg) dams, microscopic pathology showed increased (p<0.05) follicular cell height in the 

thyroid. 

P Positive findings 

N Negative findings 

NE Not examined 

 

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IV. Committee’s Assessment of Weight of Evidence 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

whether or not the responses are mediated through an endocrine pathway, therefore nonendocrine toxic responses (including but not limited to mortality or body weight changes) are 

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

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

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

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

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

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

2013). 

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

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

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

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

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

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

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

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

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

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1. Tier 1 in vivo Assays 

Systemic toxicity was not observed in the uterotrophic assay. In the Hershberger assay, there 

were no significant effects on body weights or body weight gains in the chlorpyrifos treated 

groups. Rats in the 6 and 12 mg/kg/day groups had increased (p<0.05) adrenal glands weights 

(↑15 and 20-38%, respectively); mean adrenal glands weight in the 1 mg/kg/day dose group was 

comparable to the controls. ChE activity in RBCs were significantly decreased (p<0.05) at 1 

mg/kg/day (90-91% inhibition) and below the level of detection at 6 and 12 mg/kg/day. Brain 

ChE activity levels were significantly decreased (p<0.05) at 6 and 12 mg/kg/day (60-64% and 

76-79% inhibition). 

In the pubertal rat assays, RBC ChE activity was decreased (p<0.05) in females compared to the 

control at 0.5 (↓19%) and 1.0 (↓88%) mg/kg/day, and completely inhibited at 2 mg/kg/day. 

Brain ChE activity was decreased (p<0.05) at 2 mg/kg/day (↓22%) compared to the control, but 

not affected at 0.5 or 1.0 mg/kg/day. In males, RBC ChE activity was dose-dependently 

inhibited (p<0.05) between 34-100% at all doses of chlorpyrifos. Brain ChE activity was also 

inhibited (p<0.05) by 23% at 2 mg/kg/day. 

In the FSTRA, male body weights were reduced by 17% in the 3.02 μg ai/L treatment relative to 

the negative control. The was significant decreases in ChE (40 to 92%) within brain tissue of 

females at all treatment levels and in brain tissue of males (72% and 90%) at the 0.812 and 3.02 

μg ai/L treatment groups, respectively. A 65% decrease (NS) in ChE in males was observed at 

the lowest treatment group compared to the control. Clinical signs of toxicity, including injury to 

eyes and snout (along with females reported as bloated or a side injury) were reported at this 

concentration, however, similar effects and incident rates were reported in the control fish and 

other treatment groups, therefore it is uncertain if these clinical signs are treatment-related. 

In the AMA, several tadpoles were observed swimming erratically on Day 8 in the 20 μg a.i./L 

treatment group; no other behavioral abnormalities or effects on survival were noted. Reductions 

of 21-79% (p<0.05) in ChE activity from hind limb tissue was observed in all treatment groups 

on Day 21 compared to the negative control. ChE activity was also reduced (p<0.05) in tail 

tissue in the 5 and 20 μg a.i./L treatment groups (28 and 68%, respectively) on Day 21. 

2. OSRI 

In a two-generation rat reproduction toxicity study, dose-related decreases (p<0.05) in plasma 

(↓43-64% males; ↓49-72% females) and RBC (↓67-70% males; ↓65-75% females) ChE activity 

were observed in the P and F1 adults at 1 and 5 mg/kg/day. In addition, brain ChE was inhibited 

in the high dose P and F1 males (↓48-53%) and females (↓49-58%). Additionally at the high 

dose, in the P females, very slight vacuolation (16/30 treated vs. 0 controls) consistent with 

altered tinctorial properties of the zona fasciculata (21/30 treated vs. 0 controls) of the adrenal 

gland was noted; altered tinctorial properties of the zona fasciculata (13/30 treated vs. 2/30 

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controls) of the adrenal gland was also noted in the F1 females. Very slight (19/30 treated vs. 

14/30 controls) to slight (5/30 treated vs. 1/30 control) vacuolation of the adrenal was observed 

in the adrenal glands of the P males; slight (6/30 treated vs. 1/30 controls) vacuolation of the 

adrenal glands was also found in the F1 males. It was stated that the histological changes were 

consistent with fatty changes in the males. Neonatal effects were observed only in the presence 

of maternal toxicity and consisted of reduced pup weights and increased mortality at 5 

mg/kg/day. 

In a male mouse reproductive toxicity study by Farag et al. (2010), one mid-dose (15 mg/kg/day) 

and three high-dose (25 mg/kg/day) males died during treatment. Tremors were observed on 

Day 10 in the mid-dose group (60% incidence) and on Day 5 in the high-dose group (80% 

incidence) and progressed in the same mice throughout the second half of the treatment period. 

Body weights, body weight gains, and food consumption were reduced (p≤0.05) at 25 mg/kg/day 

from Week 2 to the end of treatment, with terminal body weights decreased (p<0.05) by 21%. 

Brain and muscle ChE activities were significantly (p≤0.05) decreased in mice dosed at 15 

mg/kg/day (85% and 78%, respectively) and 25 mg/kg/day (73% and 68%, respectively). 

In a testicular toxicity study in rats by Joshi et al. (2007), there were no mortalities and body 

weights were unaffected by treatment (7.5, 12.5 or 17.5 mg/kg/day). However, levels of 

glycogen (↓63% and ↓88%) and sialic acid (↓15% and 25%) were decreased (p<0.05) in the 

testes, and cholesterol (↑42% and ↑58%) and protein (↑26% and ↑44%) were increased (p<0.05). 

When viewed as a whole, these finding were considered to be indicative of toxicity at a systemic 

level. Although ChE activity was not determined, the dose levels administered have been 

observed to cause significant inhibition of ChE activities in other studies. 

In a rat developmental neurotoxicity study, maternal toxicity at the high dose (5 mg/kg/day) was 

manifested at the end of gestation and during LD 1-5. Dams with all pups dying were increased 

at the high dose. Maternal brain ChE activity was decreased at the high dose (↓90%) and mid (1 

mg/kg/day) dose (↓18%) dams. RBC (↓41-99%) and plasma (↓43-92%) ChE were inhibited in a 

dose-dependent manner in all treated groups. For the pups, the high dose body weights were 

reduced (p<0.05; ↓8-15%) at PND 1 and 5 (pre- and post-culling). Body weights were also 

reduced from birth to PND 22 in Subset 4 high-dose animals (↓5-19%); body weight gains were 

reduced in these animals during the same period (↓5-30%). Additionally, reduced terminal body 

weights were observed in the Subset 1 (PND 12) high-dose animals (↓17-19%) and the Subset 4 

(PND 66) high-dose males (↓10%). For the F1 adults, body weights of the high dose males were 

decreased during PND 22-66 (↓11-17%). High-dose F1 adult females also weighed less than 

controls at PND 22 (↓17%), but were of similar weight at PND 66. Body weight gains were also 

decreased in the high dose males during PND 22-40 (↓13%) and PND 40-66 (↓7%). Food 

consumption was decreased immediately after weaning (PND 23-30) in high-dose males and 

females (↓13%). 

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In the first of two prenatal developmental toxicity studies in rats, mean group maternal body 

weight gain was reduced by 26% during dosing (GD 6-15) at the high dose (15 mg/kg/day). 

However, these dams had weight gain similar to the other groups during post-treatment (GD 16-

21). Clinical signs of toxicity were evident at the high dose (excessive salivation, perineal urine 

stains, periocular porphyrin deposits, vaginal bleeding, and tremors) throughout the dosing 

period. Plasma ChE activity was decreased by 90% and 97% at the mid (3 mg/kg/day) and high 

dose, respectively. RBC ChE activity was decreased by 74% and 79% at the mid and high dose, 

respectively. In the second rat developmental toxicity study, tremors were noted in 3/32 high 

dose (15 mg/kg/day) rats either on the day of dosing or one day post-dosing. High dose body 

weights were decreased (p<0.05) by 4-5% during GD 8-20, and corrected terminal body weights 

(minus gravid uterine weights) were decreased (p<0.05) by 4%. A slight increase (p<0.01) in 

post-implantation loss was noted at the high dose (9.0% treated vs. 7.0% controls). Plasma ChE 

activity was inhibited (p<0.001) by 51-94% in all treated groups on GD 15. On GD 20 (after 

cessation of treatment on GD 15), the high dose group continued to exhibit plasma ChE 

inhibition (↓28%; p<0.01). 

In the prenatal developmental toxicity study in rabbits, the high dose group (140 mg/kg/day) 

exhibited a mean body weight loss of 99 g during dosing (GD 7-19). However, there was a 

compensatory increase in body weight gains post-dosing (GD 19-29), such that both total (GD 0-

29) body weight gains and corrected (minus gravid uterine weights) terminal body weights were 

similar to controls. An increase (p<0.001) in post implantation loss was observed at the high 

dose (13.9% treated vs. 6.5% controls). Plasma ChE activity was inhibited (p<0.001) by 57-72% 

in all treated groups following 10 days of dosing. 

In a modified mouse developmental toxicity study by De Angelis et al. (2009), the dose levels 

used were selected (3 and 6 mg/kg/day) because they did not induce brain ChE inhibition in a 

previously published study; systemic toxicity was not observed is this study. 

In the rat chronic toxicity/carcinogenicity study, it was stated that body weights of the high dose 

(10 mg/kg/day) males were decreased (p<0.05) from Day 7 on to study termination, but that the 

difference never exceeded 9%, and that overall body weight gains were decreased by 

approximately 10%. Periodic decreases (p<0.05) were noted in the mid dose (1 mg/kg/day) 

males that also never exceeded 9%. Decreased RBC count and hemoglobin were noted during 

the first year at the high dose in both sexes. The absolute and relative adrenal weights were 

increased (p<0.05) in the high dose males at the 12-month interim sacrifice and in both sexes at 

the terminal sacrifice. An excess of fatty vacuolation of the zona fasciculata in the adrenal 

cortex was observed in males only at the high dose both at the interim and terminal sacrifice. In 

the mid (1 mg/kg/day) and high dose males, chlorpyrifos inhibited (p<0.05) plasma ChE activity 

at the by 39-87% and RBC ChE activity by 24-37%; brain ChE activity was inhibited (p<0.05) at 

the high dose by 56-58%. Chlorpyrifos inhibited (p<0.05) plasma ChE activity in females at the 

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mid and high dose by 60-95% and RBC ChE activity in females by 41% at the high dose; brain 

ChE activity was inhibited (p<0.05) at the high dose by 57-61%. 

In one of two mouse carcinogenicity studies, increased (p<0.05) incidences of excessive 

lachrymation were noted in the high dose [(250 ppm; approximately 45/48 mg/kg/day (M/F)] 

males and females. Body weights were decreased (p<0.05) by 4-9% in the high dose males 

generally during treatment (Weeks 1-78). Female body weights were decreased (p<0.05) only 

during Weeks 1-3, suggesting palatability of the diet as a cause. Water consumption was 

consistently decreased throughout the study in the high dose females; food consumption was 

consistently decreased throughout the study in the high dose males. Plasma ChE was decreased 

(p<0.05) at all treatment levels for both Weeks 42 and 78 by 49-98% in the males, and by 45-

99% in the females. Brain ChE was decreased (p<0.05) at the high dose for both Weeks 42 and 

78 by 80-86% in the males, and by 84-85% in the females. Brain ChE was also decreased at the 

mid dose [(50 ppm; 8.8/9.8 mg/kg/day (M/F)] for Week 42 by 46% in the females. Systemic 

toxicity was not observed in the second mouse carcinogenicity study using doses up to 15 ppm. 

In the first of two rat subchronic toxicity studies, a significant decrease in body weight gain was 

observed in high dose (200 ppm; 10 mg/kg/day) males during the first half of the study, and in 

high dose females during the first three weeks. However, body weight in exposed animals was 

similar to controls by Week 13. Food consumption in the high-dose animals was also 

significantly increased during the time of increase body weight gain. Hematological effects were 

observed in both high-dose males and females, characterized by significantly reduced packed 

cell volume (PCV), hemoglobin (HB) and erythrocyte (RBC) group means relative to controls, 

which is suggestive of anemia. Urinalysis revealed that males in the high dose group had a 

significantly reduced urine volume, increased urine pH, a higher specific gravity and a higher 

protein grading, which appear to be treatment-related. Significant and dose-related plasma ChE 

inhibitions of 22, 37, and 72% were observed in the 0.5, 10 and 200 ppm male groups (0.025, 0.5 

and 10 mg/kg/day), respectively. In females, plasma ChE was also significantly inhibited at 91 

and 57% for the 10 and 200 ppm groups, respectively, but was not inhibited in the low dose 

group. However, the registrant acknowledged the possibility that the ChE data for the 10 and 

200 ppm female groups were accidently switched. 

In the second rat subchronic toxicity study, body weights of the high dose (15 mg/kg/day) males 

were slightly decreased (p<0.05) during the first three weeks of treatment by 5-6%, with 

associated decreases (p<0.05) in body weight gains of 11-30%. It was stated that these losses 

were due to palatability of the diet. From Day 70 to study termination, body weights in the high 

dose males were decreased (p<0.05) by 5-6%, with decreased (p<0.05) body weight gains of 9-

10%, with slightly increased food consumption. It was stated that PCV were decreased (p<0.05) 

in the high dose females, platelets were increased (p<0.05) in the mid and high dose males, and 

RBC were decreased (p<0.05) in the mid dose males and high dose males and females. A slight 

increase in urine specific gravity was noted in the high dose females. High dose males displayed 

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a slight (5/10) to moderate (5/10) vacuolation in the adrenal gland (zona fasciculata), suggestive 

of lipid accumulation. At the mid-high dose (5 mg/kg/day), 8/10 males displayed very slight 

vacuolation of cells in the zona fasciculata, which was said to be in excess of the scattered 

vacuolated cells normally observed in the adrenal gland of male controls. There was a doserelated decrease in ChE activity (plasma, erythrocyte, and brain) at each noted time point 

measured in both sexes. At the interim sacrifice, animals of both sexes displayed decreases 

(p<0.05) in plasma and RBC ChE activity at the mid-low (1 mg/kg/day), mid-high (5 

mg/kg/day), and high dose levels. Low dose (0.1 mg/kg/day) females also displayed reduced 

(p<0.05) levels. At terminal sacrifice, plasma ChE activity was decreased (p<0.05) at the mid 

low, mid and high dose in both sexes. RBC ChE activity was significantly decreased at the mid 

and high dose in both sexes and in the mid low females, and brain ChE activity was significantly 

reduced in both sexes at the mid and high dose groups. 

In a subchronic inhalation toxicity study, no signs of systemic toxicity were noted using 

concentrations up to 287 μg/m3

. 

In the two avian reproduction toxicity studies on Bobwhite quail, no signs of systemic toxicity 

were observed at dietary concentrations of up to 125 or 130 ppm; but parental food consumption 

was reduced at 130 ppm. 

In the first of two avian reproduction toxicity studies on ducks, the high-dose ducks (125 ppm) 

exhibited locomotor dysfunction during Week 5 (MRID 42144902). Symptoms included high 

stepping, gate abnormalities, imbalance and lack of coordination. Males seemed slightly more 

affected than females. By Week 14, the 125 ppm birds had poor feather condition and were 

significantly thinner than controls. Food consumption was much lower than that of controls 

during the first four weeks, and a depression in body weight was evident. Mortality was 

increased (8/35) at the high dose, compared to the control (0/35). Systemic toxicity was not seen 

in the low-dose group (25 ppm). In the second duck reproduction toxicity study, there was a 

treatment-related increase in mortality during the pre-egg production period for birds in the 

highest test group (90 ppm). Additionally, there was a treatment-related reduction in body 

weight between Days 0 and 42 at this dose. Therefore, the dose was reduced to 60 ppm on Day 

49. No systemic toxicity was observed in the low and mid dose groups (10 and 30 ppm). 

In a fish full life cycle toxicity study, survival of young fry were significantly reduced in the F0 

and F1 generation at the highest treatment (1.09 μg/L). 

In the three early life-cycle studies with saltwater fish (MRID 000154718) effects on larvae 

survival were reported at concentrations ≥0.48 μg/L; effects on body weight were reported at 

higher concentrations than for survival. 

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B. Estrogen Pathway 

The Tier 1 Estrogen Receptor (ER) binding assay was negative. However, there were positive 

responses in the Tier 1 ER transactivation assay (ERTA) as well as multiple ERTA studies from 

the published literature. In the steroidogenesis assay, estradiol levels were increased at the two 

highest concentrations (10-5 to 10-4 M) of chlorpyrifos. However the in vitro effects were not 

supported by higher-tiered mammalian data such as the uterotrophic, the female pubertal and the 

fish short term reproduction assay (FSTRA). There were no estrogen-related effects observed in 

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

in the mammalian Part 158 studies in the absence of systemic toxicity. In the FSTRA, while 

there was a significant decrease (52-71%) on fecundity, there were no treatment-related changes 

in estrogen-related endpoints such as ovarian histopathology, GSI, plasma vitellogenin or plasma 

17β-estradiol; reductions in brain cholinesterase activity were observed at all test concentrations. 

In the Part 158 avian reproduction studies (n=4), all effects occurred in the presence of overt 

toxicity, except for a reduction in eggs laid for one of the studies where decreases in food 

consumption were observed. There were no effects on reproductive endpoints in the Part 158 

fish life-cycle study. Therefore, there is a lack of convincing evidence for potential interaction 

with the estrogen pathway in mammals and wildlife. 

C. Androgen Pathway 

There was an equivocal finding (at 10-4 to 10-3 M) in the Tier 1 AR binding assay, and in the 

steroidogenesis assay, testosterone levels were decreased at the high test concentrations (10-5 to 

10-4 M). However, these positive in vitro findings were not supported across the body of the 

higher-tiered in vivo data. The Tier 1 Hershberger assay was negative, and in the male pubertal 

assay, there were no treatment-related changes in age or body weight at preputial separation, 

weights of the testes, accessory sex tissue weights, pituitary weight, serum testosterone levels, or 

testicular and epididymal histopathology. In an OSRI study, (Joshi et al. 2007), effects on 

androgen-related endpoints were observed (decreased testosterone, decreased testes 

weight/altered histopathology, and altered fertility). However, signs of toxicity were reported at 

all doses tested. Furthermore, although ChE activity was not determined in this study, the dose 

levels administered (7.5, 12.5, 17.5 mg/kg/day) have been observed to cause significant 

inhibition of ChE activities in other studies (e.g. male pubertal assay at 2 mg/kg/day). In the 

FSTRA there were no treatment-related effects in androgen-related endpoints such as testes 

histopathology, plasma testosterone, or male secondary sex characteristics. There were no 

androgen-related effects in the Part 158 mammalian studies in the absence of overt toxicity; there 

were also no androgen-related effects observed in the Part 158 avian or fish reproduction studies. 

Therefore, while in vitro studies indicate some potential androgen-related effects, no significant 

corroborating effects were observed in the in vivo studies in the absence of systemic (particularly 

cholinesterase inhibition) or overt toxicity. 

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D. Thyroid Pathway 

There were no treatment-related changes in any of the thyroid parameters (i.e., thyroid weight, 

serum thyroid hormone levels or histopathology) in the male and female pubertal assays at doses 

that caused significant cholinesterase inhibition. While developmental delays were observed in 

the AMA, the lack of effects on thyroid histopathology along with a reduction in growth do not 

support a potential thyroid-related effect. Furthermore, no agonist or antagonist activity was 

seen in a receptor transactivation assay using thyroid receptor transfected Vero cells. No thyroidrelated effects were seen in the mammalian Part 158 studies. Although there were reported 

effects on thyroid hormone and histopathology in the De Angelis et al. (2009), there is less 

confidence in these findings due to uncertainty in the experimental design as previously 

discussed in Section III C. Overall, based on the available information the evidence does not 

support a potential interaction with the thyroid pathway. 

E. Conclusions 

The conclusion of the WoE evaluation is that chlorpyrifos demonstrates no convincing evidence 

of potential interaction with the estrogen, androgen, or thyroid pathways. 

V. EDSP Tier 2 Testing Recommendations 

Based on weight of evidence considerations, EDSP Tier 2 testing is not recommended for 

chlorpyrifos since there was no evidence of potential interaction with the estrogen, androgen and 

thyroid pathways. 

 

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Kojima, H; Katsura, E.; Takeuchi, S.; Niiyama, K.; and Kobayashi, K. (2004). Screening for 

Estrogen and Androgen Receptor Activities in 200 Pesticides by In Vitro Reporter Gene Assays 

Using Chinese Hamster Ovary Cells. Environmental Health Perspectives. 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. 

LeBaron, M., Schisler, M., and Visconti, N. (2011) Evaluation of chlorpyrifos in an in vitro

androgen receptor binding assay. The Dow Chemical Company, Toxicology & Environmental 

Research and Consulting, Midland, MI. Laboratory Project Study ID 111099, November 1, 

2011. MRID 48615502. Unpublished. 

LeBaron, M., Schisler, M., and Visconti, N. (2011) Evaluation of chlorpyrifos in an in vitro estrogen 

receptor binding assay. Toxicology & Environmental Research and Consulting, The Dow Chemical 

Company, Midland, MI. Laboratory Study No. 111122, October 31, 2011. MRID 48615504. 

Unpublished.

LeBaron, M., and Kan, H. (2011) Evaluation of chlorpyrifos in an in vitro estrogen receptor 

transcriptional activation assay in human cell line hERα-HELA-9903. Toxicology & 

Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. 

Laboratory Project Study ID 101190, October 27, 2011. MRID 48615505. Unpublished. 

Le Baron, M., Kan, H., and Perala, A. (2011) Evaluation of chlorpyrifos in the in vitro 

steroidogenesis assay. Toxicology & Environmental Research and Consulting, The Dow 

Chemical Company, Midland, MI. Laboratory Report No. 101189, Oct 24, 2011. MRID 

48615510. Unpublished. 

Marty, M., and Marshall, V. (2011) Chlorpyrifos: Hershberger assay in castrated male Crl:CD(SD) 

Rats. Toxicology & Environmental Research and Consulting, Dow Chemical Company, Midland, 

MI. Laboratory Project Study ID 101152, October 25, 2011. MRID 48615507. Unpublished. 

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Marty, M, Zablotny, C., and Stebbins, K. (2011) Pubertal development and thyroid function in 

intact juvenile/peripubertal female Crl:CD(SD) Rats. Dow Chemical Company, Midland, MI. 

Laboratory Project Study ID 101176, November 1, 2011. MRID 48615508. Unpublished. 

Marty, M., Andrus, A., and Hukkanen, R. (2011) Chlorpyrifos: pubertal development and 

thyroid function in intact juvenile/peripubertal male Crl:CD(SD) rats. Toxicology & 

Environmental Research and Consulting, Dow Chemical Co., Midland, MI. Laboratory Study 

ID 111077, October 28, 2011. MRID 48615509. Unpublished. 

Marty, M., Brooks, K., and Jeong, Y. (2011) Chlorpyrifos: uterotrophic assay in the immature 

female Crl:CD(SD) rat. Dow Chemical Company, Midland, MI. Laboratory Project Study ID 

111008, October 10, 2011. MRID 48615511. Unpublished. 

Ouellette, J., Dittenber, D., Kloes, P., and John, J. (1983) Oral teratology study on Fischer rats. 

Toxicology Research Laboratory, Midland, MI. Study No. not provided, July 5, 1983. MRID 

00130400. Unpublished. 

Reylea, R (2009) A cocktail of contaminants: how mixtures of pesticides at low concentrations 

affect aquatic communities. Oecologia: 159: 363-376. 

Richards, S. and Kendall.R. (2003). Physical effects of chlorpyrifos on two stages of Xenopus 

laevis. J. Toxicol. Environ. Health. Part A: Current Issues. 66(1): 75-91.. 

Rubin, Y., Gal, N., Waner, T., and Nyska, A. (1987) Pyrinex: teratogenicity study in the rat. 

Life Science Research Israel Ltd., Ness Ziona, Israel. Study Number MAK/101/PYR, July 15, 

1987. Sponsored by Makhthshim-Agan (America), Inc. MRID 40436407. Unpublished. 

Rubin, Y., Nyska, A., and Waner, T. (1987) Pyrinex: teratogenicity study in the rabbit. Life 

Science Research Israel Ltd., Ness Ziona, Israel. Study Number MAK/103/PYR, July 15, 1987. 

Sponsored by Makhthshim-Agan (America), Inc. MRID 40436408. Unpublished. 

Soto, A.M., Sonnenschein, C., Chung, K.L., Fernandez, M.F., Olea, N. and Serrano, F.O. (1995) 

The ESCREEN assay as a tool to identify estrogens: an update on estrogenic environmental 

pollutants. Environmental Health Perspectives. 103 (Suppl 7):113-122. 

Sparling, D.W. and Fellers, G.M. (2009) Toxicity of two insecticides to California, USA, 

anurans and its relevance to declining amphibian populations. Environ. Toxic. Chem. 28(8) 1696-

1703. 

Sun, H., Si, C., Bian, Q., et al. (2012) Developing in vitro reporter gene assays to assess the 

hormone receptor activities of chemicals frequently detected in drinking water. J. Appl. Toxicol.

32(8): 635-641. 

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Szabo, J., Young, J., and Grandjean, M. (1988) Chlorpyrifos: 13-week dietary toxicity study in 

Fischer-344 rats. Health and Environmental Sciences - Texas, Lake Jackson Research Center, 

The Dow Chemical Company, Freeport, TX. Laboratory Study ID TXT:K-044793-071, 

December 28, 1988. MRID 40952801. Unpublished. 

USEPA (2010) Chlorpyrifos: Report of the Endocrine Disruptor Review Team; Test Order # 

EDSP·059101·30, 059101·31, 059101.33, 059101·34, October 20, 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 Science Coordination and Policy. 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. 

Warner, S., Gerbig, G., Strebing, R., et al. (1980) Results of a two-year toxicity and oncogenic 

study of chlorpyrifos administered to CD-l mice in the diet. Dow Chemical U.S.A., Midland, 

MI. Laboratory Study ID CDL:242059-A, Mar 20, 1980. MRID 00054352. Unpublished. 

Young, J., and Grandjean, M. (1988) Chlorpyrifos: 2-year dietary chronic toxicity-oncogenicity 

study in Fischer-344 rats. Health and Environmental Sciences - Texas, Lake Jackson Research 

Center, The Dow Chemical Company, Freeport, TX. Laboratory Study ID TXT:K/044793/079, 

December 23, 1988. MRID 40952802, Unpublished. 

 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100) 

The 21-day assay (MRID 48615501) of chlorpyrifos on amphibian metamorphosis of the African 

clawed frog (Xenopus laevis) was conducted under flow-through conditions. Amphibian larvae at 

Nieuwkoop-Faber (NF) stage 51 (80/control and treatment group; 20/replicate) were exposed to 

negative and solvent (0.1 mL/L dimethylformamide; DMF) controls and chlorpyrifos (99.8% 

purity) at nominal test chemical concentrations of 0.000310, 0.00125, 0.00500, and 0.0200 mg 

a.i./L. The 21-day time-weighted average (TWA), measured concentrations were <0.0000537 

(<LOQ; controls), 0.000215, 0.000881, 0.00368, and 0.0136 mg a.i./L. The test system was 

maintained at 21.4 to 22.8°C and a pH of 7.0 to 7.6. 

Unless otherwise indicated, all effects are reported based on comparison to the negative control. 

There were no treatment-related effects on survival, which ranged from 98.3 to 100% across the 

controls and all treatment groups. Several tadpoles were observed swimming erratically on Day 8 

in the TWA 0.0136 mg a.i./L treatment group; no other behavioral abnormalities were noted. 

Chlorpyrifos had no significant effect (p>0.05) on any Day 7 growth or development parameters. 

Chlorpyrifos significantly reduced (Jonckheere-Terpstra; p<0.05) Day 21 body wet weight by 23 

and 39% at TWA 0.00368 and 0.0136 mg a.i./L, respectively, relative to the negative control. 

Day 21 snout-vent length (SVL) was also significantly reduced (Jonckheere-Terpstra; p<0.05) by 

4 to 15% at TWA 0.000881, 0.00368, and 0.0136 mg a.i./L compared to the negative control. 

At TWA 0.0136 mg a.i./L, chlorpyrifos significantly delayed (Jonckheere-Terpstra, p=0.03) Day 

21 NF developmental stage by one stage and significantly reduced (Jonckheere-Terpstra, p=0.03) 

normalized (for snout-vent length) hind-limb length (HLL) by 16.6% when compared to the 

negative control. No asynchronous development was observed. Mild follicular cell hypertrophy 

was observed in both controls and all treatment groups, with no apparent treatment-related 

response. 

Concentration-dependent significant reductions (p<0.05) of 21-79% in cholinesterase activity 

from hind limb tissue were observed in all treatment groups on Day 21 compared to the negative 

control. Cholinesterase activity was also significantly reduced (p<0.05) by 28 and 68% in tail 

tissue of the TWA 0.00368 and 0.0136 mg a.i./L treatment groups, respectively, on Day 21 

relative to the negative control. 

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Androgen Receptor (AR) Binding Assay (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48615502), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of AR. Saturation binding information was 

provided in the study profile (MRID 48682802) in which the cytosolic AR was characterized by 

duplicate saturation binding experiments. This study showed that cytosolic AR was present in 

reasonable numbers and was functioning with appropriate affinity for the radio labeled reference 

androgen (R1881). 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 

chlorpyrifos (logarithmic increase from 10-10 to 10-3 M). Ethanol was used as the vehicle at a 

final assay concentration of <3%. The assay included dexamethasone as a weak positive control, 

and R1881 as the ligand reference standard. Three independent runs were conducted with 3 

replicates per concentration per run. 

In the saturation binding experiment, the maximum binding capacity (Bmax) was 3.245 fmol/100 

μg protein and the dissociation constant (Kd) was 0.4641 nM. These values were below the 

range of values from the validation studies (0.685-1.57 nm); however, the results were 

reproducible. The Scatchard plot indicated a linear response across the concentrations of ligand 

added. Nonspecific binding as a percent of total binding was less than 20% across the entire 

concentration range in the saturation binding assays (range 6.2-19.8%, with the exception of the 

high concentration (10 nM) in one assay, which was 24.6%). 

In the competitive binding experiment, the mean log IC50s for R1881 and the weak positive 

control (dexamethasone) were -9.0 and -4.5 M, respectively, and the mean relative binding 

affinity (RBA) for the weak positive control was 0.0034%. The solvent control responses 

indicated no drift in the study assay. All performance criteria were met, with the exception of 

the bottom (% binding) of R1881 in Assay #2 which was slightly low (-2.1%), and is considered 

a minor deviation. 

Based on the responses of three independent competitive binding assays, the bottom of the curve 

for percent of total binding at the 95% confidence interval for chlorpyrifos was between 50 and 

75% in all three runs (53.7%, 57.2%, and 59.3% in runs 1, 2, and 3, respectively) with an 

average of 58.2 ± 1.1%. At the next highest concentration of 10-4 M, the mean binding was 74.5 

± 1.0%, which was also in the equivocal range. An IC50 and RBA could not be calculated for 

chlorpyrifos. The Hill slopes for each of the three runs were -1.132, -1.367, and -1.541, 

suggesting a slightly greater than normal steepness (-1). 

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

Receptor Binding Assay. 

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Aromatase Assay (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48615503), chlorpyrifos (99.8% a.i., Lot # 

KC28161419, TSN101285) in ethanol was incubated with human recombinant aromatase and 

tritiated androstenedione ([1β3

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

concentrations from 10−10 to 10−3

 M for 15 minutes at 37°C to assess the potential of chlorpyrifos 

to inhibit aromatase activity. 

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

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

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

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

(10−10 M to 10−5

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

test chemical series (10−10 M to 10−3 M) with 3 repetitions per concentration. In Run #1, the 

average aromatase activity of the full activity controls was 0.0869 nmol·mg-protein−1·min−1, 

indicating that the microsomal lot used in this particular run (Lot # 85585) had relatively low 

aromatase activity per mg protein. The report stated that a different lot of microsomes (Lot # 

74101) was selected for use in the subsequent runs of the assay. Due to the fact that the full 

activity levels were lower than the assay performance criteria of 0.1 nmol·mg-protein−1

·min−1, 

Run #1 was not included in the interpretation of the study. 

Aromatase activity in the full activity controls ranged from 0.121 to 0.214 nmol·mgprotein−1

·min−1 for the 3 successful test runs, with a mean and standard deviation of 0.164±0.036 

nmol·mg-protein−1·min−1. Activity in the background controls ranged from 2.25 to 2.66% of the 

full activity controls. The response of the full activity controls and background controls was 

acceptable for each run. 

Results for the positive control were generally within the recommended ranges for the top of the 

curve, bottom curve, hill slope, log IC50, and coefficient of variation for replicates of each 

concentration within runs. For 4-OH ASDN, the estimated log IC50 averaged −7.17 M, and the 

Hill slope was −0.97. 

For chlorpyrifos, aromatase activity averaged 0.164±0.036 nmol·mg-protein−1

·min−1 at the 

lowest tested concentration of 10−10 M and 0.142±0.030 nmol·mg-protein−1

·min−1 at the highest 

tested concentration of 10−3

 M. The data for chlorpyrifos were modeled; however, the goodness 

of fit (R2

) was only 0.57–0.77. Because the data failed to model adequately, valid log IC50 and 

Hill slope values could not be determined for chlorpyrifos. The average dose-response curve 

indicated that chlorpyrifos had no effect on aromatase activity at concentrations of 10−10 to 

10−6 M. However, chlorpyrifos reduced aromatase activity to approximately 93% at 10−5

 M and 

to approximately 87% at 10−4 M and 10−3 M. Aromatase activity was >75% at test 

concentrations up to 10−3 M. 

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Based on the data from the average response curve, chlorpyrifos is classified as a Non-inhibitor 

of aromatase activity in this assay. 

Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48615504), uterine cytosol from Sprague 

Dawley rats was used as the source of estrogen receptors (ER) to evaluate the potential for 

chlorpyrifos to displace the bound reference estrogen, radiolabeled [3

H]-17β-estradiol from the 

ER. A saturation binding experiment was conducted prior to the competitive binding experiment 

to demonstrate that the ER in the uterine cytosol was present in adequate numbers and 

functioning with the appropriate affinity for the radiolabeled ligand. Saturation binding data 

were not presented in the study report; however, saturation binding data was presented in the 

study profile submitted separately by the test order recipient (study profile, MRID 48682804). 

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

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

chlorpyrifos (logarithmic increase from 10-10 to 10-3 M). Ethanol was used as test substance 

vehicle at a final concentration of <3%. The assay included 19-norethindrone as a weak positive 

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

reference material. Three independent runs were conducted with 3 replicates per concentration 

per run. 

In the saturation binding experiment, the maximum binding capacity (Bmax) was 59.28 fmol/100 

μg protein and the dissociation constant (Kd) was 0.1032 nM. The Bmax fell within the expected 

range of 10 to 150 fmol/100 μg protein and the Kd for the run was within the expected range of 

0.03 to 1.5 nM. Nonspecific binding as a percent of total binding was 1.7%-8.6% across the 

entire concentration range in the saturation binding assay. Only a single run of the saturation 

binding experiment was conducted on the batch of cytosol preparation used for the competitive 

binding experiment. 

In the competitive binding experiment, the bottom of the curve for percent of total binding at the 

95% confidence interval for chlorpyrifos was 81.7%, 92.5%, and 77.2% in Assays 1, 2, and 3, 

respectively. As the minimum binding observed with chlorpyrifos was >75% at concentrations 

up to 10−3 M, an IC50 and relative binding affinity (RBA) were not calculated. 

 

The performance criteria were met for chlorpyrifos, and the reference compounds performed as 

expected in the assay. The negative control, octyltriethoxysilane, had no effect on binding, and 

the binding curves for 17β-estradiol and 19-norethindrone showed that increasing concentrations 

of each compound displaced [3

H]-17β-estradiol in a manner consistent with one-site binding. 

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CHLORPYRIFOS FINAL 

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The mean log IC50 was -5.5 M for 19-norethindrone and -8.9 M for 17β-estradiol, and the mean 

RBA was 0.034% for 19-noirethindrone compared to the natural ligand. 

Based on the results from the three competitive runs, chlorpyrifos is classified as Not Interactive 

in the Estrogen Receptor Binding Assay. 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48615505), hERα-HELA-9903 

cells cultured in vitro were exposed to chlorpyrifos (99.8% a.i., Lot# KC28161419) at 

logarithmically increasing concentrations from 10−10 to 10−4

 M in DMSO (final concentration 

0.1%) for 24 hours. The experiments were performed using 96-well plates and each chlorpyrifos 

concentration was tested in triplicate (3 wells/plate). Cells were exposed to the test agents for 24 

hours to induce reporter (luciferase) gene products. Luciferase expression in response to 

activation of the estrogen receptor by chlorpyrifos was measured upon addition of a luciferase 

substrate and detection with a luminometer with acceptable sensitivity. 

Chlorpyrifos was tested up to the limit of solubility, 10−4 M. 

The mean RPCMax for chlorpyrifos was 25.4% in the first run, 10.2% in the second run and 

19.5% in the third run, and the associated PCMax was 10−4

 M for the first and third run, and 

10−5 M for the second run. Acceptance criteria were met for all reference chemicals, and the 

assay displayed slightly increased sensitivity to very weak agonists like 17α-methyltestosterone. 

This does not negatively impact the validity of the study. 

Because the RPCMax > PC10 in all three assay runs, chlorpyrifos was considered positive for 

estrogen receptor transcriptional activation in this test system. 

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350) 

The 21-day short-term reproduction assay (MRID 48615506) of chlorpyrifos technical with 

fathead minnows (Pimephales promelas) was conducted under flow-through conditions. Adult 

fish (16 spawning groups; 2 males and 4 females in each group; 1 group per replicate tank and 4 

replicates per treatment level; 6 months old) were exposed to chlorpyrifos (99.8% purity) at 

nominal concentrations of 0 (negative control), 0.0002, 0.00064, and 0.002 mg a.i./L; timeweighted average (TWA), measured concentrations were <0.0000312 (<LOQ), 0.000251, 

0.000812, and 0.00302 mg a.i./L. The test system was maintained at 24.8 to 25.3oC and a pH of 

7.3 to 7.8. 

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There was no significant effect (p>0.05) on fish mortality; overall mean survival values were 

83.3, 91.6, 87.5 and 91.6% in the negative control, low, mid, and high treatment groups, 

respectively. Clinical signs of toxicity were observed in both the negative control and treatment 

groups with similar lesions (e.g., injury to eyes, body) and incident rates. Male body weights 

were significantly reduced (p<0.05) by 17% in the high treatment group relative to the negative 

control. 

Spawning in the negative control occurred at least every four days, and fecundity averaged 34.2 

eggs/female/day; fertilization success in the negative control was 98.9%. There were significant 

reductions (Jonckheere-Terpstra; p<0.05) in fecundity of 52 to 71% at all treatment levels 

compared to the negative control. Fertility was not significantly different (p>0.05) for the 

treatment groups compared to the negative control. 

There were no significant differences (p>0.05) in male tubercle scores or male or female gonadosomatic index (GSI) compared to the negative control. Tubercles were not observed in females. 

Plasma vitellogenin, testosterone, and estradiol for the treatment groups were not significantly 

different (p>0.05) when compared to the negative control. 

Although not analyzed statistically, there was a marginally higher incidence of oocyte atresia (all 

severities) in treated females compared to negative control females; however, this may have been 

due to granulomatous inflammation and was interpreted by the study authors as a spontaneous 

alteration not associated with exposure to chlorpyrifos due to the lack of a treatment-related 

response and bacterial etiology. There were also incidences of egg debris in the oviduct of 

females in the negative control and treatment groups. Similarly, incidences of granulomatous 

inflammation that were also attributed to an infectious agent were observed in the negative 

control and treatments groups. Overall, these histopathological findings were not attributed to 

chlorpyrifos exposure. 

The study authors’ analysis detected significant decreases of cholinesterase activity in brain 

tissue of females at all treatment levels (40 to 92%) and in brain tissue of males at the mid and 

high treatment levels (72% and 90%, respectively). While not significant, a 65% decrease in 

cholinesterase activity in males was observed at the low treatment level compared to the negative 

control. 

Hershberger Assay (OCSPP 890.1400) 

In a Hershberger assay (MRID 48615507) screening for androgenic activity, chlorpyrifos (99.8% 

a.i., lot# KC28161419) in corn oil was administered daily via oral gavage to seven 55-day old, 

castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 1, 6, or 12 mg/kg/day. An 

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CHLORPYRIFOS FINAL 

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androgenic positive control group consisted of seven castrated rats exposed to 0.4 mg/kg/day of 

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

To screen for potential anti-androgenic activity, chlorpyrifos (99.8%, lot# KC28161419) in corn 

oil was administered daily via oral gavage to seven 55-day old, castrated male Sprague Dawley 

rats at dose levels of 0 (vehicle), 1, 6, or 12 mg/kg/day in conjunction with a daily dose of 

reference androgen TP at 0.4 mg/kg/day by s.c. injection. The anti-androgenic positive control 

group consisted of seven castrated rats exposed to 0.4 mg/kg/day TP and 3 mg/kg/day flutamide 

(FT). TP alone was used as the anti-androgenic negative control. 

For both components of the assay, body weights were determined daily, and food consumption 

was measured at 3- to 4-day intervals during dosing. The animals were dosed for 10 consecutive 

days and terminated approximately 24 hours after the final dose administration. At necropsy, the 

five androgen-dependent tissues, liver, adrenals, and kidneys were collected and weighed. Brain 

and red blood cell (RBC) samples were also collected for determination of cholinesterase (ChE) 

activity. 

All animals survived until scheduled termination. There were no clinical signs of toxicity and no 

treatment-related gross pathological findings. 

In the androgen agonist assay, there were no significant effects on body weights or body weight 

gains in the chlorpyrifos treated groups. Rats dosed with TP (positive control) had increased 

(p<0.05) terminal body weights (↑6%), resulting in increased body weight gains (↑29%) 

compared to the controls. Food consumption for Days 4-7 was increased (p<0.05) by 7% in the 

12 mg/kg/day group; this followed a period of decreased body weight gain (not statistically 

significant, NS) in this group, and thus reduced the effects on body weight gain. Food 

consumption for Days 7-11 was decreased (p<0.05) in the 6 mg/kg/day group (↓5%); however, 

this change was not considered biologically meaningful because it was not dose related. 

Kidney and liver weights in all chlorpyrifos groups were comparable to vehicle controls. Rats in 

the 12 mg/kg/day group had increased (p<0.05) adrenal glands weights (↑38%); adrenal glands 

weights in the 1 and 6 mg/kg/day dose groups were comparable to the controls. ChE activity in 

RBC was significantly decreased (p<0.05) at 1 mg/kg/day (90% inhibition) and below the level 

of detection at 6 and 12 mg/kg/day. Brain ChE activity levels were significantly decreased 

(p<0.05) at 6 and 12 mg/kg/day (64% and 79% inhibition). There were no increases in accessory 

sex organ weights at any dose in the chlorpyrifos treated animals. Rats in the positive control 

(TP) group responded properly with accessory sex organ weight increases (p<0.05) in all five 

target tissues. 

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In the anti-androgen assay, there were no effects on body weights, body weight gains, or food 

consumption in the chlorpyrifos + TP treated groups when compared to the TP control. Rats in 

the 6 and 12 mg/kg/day had increased (p<0.05) adrenal glands weights (↑15% and ↑20%) 

compared to controls. Adrenal weights in the 1 mg/kg/day group, and kidney and liver weights 

in all chlorpyrifos treatment groups were comparable to controls. RBC ChE activity levels were 

significantly decreased (p<0.05) at 1 mg/kg/day (91% inhibition) and were below the level of 

detection at 6 and 12 mg/kg/day. Brain ChE activity levels were significantly decreased 

(p<0.05) at 6 and 12 mg/kg/day (60% and 76% inhibition). Rats in the 6 and 12 mg/kg/day 

groups had decreased (p<0.05) glans penis weights (↓6% and ↓8%) compared to controls; glans 

penis weights in the 1 mg/kg/day group were comparable to controls. The weights of the four 

other accessory sex organs in the chlorpyrifos treatment groups were comparable to vehicle 

controls. Rats in the positive control (TP + FT) group responded appropriately with significant 

decreases in all five of the target accessory sex organ weights. All CV values were less than the 

maximum recommended values for each organ, with the exception of levator anibulbocavernosus (LABC) in the 12 mg/kg/day group (33% CV compared to maximum 

recommended CV of 20%). 

The doses tested were judged to be adequate based on the observed RBC and brain 

cholinesterase activity. 

Statistically significant changes were not seen in two or more of the five androgen sensitive 

tissue weights. Chlorpyrifos was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

Female Pubertal Assay (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 46815508), 16 Sprague Dawley [Crl:CD(SD)] rats/dose 

group were treated daily via oral gavage with chlorpyrifos (99.8% a.i., lot# KC28161419, 

TSN101285) in corn oil at doses of 0, 0.5, 1.0, or 2.0 mg/kg/day from post-natal day (PND) 22 to 

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

and weight at day of attainment was recorded. Following sacrifice on PND 42, total thyroxine 

(T4) and thyroid stimulating hormone (TSH) levels were determined using chemiluminescent 

immunoassay and radioimmunoassay, respectively. Weights were recorded for the liver, 

kidneys, urogenital organs, pituitary, thyroid and adrenal glands and microscopic examinations 

were performed on the ovaries, uterus, thyroid, and kidneys. Cholinesterase (ChE) activity was 

determined in red blood cells (RBC) and the brain. 

There were no effects of treatment on mortality, clinical signs of toxicity, body weights, body 

weight gains, age of attainment of VO, body weight at VO, mean age at first estrus, mean cycle 

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length, percent cycling, percent regular cycling, organ weights, serum T4 and TSH levels, clinical 

chemistry parameters, or gross or microscopic pathology. 

RBC ChE activity was decreased (p<0.05) compared to the control at 0.5 (↓19%) and 1.0 (↓88%) 

mg/kg/day, and completely inhibited at 2 mg/kg/day. Brain ChE activity was decreased (p<0.05) 

at 2 mg/kg/day (↓22%) compared to the control, but not affected at 0.5 or 1.0 mg/kg/day. The 

doses tested were considered adequate based on the observed RBC and brain cholinesterase 

activity.

Male Pubertal Assay (OCSPP 890.1500) 

In a male pubertal assay (MRID 48615509), 16 Sprague-Dawley (Crl:CD[SD]) rats/dose group 

were treated daily via oral gavage (4 mL/kg) with chlorpyrifos (99.8% a.i., Lot # KC28161419, 

TSN101285) in corn oil at doses of 0, 0.5, 1.0, or 2.0 mg/kg/day from post-natal day (PND) 23 to 

53. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and the 

age and 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 using 

radioimmunoassays (TSH) or chemiluminescent assays (testosterone and T4). Additionally, 

standard clinical chemistry parameters were evaluated, along with red blood cell (RBC) and 

brain cholinesterase (ChE) activities. Weights were recorded for the liver, kidneys, urogenital 

organs, pituitary, thyroid and adrenal glands, and microscopic examinations were performed on 

the testes, epididymides, thyroid, and kidneys. 

At chlorpyrifos doses up to 2 mg/kg/day, no treatment-related effects were observed on 

mortality, clinical signs, body weights, body weight gains, age or weight at attainment of PPS, 

serum hormone (T4, TSH, or testosterone) levels, organ weights, and gross or histopathological 

parameters. RBC ChE activity was dose-dependently inhibited (p<0.05) between 34-100% at all 

doses of chlorpyrifos. Brain ChE activity was also inhibited (p<0.05) by 23% at 2 mg/kg/day. 

The doses tested were judged to be adequate based on the observed RBC and brain 

cholinesterase activity. 

Steroidogenesis Assay (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48615510), H295R cells cultured in vitro in 24-well plates 

were incubated with chlorpyrifos (99.8% purity, Lot # KC28131419, TSN101285 at 

concentrations of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 μM in triplicate for 48 hours. 

Dimethyl sulfoxide (DMSO) was used as a vehicle, at a final concentration of 0.1%. 

Testosterone and estradiol levels were measured using LC/APPI-MS/MS. Three independent 

experiments were performed. A Quality Control (QC) plate was run concurrently with each 

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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 the known 

inhibitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production. 

Guideline recommendations were met including: lack of cytotoxicity, adequate production of 

testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction and 

inhibition with positive controls. 

Both testosterone and estradiol concentrations were affected by chlorpyrifos. In each of the three 

independent runs of the assay at 10 and 100 μM, statistically significant inhibition of testosterone 

was observed, and statistically significant increases of estradiol were observed. The average 

decrease in testosterone concentration was 0.6-fold at 10 μM and 0.4-fold at 100 μM. The 

average increase in estradiol concentration was 2.3-fold at 10 μM and 2.4-fold at 100 μM. No 

changes in hormone production were noted at ≤ 1 μM of chlorpyrifos. 

Based on the hormone responses in each of the three independent runs, chlorpyrifos treatment 

resulted in statistically significant and reproducible decreases in testosterone production and 

increases in estradiol production. 

Uterotrophic Assay (OCSPP 890.1600) 

In a uterotrophic assay (MRID 48615511) conducted to screen for potential estrogenic activity, 

chlorpyrifos (99.8% a.i., lot # KC28161419) in corn oil was administered daily via oral gavage 

to groups of 6 immature female, Sprague-Dawley rats at dose levels of 0 (vehicle), 0.5, 1.5, or 4 

mg/kg/day on post-natal days (PND) 19−21. A positive control group was treated with 17αethynyl estradiol (EE) in corn oil by gavage at a dose level of 10 μg/kg/day. All animals were 

terminated and necropsied approximately 24 hours after the final dose on PND 22 to determine 

wet and blotted uterine weights. 

All animals survived until scheduled termination. No clinical signs of toxicity were observed in 

any animals for any chlorpyrifos treated groups. No precocious vaginal opening was observed in 

treated females. Body weights and overall body weight gains (Days 1-4) in the chlorpyrifos 

treated groups were comparable to the controls throughout the study. Uterine weights in the 

chlorpyrifos treated groups were also comparable to the controls. Absolute wet and blotted 

uterus weights for the positive EE group were increased (p<0.05) by 528% and 408%, 

respectively, as expected. 

The dose levels tested in this study are adequate based on the results of a probe study which 

showed significant decreases in brain ChE at doses ≥2 mg/kg/day. 

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No statistically significant changes were seen in uterine weight in this assay. Chlorpyrifos was 

negative in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Receptor Transactivation and Proliferation Assays 

In a published study by Andersen et al. (MRID 48033001), chlorpyrifos was tested for 

interactions with the ER and AR and microsomal aromatase in vitro. Agonist and antagonist 

interactions with the ER were examined using cultured human breast cancer MCF-7 cells in a 

cell proliferation assay. ER activation was determined with an ERTA using transfected MCF-7 

cells, and AR activation was determined with an ARTA using transfected Chinese hamster ovary 

(CHO) cells. Effects on aromatase activity were examined using a microsomal aromatase assay. 

Finally, cytotoxicity was determined in both MCF-7 and CHO cells. The cell proliferation assay, 

ERTA, and ARTA were conducted using chlorpyrifos at concentrations ranging from 10−8

 M to 

10−3

 M, while the aromatase assay was performed at a single concentration of 50 μM. 

Chlorpyrifos induced a weak estrogenic response in both the cell proliferation assay (36% of the 

maximum 17β-estradiol response at 50 μM), and the ERTA (25% of the maximum 17β-estradiol 

response at 50 μM, but had no antagonistic effects on 17β-estradiol-induced cell proliferation. 

Chlorpyrifos had no agonist effects in the ARTA, and no effects in the aromatase assay. 

In a published non-guideline study, Soto et al, 1995, using the E-SCREEN assay assessed the 

potential estrogenicity of environmental chemicals using the proliferative effect of estrogen and 

estrogenic chemical on and estrogen-sensitive target cell line. This quantitative in vitro assay 

compares the cell number achieved by similar inocula of human breast cancer estrogen-sensitive 

(MCF-7) cells in the absence of estrogens (negative control), and in the presence of 17β-estradiol 

(E2, positive control) and a range of concentrations of chemicals suspected to be estrogenic. The 

assay was developed based on the premise that a human serum-borne molecule specifically 

inhibits the proliferation of human estrogen sensitive cells and estrogens or estrogen-like 

chemicals induce cell proliferation by blocking this inhibitory effect. The aim of the work was 

to: 1) validate the E-SCREEN Assay, 2) screen a variety of chemicals, 3) examine whether the 

effects of different chemicals act cumulatively and 4) assess the reliability of the assay for use as 

a screening tool. 

Proliferation rates are measured by comparing the cell yield achieved by similar inocula 

harvested simultaneously during the late exponential phase of proliferation. The proliferative 

effect is measured as the ratio between the highest cell yield obtained with the test chemical and 

the hormone free control. In this experimental design, MCF-7 cell yields were measured after 6 

days of exposure and differences between treatment and positive (E2) control were apparent after 

4 days. Estrogenic activity was assessed by 1) relative proliferative potency (RPP) which is the 

ratio between the minimal concentration of estradiol needed for maximum cell yield and the 

minimum dose of the test compound needed to achieve a similar effect, and 2) measuring the 

relative proliferative effect (RPE) which is 100 times the ratio between the highest cell yield 

obtained with the chemical and with E2l. The RPE allows one to quantitatively compare the 

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agonist effect relative to E2. Results were expressed as mean ± SE. Proliferation yield 

experiments were conducted in duplicate wells with a minimum of 5 replicated. Differences 

between groups were assessed by analysis of variance and the a posteriori Shaffe’s test. A p 

value ≤ 0.05 was regarded as significant. Chlorpyrifos was tested over a concentration range of 

10-9 to 10-3 M and found to have no activity in the E-SCREEN Assay. 

Receptor Transactivation Assays 

In a published study by Sun et al. (2012), receptor-mediated luciferase reporter gene assays were 

developed to evaluate and compare ER, AR, and thyroid hormone receptor (TR) activities of 

target chemicals. The assays were tested using 2,4-dichlorophenoxyacetic acid (2,4-D), 

chlorpyrifos, di-2-ethylhexyl-phthalate, and bisphenol A. All the assays utilized Vero (African 

green monkey kidney) cells which do not contain the endogenous receptors. For the ER 

Reporter Gene Assay, the cultured, plated cells were transfected and following 12 hours of 

incubation, were subjected to treatment with the test chemical at various concentrations, with 

17β-estradiol (10–9 to 10–3 mg/L; positive control), or with 0.1% DMSO (negative control) to 

assess estrogenic activity. To assess anti-estrogenic activity, cells were treated with various 

concentrations of the test chemical mixed with 1×10-6 mg/L E2. For the AR Reporter Gene 

Assay, the cultured, plated cells were transfected and following 12 hours of incubation, were 

subjected to treatment with the test chemical at various concentrations, with testosterone (10–7 to 

10–1 mg/L; positive control), or with 0.1% DMSO to assess AR agonistic activity. To assess AR 

antagonistic activity, cells were treated with various concentration of the test chemical mixed 

with 5×10–5 mg/L testosterone. For the TR Reporter Gene Assay, the cultured cells were 

transfected and following 12 hours of incubation, the cells were subjected to treatment with the 

test chemical at various concentrations, with triiodothyronine (T3, 10–5 to 1 mg/L; positive 

control), or with 0.1% DMSO to assess TR agonistic activity. Cells were treated with various 

concentrations of the test chemical mixed with 5×10–4 mg/L T3 to assess TR antagonistic 

activity. The cells from each assay were harvested after 24 hours of exposure to the treatment, 

lysed, and immediately analyzed using a 96-well plate luminometer. A luciferase reporter assay 

system kit was used for quantitation. Relative transcriptional activity was converted into the fold 

induction above the corresponding vehicle control (n-fold). Triplicate analyses were conducted 

for each treatment. The cytotoxicity of the test chemicals was determined prior to conducting the 

receptor assays, and the various test chemicals did not affect the viability or proliferation of the 

Vero cells with or without E2, testosterone, or T3. 

All three positive controls exhibited appropriate concentration-dependent responses in the 

respective assays. Chlorpyrifos had no estrogenic activity at 0.003-3.0 mg/L but did show 

significant (p<0.01) suppression of luciferase at 0.3 and 3.0 mg/L compared to E2, indicating 

antagonistic activity. The RIC20 for chlorpyrifos was 1.15×10-6 M (13.44x MCL). Chlorpyrifos 

had no androgen agonist activity at 0.003-3.0 mg/L but did show significant antagonist activity at 

0.3 mg/L (p<0.05) and 3.0 mg/L (p<0.01) compared with testosterone alone. The RIC20 for 

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chlorpyrifos was 6.43×10-7 M (7.51x MCL). Chlorpyrifos showed no TR agonist or anti-agonist 

activity at 0.003-3.0 mg/L. 

In a published study by Kojima et al. (2004) 200 pesticides, covering nine chemical classes 

were evaluated for their potential to interact with two human estrogen receptor subtypes (hERα 

and hERβ) and the human androgen receptor (hAR) in transactivation assays in Chinese hamster 

ovary cells (CHO-K1 cells) using a luciferase reporter construct. 

Agonistic activities were evaluated by relative activity expressed as REC20 (20% relative 

effective concentration). The REC20 is the concentration of the compound showing 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 by relative activity expressed as RIC20 (20% relative 

inhibitory concentration). The RIC20 is the concentration of the compound showing 20% of the 

inhibition of the activity of 10-10 M E2, 10-9 M E2 or 10-9 M DHT for ERα, ERβ and AR, 

respectively. The range of concentrations tested was ≈ 10-8 to 10-5 M; all pesticides were tested 

at concentrations ≤ 10-5 M to avoid cytotoxicity. When the activity of the test compound was 

higher than the REC20 the pesticide was judged to be positive for activity. When the activity of 

the test compound was higher than the RIC20 the pesticide was judged to be positive for 

inhibitory activity. The statistical significance of differences was evaluated with a two-tailed 

Student’s t test with a nominal level of significance of p < 0.05. 

Chlorpyrifos was tested and found to be positive for ERα transactivation with a REC20 value of 

7.5 x 10-6 M with a relative luciferase activity (RLA) of 27%. Chlorpyrifos was negative for 

ERβ and AR transactivation in this test system. 

The study authors concluded that chlorpyrifos at 0.05 μM induced cell proliferation through ERα

activation in hormone-dependent breast cancer cells. In contrast, chlorpyrifos at 50 μM was 

found to induce cell cycle arrest in S-phase modifying checkpoints proteins, through a 

mechanism that may involve changes in redox balance in MCF-7. In MDA-MB-231 cell, it was 

found that chlorpyrifos at 50 μM produced an arrest in G2/M phase which could be related to the 

capacity of the pesticide for binding to tubulin sites altering microtubule polymerization. 

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, chlorpyrifos had a 

reported purity of >96%. 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 

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

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. 

Chlorpyrifos did not show estrogenic activity in this assay system. 

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800) 

In a two-generation reproduction toxicity study (MRID 41930301), chlorpyrifos (97.8-98.5% 

purity) was administered to 30 Sprague Dawley rats/sex/dose via the diet at dose levels 

equivalent to 0, 0.1, 1 and 5 mg/kg/day during the pre-mating period of 10 or 12 weeks (P or F1

generation, respectively); with exposure continuing in dams through gestation, lactation and 

weaning. The P generation rats were mated once to produce F1 litters. In addition to the 

standard reproductive study parameters, plasma, red blood and brain ChE activity were 

determined for the first 10 P and F1 adult rats/sex/dose at the scheduled necropsy. 

There were no treatment related effects on mortality, body weight, food consumption or clinical 

signs in either P or F1 animals. Dose-related decreases (p<0.05) in plasma (↓43-64% males; ↓49-

72% females), and RBC (↓67-70% males; ↓65-75% females) ChE activity were observed in the 

P and F1 adults at 1 and 5 mg/kg/day. In addition, brain ChE was inhibited in the high dose P 

and F1 males (↓48-53%) and females (↓49-58%). Additionally at the high dose, in the P females, 

very slight vacuolation (16/30 treated vs. 0 controls) consistent with altered tinctorial properties 

of the zona fasciculata (21/30 treated vs. 0 controls) of the adrenal gland was noted; altered 

tinctorial properties of the zona fasciculata (13/30 treated vs. 2/30 controls) of the adrenal gland 

was also noted in the F1 females. Very slight (19/30 treated vs. 14/30 controls) to slight (5/30 

treated vs. 1/30 control) vacuolation of the adrenal was observed in the adrenal glands of the P 

males; slight (6/30 treated vs. 1/30 controls) vacuolation of the adrenal glands was also found in 

the F1 males. It was stated that the histological changes were consistent with fatty changes in the 

males. 

Neonatal effects were observed only in the presence of maternal toxicity and consisted of 

reduced pup weights and increased mortality at 5 mg/kg/day. No treatment-related effects were 

seen on reproductive parameters, such as female fertility indices, lengths of gestation, time to 

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mating, pup sex ratio, pup survival or litter size in the first (P adults/F1 litters) or the second (F1

adults/F2 litters) generations. No treatment-related histopathological lesions were seen in the 

testes, epididymides, and seminal vesicle, coagulating glands, prostate, uterus, ovaries, oviducts, 

vagina, cervix, or pituitary glands. 

The NOAEL and LOAEL for parental toxicity are 0.1 and 1 mg/kg/day, respectively based on 

significant plasma, and RBC ChE inhibition. The NOAEL and LOAEL for neonatal effects are 1 

and 5 mg/kg/day, respectively, based on decreased pup weight and increased pup mortality. 

Testicular Toxicity (Rat) 

In a published testicular toxicity study by Joshi et al. (MRID 48175902), chlorpyrifos (purity 

not reported) in olive oil was administered via oral gavage to groups of six male Wistar 

rats/group at 0, 7.5, 12.5, or 17.5 mg/kg/day for 30 days. On Day 25, the males were cohabited 

with proestrus females (1 male with 3 females) until Day 30. Females were checked for the 

presence of a vaginal plug and the presence of sperm in a vaginal smear, then separated, and 

allowed to litter normally. Fertility indices were calculated for all groups. On Day 30, the males 

were weighed and then euthanized and necropsied. Sperm motility in the cauda epididymis and 

the density of testis and cauda epididymis suspended sperm were calculated. The wet weight of 

testes was recorded after removing the adherent tissue and frozen for measurement of glycogen, 

cholesterol, protein, and sialic acid. Blood was collected by cardiac puncture arid serum was 

separated by centrifugation. Testosterone concentration was measured by radioimmunoassay; 

ChE activity in plasma and RBC was not determined. One of the paired testes of each animal 

was fixed in Bouin's fixative, processed, and stained with hematoxylin and eosin. 

Photomicrographs of the sections were taken. 

There were no mortalities and body weights were unaffected by treatment. There were no effects 

of treatment at the low dose; however, findings suggestive of testicular toxicity were observed at 

the mid and high doses. Testes weights were decreased (p<0.05) by 18% and 28%, respectively. 

Sperm motility in the cauda epididymides was reduced (p<0.05) by 58% and 83%, respectively. 

Testes sperm counts (↓71% and ↓80%) and epididymal sperm counts (↓41% and ↓67%) were 

decreased (p<0.05), resulting in reduced fertility (↓80% and ↓85%). In the testes, levels of 

glycogen (↓63% and ↓88%) and sialic acid (↓15% and 25%) were decreased (p<0.05), and 

cholesterol (↑42% and ↑58%) and protein (↑26% and ↑44%) were increased (p<0.05). Serum 

testosterone levels were decreased (p<0.01) in a dose-dependent manner (presented graphically). 

Additionally at the mid and high doses, histopathology of the testes showed severe degenerative 

changes marked with damaged epithelium, irregular and incomplete spermatogenesis, and lumen 

filled with cellular debris and devoid of sperm. 

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Developmental Neurotoxicity (Rat, OCSPP 870.6300) 

In a developmental neurotoxicity study (MRID 44556901), 25 pregnant Sprague-Dawley 

rats/group were administered chlorpyrifos (99.8% purity) by gavage from GD 6 through LD 11 

at 0, 0.3, 1, or 5 mg/kg/day. An additional 5 pregnant females/group were dosed at the same 

levels for the ChE phase of the study, and were sacrificed 4-5 h post-dosing on GD 20 for ChE 

analyses to be performed on brain, plasma, and erythrocytes. Four subsets of pups were 

examined as follows: (i) fixed brain weight measurements and neuropathological evaluations 

including morphometrics (Subset 1); (ii) learning and memory (Subset 2); (iii) motor activity and 

auditory startle habituation (Subset 3); and (iv) fixed brain weights neuropathological 

examinations (Subset 4). 

Maternal toxicity at the high dose (5 mg/kg/day) was manifested as increased signs of autonomic 

function toxicity, apparent at the end of gestation as fasciculations (6/25 treated vs. 0/25 

controls), and during LD 1-5 as fasciculations (16/24 treated vs. 0/25 controls), hyperpnea (8/24 

treated vs. 0/25 controls), and hyperreactivity (17/24 treated vs. 2/25 controls). Dams with all 

pups dying were increased at the high dose (3/23 treated vs. 0/25 controls). There were no 

significant effects on body weights, food consumption, or pregnancy parameters. There were no 

unscheduled deaths in the maternal animals. 

Brain ChE activity was decreased in dams at the high dose (↓90%) and mid dose (↓18%). RBC 

(↓41-99%) and plasma (↓43-92%) ChE were inhibited in a dose-dependent manner in all treated 

groups. 

The maternal toxicity NOAEL was not observed. The maternal LOAEL was 0.3 mg/kg/day, 

based on plasma and RBC ChE inhibition. 

For the pups, the high dose body weights were reduced (p<0.05; ↓8-15%) at PND 1 and 5 (preand post-culling). Body weights were also reduced from birth to PND 22 in Subset 4 high-dose 

animals (↓5-19%); body weight gains were reduced in these animals during the same period (↓5-

30%). Additionally, reduced terminal body weights were observed in the Subset 1 (PND 12) 

high-dose animals (↓17-19%) and the Subset 4 (PND 66) high-dose males (↓10%). For the F1

adults, body weights of the high dose males were decreased during PND 22-66 (↓11-17%). 

High-dose F1 adult females also weighed less than controls at PND 22 (↓17%), but were of 

similar weight at PND 66. Body weight gains were also decreased in the high dose males during 

PND 22-40 (↓13%) and PND 40-66 (↓7%). Food consumption was decreased immediately after 

weaning (PND 23-30) in high-dose males and females (↓13%). 

Development as assessed by pinna unfolding was slightly delayed (Day 4.0 treated vs. Day 3.5 

controls) at the high dose. At the high dose, female pups also had reduced body weight gains for 

PND 18-22 (↓30%) compared to the control, as well as a biologically insignificant delay in VO 

(↑1 d; PND 33.4 treated vs. PND 32.4 controls). The minor delay in VO may have been due to 

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the significant decreased body weights in the pups observed during PND 4-21. Male pups also 

had reduced body weight gains for PND 18-22 (↓27%), as well as a biologically insignificant 

delay in PPS (↑2.8 d; PND 47.0 treated vs. PND 44.2 controls) at the high dose compared to 

controls. The minor delays may again have been due to the decreased body weight in the pups 

observed during PND 22-66. 

Pup viability was reduced at the high dose as follows: surviving pups/ litter (↓27%); live litter 

size at culling (↓16%); pup viability index (↓29%); and pups found dead or presumed 

cannibalized (PND 1: 7.2 % treated vs. 0 controls; PND 2-5: 24.7% treated vs. 1.3% controls). 

Motor activity was decreased in high dose male and female pups on PND 14 (↓56% in males and 

↓37% in females), and increased in high dose females on PNDs 18 and 22 (↑51% on both days). 

On PND 61, motor activity was increased for both sexes (↑16-17%). There was a statistically 

significant increase (↑16-25%) in the latency to peak response during the auditory startle 

habituation assessments on PND 23 in the high-dose animals compared to concurrent controls. 

At PND 62, the latency to peak response in the high-dose animals was 10-12% higher than in the 

controls. Additionally, the peak response amplitudes in the high-dose animals were decreased by 

9 to 29% on PNDs 23 and 62 (not statistically significant) compared to the controls. 

There were no gross or microscopic lesions of the nervous system in Subset 1 or 4 offspring. 

Subset 1 high-dose males at PND 12 had reduced absolute brain weights (↓9% vs controls), 

increased relative brain weights (↑13% vs controls), reduced anterior to posterior measurement 

of the cerebellum (↓24% vs controls), reduced height of the cerebellum (↓14% vs controls), 

decreased thickness of the parietal cortex (↓6% vs controls), and decreased thickness of the 

hippocampal gyrus (↓9% vs controls). High-dose female pups had reduced absolute brain 

weights (↓9% vs controls), increased relative brain weights (↑14% vs controls), decreased 

thickness of the parietal cortex (↓6% vs controls), decreased width of the caudate-putamen 

(↓10% vs controls), and decreased thickness of the hippocampal gyrus (↓12% vs controls). In 

Subset 4 F1 animals, killed on PND 66, morphometric analysis revealed significantly decreased 

parietal cortex measurements in high-dose (↓5%) and mid-dose (↓4%) females, as compared to 

controls. Decreases in the thickness of the hippocampal gyrus in high-dose females (↓7%) 

resulted in contradictory statistical results when compared to controls; decreases in mid-dose 

(↓4%) females as compared to control were not found to be statistically significant. There was 

no evaluation of the morphometric data for low dose females at PND 66. Brain weight in high 

dose females was similar to control brain weight at day 66 (↓0.3%). 

It is not possible to definitively classify findings in the pre-weaning offspring as having 

originated with pre- or post-natal exposure, nor as resulting from developmental perturbation 

versus direct systemic toxicity or neurotoxicity. However, adverse findings in the adult (~PND 

66) offspring, i.e., alterations in motor activity, auditory startle response, and brain structure 

(decreased measurements of the parietal cortex and hippocampal gyrus, in the absence of brain 

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weight deficits) can be interpreted to represent the long-term sequelae of developmental 

exposure to chlorpyrifos. 

Adverse effects in the offspring have been identified at the MDT of 1.0 mg/kg/day; these include 

a significant treatment-related decrease in the measurement of the parietal cortex, supported by 

possible (although non-significant) alterations in the hippocampal gyrus, in the brain of female 

rats at PND 66. However, due to the lack of morphometric data for low-dose (0.3 mg/kg/day) 

female rats at PND 66, the offspring NOAEL and LOAEL cannot be determined. 

Developmental Toxicity (Rat, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 00130400) groups of 31-33 pregnant Fischer 

344 rats were administered chlorpyrifos (96.6% purity) in corn oil by oral gavage at dose levels 

of 0, 0.1, 3.0, or 15 mg/kg/day during GD 6-15. There were 31 rats each in the control and the 

0.1 mg/kg/day (low dose) groups and 32 in the 3.0 mg/kg/day (mid dose) group and 33 in the 15 

mg/kg/day (high dose) group. On GD 15, 10 rats/dose level were euthanized approximately 4 

hours after dosing, and blood was obtained by cardiac puncture for determination of plasma and 

RBC ChE activity. The remaining rats were euthanized on GD 21. 

There were no deaths in any group. Food and water consumption were not altered by compound 

exposure and liver weights (the only organ for which weights were obtained) was not altered in 

dosed groups compared to controls. Mean group maternal body weight gain was reduced by 

26% during dosing (GD 6-15) at the high dose. This effect appeared to be transient, as the high 

dose dams had weight gain similar to the other groups during post-treatment (GD 16-21). 

Clinical signs of toxicity were evident at the high dose only. Excessive salivation, perineal urine 

stains, periocular porphyrin deposits, vaginal bleeding, and tremors were noted throughout the 

dosing period at the high dose. Most cesarean section parameters were not altered by compound 

exposure. Inhibition of ChE activity was seen at the mid and high dose. Plasma ChE activity 

was decreased by 90.4% and 96.5% at the mid and high dose, respectively. RBC ChE activity 

was decreased by 74.3% and 79% at the mid and high dose, respectively. ChE activities at the 

low dose were similar to controls. 

The maternal LOAEL is 3.0 mg/kg/day based on statistically significant decreases in RBC and 

plasma ChE activity. The maternal NOAEL is 0.1 mg/kg/day. 

No treatment-related changes were seen in post implantation loss, fetal viability, sex ratio, or 

external, visceral, or skeletal examinations. There were no treatment-related effects in 

developmental parameters noted at any dose. 

The developmental NOAEL is 15 mg/kg/day. The developmental LOAEL was not determined. 

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Developmental Toxicity (Rat, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 40436407) groups of 32 pregnant Sprague 

Dawley rats were administered chlorpyrifos (96.1% purity) in corn oil by oral gavage at dose 

levels of 0, 0.5, 2.5, or 15 mg/kg/day during GD 6-15. Plasma ChE activity was determined 

from 10 females/group on GD 15 and on 10 rats/group immediately prior to necropsy on GD 20. 

All rats survived to scheduled termination. Tremor was noted in 3/32 high dose rats either on the 

day of dosing or one day post-dosing. High dose body weights were decreased (p<0.05) by 4-

5% during GD 8-20, and corrected terminal body weights (minus gravid uterine weights) were 

decreased (p<0.05) by 4%. Food consumption was unaffected by treatment. A slight increase 

(p<0.01) in post-implantation loss was noted at the high dose (9.0% treated vs. 7.0% controls). 

Historical control means ranged from 4.4% to 6.3%, indicating that the control and high-dose 

values are both above the historical values. No treatment-related changes were seen in fetal 

viability, sex ratio, or soft tissue variations/malformations. Plasma ChE activity was inhibited 

(p<0.001) by 51-94% in all treated groups on GD 15. On GD 20 (after cessation of treatment on 

GD 15), the high dose group continued to exhibit plasma ChE inhibition (↓28%; p<0.01). 

Developmental Toxicity (Rabbit, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 40436408), groups of 14 pregnant New 

Zealand White rabbits were administered chlorpyrifos (96.1% purity) in corn oil by oral gavage 

at dose levels of 0, 1, 9, 81, or 140 mg/kg/day during GD 7-19. Plasma ChE activity was 

determined prior to mating and following ten doses of chlorpyrifos. Surviving rabbits were 

euthanized on GD 29. 

No mortalities or treatment-related clinical observations were noted during the study. The high 

dose group (140 mg/kg/day) exhibited a mean body weight loss of 99 g during dosing (GD 7-

19). However, there was a compensatory increase in body weight gains post-dosing (GD 19-29), 

such that both total (GD 0-29) body weight gains and corrected (minus gravid uterine weights) 

terminal body weights were similar to controls. There were no apparent treatment-related effects 

on food consumption during this study. No treatment-related changes were seen in fetal 

viability, sex ratio, or soft tissue variations/malformations. An increase (p<0.001) in post 

implantation loss was observed at the high dose (13.9% treated vs. 6.5% controls). Plasma ChE 

activity was inhibited (p<0.001) by 57-72% in all treated groups following 10 days of dosing. 

Modified Developmental Toxicity (Mouse)

In a published modified developmental toxicity study by De Angelis et al. (MRID 48175901), 

chlorpyrifos (98.8% purity) in peanut oil was administered via oral gavage to groups of five 

pregnant CD 1 mice on GD 15-18 at dose levels of 0, 3, or 6 mg/kg/day. Females were allowed 

to litter normally, and selected offspring were administered chlorpyrifos via s.c. injection on 

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PND 11-14 at dose levels of 0, 1, and 3 mg/kg/day. A split-litter design was used, such that one 

pup/sex/dose group from each litter in the three maternal treatment groups was randomly 

assigned to each of the three offspring treatment groups. Therefore, a total of nine overall 

treatment groups were examined. Serum T4 levels were determined in dams immediately after 

the last treatment on GD 18, and in offspring on PND 15 and 150, and serum T3 levels were also 

determined in offspring on PND 150. Adrenal and thyroid histology and histomorphometry were 

evaluated in dams and in offspring on PND 2 and 150. It was stated that the dose levels used 

were selected because they did not induce brain ChE inhibition in a previously published study. 

Therefore, brain ChE measurements were not performed. 

There were no effects of treatment on mortality, clinical signs, body weights, or food 

consumption in the dams. The numbers of pups delivered, sex ratio, and liter weights were 

unaffected by treatment. In the 6 mg/kg dams, T4 levels were decreased (p<0.05) by 34%. 

Microscopic pathology showed a non-significant trend toward increased colloid foaminess in the 

thyroid. Thyroid histomorphometry showed increased (p<0.05) follicular cell height. Adrenal 

histopathology showed a slightly increased vacuolation in the X-zone; histomorphometry did not 

show any changes in adrenal structure. 

In the PND 2 pups, no major changes in thyroid histopathology were seen. Thyroid 

histomorphometry showed higher follicular density at 3 mg/kg/day. Also at this dose, follicular 

size and in particular colloid area were significantly reduced. No effects were seen in the adrenal 

glands. 

In the PND 15 pups, no changes in serum T4 levels were seen in either sex at any dose. 

In the PND 150 offspring, male T3 levels were reduced (p<0.05) by 17% in the litters treated 

with 3 mg/kg/day, regardless of maternal treatment, compared with litters that were treated with 

vehicle, regardless of maternal treatment. Female T3 levels were increased (p<0.05) by 12% in 

the litters treated with 3 mg/kg/day, regardless of maternal treatment, compared with litters that 

were treated with vehicle, regardless of maternal treatment. Male T4 levels were reduced 

(p<0.05) by 11% in the litters treated with 3 mg/kg/day, regardless of maternal treatment, 

compared with litters that were treated with vehicle, regardless of maternal treatment. Female T4

levels were unaffected by treatment. Histopathology showed a dose-dependent higher number of 

necrotic follicular epithelial cells exfoliated into the lumen and colloidal fluid when compared to 

prenatal treatment. No dose-related effects were seen after postnatal treatment. 

Histomorphometry showed a dose-dependent decreased follicular density in males exposed 

prenatally at 3 and 6 mg/kg. No significant differences were seen in females. No treatmentrelated effects were seen in the adrenals. 

These effects were seen at dose levels that did not cause brain ChE inhibition. No 

histopathological changes were seen in the adrenal glands at any dose level. 

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Male Developmental Toxicity (Mouse) 

In a published study by Farag et al. (2010), the reproductive toxicity of chlorpyrifos in male 

mice at doses that caused developmental toxicity in female rats in previous studies was 

investigated. Groups of 25 male ICR (CD-1) mice were administered chlorpyrifos (99.2% 

purity) in corn oil by oral gavage at dose levels of 0 (vehicle only), 5, 15, or 25 mg/kg/day for 5 

days/week for 4 weeks. Mice were examined daily for signs of toxicity, and body weight, body 

weight gain, and food consumption were recorded weekly. Twenty-four hours after the last 

dose, the males were mated (1:1) with untreated females for 7 days to evaluate fertility and 

reproductive performance. The day that a vaginal plug was observed was considered GD 0. 

Pregnant females were anesthetized with diethyl ether on GD 14, and fertility and mating 

indexes, number of implantation sites, resorptions, and dead and live fetuses were recorded. 

After the mating period, the males were weighed and euthanized. Weights of testes, 

epididymides, seminal vesicles, cauda epididymides, prostate, brain, liver, and kidneys were 

recorded. Sperm counts, motility, and morphology analyses were performed. Brain and foreleg 

muscle were collected and analyzed for protein levels and ChE activity. Blood samples were 

collected from the abdomen aorta at necropsy and serum testosterone levels were quantified. 

The brain, foreleg muscle, testis, and epididymis were examined for histopathological changes 

by light microscopy. 

One 15 mg/kg and three 25 mg/kg males died during treatment. Clinical signs of toxicity were 

also observed at these doses. Tremors, starting 60 minutes after dosing, were observed on Day 

10 in the 15 mg/kg group (60% incidence) and on Day 5 in the 25 mg/kg group (80% incidence) 

and progressed in the same mice throughout the second half of the treatment period. Body 

weights, body weight gains, and food consumption were reduced (p≤0.05) at 25 mg/kg from 

Week 2 to the end of treatment, with terminal body weights decreased (p<0.05) by 21%. No 

statistically significant differences in the absolute or relative brain and kidney weights were 

observed. Absolute liver weight was decreased (p≤0.05) by 13% in the 25 mg/kg group, but no 

change was seen for the relative liver weight. Additionally at 25 mg/kg/day, the absolute 

weights of the reproductive organs were reduced (p<0.05) as follows: testes 29%; epididymides 

43%; cauda epididymides 67%; prostate 31%; and seminal vesicles 50%. 

Reproductive effects were noted at the mid and high doses (15 and 25 mg/kg/day). A reduction 

(p≤0.05) in the mating and fertility indices of male mice was observed; the reproductive indices 

were also affected for the untreated females mating with the treated males. There were no 

effects seen on the number of resorptions and live fetuses at the 15 mg/kg dose, but the number 

of dead fetuses was increased (p≤0.05) in this group. The number of live fetuses was decreased 

(p≤0.05) and the number of resorptions and dead fetuses was increased (p≤0.05) in the 25 mg/kg 

group. Spermatid and spermatozoa counts and sperm motility were decreased (p<0.05), and the 

rate of morphological abnormal sperm was also increased at the mid and high doses. 

Additionally, testosterone levels were significantly (p≤0.01) decreased by 52 and 60% at the mid 

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and high doses, respectively. Mild changes in spermatogenic cells in the testis were noted in the 

form of focal areas of irregular seminiferous tubules in the 5 mg/kg group, and edema and wide 

separation of seminiferous tubules were also noted. Seminiferous tubules appeared wider and 

more separated with evidence of congestion in blood vessels due to accumulation of edema in 

the 15 and 25 mg/kg groups; degeneration of spermatogenic cells was also observed in these 

groups. Mild to severe vacuolation of the epithelium lining and congestion of blood vessels in 

the epididymal tubules were noted in a dose-dependent manner; edema and congestion between 

the epididymis tubules were seen in the 15 and 25 mg/kg groups, compared to controls. 

Histological changes in the cerebral cortex of male mice were observed in a dose-dependent 

manner for all treated groups; mild to severe degeneration of neurons with pyknotic nuclei, 

vacuolated cytoplasm, and congestion of blood vessels were noted. At 25 mg/kg, 

disorganization and destruction of the molecular layer in the cerebral cortex of the brain was 

observed. Cellular infiltration, congestion of blood vessels, and alteration and degeneration of 

muscle fibers were also observed in a dose dependent manner. A wide separation of muscle 

fibers and fragmentation of muscles was seen at 25 mg/kg. Brain and muscle ChE activities 

were significantly (p≤0.05) decreased in mice dosed at 15 mg/kg (↓15% and 22%, respectively) 

and 25 mg/kg (↓27% and 32%, respectively), but ChE activities at 5 mg/kg/day were similar to 

the control. 

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300) 

In a combined chronic toxicity/carcinogenicity study (MRID 40952802), groups of 50 Fischer 

344 rats/sex were fed diets containing chlorpyrifos (98.5% purity) at levels equivalent to doses of 

0, 0.05, 0.1, 1, or 10 mg/kg/day for 104 weeks. Ten additional rats/sex/group were randomly 

allocated for a 12-rnonth sacrifice. Plasma and RBC ChE activity was assayed from 10 

rats/sex/dose level at 6, 12, 18, and 24 months. Brain ChE activity was determined from a halfbrain samples obtained at the scheduled 12-rnonth (10 rats/sex/dose) and 24-month (20 

rats/sex/dose) sacrifices. 

There were no treatment-related mortalities or clinical signs of toxicity. It was stated that body 

weights of the high dose (10 mg/kg/day) males were decreased (p<0.05) from Day 7 on to study 

termination, but that the difference never exceeded 9%, and that overall body weight gains were 

decreased by approximately 10%. Periodic decreases (p<0.05) were noted in the mid dose (1 

mg/kg/day) males that also never exceeded 9%. Body weights were decreased (p<0.05) in the 

high dose females during Days 56-231, but these decreases never exceeded 4%. Food 

consumption was unaffected by treatment. Decreased RBC count and hemoglobin were noted 

during the first year at the high dose in both sexes. In males, both the absolute and relative 

adrenal weights were increased (p<0.05) at the high dose both at the 12-month interim sacrifice 

and at termination. In females, the absolute and relative adrenal weights were increased only at 

the terminal sacrifice. No treatment-related changes were seen in the testes weights or ovary 

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weights. No treatment-related histopathological lesions were seen in the testes, epididymides, 

and seminal vesicle, coagulating glands, prostate, uterus, ovaries, oviducts, vagina, cervix, 

mammary, thyroid or pituitary glands. An excess of fatty vacuolation of the zona fasciculata in 

the adrenal cortex was observed in males only at the high dose both at the interim (12 months) 

and terminal sacrifice. The increase observed was an exacerbation of this vacuolation without 

obvious concomitant necrosis or cellular degeneration. It should be noted that the increases in 

adrenal weights were seen in males at this dose. 

Chlorpyrifos inhibited (p<0.05) plasma ChE activity in males at the mid and high dose as 

follows: 39-56% at 6 months; 71-87% at 12 months; 63-77% at 18 months; and 60-80% at 24 

months. Chlorpyrifos inhibited (p<0.05) RBC ChE activity in males as follows: 24% (mid and 

high dose) at 6 months; 33-37% (mid and high dose) at 12 months; 29-34% (mid and high dose) 

at 18 months; and 26% (high dose) at 24 months. Chlorpyrifos inhibited (p<0.05) brain ChE 

activity in males at the high dose as follows: 58% at 12 months; and 56% at 24 months. 

Chlorpyrifos inhibited (p<0.05) plasma ChE activity in females at the mid and high dose as 

follows: 65-83% at 6 months; 86-95% at 12 months; 14-88% (mid-low, mid, and high dose) at 

18 months; and 60-82% at 24 months. Chlorpyrifos inhibited (p<0.05) RBC ChE activity in 

females by 41% at the high dose at 12 months; all other points were not significant. Chlorpyrifos 

inhibited (p<0.05) brain ChE activity in females at the high dose as follows: 61% at 12 months; 

and 57% at 24 months. 

Carcinogenicity (Mouse, OCSPP 870.4200) 

In a carcinogenicity study (MRID 00054352), groups of 56 CD-1 mice/sex were fed diets 

containing chlorpyrifos at 0, 0.5, 5.0, or 15 ppm for approximately 105 weeks, equivalent to 

doses of approximately 0.075, 0.75 and 2.25 mg/kg/day. Cholinesterase measurements were not 

performed. 

No effects of treatment were observed on mortality, clinical signs, body weights, food 

consumption, or blood smears. Weights of the kidneys, liver, or testes were unaffected by 

treatment. No treatment-related histopathological lesions were seen in the testes, epididymides, 

prostate and/or accessory sex glands, preputial or clitoral gland, uterus, ovaries, adrenal, thyroid 

and pituitary glands. Chlorpyrifos was not considered oncogenic under the conditions of this 

study. 

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Carcinogenicity (Mouse, OCSPP 870.4200) 

In a carcinogenicity study (MRID 42534201), groups of 59 CD-1 mice/sex were fed diets 

containing chlorpyrifos at 0, 5, 50, or 250 ppm (equivalent to 0/0, 0.89/0.94, 8.84/9.79, and 

45.2/48.1 mg/kg/day in males/females) for 78 weeks. Additional groups of 5 mice/sex were 

dosed as above for determination of plasma, RBC, and brain ChE activities during Week 42 and 

78. NOTE: RBC ChE activities were not presented, as two different methods were used at the 

two time points resulting in greatly differing values. 

No effects of treatment were observed on mortality, differential white blood cell counts, organ 

weights, or gross pathology. Increased (p<0.05) incidences of excessive lachrymation were 

noted in the high dose males and females. Increased (p<0.05) ocular opacity was seen in the mid 

and high dose males and high dose females. Increased (p<0.05) hair loss on the head and around 

the eyes was observed in the high dose males. Body weights were decreased (p<0.05) by 4-9% 

in the high dose males generally during treatment (Weeks 1-78). Female body weights were 

decreased (p<0.05) only during Weeks 1-3, suggesting palatability of the diet as a cause. Water 

consumption was consistently decreased throughout the study in the high dose females; food 

consumption was consistently decreased throughout the study in the high dose males. A number 

of non-neoplastic microscopic findings were observed at the high dose in both males and 

females: keratitis, retinal atrophy, and pano- and endophthalmitis of the eyes; hepatocytic fatty 

vacuolation and pericholangitis of the liver; lymphocytic infiltration of the salivary glands; and 

accumulation of alveolar macrophages and peribronchial lymphoid hyperplasia of the lungs. No 

treatment-related changes were seen in testes or adrenal weights. No treatment-related 

histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, uterus, 

ovaries, cervix, mammary, adrenal, thyroid or pituitary glands. Chlorpyrifos was not considered 

oncogenic under the conditions of this study. 

Plasma ChE was decreased (p<0.05) at all treatment levels for both Weeks 42 and 78 by 49-98% 

in the males, and by 45-99% in the females. Brain ChE was decreased (p<0.05) at the high dose 

for both Weeks 42 and 78 by 80-86% in the males, and by 84-85% in the females. Brain ChE 

was also decreased at the mid dose for Week 42 by 46% in the females. 

Subchronic Toxicity (Rat, OCSPP 870.3100) 

In a subchronic toxicity study (MRID 40436406), groups of 20 rats/sex were fed diets 

containing chlorpyrifos (95.5% purity) at 0, 0.5, 10 or 200 ppm (equivalent to 0, 0.025, 0.5 or 10 

mg/kg/day) for 13 weeks. In addition to the parameters normally examined, plasma ChE 

activities were determined; RBC and brain ChE activity were not evaluated in this study. 

There were no treatment related effects on mortality, clinical signs, organ weights, or 

histopathology. A significant decrease in body weight gain was observed in high dose males 

during the first half of the study, and in high dose females during the first three weeks. 

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However, body weight in exposed animals was similar to controls by Week 13. Food 

consumption in the high-dose animals was also significantly increased during the time of 

increase body weight gain. Hematological effects were observed in both high-dose males and 

females, characterized by significantly reduced packed cell volume (PCV), hemoglobin (HB) 

and erythrocyte (RBC) group means relative to controls, which is suggestive of anemia. 

However, these parameters were within the normal range. Urinalysis revealed that males in the 

high dose group had a significantly reduced urine volume, increased urine pH, a higher specific 

gravity and a higher protein grading, which appear to be treatment-related. No treatment-related 

changes were seen in the weights of the testes or adrenals. No treatment-related 

histopathological lesions were seen in the testes, prostate, uterus, ovaries, adrenal, thyroid or 

pituitary glands. 

No biologically or significant or treatment-related differences were noted for clinical chemistry 

parameters, with the exception of ChE inhibition. Significant and dose-related plasma ChE 

inhibitions of 22, 37, and 72% were observed in the 0.5, 10 and 200 ppm male groups, 

respectively. In females, plasma ChE was also significantly inhibited at 91 and 57% for the 10 

and 200 ppm groups, respectively, but was not inhibited in the low dose group. However, the 

study author acknowledged the possibility that the ChE data for the 10 and 200 ppm female 

groups were accidently switched. 

The LOAEL for plasma ChE inhibition is 0.5 ppm (0.025 mg/kg/day) for males, which was the 

lowest dose tested. No NOAEL was observed for ChE inhibition. The systemic NOAEL and 

LOAEL are 10 and 200 ppm, respectively (0.5 and 10 mg/kg/day, respectively) based on 

decreased body weight gains and possible anemia. 

Subchronic Toxicity (Rat; OCSPP 870.3100) 

In a subchronic toxicity study (MRID 40952801), groups of 10 rats/sex were fed diets 

containing chlorpyrifos (95.7-98.5% purity) at levels equivalent to 0, 0.1, 1, 5, or 15 mg/kg/day 

for 13 weeks. In addition to the parameters normally examined, plasma, RBC, and brain ChE 

activities were determined. 

There were no treatment related effects on mortality, clinical signs, food consumption, or organ 

weights. For the high dose males, body weights were slightly decreased (p<0.05) during the first 

three weeks of treatment by 5-6%, with associated decreases (p<0.05) in body weight gains of 

11-30%. It was stated that these losses were due to palatability of the diet. From Day 70 to 

study termination, body weights in the high dose males were decreased (p<0.05) by 5-6%, with 

decreased (p<0.05) body weight gains of 9-10%, with slightly increased food consumption. It 

was stated that PCV were decreased (p<0.05) in the high dose females, platelets were increased 

(p<0.05) in the mid and high dose males, and RBC were decreased (p<0.05) in the mid dose 

males and high dose males and females. Several changes (p<0.05) were observed in clinical 

chemistry parameters that were minor or occurred in a non-dose-dependent manner. A slight 

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increase in urine specific gravity was noted in the high dose females. No treatment-related 

changes were seen in the testes or ovary weights. No treatment-related histopathological lesions 

were seen in the testes, epididymides, seminal vesicle, coagulating glands, prostate, uterus, 

ovaries, oviducts, vagina, cervix, mammary, adrenal, thyroid or pituitary glands. High dose 

males displayed a slight (5/10) to moderate (5/10) vacuolation in the adrenal gland (zona 

fasciculata), suggestive of lipid accumulation. At the mid dose, 8/10 males displayed very slight 

vacuolation of cells in the zona fasciculata, which was said to be in excess of the scattered 

vacuolated cells normally observed in the adrenal gland of male controls. 

There was a dose-related decrease in ChE activity (plasma, erythrocyte, and brain) at each time 

point measured in both sexes (values not include in review). At the interim sacrifice, animals of 

both sexes displayed decreases (p<0.05) in plasma and RBC ChE activity at the mid low, midhigh, and high dose levels. Low dose females also displayed reduced (p<0.05) levels. At 

terminal sacrifice, plasma ChE activity was decreased (p<0.05) at the mid low, mid-high and 

high dose in both sexes. RBC ChE activity was significantly decreased at the mid-high and high 

dose in both sexes and in the mid low females, and brain ChE activity was significantly reduced 

in both sexes at the mid and high dose groups. 

Subchronic Inhalation Toxicity (Rat, OCSPP 870.3465)

In a subchronic nose-only inhalation study (MRIDs 40013901 and 40166501), groups of Fischer 

344 rats (10 rats/sex/concentration) were exposed nose only to chlorpyrifos (100% purity) at 

vapor concentrations of 0, 5.2, 10.3, or 20.6 ppb (equivalent to 0, 72, 143 or 287 μg/m3

, 

respectively) 6 hours/day, 5 days/week for 13 weeks. ChE activity was measured at study 

termination. The maximum dose to rats in the 20.6 ppb group was estimated to be 0.044-0.082 

mg/kg/day based on average study specific body weights of 0.15 and 0.282 kg for female and 

male control animals, respectively and the EPA default rat ventilation rate of 0.00715 m3/h 

(average for males and females). 

There were no treatment-related effects on body weight, clinical signs, urinalysis, hematology, 

clinical chemistry, gross or microscopic pathology, or plasma, red blood cell or brain ChE 

activities. Although female rats of all treatment groups had a slight (<4%) but significant 

decrease in red blood cell count, and males of all treatment groups had slightly elevated 

(approximately 13%) serum urea nitrogen, these observations were not considered treatmentrelated due to a lack of dose-response, and all values were within the historical control range. 

No treatment-related changes were seen in testes weights. No treatment-related 

histopathological lesions were seen in the testes, epididymides, and seminal vesicle, prostate, 

uterus, ovaries, oviducts, vagina, cervix, mammary, coagulating, adrenal, thyroid or pituitary 

glands. 

No LOAEL was identified in this study. Therefore, the NOAEL for systemic toxicity and ChE 

inhibition exceeds 20 ppb or 0.082 mg/kg/day.

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Avian Reproduction Toxicity (Quail, OCSPP 850.2300) 

In an avian reproduction toxicity study (MRID 00046957), groups of 36 bobwhite quail (Colinus 

virginianus) were fed diets containing chlorpyrifos at 0, 25, or 125 ppm. The birds were fed the 

diets prior to the onset of egg laying (duration of treatment not provided), and then during egg 

laying (duration of treatment not provided). The total duration of the study was not provided. 

The birds were housed with one male and two females per pen, with 12 pens per dose group. 

Adult quail showed no symptoms of toxicity or behavioral abnormalities. There were no 

mortalities in the control group. In the 25 ppm group one hen died during Week 12, one hen 

during Week 13, one hen during Week 14, one hen during Week 15, and two hens died during 

Week 17. There was one hen mortality in the 125 ppm group during Week 9. No gross 

abnormalities were found in any of the birds upon necropsy; therefore all deaths were considered 

to be incidental to treatment. 

A high proportion of eggshell cracks was reported in this study. The authors considered this to 

have occurred because the birds were not debeaked. A reviewer of this study suggested that the 

high number of eggshell cracks and mortalities at 25 ppm resulted from substandard housing 

conditions (crowding). However, there were no apparent effects on egg weights or eggshell 

thickness. 

The original reviewer concluded that the study was scientifically sound and fulfilled the 

requirement for an avian reproduction study on an upland species. Bobwhite quail fed 25 and 

125 ppm chlorpyrifos in their diet, showed no impairment in any of the reproductive parameters 

tested. 

In another avian reproduction toxicity study (MRID 42144902, 1990), groups of 40 bobwhite 

quail (Colinus virginianus) were fed diets containing chlorpyrifos at 0, 10, 40, or 130 ppm. The 

birds were fed the diets for 12 weeks prior to the onset of egg laying (calculated by reviewer), 

and then during a 12 week egg laying period. The total duration of the study was 24 weeks. The 

birds were housed with one male and one female per pen, with 20 pens per dose group. 

Prior to Week 13, three birds (two at 10 ppm, one at 130 ppm) were sacrificed due to injury or 

poor condition. These birds were replaced with spare birds fed the same diet. From Week 13 

until the end of the test, two mortalities occurred at 10 ppm and one at 40 ppm. These birds were 

not replaced since this was during the egg production phase of the test. These deaths were not 

considered treatment-related. Mean body weights were unaffected by treatment. Analysis of 

food consumption (g/bird/day) over the entire test period indicated that values in the 130 ppm 

group were significantly lower than controls. However, when the pre-egg laying and the egg 

laying periods were analyzed separately, no significant differences were observed. Significantly 

fewer eggs were laid by the 130 ppm hens (842 treated vs. 1148 controls), but no significant 

differences were observed in the other reproductive parameters reported (egg weights, cracked or 

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broken eggs, infertile eggs per eggs set, early embryonic deaths per fertile eggs, late embryonic 

deaths per fertile eggs, eggs hatched per fertile eggs, and 14-day survivors per eggs set). A high 

percentage of cracked eggs was noted in all groups, but eggshell thickness was unaffected by 

treatment. Chick body weights at hatching were unaffected by treatment; chick body weights at 

14 days post-hatching were slightly statistically increased in all treatment groups compared to 

controls. These increases were not considered treatment-related, but to have resulted from a 

slightly lower fitness observed in the control group. 

The NOAEC was 40 ppm. 

Avian Reproduction Toxicity (Duck, OCSPP 850.2300) 

In an avian reproduction toxicity study (MRID 00046952), groups of 35 Mallard ducks (Anas 

platyrhyncos) were fed diets containing chlorpyrifos at 0, 25, or 125 ppm. The birds were fed 

the diets prior to the onset of egg laying (duration of treatment not provided), and then during 

egg laying (duration of treatment not provided). The total duration of the study was not 

provided. The birds were housed with two male and five females per pen, with five pens per 

dose group. 

Ducks within the control groups and the 25 ppm group did not show symptoms of toxicity or 

behavioral abnormities during the study. No mortalities occurred in either of these groups. 

Ducks fed 125 ppm exhibited locomotor dysfunction during Week 5. Symptoms included high 

stepping, gate abnormalities, imbalance and lack of coordination. Males seemed slightly more 

affected than females. By Week 14, the 125 ppm birds had poor feather condition and were 

significantly thinner than controls. Food consumption was much lower than that of controls 

during the first four weeks, and a depression in body weight was evident. The following 

mortalities occurred at the 125 ppm dose level: one drake during Week 5; one drake during 

Week 7; one hen and one drake during Week 8; one hen and one drake during Week 10; one hen 

during Week 12; and one hen during Week 15. Necropsy of these birds showed a depletion of 

body fat and a decrease in body muscle mass. 

At the 25 ppm dosage level, chlorpyrifos caused no impairment in the reproductive 

characteristics measured. At the 125 ppm dose level, statistically significant impairment was 

seen in: significant decreases in egg laid (84%), viable embryos/hatchlings, and 14-d old 

offspring (92%) were observed along with significant decreases in egg weight (14%) and 

eggshell thickness (9%) at 125 mg/kg-diet. 

In another avian reproduction toxicity study (MRID 42144901), groups of 42 Mallard ducks 

(Anas platyrhyncos) were fed diets containing chlorpyrifos at 0, 10, 30, or 90 ppm. The 90 ppm 

diet was changed to 60 ppm at the beginning of Week 8 due to bodyweight loss and mortality. 

The birds were fed the diets for 10 weeks prior to the onset of egg laying, and then during a 12 

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week egg laying period. The total duration of the study was 22 weeks. The birds were housed 

with two male and five females per pen, with six pens per dose group. 

One bird died during the acclimation period and was replaced. Adult mortality during the preegg laying period (Weeks 1-10) was as follows: 1 control bird, 1 at 10 ppm, 2 at 30 ppm, and 11 

at 90/60 ppm. Adult mortality during the egg laying (Weeks 11-22) period was as follows: 1 

control bird, 1 at 10 ppm, 5 at 30 ppm, and 4 at 90/60 ppm. Eight of the birds were replaced 

during the pre-egg laying period and no replacements were made during egg production. There 

was evidence of a treatment-related increase in mortality during the pre-egg production period 

for birds in the highest test group (90 ppm). Additionally, there was a treatment-related 

reduction in body weight between Days 0 and 42 at this dose. Therefore, the dose was reduced 

to 60 ppm on Day 49. By Day 56, the birds in this group showed an increase in body weight 

gain. When compared to the control group, there was a significant reduction in body weight in 

the highest treatment group during the pre-egg production period (Days 1-70). 

When compared to the control group, food consumption in the 30 and 90/60 ppm groups was 

significantly greater during the egg production period. During Week 1, food consumption in the 

90 ppm group was depressed. 

When compared to the control group, there was a significant reduction in the number of eggs laid 

in the 90/60 ppm treatment group. When compared to the control group, there were no 

significant differences in cracked or broken eggs, egg weights, egg shell thickness, infertile eggs, 

or early or late embryonic deaths at any concentration. When compared to the control group, 

there was a significantly higher proportion of fertile eggs that hatched in the 90/60 ppm group. 

When compared to the control group, there were no significant differences in the ratio of 14-day 

survivors/eggs hatched. 

There were no significant differences in offspring bodyweight among groups for weight at hatch 

or at 14 days of age. No abnormalities were detected in postmortem examinations of ducklings 

that died during the 14-day observation period. 

Fish Full Life Cycle Toxicity (Fathead minnow, OCSPP 850.1500) 

In a fish full life cycle toxicity study (MRID 42834401), groups of 80 fathead minnow 

(Pimephales promelas) embryos (20 embryos/cup; 2 cups/aquarium; 2 aquaria/dose level) were 

exposed to chlorpyrifos at nominal concentrations of 0 (negative and solvent controls), 63, 125, 

250, 500, or 1000 ng/L under flow-through conditions for 238 days. Mean measured 

concentrations of chlorpyrifos were 82.6, 143.8, 300.1, 568.3, and 1093.2 ng/L. At study 

initiation, the embryos were approximately 24 h post-fertilization. On Day 25, the larvae were 

randomly culled to 25 larvae/aquaria. Spawning substrates were placed in the aquaria on Day 

80, after which time the fish were observed for secondary sex characteristics and breeding 

behavior. On Day 105, the grow-out section of each aquarium was partitioned into four 

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spawning cells, each containing a single spawning substrate. One male and two females were 

placed in each of four spawning cells per replicate. The remaining fish were placed in the larval 

growth section of-the tank and held until Day 199. The spawning substrates were checked daily. 

For each spawning group, the number of eggs spawned was recorded. Hatching success was 

determined by observing groups of 25 embryos from each spawning unit. The viability of at 

least three sets of embryos for 70% of the spawning sets were evaluated. Observations of 

reproductive effects continued until 88% of the water controls and 75% of all breeding pairs had 

spawned 5 times. Observations of sublethal effects were made weekly. Mortality was recorded 

when observed. 

The F1 embryos were observed, selected, and assigned to hatching cups using the same 

procedure as that followed for P embryos. Eighty F1 embryos, 40 from a single spawning cell 

for each replicate, were hatched, and growth and survival observed until 28 days post-day-tomean-hatch of the controls. At termination, the length (to the nearest 1 mm) and weight (to the 

nearest 0.01 g) of all surviving fish were recorded. 

There was no significant difference in the percentage hatch or the percentage normal at hatch. 

However, percentage hatch on Day 4 at 568 and 1093 ng/L was significantly decreased 

compared to the solvent control. This difference was not evident on Day 5. On Days 12, 19, and 

25, a significant increase in mortality was observed at 1093 ng/L when compared to both the 

dilution water control and the solvent control. No other significant increase in mortality was 

determined. 

There was no significant decrease in P growth after 31 days of exposure. After 61 days of 

exposure, a significant decrease was demonstrated at 300 ng/L when compared to the solvent 

control; however, no effect was detected above or below this concentration. After 199 days of 

exposure, there was a significant difference in weights of the non-breeders at 568 ng/L and in the 

water control when compared to the solvent control. Here again there was no dose-related 

response and these observations are considered spurious. After 216 days of exposure, there was 

no significant difference in growth (length and weight) of the breeders when compared to the 

solvent control. Sublethal effects were demonstrated by one fish in the two highest test 

concentrations and in three fish of the dilution water control. 

The reproduction data (mean number of spawns, mean number of eggs per spawn per breeding 

unit, and mean egg production) indicated that the water controls had relatively higher 

reproductive performance, however, the reproductive data for the acetone controls was consistent 

with the remainder of the treatment levels and there was no statistically significant treatmentrelated effects on any of the reproductive parameters analyzed. 

For the F1 generation, there was no significant difference in growth, percentage hatched, or 

percentage normal at hatch between the controls and treatments. Mortality at 1093 ng/L was 

statistically higher on Days 8, 11, 14, 19, 22, 27, and 32. 

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The LOEAC is 300 ng/L and the NOEAC is 144 ng/L. 

Estuarine/marine fish early life-cycle 

Early life-stage toxicity testing (MRID 00154718) (continuous exposure from embryonic stage 

to 3 weeks or more into the exogenous feedinq staqe) was conducted on three estuarine species 

of atherinid fish (Menidia beryllina; M. menidia; M.-peninsulae). Nominal concentrations of 

chlorpyrifos were established at 0.12, 0.25, 0.50, 1.0, and 2.0 μg/L. Carrier solvent control and 

negative controls were also set up. Temperature was maintained at 25 to 28 °C and photoperiod 

was 12L:12D. Chlorpyrifos exposure decreased survival of hatched fish but not embryos. 

Survival of embryos to hatching was ≥ 80 percent. Measured chlorpyrifos concentrations that 

caused significant reductions of hatched fish survival were: 1.8 μg/L for M. beryllina; ≥ 0.48 

μg/L for M. menidia; ≥ 0.78 μg/L for M. peninsulae. Most deaths occurred in the first 10 days 

after hatching began. Higher chlorpyrifos concentrations produced reduced weight gain relative 

to controls. When chlorpyrifos was detected in test fish, bioconcentration factors averaged 220 

for M. beryllina and 460 for M. peninsulae. Chlorpyrifos was not detected (<0.20 μg/g in M. 

menidia that survived testing. The highest concentration that caused no significant effect and the 

lowest concentration that caused a statistically significant adverse effect were similar for all three 

species. These values are as follows: M. beryllina, 0.75 and 1.8 μg/L; M. menidia, 0.28 and 0.48 

μg/L; M. peninsulae, 0.38 and 0.78 μg/L. 

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APPENDIX 3: References Not Utilized in the Chlorpyrifos 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 Chlorpyrifos 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. 

Abbott, D, Padmanabhan, V, and Dumesic, D (2006) Contributions of androgen and estrogen to 

fetal programming of ovarian dysfunction. Reprod. Biol. Endocrinol. 4: 17. 

Akhtar, N., Srivastava, M., and Raizada, R. (2009) Assessment of chlorpyrifos toxicity on 

certain organs in rat, Rattus norvegicus. J. Environ. Biol. 30(6): 1047-1053. 

Barr, D, Allen, R, Olsson, A, et al. (2005) Concentrations of selective metabolites of 

organophosphorus pesticides in the United States population. Environ. Res. 99: 314-326. 

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Bernabò, I., Gallo, L., Sperone, E., et al. (2011) Survival, development, and gonadal 

differentiation in Rana dalmatina chronically exposed to chlorpyrifos. J. Exp. Zool. 315(5): 

314-327 

Bowman, C (2005) Validation of the pubertal male assay in rats (WA 4-15). WIL Research 

Laboratories, LLC, Ashland, OH. Laboratory Study No. WIL-431005, December 13, 2005. 

Breslin, W, Liberacki, A, Dittenber, D, et al. (1996) Evaluation of the developmental and 

reproductive toxicity of chlorpyrifos in the rat. Fundam. Appl. Toxicol. 29: 119-130. 

Buratti, F, De Angelis, G, Ricceri, L, et al. (2011) Fetal and neonatal exposure to chlorpyrifos: 

biochemical and metabolic alterations in the mouse liver at different developmental stages. 

Toxicology 280(3): 98-108. 

Canesi, L, Negri, A, Barmo, C, et al. (2011) The organophosphate chlorpyrifos interferes with 

the responses to 17β-estradiol in the digestive gland of the marine mussel Mytilus 

galloprovincialis. PLoS One 6(5): e19803. 

Carney, E, Zablotny, C, Marty, S et al. (2004) The effects of feed restriction during in utero and 

postnatal development in rats. Toxicol. Sci. 82: 237-249. 

Chandrasekara, L, and Pathiratne, A (2006) Body size-related differences in the inhibition of 

brain acetylcholinesterase activity in juvenile Nile tilapia (Oreochromis niloticus) by 

chlorpyrifos and carbosulfan. Ecotoxicol. and Environ. Saf. 67: 109-119. 

Corley, R, Calhoun, L, Dittenber, D, et al. (1989) Chlorpyrifos: a 13-week nose-only vapor 

inhalation study in Fischer 344 rats. Fundam. Appl. Toxicol. 13: 616-618. 

Deacon, M, Murray, J, Pilny, M, et al. (1979) The effects of orally administered chlorpyrifos on 

embryonal and fetal development in mice. Toxicology Research Laboratory, Health & 

Environmental Sciences, Dow Chemical U.S.A, Midland, MI. Laboratory report number (not 

provided), July 24, 1979. MRID 00095268. Unpublished. 

Della Seta, D, Farabollini,F, Dessi-Fulgheri, F, et al. (2008) Environmental-like esposure to low 

levels of estrogen affects sexual behavior and physiology of female rats. Endocrinology 149 

(11): 5592-5598. 

Fortenberry, G, Hu, H, Turyk, M, et al. (2012) Association between urinary 3, 5, 6-trichloro-2-

pyridinol, a metabolite of chlorpyrifos and chlorpyrifos-methyl, and serum T4 and TSH in 

NHANES 1999-2002. Science of the Total Environment. 424: 351-355. 

Goodman, L, Hansen, D, Middaugh, D, et al. (1984) Method for early life-stage toxicity test 

using three atherinid fishes and results with chlorpyrifos. MRID 00154718. Published: Aquatic 

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Toxicology and Hazard Assessment: Seventh Symposium, ASTM STP 854, R.D. Cardwell, R. 

Purdy, and R.C. Bahner, Eds., American Society for Testing and Materials, Philadelphia, 1985, 

pp 145- 154. 

Gray, L, Ostby, J, Furr, J, et al. (2001) Effects of environmental antiandrogens on reproductive 

development in experimental animals. Human Reprod. Update 7 (3): 248-264. 

Haviland, J., Butz, D., and Porter, W. (2010) Long-term sex selective hormonal and behavior 

alterations in mice exposed to low doses of chlorpyrifos in utero. Reprod. Toxicol. 29(1): 74-

79. 

Horsley, E (2005) Assessment of pubertal development and thyroid function in juvenile male 

rats. Huntingdon Life Sciences, East Millstone, NJ. Laboratory Study No. 04-4276, December 

22, 2005. 

Klimisch, H-J, Andreae, M, and Tillmann, U (1997) A systematic approach for evaluating the 

quality of experimental toxicological and ecotoxicological data. Reg. Toxicol. Pharmacol. 25: 

1-5. 

Lund, T, Munson, D, Haldy, M, et al. (2004) Equol is a novel anti-androgen that inhibits prostate 

growth and hormone feedback. Biol. Reprod. 70: 1188-1195. 

Mandal, T., and Das, N. (2011) Correlation of testicular toxicity and oxidative stress induced by 

chlorpyrifos in rats. Hum. Exp. Toxicol. 30(10): 1529-1539. 

Matsumoto, K, Hosokawa, M, Kuroda, K, et al. (2009) Toxicity of Agrichemicals in Daphnia 

magna. Osaka City Med. J.: 55: 89-97. 

McCollister, S, Kociba, R, Humiston, C, et al. (1974) Studies of the acute and long-term oral 

toxicity of chlorpyrifos (O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate). Cosmet. 

Toxicol. 12: 45-61. 

Mohammad, F, Al-Badrany, Y, and Al-Jobory, M (2008) Acute toxicity and cholinesterase 

inhibition in chicks dosed orally with organophosphate insecticides. Arh. Hig. Rada Toksikol. 

59: 145-151. 

O’Connor, J, Frame, S, and Ladics, G (2002) Evaluation of a 15-day screening assay using intact 

male rats for identifying anti-androgens. Toxicol. Sci. 69: 92-108. 

Pathiratne, A, Chandrasekera, L, and De Seram, P (2008) Effects of biological and technical 

factors on brain and muscle cholinesterases in Nile tilapia, Oreochromis niloticus: implications 

for biomonitoring neurotoxic contaminations. Arch. Environ. Contam. Toxicol. 54: 309-317. 

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Ricceri, L, Venerosi, A, Capone, F, et al. (2006) Developmental neurotoxicity of 

organophosphorous pesticides: fetal and neonatal exposure to chlorpyrifos alters sex-specific 

behaviors at adulthood in mice. Toxicol. Sci. 93 (1): 105-113. 

Rider, C, Furr, J, Wilson, V, et al. (2008) A mixture of seven antiandrogens induces reproductive 

malformations in rats. Internat. J. Androl. 31: 249-262. 

Rivadeneira, P., Agrelo, M., Otero, S., et al. (2013) Different effects of subchronic exposure to 

low concentrations of the organophosphate insecticide chlorpyrifos in a freshwater gastropod. 

Ecotoxicol. Environ. Saf. 90: 82-88.

Sawaki, M, Noda, S, Muroi, T et al. (2003) In utero through lactational exposure to ethynyl 

estradiol induces cleft phallus and delayed ovarian dysfunction in the offspring. Toxicol. Sci. 75: 

402-411. 

Shin, J-H, Kim, H, Moon, HJ, et al. (2002) Effects of flutamide on puberty in male rats: an 

evaluation of the protocol for the assessment of pubertal development and thyroid function. J. 

Toxicol. Environ. Health A, 65 (5): 433-445. 

Slotkin, T., Cooper, E., Stapleton, H., et al. (2013) Does thyroid disruption contribute to the 

developmental neurotoxicity of chlorpyrifos? Environ. Toxicol. Pharmacol. 36(2): 284-287. 

Sotomayor, V, Lascano, C, Pechen de D'Angelo, A, et al. (2012) Developmental and polyamine 

metabolism alterations in Rhinella arenarum embryos exposed to the organophosphate 

chlorpyrifos. Environ. Toxicol. Chem. 31(9): 2052-2058. 

Terry, K, Cappon, G, Hurtt, M, et al. (2004) Reproductive toxicity assessment of lasofoxifene, a 

selective estrogen receptor modulator (SERM), in female rats. Birth Defects Res. B, 71: 150-

160. 

van der Schoot, P (1992) Disturbed testicular descent in the rat after prenatal exposure to the 

antiandrogen flutamide. J. Reprod. Fert. 96: 483-496. 

Vanage, G, Dao, B, Li, X-J, et al. (1997) Effects of anordiol, an antiestrogen, on the reproductive 

organs of the male rat. Arch. Androl. 38: 13-21. 

Ventura, C., Núñez, M., Miret, N., et al. (2012) Differential mechanisms of action are involved 

in chlorpyrifos effects in estrogen-dependent or -independent breast cancer cells exposed to low 

or high concentrations of the pesticide. Toxicol Lett. 213(2): 184-193. 

Viswanath, G., Chatterjee, S., Dabral, S., et al. (2010) Anti-androgenic endocrine disrupting 

activities of chlorpyrifos and piperophos. J. Steroid. Biochem. Mol. Biol. 120(1): 22-29. 

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York, R (2006) Interlaboratory validation of the pubertal male assay. CR-DDS Argus Division, 

Horsham, PA. Laboratory Project ID RTP00002, January 3, 2006. 

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