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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

## ~ S

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

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

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

.

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

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

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

CHEMICAL: CHLORPYRIFOS

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYCHLORPYRIFOS FINAL

ii TABLE OF CONTENTS

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

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

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

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

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

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

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

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................8
1. Effects on Estrogen Pathway.....................................................8
2. Effects on Androgen Pathway..................................................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-Estroge nic Pathway for Chlorpyrifos..................................11
Table 3. Androgenic/Anti-Androgeni c 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......................70CHLORPYRIFOS FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

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

CG Cowper’s Gland

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

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

iv Abbreviation Terminology

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

K OW Octanol/Water Partition Coefficient

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

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

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

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated ConcentrationCHLORPYRIFOS FINAL

v Abbreviation Terminology

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

OPP Office of Pesticide Programs

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

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

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

T4 Thyroxine (tetraiodothyronine)CHLORPYRIFOS FINAL

vi Abbreviation Terminology

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

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

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

Page 1 of 74 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the potentia l of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pathways. Th is interaction includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitarygonadal (HPG) and hypothalamic-pitu itary thyroid (HPT) axes. In addi tion to the available Tier 1 data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were co nsidered in this weight of evidence (WoE)

assessment.

In determining whether chlorpyrifos interacts w ith E, A or T hormone pa thways, the number and type of effects induced, the magnitude and pattern of responses observed acr oss studies, taxa and
sexes were considered. Additiona lly, the conditions under which effe cts occur were considered, in particular, whether or not endocri ne-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 Offi ce of Science Coordination and Policy (OSCP) conducted a weight-of-evidence analysis (WoE) of th e 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 assa y (ERTA) as well as multiple ERTA studies from the published literature. In the st eroidogenesis 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 assa y (FSTRA). There were no estroge n-related effects observed in the Tier 1 uterotrophic assay or the female pube rtal assay. There were no estrogen-related effects
in the mammalian Part 158 studies in the absenc e of systemic toxicit y. In the FSTRA, while there was a significant decrease (52-71%) on fec undity, there were no treatment-related changes in estrogen-related endpoints such as ovarian histopathology, GSI, plasma vitellogenin or plasma

17 β -estradiol; reductions in brain ch olinesterase 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 eff ects on reproductive endpoints in the Part 158 fish life-cycle study. Therefore, there is a lack of convincing ev idence for potential interaction with the estrogen pathway in mammals and wildlife.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, test osterone 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 treatme nt-related changes in age or body weight at preputial separation, weights of th e testes, accessory sex tissue weights, pituitary weight, serum testosterone levels, or testicular and epididymal histopathol ogy. In an OSRI study (Joshi et al

. 2007), effects on androgen-related endpoints were observed (decreas ed testosterone, decreased testes weight/altered histopathol ogy, and altered fertility). Howe ver, signs of toxicity were reported at all doses tested. Furt hermore, 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 Pa rt 158 mammalian studies in the absence of overt toxicity; there were also no androgen-related e ffects observed in the Part 158 avia n or fish reproduction studies. Therefore, while in vitro studies indicate some potential andr ogen-related effects, no significant corroborating effects were observed in the in vivo studies in the absence of systemic (e.g.,
cholinesterase inhibiti on) 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 fe male pubertal assays at doses that caused significant cholineste rase inhibition. While developmen tal delays were observed in the AMA, the lack of effects on thyroid histop athology along with a reduction in growth do not support a potential thyroi d-related effect. Furthermore, no agonist or antagonist activity was seen in a receptor transactiva tion assay using thyroid receptor tr ansfected Vero cel ls. No thyroidrelated effects were seen in the mammalian Part 158 studies. Alt hough there were reported effects on thyroid hormone and hi stopathology 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 suppo rt a potential interact ion 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 poten tial interaction with th e estrogen, androgen and thyroid pathways.CHLORPYRIFOS FINAL

Page 3 of 74 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the potentia l of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pathways. Th is interaction includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitarygonadal (HPG) and hypothalamic-pitu itary thyroid (HPT) axes. In addi tion to the available Tier 1 data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were co nsidered in this weight of evidence (WoE)

assessment.

In determining whether chlorpyrifos interacts with E, A or T hormone path ways, the number and type of effects induced, the magnitude and pattern of responses observed acr oss studies, taxa and
sexes were considered. Additiona lly, the conditions under which effe cts occur were considered, in particular, whether or not endocri ne-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 Offi ce of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE ) analysis of the pot ential interaction of Chlorpyrifos with the E, A or T hormone pathways.

The water solubility of chlorpyrif os 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 biodegrada tion. Abiotic hydrolysis unde r neutral and aci dic conditions, photodegradation, and volatilization do not seem to play a significant role in the dissipation process. Under alkaline conditions (pH 9), hydrolysis of chlorpyrif os occurs more rapidly with a half-life of approximately 2 weeks. Based on av ailable data, chlorpyrifos appears to degrade slowly in soil under both aerobic and anaerobic co nditions. 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) depe nding on soil type, environmental conditions and possibly previous use history at the treat ment site. Information on leaching and adsorption/desorption indicate that parent chlorpyr ifos 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 determ ining the potential intera ction 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 st udies and other published
articles. These data are summarized in Sections III.A through III.C. An analysis of the dataCHLORPYRIFOS FINAL

Page 4 of 74 submitted to the Agency, using the WoE appro ach outlined by the Agency (USEPA, 2011), is presented in Section IV. The ED SP 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 s hown below in Table 1. Executive Summaries are presented in Appendix 1.

B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 Screening assays were submitted as OSRI by the Te st Order recipients and/or the public. This included studies published in the open literatu re 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 s earch of available scientific literature for any
additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies c onsidered but not util ized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

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

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to 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 th ese 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 wh ether additional testing is n eeded based on the evidence and
conclusions described above.CHLORPYRIFOS FINAL

Page 5 of 74 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 in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

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

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

Page 6 of 74 toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.

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

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

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

Page 7 of 74 The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substan ce 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 pros tate cytosol) OSCPP 890.1150 Requirement Satisfied (MRID No. 48615502)

ER Binding Assay (Rat uter ine cytosol) OSCPP 890.1250 Requirement Satisfied (MRID No. 48615504)

ER α 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)CHLORPYRIFOS FINAL

Page 8 of 74 B. Effects on Hypothalamic-P ituitary-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 estrogen ic, anti-estrogenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

Chlorpyrifos was classified as not interactive (negative) in the Tier 1 in vitro estrogen receptor (ER) binding assay, and as negati ve in the Tier 1 aromatase assa y up to the limit concentration (1 mM). As summarized below, chlorpyrifos was positive in the Tier 1 ER α transcriptional activation assay and positive in seve ral ER transcriptional activation assays considered as OSRI.

In the Tier 1 ERTA, the mean RPC max was 25.4% in the first run, 10.2% in the second run and 19.5% in the third run; the associated PC max was 10

− 4 M for the first and third runs, and 10

− 5 M
for the second run. Because the RPC max ≥ PC 10 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 estr ogen receptor subtypes (hER α and hER β ) in transactivation assays in Chinese hamster ovary cells (CHO-K1 cells). In this study, chlorpyrif os was reported to be positive for ER α (agonist) transactivation with a REC 20 value of 7.5 x 10 -6 M and negative as an

ER α antagonist. Chlorpyrifos was also negative for ER β activity (agonist and antagonist). Howe

In another study (Andersen et al ., 2002), chlorpyrifos was positive in a non-guideline ER α

transcriptional activation assay in MCF7 cells . In this study, chlorpyrifos induced a weak estrogenic response in both the cell pro liferation assay (36% of the maximum 17 β -estradiol response at 50 μM), and the ERTA (25% of the maximum 17 β -estradiol response at 50 μM), but had no antagonistic effects on 17 β -estradiol-induced cell prolifer ation. 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-guide line ERTA study (Sun et al ., 2012), chlorpyrifos dem onstrated antagonistic activity in an ER reporter gene assay. Chlor pyrifos had no estrogenic activity at 0.003-3.0 mg/L but did show significant suppression of lucifera se at 0.3 and 3.0 mg/L compared to estradiol, suggesting antagonistic activity. The re lative 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).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 concentra tions in females nor treatment-related effects on gonadal histopathology or GSI. There was significan t 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 a ny treatment. Clinical signs of toxicity, including injury to eyes and snout (along with females reported as bloated or a side injury) were reporte d at this concentration, however, sim ilar effects and incident rates were reported in the control fish and other treatment gr oups, therefore it is uncer tain 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); howe ver, 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. Additi onally, 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 st udy, no treatment-related reproduc tive 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, ho wever, there were no effects on body weight, mortality or clinical si gns 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 lo comotor dysfunction, reduced food consumption and body weights, and 8 mortalities (out of 35 expos ed) were observed. In the second mallard duck study, there was a significant reducti on (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.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.CHLORPYRIFOS FINALPage 11 of 74Table 2: Estrogenic/Anti-Estrogenic Pathway for ChlorpyrifosLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Chlorpyrifos1Study Type/ LiteratureCitation 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) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48615504)NERTA(MRID 48615505)PAromatase(MRID 48615503)NSteroidogenesis(MRID 48615510)PUterotrophic(MRID 48615511)NNFemale Pubertal Rat(MRID 48615508)NNNNNN↓ChE(L, M, H)NFSTRA(MRID 48615506)NNNNFcd↓52-71%(L, M, H)NChEbrain↓52-71%(L, M, H)N4OSRIER Transactivation(Andersenet al., 2002)PER Transactivation(Kojimaet al., 2004)PER Transactivation(Kojimaet al., 2005)NER Transactivation(Sunet al., 2012)PCHLORPYRIFOS FINALPage 12 of 74Table 2: Estrogenic/Anti-Estrogenic Pathway for ChlorpyrifosLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Chlorpyrifos1Study Type/ LiteratureCitation 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) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Two-generationreproduction (Rat;MRID 41930301)NENENNENNX(M, H)NDevelopmentalneurotoxicity (Rat;MRID 44556901)NNX(L, M, H)NDevelopmentaltoxicity (Rat; MRID00130400)NENX(M, H)X (H)Developmentaltoxicity (Rat; MRID40436407)NENX(L, M, H)NDevelopmentaltoxicity (Rabbit;MRID 40436408)NENX(L, ML,MH, H)X (H)Chronic toxicity/carcinogenicity (Rat;MRID 40952802)NENNNEX(M, H)NOncogenicity (Mouse;MRID 00054352)NENENNEN NCarcinogenicity(Mouse; MRID42534201)NENENNEX(L, M, H)NSubchronic toxicity(Rat; MRID40436406)NENENNEX(M, H)NSubchronic toxicity(Rat; MRID40952801)NE N N NEX(L, ML,MH, H)NCHLORPYRIFOS FINALPage 13 of 74Table 2: Estrogenic/Anti-Estrogenic Pathway for ChlorpyrifosLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Chlorpyrifos1Study Type/ LiteratureCitation 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) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Subchronic inhalationtoxicity (Rat; MRID40013901)NENENNEN NAvian reproduction(Quail; MRID00046957)NN NAvian reproduction(Quail; MRID42144902)P5(H)N NAvian reproduction(Duck; MRID00046952)NNX(H)Avian reproduction(Duck; MRID42144901)NNX(H)Fish Full Life CycleToxicity (Minnow,MRID 42834401)NX(H)1.Key to responses: L=Low treatment, ML=Medium-low treatment,M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows (↓or↑) indicate the direction of the response. A shaded cell indicatesthat is parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated. ChE=Cholinesterase inhibition2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4.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 reportedas bloated or a side injury) were reported at this concentration, however, similar effects andincident rates were reported in the control fish and other treatment groups, therefore it is uncertain if these clinical signsare treatment-related5.Significant reduction of 27% in the number ofeggs laid at the high dose (130 ppm)CHLORPYRIFOS FINALPage 14 of 74P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedCHLORPYRIFOS FINAL

Page 15 of 74 2. Effects on Androgen Pathway

The potential interactions of chlorpyrifos with the androgen pathwa y are summarized in Table 3. The various targets of the androgen pathway across the relevant Ti er 1 assays are delineated so as to facilitate determination of potential for a ndrogenic, 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 di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

Chlorpyrifos was classifi ed 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 bindi ng was noted at concentrations ≤ 10

− 5 M. In the Tier 1 steroidogenesis assay, significant inhibition of testosterone pr oduction (0.4- to 0.6-fold) was observed at 10 and 100 μM. Three published studies were also av ailable examining effects of chlorpyrifos on AR transactivati on and/or steroidogenesis. A lthough chlorpyrifos had no agonist activity in an AR transcriptional activation (A RTA) assay using Chinese hamster ovary (CHO)
cells at 10 -8 to 10 -3 M (Andersen et al ., 2002) , chlorpyrifos demons trated 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 th e 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 an ti-androgenic activity in the Hershberger assay, and no androgen-related eff ects were observed in the male pubertal assay up to doses that resulted in si gnificant inhibition of both brain a nd RBC cholinesterase activity ( ≥ 2 mg/kg/day). In the FSTRA, there were no tr eatment-related effects on male GSI, VTG, or fertility. There was significant decreases in ch olinesterase in brain ti ssue of males (72% and 90%) at the 0.000812 and 0.00302 mg ai/L treatment gr oups, 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. Clin ical signs of t oxicity, including injury to eyes and snout (along with females reporte d as bloated or a side injury) were reported at this concentration, however, similar effects and in cident rates were reporte d in the control fish and other treatment groups, therefor e it is uncertain if th ese clinical signs ar e 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, thr ee published studies were reviewed that examined effects of chlorpyrifos on testes and reproductive parameters in rodent s. In a testicular toxicity study (Joshi et al ., 2007), male rats dosed for 30 days by or al gavage with chlorpyrifos (purityCHLORPYRIFOS FINAL

Page 16 of 74 not reported) at 12.5 or 17.5 mg/kg/day (mid a nd high doses) dose-dependently decreased serum testosterone levels, testes weights, sperm count s in the testes and epididymis, and sperm motility, resulting in reduced fertility in males. Additionally, histopathology of the te stes showed severe degenerative changes marked with damage d epithelium, irregular and incomplete spermatogenesis and lumen filled with cellular debr is and devoid of sperm. No mortality or changes in body weights were observed in this st udy; however, it was repo rted that all treated animals exhibited toxicity symptoms (types of symptoms not reported). ChE inhibition was not evaluated in the study. In another study examini ng 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 decrea sed at the mid and high doses and the weights of the testes and several secondary accessory sex tissues were de creased at the high dose. In addition, male fertility was reduced at the mid and high doses, and histopathological cha nges were noted in all treatment groups. However, clinic al 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 re production; survival of young fry were significantly reduced in the F0 and F1 gene ration at the highest tr eatment (1.09 μg/L).CHLORPYRIFOS FINALPage 17 of 74Table 3: Androgenic/Anti-Androgenic Pathway for ChlorpyrifosLines of Evidence Indicating Potential Interaction withthe 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 Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48615502)ESteroidogenesis(MRID 48615510)PHershberger(MRID 48615507)NENAW↑15%,↑20-38%(M, H);↓ChE(L, M, H)NMale Pubertal Rat(MRID 48615509)NNNNNNNN↓ChE(L, M, H)NFSTRA(MRID 48615506)NNNNNNChE (brain)↓65, 72, 90%(L (NS), M, H)N4(M, H)OSRIAR Transactivation(Andersenet al.,2002)NAR Transactivation(Kojimaet al.,2004)NAR Transactivation(Sunet al., 2012)PTwo-GenerationReproduction (Rat;MRID 41930301)NNENNENNENX(M, H)NCHLORPYRIFOS FINALPage 18 of 74Table 3: Androgenic/Anti-Androgenic Pathway for ChlorpyrifosLines of Evidence Indicating Potential Interaction withthe 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 Systemic Toxicity Observed 2 Overt Toxicity Observed 3Testicular Toxicity(Rat; Joshiet al.,2007)↓(M. H)↓18,28%(M, H)P5(M, H)NEP5(M, H)NENEFrt:↓80, 85%(M, H)5X(M, H)N6Male ReproductiveToxicity (Mouse;Faraget al., 2010)NNP7(L)NNNENNX(L, M, H)X(M, H)DevelopmentalNeurotoxicity (Rat;MRID 44556901)NENENENENENENX(H)X(H)Chronic Toxicity /Carcinogenicity(Rat; MRID40952802)NNNENNENEX(L, ML,MH, H)NCarcinogenicity(Mouse; MRID00054352)NNNENNENEN NCarcinogenicity(Mouse; MRID42534201)NNENNENENEX(L, M, H)NSubchronic Toxicity(Rat; MRID40436406)NNNENENENEX(L, M, H)NSubchronic Toxicity(Rat; MRID40952801)NNNENNENEX(ML, MH, H)NSubchronicInhalation Toxicity(Rat; MRID40013901)NNNENENENEN NAvian reproduction(Quail; MRID00046957)NN NCHLORPYRIFOS FINALPage 19 of 74Table 3: Androgenic/Anti-Androgenic Pathway for ChlorpyrifosLines of Evidence Indicating Potential Interaction withthe 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 Systemic Toxicity Observed 2 Overt Toxicity Observed 3Avian reproduction(Quail; MRID42144902)NNE NAvian reproduction(Duck; MRID00046952)NNX(H)Avian reproduction(Duck; MRID42144901)NNX(H)Fish Full Life CycleToxicity (Minnow,MRID 42834401)NX(H)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. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay.Changes in weight are absolute unless otherwise indicated. ChE=Cholinesterase inhibition2. 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 thiscolumn. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For details seeSection IV. A4. At the highest dose (3.02 μg/L), male body weights were significantly reduced (17%); survivalwas not affected at any treatment. Clinical signs of toxicity,including injury to eyes and snout (along with females reportedas bloated or a side injury) were reported at this concentration, however, similar effects andincident rates were reported in the control fish and other treatment groups, therefore it is uncertain if these clinical signsare treatment-related.5. At the mid and high doses (12.5 and 17.5 mg/kg/day), histopathology of the testes showed severe degenerative changes markedwith damaged epithelium,irregular and incomplete spermatogenesis, and lumen filled with cellular debris and devoid of sperm. Sperm motility in the cauda epididymides was reduced(p<0.05), and testes and epididymal sperm counts were decreased (p<0.05;↓71-80% and 41-67%, respectively), resulting in reduced fertility.6. No mortality or changes in body weights were observed in this study, however it was reported that all treated animals exhibited toxicity symptoms (types ofsymptoms 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 seminiferous tubules, and edemaand wide separation of seminiferous tubules were also noted. Mild to severe vacuolation of the epithelium lining and congestionof blood vessels in the epididymaltubules were noted in a dose-dependent manner.CHLORPYRIFOS FINALPage 20 of 74P Positive findingsN Negative findings (in vitro)/No effect (in vivo)E Equivocal assay responseNE Not examinedCHLORPYRIFOS FINAL

Page 21 of 74 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

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

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

There were no effects of chlorpyrifos noted on serum T 4 or TSH levels, pituitary or thyroid weights, or thyroid hist opathology in either the male pubertal or female pubertal assays up to a dose of 2.0 mg/kg/day that result ed in significant reductions ( ≥ 22%) in brain cholinesterase activity.

In the AMA, the highest test concentration of chlo rpyrifos (13.6 μg a.i./L ) was associated with a significant stage delay in 21-day NF developmen tal 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 con centrations (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 ta dpoles were observed swimming erratically on Day 8 in the high concentration group.

There were no thyroid-related e ffects observed in the mammalian Part 158 studies. In a journal article considered as OSRI, chlorpyrifos had no agonist or antagonist ac tivity 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 gestati on days 15-18. After delivery the pups were dosed via subcutaneous injection (sc) during postnatal days 11-14. T 4 levels were decreased in dams at an oral gavage dose of 6 mg/kg, and microscopi c pathology showed increased colloid foaminess (NS) and increased (p<0.05) follicular cell heig ht 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, follic ular size and in particular colloid area were reduced. In the PND 150 offspring, T 3 levels in the 3 mg/kg/da y (sc) dose were reduced (p<0.05) by 17% in males and increased (p<0.05) by 12% in females. Also, T 4 levels in the 3 mg/kg/day (sc) male were reduced (p<0.05) by 11%. Histopathology showed a dose-dependentCHLORPYRIFOS FINAL

Page 22 of 74 higher number of necrotic follicular epithelial cells exfoliated into the lumen and colloidal fluid. Histomorphometry showed a dose-dependent decrea sed follicular density at 1 and 3 mg/kg/day dose in males. However, it is unclea r 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 un certainty in the expe rimental dosing design, particularly for the pups and consequently the re ported results. Although only the findings in dams and PND 2 pups were considered of this evaluation, there is reduc ed confidence in the study. While there were no clinical signs of cholinergic inhibiti on in this study, ChE activity was
not assessed.

Several open literature studies on amphibian development for chlorp yrifos 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 studi es are limited in their ability to inform about potential thyr oid-interactions (e.g., Sparli ng and Fellers, 2009; Reylea 2009,
Bonfanti et al . 2004; Richards and Kendall 2003).CHLORPYRIFOS FINAL

Page 23 of 74 Table 4. Thyroid Pathway for Chlorpyrifos.

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

Study Type/ Literature CitationThyroid receptorsThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth(BW, SVL)Systemic Toxicity2Overt ToxicityObserved3

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) N 4

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 NCHLORPYRIFOS FINAL

Page 24 of 74 Table 4. Thyroid Pathway for Chlorpyrifos.

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

Study Type/ Literature CitationThyroid receptorsThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth(BW, SVL)Systemic Toxicity2Overt ToxicityObserved3

Modified Developmental Toxicity (Mouse; De Angelis et al

., 2009) NE P 5 (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 examinedCHLORPYRIFOS FINAL

Page 25 of 74 IV. Committee’s Assessment of Weight of Evidence

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

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

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses are mediated through an endocrine pathway, therefore nonendocrine toxic responses (incl uding but not limited to mortalit y 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, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All effects in endocrine se nsitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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 eff ects observed which may be considered overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. However, in other instances, mo re than one parameter was needed to determine overt toxicity. Effects which were considered to be signs of system ic toxicity were generally less severe forms of toxicity (e.g., changes in organ weights or blood chemistry).CHLORPYRIFOS FINAL

Page 26 of 74 1. Tier 1 in vivo Assays

Systemic toxicity was not observed in the uterot rophic assay. In the Hershberger assay, there were no significant effects on body weights or bo dy 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 gla nds weight in the 1 mg/kg/day dose group was comparable to the controls. ChE activity in RBCs were significantly de creased (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 signi ficantly 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 d ecreased (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 decrease d (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 μ g ai/L treatment relative to the negative control. The was significant decreas es 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

μ g ai/L treatment groups, respectively. A 65% decr ease (NS) in ChE in males was observed at the lowest treatment group compared to the control. Clinical signs of toxic ity, including injury to eyes and snout (along with females reported as bl oated 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 th ese clinical signs ar e treatment-related.

In the AMA, several tadpoles were observ ed swimming erratically on Day 8 in the 20 μ g 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 lim b tissue was observed in all treatment groups on Day 21 compared to the negative control. ChE activity was also re duced (p<0.05) in tail
tissue in the 5 and 20 μ g a.i./L treatment groups (28 a nd 68%, respectively) on Day 21.

2. OSRI

In a two-generation rat reproduction toxicity st udy, 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 F 1 adults at 1 and 5 mg/kg/day. In addition, brain ChE was inhibited in the high dose P and F 1 males ( ↓ 48-53%) and females ( ↓ 49-58%). Additionally at the high dose, in the P females, very slight vacuolati on (16/30 treated vs. 0 cont rols) consistent with altered tinctorial properties of the zona fasciculata ( 21/30 treated vs. 0 cont rols) of the adrenal gland was noted; altered tinctorial properties of the zona fasciculata (13/30 treated vs. 2/30CHLORPYRIFOS FINAL

Page 27 of 74 controls) of the adrenal gla nd was also noted in the F 1 females. Very slight (19/30 treated vs. 14/30 controls) to slight (5/30 tr eated vs. 1/30 control) vacuolati on of the adrenal was observed in the adrenal glands of the P males; slight (6 /30 treated vs. 1/30 contro ls) vacuolation of the adrenal glands was also found in the F 1 males. It was stated that the histological changes were consistent with fatty changes in the males. Ne onatal effects were observed only in the presence of maternal toxicity and consisted of redu ced pup weights and increased mortality at 5 mg/kg/day.

In a male mouse reproduc tive 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, an d food consumption were reduced (p ≤ 0.05) at 25 mg/kg/day from Week 2 to the end of treatment, with terminal body weights decr eased (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). Ho wever, 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 consider ed to be indicative of toxicity at a systemic level. Although ChE activity was not determin ed, the dose levels administered have been observed to cause significant inhibiti on of ChE activities in other studies.

In a rat developmental neurotoxic ity study, maternal toxicity at th e high dose (5 mg/kg/day) was manifested at the end of gesta tion and during LD 1-5. Dams w ith all pups dying were increased at the high dose. Maternal brain ChE ac tivity 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 Subs et 1 (PND 12) high-dose animals ( ↓ 17-19%) and the Subset 4 (PND 66) high-dose males ( ↓ 10%). For the F 1 adults, body weights of the high dose males were decreased during PND 22-66 ( ↓ 11-17%). High-dose F 1 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%).CHLORPYRIFOS FINAL

Page 28 of 74 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 (ex cessive salivation, perineal urine stains, periocular porphyrin deposits, vaginal bleeding, and tr emors) 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 decrea sed by 74% and 79% at the mid and high dose, respectively. In the second ra t 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 corre cted 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 dos e 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 duri ng 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% cont rols). Plasma ChE activity was inhibited (p<0.001) by 57-72% in all treated groups fo llowing 10 days of dosing.

In a modified mouse developmenta l 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 t oxicity was not observed is this study.

In the rat chronic toxicity/car cinogenicity study, it was stated that body weights of the high dose (10 mg/kg/day) males were decreased (p<0.05) fr om Day 7 on to study termination, but that the difference never exceeded 9%, and that ove rall 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 a nd 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 ex cess 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, chlo rpyrifos 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 theCHLORPYRIFOS FINAL

Page 29 of 74 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 carcinogeni city studies, increased (p<0.0 5) 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 decr eased (p<0.05) by 4-9% in the high dose males generally during treatment (Weeks 1-78). Fema le body weights were decreased (p<0.05) only during Weeks 1-3, suggesting palatability of th e 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 th e high dose males. Plasma ChE was decreased (p<0.05) at all treatment levels for both Week s 42 and 78 by 49-98% in the males, and by 45-
99% in the females. Brain ChE was decrease d (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 fe males. 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 firs t 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 ti me 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 erythr ocyte (RBC) group means relative to controls, which is suggestive of anemia. Urinalysis re vealed that males in the high dose group had a significantly reduced urine volume, increased ur ine pH, a higher specific gravity and a higher protein grading, which appear to be treatment-re lated. 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, pl asma ChE was also signi ficantly inhibited at 91 and 57% for the 10 and 200 ppm groups, respective ly, 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 fi rst three weeks of tr eatment 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 th e diet. From Day 70 to study te rmination, 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 th at PCV were decreased (p<0.05) in the high dose females, platelets were increas ed (p<0.05) in the mid and high dose males, and RBC were decreased (p<0.05) in th e 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 displayedCHLORPYRIFOS FINAL

Page 30 of 74 a slight (5/10) to moderate (5/ 10) vacuolation in the adrenal gl and (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 adre nal 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 sacrif ice, 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 al so 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 fe males, 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/m 3

.
In the two avian reproduction toxi city studies on Bobwhite quail, no signs of systemic toxicity were observed at dietary concen trations 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 feat her condition and were significantly thinner than controls. Food consumption was mu ch lower than that of controls during the first four weeks, and a depression in body weight was evid ent. 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 sec ond duck reproduction toxicity study, there was a treatment-related increase in mortality during the pre-egg production pe riod 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. Th erefore, 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 highe st 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.CHLORPYRIFOS FINAL

Page 31 of 74 B. Estrogen Pathway

The Tier 1 Estrogen Receptor (ER) binding assay was negative. However, there were positive responses in the Tier 1 ER transactivation assa y (ERTA) as well as multiple ERTA studies from the published literature. In the st eroidogenesis 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 assa y (FSTRA). There were no estroge n-related effects observed in the Tier 1 uterotrophic assay or the female pube rtal assay. There were no estrogen-related effects
in the mammalian Part 158 studies in the absenc e of systemic toxicit y. In the FSTRA, while there was a significant decrease (52-71%) on fec undity, there were no treatment-related changes in estrogen-related endpoints such as ovarian histopathology, GSI, plasma vitellogenin or plasma

17 β -estradiol; reductions in brain ch olinesterase 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 eff ects on reproductive endpoints in the Part 158 fish life-cycle study. Therefore, there is a lack of convincing ev idence 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 th e 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 wa s negative, and in the male pubertal assay, there were no treatment-re lated changes in age or body we ight at preputial separation, weights of the testes, accessory sex tissue weights, pituitary weight, serum testosterone levels, or testicular and epididymal histop athology. In an OSRI study, (Joshi et al . 2007), effects on androgen-related endpoints were observed (d ecreased testosterone, decreased testes weight/altered histopathology, and a ltered fertility). However, signs of toxicity were reported at all doses tested. Furthermore, although ChE activ ity was not determined in this study, the dose levels administered (7.5, 12.5, 17.5 mg/kg/day) have been obs erved 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 endpoi nts such as testes histopathology, plasma testoster one, or male secondary sex ch aracteristics. There were no androgen-related effects in the Pa rt 158 mammalian studies in the absence of overt toxicity; there were also no androgen-related e ffects observed in the Part 158 avia n or fish reproduction studies. Therefore, while in vitro studies indicate some potential andr ogen-related effects, no significant corroborating effects were observed in the in vivo studies in the absence of systemic (particularly
cholinesterase inhibiti on) or overt toxicity.CHLORPYRIFOS FINAL

Page 32 of 74 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 fe male pubertal assays at doses that caused significant cholineste rase inhibition. While developmen tal delays were observed in the AMA, the lack of effects on thyroid histop athology along with a reduction in growth do not support a potential thyroi d-related effect. Furthermore, no agonist or antagonist activity was seen in a receptor transactiva tion assay using thyroid receptor tr ansfected Vero cel ls. No thyroidrelated effects were seen in the mammalian Part 158 studies. Alt hough there were reported effects on thyroid hormone and hi stopathology 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 th e available information the evidence does not support a potential in teraction with the thyroid pathway.

E. Conclusions

The conclusion of the WoE evaluation is that chlorpyrifos demonstrates no convincing evidence of potential interaction with the estrog en, 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 poten tial interaction with th e estrogen, androgen and thyroid pathways.CHLORPYRIFOS FINAL

Page 33 of 74 VI. References

Andersen, H., Vinggard, A., Rasmussen, T., et al . (2002) Effects of current ly used pesticides in assays for estrogenicity, androge nicity, and aromatase activity in vitro . MRID 48033001.
Published: Toxicol. Appl. Pharmacol. 179 : 1-12.

Beavers, J., and Fink, R. (1978) One-generation reproduction study - Mallard Duck - chlorpyrifos. Final report. Wildlife Intern ational, Easton, MD. Laboratory Study ID not provided, December 16, 1978. Submitted by Dow Chemical Co. MRID 00046952. Unpublished.

Beavers, J., and Fink, R. (1978) One-gene ration reproduction study - bobwhite quail - Chlorpyrifos. Final Report. Wildlife Intern ational, Easton, MD. Laboratory Study ID not provided, October 30, 1978. Sponsored by Dow Chemical Co. MRID 00046957. Unpublished.

Bonfanti P, Colombo A, Orsi F, Pizzetto I, Andrioletti M, Bacchetta R, Manteca P, Fascio U, Vailati G, Vismara C (2004) Comparative terato genicity of Chlorpyrifos and Malathion on Xenopus laevis development. Aquat Toxicol 70:189–200

Breslin, W., Liberacki, A., Dittenber, D., et al . (1991) Chlorpyrifos: two-generation dietary reproduction study in Sprague-Dawley rats. The Toxicology Research Laboratory, Health and Environmental Sciences, The Dow Chemical Company, Midland, MI. Laboratory Study Number K-044793-088, June 5, 1991. MRID 41930301. Unpublished.

Coady, K., Lehman, C., Hutchinson, K., et al . (2011) Chlorpyrifos : the amphibian metamorphosis assay using the African clawed frog, Xenopus laevis . Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Laboratory Report No. 101127, August 8, 2011. MRID 48615501. Unpublished.

Coady, K., and Sosinski, L. (2011) Evaluation of chlorpyrifos in the human recombinant aromatase assay. Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. La boratory Study Number 101142, October 21, 2011. MRID 48615503. Unpublished.

Corley, R.A., et al . (1986) Chlorpyrifos: 13-week nose-only vapor i nhalation exposure study in Fischer 344 rats. Toxicology Research Laborat ory, Dow Chemical Comp any. Laboratory Study No. K-044793-077, November 13, 1986. MRIDs 40013901 and 40166501. Unpublished.CHLORPYRIFOS FINAL

Page 34 of 74 Currie, R., Louch, D., Coady, K., Fiting, J., et al . 2011 Chlorpyrifos: a fish short-term reproduction assay with the fathead minnow, Pimephales promelas . Toxicology & Environmental Research Consulting, The Dow Chemical Company, Midland, MI. Laboratory Study No. 101123, October 24, 2011. MRID 48615506. Unpublished.

Crown, S., Gur, E., Nyska, A., et al . (1985) Toxicity in dietary administration to rats for 13 weeks [Pyrinex (chlorpyrifos) Insecticide]. Life Science Research Israel, Ltd., Ness Ziona, Israel. Laboratory Project ID MAK/058/PYRA ., October 28, 1985. Sponsored by MakhthshimAgan (America), Inc. MRID 40436406. Unpublished.

De Angelis, S., Tassinari, R., Maranghi, F., et al . (2009) Developmental exposure to chlorpyrifos induces alterations in thyroid a nd thyroid hormone levels without other toxicity signs in CD-I
mice. MRID 48175901. Published: Toxicol. Sci

. 108 (2) : 311-319.

Farag, A., Radwan, A., Sorour, F., et al . (2010) Chlorpyrifos induced reproductive toxicity in male mice. Reprod. Toxicol. 29(1): 80-85.

Goodman, L.R., D.J. Hansen, D.P. Middaugh, G.M. Cripe and J.C. Moore. (1984). Method for Early Life-Stage Toxicity Test Using Three At herinid Fishes and Results with Chlorpyrifos. Aquatic Toxicology and Hazard Assessment: Seventh Symposium, ASTM STP 854, R.D. Cardwell, R. Purdy, and R.C. Bahner, Eds., Am erican Society for Testing and Materials, Philadelphia, 1985, pp 145-154.

Gur, E. (1992) Pyrinex technica l: oncogenicity study in the mouse. Life Science Research Israel, Ltd., Ness Ziona, Israel. Study number MAK/106/PYR, October 15, 1992. Sponsored by Makhthshim-Agan (America), In c. MRID 42534201. Unpublished.

Hakin, B. (1990) The effect of dietary inclusion of chlorpyrifos on reproduction in the Mallard duck. Huntingdon Research Centre Ltd., Huntin gdon, Cambridgeshire, UK. Laboratory Project ID. MBS 28/881667, date not provided. Sponsored by Makhteshim-Agan (America) Inc. MRID 42144901. Unpublished.

Hakin, B. (1990) The effect of dietary inclusion of chlorpyrifos on reproduction in the bobwhite quail. Huntingdon Research Centre, Ltd., Hunt ingdon, Cambridgeshire, UK. Laboratory Report No. MBS 27/881666, date not provided. Sponsor ed by Makhteshim-Agan (America), Inc. MRID 42144902. Unpublished.

Hoberman, A. (1998) Developmental neurotoxicity study of chlorpyrifos administered orally via gavage to Crl:CD®BR VAF/Plus® presumed pregnant rats. Argus Research Laboratories, Inc., Horsham, PA. Laboratory study No. 304-001, May 1, 1998. MRID 44556901. Unpublished.CHLORPYRIFOS FINAL

Page 35 of 74 Joshi, S., Mathur, R., and Gulati, N. (2007) Testicular toxicity of chlorpyrifos (an organophosphate pesticide) in albino rat. MRID 48175902. Published: Toxicol. Ind. Health 23 (7) : 439-44.

Juberg, D.R., Ghen, S.C., Kramer, V.J., Lu, H., Marty, S., Coady, K.K. (2011). Chlorpyrifos: Weight of Evidence Evaluation of Potential Inte raction with the Estrogen, Androgen or Thyroid Pathways. Dow AgroSciences, LLC, Indian apolis, Indiana. Study Number 110687, MRID
48670001.

Kojima, H; Katsura, E.; Takeuchi, S.; Niiyama, K.; and Kobayashi, K. (2004). Screening for Estrogen and Androgen Receptor Ac tivities 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 us ing 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. La boratory 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 & Environmen tal 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 hER α -HELA-9903. Toxicology & Environmental Research and Consulting, Th e Dow Chemical Company, Midland, MI. Laboratory Project Study ID 101190, Oct ober 27, 2011. MRID 48615505. Unpublished.

Le Baron, M., Kan, H., and Perala, A. (2011) Ev aluation of chlorpyrifos in the in vitro steroidogenesis assay. Toxicology & Envir onmental Research and Consulting, The Dow Chemical Company, Midland, MI. Labor atory 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.CHLORPYRIFOS FINAL

Page 36 of 74 Marty, M, Zablotny, C., and Stebbi ns, K. (2011) Pubertal devel opment and thyroid function in intact juvenile/peripubertal female Crl:CD(SD ) Rats. Dow Chemical Company, Midland, MI. Laboratory Project Study ID 101176, Nove mber 1, 2011. MRID 48615508. Unpublished.

Marty, M., Andrus, A., and Hukkanen, R. (2011) Chlorpyrifos: pubertal development and thyroid function in intact j uvenile/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: uterot rophic assay in the immature female Crl:CD(SD) rat. Dow Chemical Comp any, Midland, MI. Labor atory 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 Laborator y, 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. Pa rt A: Current Issues. 66(1): 75-91..

Rubin, Y., Gal, N., Waner, T., and Nyska, A. (1987) Pyrinex: teratogenici ty 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, Isra el. Study Number MAK/ 103/PYR, July 15, 1987. Sponsored by Makhthshim-Agan (Ameri ca), 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 iden tify 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 repor ter gene assays to assess the hormone receptor activities of chemicals fr equently detected in drinking water. J. Appl. Toxicol. 32(8) : 635-641.CHLORPYRIFOS FINAL

Page 37 of 74 Szabo, J., Young, J., and Grandjean, M. (1988) Chlo rpyrifos: 13-week diet ary toxicity study in
Fischer-344 rats. Health and Environmental Scie nces - 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 E ndocrine 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. Envir onmental Protection Agency, Washington, DC.

USEPA (2011) Endocrine Disrupt or 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 Coor dination and Policy. U.S. Environmental Protection Agency, Washington, DC.

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

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

Warner, S., Gerbig, G., Strebing, R., et al . (1980) Results of a two-ye ar toxicity and oncogenic study of chlorpyrifos administered to CD-l mi ce 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 Environmen tal Sciences - Texas, Lake Jackson Research Center, The Dow Chemical Company, Freepor t, TX. Laboratory Study ID TXT:K/044793/079, December 23, 1988. MRID 40952802, Unpublished.CHLORPYRIFOS FINAL

Page 38 of 74 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 fl ow-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 dimethylformamid e; DMF) controls a nd chlorpyrifos (99.8% purity) at nominal test chemical c oncentrations of 0.000310, 0.00125, 0.00500, and 0.0200 mg a.i./L. The 21-day time-weighted average (T WA), 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 eff ects are reported based on comparis on to the negative control.

There were no treatment-related effects on surv ival, which ranged from 98.3 to 100% across the controls and all treatment groups . Several tadpoles were observe d 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 redu ced (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, respec tively, 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 ne gative control.

At TWA 0.0136 mg a.i./L, chlorpyrifos significa ntly delayed (Jonckheere-Terpstra, p=0.03) Day 21 NF developmental stage by one stage and sign ificantly 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 follic ular cell hypertrophy
was observed in both controls and all treatment groups, with no apparent treatment-related

response.

Concentration-dependent signifi cant reductions (p<0.05) of 21-79% in cholinesterase activity
from hind limb tissue were observed in all treatme nt groups on Day 21 compared to the negative control. Cholinesterase activity was also sign ificantly 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 nega tive control.CHLORPYRIFOS FINAL

Page 39 of 74 Androgen Receptor (AR) Bi nding 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 (M RID 48682802) in which the cytoso lic AR was characterized by duplicate saturation binding experiments. This study showed that cytosolic AR was present in reasonable numbers and was functioning with appropr iate affinity for the radio labeled reference androgen (R1881). The competitive binding experi ment 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 dexamethas one as a weak pos itive control, and R1881 as the ligand reference standard. Th ree independent runs were conducted with 3 replicates per con centration per run.

In the saturation binding experiment, the maximum binding capacity (B max ) was 3.245 fmol/100 μg protein and the dissociation constant (K d ) 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 satu ration 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 IC 50 s 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 cont rol was 0.0034%. The solven t control responses indicated no drift in the study a ssay. 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 co mpetitive 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 ne xt highest concentration of 10 -4 M, the mean binding was 74.5 ± 1.0%, which was also in th e equivocal range. An IC 50 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 th an normal steepness (-1).

Based on the results from the three runs, chlorpyrif os is classified as E quivocal in the Androgen Receptor Binding Assay.CHLORPYRIFOS FINAL

Page 40 of 74 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 incubate d with human recombinant aromatase and tritiated androstenedione ([1 β

3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]-ASDN) at logarithmic
concentrations from 10

− 10 to 10

− 3 M for 15 minutes at 37°C to asse ss 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 con centration 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 bac kground activity control, a positive control series (10

− 10 M to 10

− 5 M) using a known inhib itor (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 fu ll activity controls was 0.0869 nmol

· mg-protein

− 1

· min

− 1

, indicating that the microsomal lo t used in this particular run (Lot # 85585) had relatively low aromatase activity per mg protein. The report stat ed 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 lowe r than the assay perfor mance 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 contro ls and background controls was acceptable for each run.

Results for the positive control were generally within the recommended ra nges for the top of the curve, bottom curve, hill slope, log IC 50 , and coefficient of varia tion for replicates of each
concentration within runs. Fo r 4-OH ASDN, the estimated log IC 50 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 (R 2 ) was only 0.57–0.77. Because the data faile d to model adequately, valid log IC 50 and Hill slope values could not be determined for chlorpyrifos. The average dose-response curve indicated that chlorpyr ifos had no effect on aromatase act ivity at concentrations of 10

− 10 to
10

− 6 M. However, chlorpyrifos reduced arom atase 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.CHLORPYRIFOS FINAL

Page 41 of 74 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) Bi nding Assay (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48615504), uterine cytosol from Sprague Dawley rats was used as the s ource of estrogen receptors (ER) to evaluate the potential for chlorpyrifos to displace the bound re ference estrogen, radiolabeled [ 3 H]-17 β -estradiol from the ER. A saturation binding experiment was conduct ed 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 th e 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 [ 3 H]-17 β -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 replic ates per concentration

per run.

In the saturation binding experime nt, the maximum binding capacity (B max ) was 59.28 fmol/100 μg protein and the di ssociation constant (K d ) was 0.1032 nM. The B max fell within the expected range of 10 to 150 fmol/100 μg protein and the K d for the run was within the expected range of 0.03 to 1.5 nM. Nonspecific binding as a per cent of total binding was 1.7%-8.6% across the entire concentration range in the saturation bind ing 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 w ith chlorpyrifos was >75% at concentrations up to 10

− 3 M, an IC 50 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, ha d no effect on binding, and the binding curves for 17 β -estradiol and 19-norethi ndrone showed that increasing concentrations of each compound displaced [ 3 H]-17 β -estradiol in a manner consis tent with one-site binding.CHLORPYRIFOS FINAL

Page 42 of 74 The mean log IC 50 was -5.5 M for 19-norethindrone and -8.9 M for 17 β -estradiol, and the mean
RBA was 0.034% for 19-noirethindrone co mpared 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.

ER α Transcriptional Activatio n (ERTA) Assay (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48615505), hER α -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 perfor med using 96-well plates and each chlorpyrifos concentration was tested in tripli cate (3 wells/plate). Cells were exposed to the test agents for 24 hours to induce reporter (lucifer ase) gene products. Luciferase expression in response to activation of the estrogen receptor by chlorpyrif os was measured upon addition of a luciferase
substrate and detection with a lumi nometer with acceptable sensitivity.

Chlorpyrifos was tested up to the limit of solubility, 10

− 4 M.

The mean RPC Max for chlorpyrifos was 25.4% in the fi rst run, 10.2% in the second run and 19.5% in the third run, and the associated PC Max 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 se nsitivity to very weak agonists like 17 α -methyltestosterone. This does not negatively impact the validity of the study.

Because the RPC Max > PC 10 in all three assay runs, chlorpyr ifos 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 (MRI D 48615506) of chlorpyrifos technical with fathead minnows ( Pimephales promelas) was conducted under flow-t hrough 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) we re 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 c oncentrations were <0.0000312 (<LOQ), 0.000251, 0.000812, and 0.00302 mg a.i./L. The test system was maintained at 24.8 to 25.3 o C and a pH of

7.3 to 7.8.CHLORPYRIFOS FINAL

Page 43 of 74 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 negati ve control and treatment groups with similar lesions ( e.g. , injury to eyes, body) and in cident rates. Male body weights were significantly reduced (p<0.05) by 17% in th e high treatment group relative to the negative
control.

Spawning in the negative control occurred at leas t every four days, and fecundity averaged 34.2 eggs/female/day; fertilization su ccess in the negative control was 98.9%. There were significant reductions (Jonckheere-Terpstra; p<0.05) in fecund ity 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 differenc es (p>0.05) in male tubercle sc ores or male or female gonadosomatic index (GSI) compared to the negative control. Tubercles were not observed in females. Plasma vitellogenin, testosterone , and estradiol for th e treatment groups were not significantly different (p>0.05) when compared to the negative control.

Although not analyzed sta tistically, there was a marginally higher incidence of oocyte atresia (all severities) in treated females compared to negativ e control females; however, this may have been due to granulomatous inflammation and was inte rpreted by the study auth ors as a spontaneous
alteration not associated with e xposure to chlorpyrifos due to th e lack of a treatment-related response and bacterial etiology. There were also incidences of egg debr is 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 hist opathological findings we re not attributed to chlorpyrifos exposure.

The study authors’ analysis det ected significant decreases of c holinesterase 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%, respectivel y). 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 leve ls of 0 (vehicle), 1, 6, or 12 mg/kg/day. AnCHLORPYRIFOS FINAL

Page 44 of 74 androgenic positive control group consisted of seve n castrated rats exposed to 0.4 mg/kg/day of testosterone propionate (TP) by s ubcutaneous (s.c.) injection.

To screen for potential anti-a ndrogenic activity, 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. in jection. The anti-androg enic 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 th e anti-androgenic ne gative 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 collect ed and weighed. Brain and red blood cell (RBC) samples we re also collected for determin ation of cholinesterase (ChE) activity.

All animals survived until schedul ed termination. There were no c linical signs of toxicity and no treatment-related gross pathological findings.

In the androgen agonist assay, there were no si gnificant 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 incr eased body weight gains ( ↑ 29%) compared to the controls. Food consumption fo r Days 4-7 was increased (p<0.05) by 7% in the 12 mg/kg/day group; this followed a period of decreased body wei ght gain (not statistically significant, NS) in this group, and thus redu ced 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 me aningful because it was not dose related.

Kidney and liver weights in all chlo rpyrifos 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% i nhibition) 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 (6 4% 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 or gan weight increases (p<0.05) in all five target tissues.CHLORPYRIFOS FINAL

Page 45 of 74 In the anti-androgen assay, there were no eff ects on body weights, body we ight 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 th e 1 mg/kg/day group, and ki dney and liver weights in all chlorpyrifos treatment groups were compar able to controls. RBC ChE activity levels were significantly decreased (p<0.05) at 1 mg/kg/day (91% i nhibition) and were below the level of detection at 6 and 12 mg/kg/day. Brain ChE act ivity levels were significantly decreased (p<0.05) at 6 and 12 mg/kg/day (60% and 76% inhibition). Ra ts 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 comp arable to controls. Th e 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. A ll CV values were less than the maximum recommended values for each orga n, with the exception of levator anibulbocavernosus (LABC) in the 12 mg/kg/da y group (33% CV compared to maximum recommended CV of 20%).

The doses tested were judged to be ade quate 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 fo r 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 or al gavage with chlorpyrif os (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 openi ng (VO) daily beginning on PND 22, and the age and weight at day of attainment was recorde d. Following sacrifice on PND 42, total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) leve ls were determined using chemiluminescent immunoassay and radioimmunoassay, respectively. Weights were recorded for the liver, kidneys, urogenital organs, pituit ary, thyroid and adrenal glands and microscopic examinations were performed on the ovaries, uterus, thyroid, a nd kidneys. Cholinesterase (ChE) activity was determined in red blood cells (RBC) and the brain.

There were no effects of treatment on mortalit y, clinical signs of t oxicity, body weights, body weight gains, age of attainment of VO, body weight at VO, mean age at first estrus, mean cycleCHLORPYRIFOS FINAL

Page 46 of 74 length, percent cycling, pe rcent regular cycling, or gan weights, serum T 4 and TSH levels, clinical chemistry parameters, or gro ss or microscopic pathology.

RBC ChE activity was decrea sed (p<0.05) compared to the control at 0.5 ( ↓ 19%) and 1.0 ( ↓ 88%) mg/kg/day, and completely inhibi ted 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 base d on the observed RBC and brain cholinesterase activity.

Male Pubertal Assay (OCSPP 890.1500)

In a male pubertal assay (MRID 48615509), 16 Sp rague-Dawley (Crl:CD[SD]) rats/dose group were treated daily via oral gavage (4 mL/kg) with chlor pyrifos (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 se paration (PPS) daily beginning on PND 30, and the age and weight at day of attainment was record ed. Following sacrifice on PND 53, total serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were analyzed using radioimmunoassays (TSH) or chemilumi nescent assays (testosterone and T 4 ). 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 ad renal glands, and microscopic ex aminations 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 wei ghts, body weight gains, age or we ight at attainment of PPS, serum hormone (T 4 , TSH, or testosterone) levels, organ we ights, and gross or histopathological parameters. RBC ChE activity was dose-dependen tly 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 ade quate based on the observed RBC and brain cholinesterase activity.

Steroidogenesis Assay (OCSPP 890.1550)

In a steroidogenesis assay (M RID 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 leve ls were measured using LC/A PPI-MS/MS. Three independent experiments were performed. A Quality Contro l (QC) plate was run concurrently with eachCHLORPYRIFOS FINAL

Page 47 of 74 independent run of a test chemical plate to de monstrate that the assay responded properly to positive control agents at two concentration le vels. The positive controls included the known inhibitor (prochloraz) and i nducer (forskolin) of estradiol and test osterone production.

Guideline recommendations were met including: l ack of cytotoxicity, ad equate production of testosterone and estradiol, accep table reproducibility (low %CV) , and appropriate induction and inhibition with posi tive 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 signifi cant inhibition of testosterone was observed, and statistically si gnificant increases of estradiol were observed. The average
decrease in testosterone c oncentration was 0.6-fold at 10 μ M and 0.4-fold at 100 μ M. The average increase in estradiol c oncentration was 2.3-fold at 10 μ M and 2.4-fold at 100 μ M. No changes in hormone production were noted at ≤ 1 μ M of chlorpyrifos.

Based on the hormone responses in each of the th ree independent runs, ch lorpyrifos treatment resulted in statistically signi ficant 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 potenti al 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 ra ts 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 17 α

ethynyl 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 we ight gains (Days 1-4) in the chlorpyrifos treated groups were comparable to the contro ls 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.CHLORPYRIFOS FINAL

Page 48 of 74 No statistically significant changes were seen in uterine weight in this assay. Chlorpyrifos was negative in the uterotrophic assay.CHLORPYRIFOS FINAL

Page 49 of 74 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 ac tivation 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 MC F-7 and CHO cells. The cell proliferation assay, ERTA, and ARTA were conducted using chlorp yrifos at concentrations ranging from 10

− 8 M to
10

− 3 M, while the aromatase assay was perfor med at a single concentration of 50 μM.

Chlorpyrifos induced a weak estrogenic response in both the cell proliferat ion assay (36% of the
maximum 17 β -estradiol response at 50 μM), and the ERTA (25% of the maximum 17 β -estradiol response at 50 μM, but had no antagonistic effects on 17 β -estradiol-induced ce ll 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 chemical s using the proliferative effect of estrogen and estrogenic chemical on and estrogen-sensitiv e target cell line. This quantitative in vitro assay compares the cell number achieved by similar inoc ula of human breast can cer estrogen-sensitive (MCF-7) cells in the absence of estrogens (n egative control), and in the presence of 17 β -estradiol (E 2 , 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 eff ect. The aim of the work was to: 1) validate the E-SCREEN A ssay, 2) screen a variety of chem icals, 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 compari ng the cell yield achieved by similar inocula harvested simultaneously during the late exponentia l 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 treatmen t and positive (E2) contro l were apparent after 4 days. Estrogenic activity was assessed by 1) rela tive proliferative poten cy (RPP) which is the
ratio between the minimal concentration of es tradiol 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 (R PE) which is 100 times the ratio between the highest cell yield
obtained with the chemical and with E2l. Th e RPE allows one to quantitatively compare theCHLORPYRIFOS FINAL

Page 50 of 74 agonist effect relative to E2. Results were expressed as mean ± SE. Proliferation yield experiments were conducted in duplicate wells w ith a minimum of 5 replicated. Differences between groups were assessed by analysis of va riance and the a posteriori Shaffe’s test. A p
value ≤ 0.05 was regarded as significant. Chlorpyr ifos 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 bisphe nol 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 tr ansfected and following 12 hours of incubation, were subjected to treat ment with the test chemical at various concentrations, with

17 β -estradiol ( 10 –9 to 10 –3 mg/L; positive control), or with 0.1% DMSO (negative control) to assess estrogenic activity. To assess anti-estr ogenic activity, cells were treated with various
concentrations of the test chemical mixed with 1×10 -6 mg/L E 2 . For the AR Reporter Gene Assay , the cultured, plated cells were transfec ted and following 12 hours of incubation, were subjected to treatment with the test chemical at various concentrati ons, 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 tr eated with various concentrati on 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, th e cells were subjected to treatment with the
test chemical at various concen trations, with triiodothyronine (T 3 , 10 –5 to 1 mg/L; positive control), or with 0.1% DMSO to assess TR agonistic activity. Ce lls were treated with various
concentrations of the test chemical mixed with 5×10 –4 mg/L T 3 to assess TR antagonistic activity. The cells from each assay were harves ted after 24 hours of exposure to the treatment, lysed, and immediately analyzed us ing a 96-well plate luminometer. A luciferase reporter assay system kit was used for quantitation. Relative tran scriptional activity was c onverted into the fold induction above the corresponding vehicle control (n -fold). Triplicate an alyses were conducted for each treatment. The cytotoxi city 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 E 2 , testosterone, or T 3

.
All three positive controls exhibited appropria te concentration-dependent responses in the respective assays. Chlorpyrif os had no estrogenic activity at 0.003-3.0 mg/L but did show significant (p<0.01) suppression of lucifera se at 0.3 and 3.0 mg/L compared to E 2 , indicating antagonistic activity. The RIC 20 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) compar ed with testosterone alone. The RIC 20 forCHLORPYRIFOS FINAL

Page 51 of 74 chlorpyrifos was 6.43×10 -7 M (7.51x MCL). Chlorpyrifos show ed 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 (hER α and hER β ) and the human androgen receptor (hAR) in tr ansactivation assays in Chinese hamster ovary cells (CHO-K1 cells) using a luciferase reporter construct.

Agonistic activities were evaluated by relative ac tivity expressed as REC 20 (20% relative effective concentration). The REC 20 is the concentration of the compound showing 20% of the activity of 10 -10 M E 2 , 10 -9 M E 2 , or 10 -9 M DHT for ER α , ER β and AR, respectively. Antagonistic activities were evaluated by relative activity expressed as RIC 20 (20% relative inhibitory concentration). The RIC 20 is the concentra tion of the compound showing 20% of the inhibition of the activity of 10 -10 M E 2 , 10 -9 M E 2 or 10 -9 M DHT for ER α , ER β and AR, respectively. 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 th e activity of the test compound was higher than the REC 20 the pesticide was judged to be positiv e for activity. When the activity of the test compound was higher than the RIC 20 the pesticide was judged to be positive for inhibitory activity. The statistical significance of differences was eval uated with a two-tailed
Student’s t test with a nomina l level of significance of p < 0.05.

Chlorpyrifos was tested a nd found to be positive for ER α transactivation with a REC 20 value of

7.5 x 10 -6 M with a relative luciferase activity (RLA ) of 27%. Chlorpyrifos was negative for

ER β and AR transactivation in this test system.

The study authors concluded that chlorpyrifos at 0.05 μM indu ced cell proliferation through ER α activation in hormone-dependent breast cancer cells. In contra st, 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 ba lance 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 microt ubule polymerization.

In a published study by Kojima et al . (2005) , the response of an in vitro ER–related reporter gene assay was investigated af ter exposure to thirty two pest icides either alone or in combination. The source of the test compounds was not reported; however , 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 pr omoter, confer estrogen responsiveness to the viral promoter and reporter gene. The transfected cells are made st able by an antibiotic resistanceCHLORPYRIFOS FINAL

Page 52 of 74 gene resulting in the cell lin e used in the assay (BG1Luc4E 2 ). Compounds that interact with estrogen-responsive DNA are identif ied by the luciferase gene re sponse and quantified in terms of Relative Light Units (RLU).

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

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a two-generation repr oduction 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 F 1 generation, respectively); with exposure continui ng in dams through gestation, lactation and weaning. The P generation rats were mated once to produce F 1 litters. In addition to the standard reproductive study parameters, plas ma, red blood and brain ChE activity were
determined for the first 10 P and F 1 adult rats/sex/dose at the scheduled necropsy.

There were no treatment related effects on mort ality, body weight, food consumption or clinical signs in either P or F 1 animals. Dose-related decr eases (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 F 1 adults at 1 and 5 mg/kg/day. In addition, brain ChE was inhibited in the high dose P
and F 1 males ( ↓ 48-53%) and females ( ↓ 49-58%). Additionally at the high dose, in the P females, very slight vacuolation (16/30 treated vs. 0 contro ls) consistent with altered tinctorial properties of the zona fasciculata (21/30 treated vs. 0 c ontrols) 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 F 1 females. Very slight (19/30 treated vs. 14/30 controls) to slight (5/30 treated vs. 1/30 control) vacuola tion of the adrenal was observed in the adrenal glands of the P males; slight (6/30 treated vs. 1/30 controls) vacuolati on of the adrenal glands was also found in
the F 1 males. It was stated that the histological ch anges were consistent with fatty changes in the
males.

Neonatal effects were observed only in the pres ence of maternal toxici ty 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 fe rtility indices, lengths of gestation, time toCHLORPYRIFOS FINAL

Page 53 of 74 mating, pup sex ratio, pup survival or litter size in the first (P adults/F 1 litters) or the second (F 1 adults/F 2 litters) generations. No treatment-related histopathological lesions were seen in the testes, epididymides, and seminal vesicle, coagul ating 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 decrea sed 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 vi a 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 al l 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 suspen ded sperm were calculated. The wet weight of
testes was recorded after rem oving the adherent tissue and frozen for measurement of glycogen, cholesterol, protein, and sialic acid. Blood was collected by ca rdiac puncture arid serum was separated by centrifugation. Te stosterone 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 sugge stive of testicular toxi city were observed at the mid and high doses. Testes weights were d ecreased (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), resulti ng 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 decrea sed (p<0.01) in a dose-dependent ma nner (presented graphically). Additionally at the mid and high doses, histopathology of the testes showed severe degenerative changes marked with damaged epithelium, irregu lar and incomplete spermatogenesis, and lumen filled with cellular debris and devoid of sperm.CHLORPYRIFOS FINAL

Page 54 of 74 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 pr egnant 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 br ain, 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 wei ghts neuropathological examinations (Subset 4).

Maternal toxicity at the high dos e (5 mg/kg/day) was manifested as increased signs of autonomic function toxicity, apparent at the end of gestation as fascic ulations (6/25 treated vs. 0/25 controls), and during LD 1-5 as fasciculations (16/24 treated vs. 0/25 co ntrols), 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 (3/2 3 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 decrease d 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 obser ved. The maternal LOAEL was 0.3 mg/kg/day, based on plasma and RBC ChE inhibition.

For the pups, the high dose body we ights were reduced (p<0.05; ↓ 8-15%) at PND 1 and 5 (preand post-culling). Body weights we re 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 weight s 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 F 1 adults, body weights of the high dose ma les were decreased during PND 22-66 ( ↓ 11-17%). High-dose F 1 adult females also weighed le ss than controls at PND 22 ( ↓ 17%), but were of similar weight at PND 66. Body weight gains we re 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 toCHLORPYRIFOS FINAL

Page 55 of 74 the significant decreased body we ights 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 contro ls) 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 dos e 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% treate d 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 re sponse during the auditory startle
habituation assessments on PND 23 in the high-dose animals compar ed 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 microscopi c lesions of the nervous system in Subset 1 or 4 offspring. Subset 1 high-dose males at PND 12 ha d reduced absolute brain weights ( ↓ 9% vs controls), increased relative brain weights ( ↑ 13% vs controls), reduced ante rior to posterior measurement of the cerebellum ( ↓ 24% vs controls), reduced height of the cerebellum ( ↓ 14% vs controls),
decreased thickness of th e parietal cortex ( ↓ 6% vs controls), and de creased thickness of the hippocampal gyrus ( ↓ 9% vs controls). High-dose fema le 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, morphomet ric 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 c ontradictory statistical results wh en compared to controls; decreases in mid-dose ( ↓ 4%) females as compared to control were not fo und 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 definitiv ely classify findings in the pr e-weaning offspring as having originated with pre- or post-natal exposure, nor as resulting from developmental perturbation versus direct systemic toxicity or neurotoxic ity. However, adverse findings in the adult (~PND 66) offspring, i.e., alterations in motor activity, auditory startle respon se, and brain structure (decreased measurements of the parietal cortex and hippocampal gyrus, in the absence of brainCHLORPYRIFOS FINAL

Page 56 of 74 weight deficits) can be interpreted to repr esent the long-term seque lae of developmental exposure to chlorpyrifos.

Adverse effects in the offspring have been identified at the MD T 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) a lterations in the hippocampal gyr us, 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 developm ental 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. Ther e 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 cardi ac 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 consumpti on were not altered by compound exposure and liver weights (the only organ for wh ich weights were obtaine d) 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 app eared to be transient, as the high dose dams had weight gain similar to the ot her groups during post-treatment (GD 16-21). Clinical signs of toxicity were evident at the high dose only. Ex cessive salivation, perineal urine stains, periocular porphyrin de posits, vaginal bleeding, and trem ors 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 a nd high dose, respectively. RBC ChE activity was decreased by 74.3% and 79% at the mid and hi gh dose, respectively. ChE activities at the
low dose were similar to controls.

The maternal LOAEL is 3.0 mg/kg/day based on statistically significan t decreases in RBC and plasma ChE activity. The ma ternal 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. Th e developmental LOAEL was not determined.CHLORPYRIFOS FINAL

Page 57 of 74 Developmental Toxicity (Rat, OCSPP 870.3700)

In a prenatal developm ental toxicity study ( MRID 40436407 ) groups of 32 pregnant Sprague Dawley rats were administered ch lorpyrifos (96.1% purity) in co rn 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/g roup 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 th e high dose (9.0% treated vs. 7.0% controls). Historical control means ranged from 4.4% to 6.3%, indicating that th e control and high-dose
values are both above the historical values. No treatment-related changes were seen in fetal viability, sex ratio, or soft tissu e variations/malformations. Pl asma 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 developm ental toxicity study ( MRID 40436408 ), groups of 14 pregnant New Zealand White rabbits were administered chlorpyr ifos (96.1% purity) in corn oil by oral gavage at dose levels of 0, 1, 9, 81, or 140 mg/kg/da y 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-re lated 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 increa se 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 tr eatment-related changes were seen in fetal viability, sex ratio, or soft ti ssue 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 ad ministered via oral gava ge 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 ad ministered chlorpyrifos via s.c. injection onCHLORPYRIFOS FINAL

Page 58 of 74 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 thr ee maternal treatment groups was randomly assigned to each of the three offspring treatmen t groups. Therefore, a total of nine overall treatment groups were examined. Serum T 4 levels were determined in dams immediately after the last treatment on GD 18, and in offspring on PND 15 and 150, and serum T 3 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 brai n ChE inhibition in a previously published study. Therefore, brain ChE measurements were not performed.

There were no effects of treatment on mortal ity, 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, T 4 levels were decreased (p<0.05) by 34%. Microscopic pathology showed a non- significant trend toward increa sed colloid foaminess in the thyroid. Thyroid histomorphometry showed increas ed (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 th yroid histopathology we re 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 T 4 levels were seen in either sex at any dose.

In the PND 150 offspring, male T 3 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 T 3 levels were increased (p<0.05) by 12% in the litters treated with 3 mg/kg/da y, regardless of maternal treatment, compared with litters that were treated with vehicle, regardless of maternal treatment. Male T 4 levels were reduced (p<0.05) by 11% in the litters tr eated with 3 mg/kg/day, regard less of maternal treatment, compared with litters that were treated with vehi cle, regardless of maternal treatment. Female T 4 levels were unaffected by treatment. Histopath ology showed a dose-dependent higher number of necrotic follicular epithelial cells exfoliated into the lumen and colloidal fluid when compared to prenatal treatment. No do se-related effects were seen after postnatal treatment. Histomorphometry showed a dose-dependent decr eased follicular density in males exposed prenatally at 3 and 6 mg/kg. No significant diffe rences were seen in females. No treatmentrelated effects were seen in the adrenals.

These effects were seen at dose levels th at did not cause brain ChE inhibition. No histopathological changes we re seen in the adrenal glands at any dose level.CHLORPYRIFOS FINAL

Page 59 of 74 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 record ed 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 vagi nal plug was observed was considered GD 0. Pregnant females were anesth etized with diethyl ether on GD 14, and fertility and mating indexes, number of implantation sites, resorptio ns, and dead and live fetuses were recorded. After the mating period, the males were wei ghed and euthanized. Weights of testes, epididymides, seminal vesicles, cauda epididym ides, prostate, brain, liver, and kidneys were recorded. Sperm counts, motility, and morphology analyses were performed. Brain and foreleg muscle were collected and analyzed for protei n levels and ChE activity. Blood samples were collected from the abdomen aorta at necropsy an d 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 ma les died during treatment. Clin ical 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 tr eatment 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 termin al body weights decreased (p<0.05) by 21%. No statistically significant differences in the absolu te or relative brain a nd 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%; prosta te 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 ma le 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 sp erm motility were decreased (p<0.05), and the rate of morphological abnormal sperm was al so increased at the mid and high doses. Additionally, testosterone le vels were significantly (p ≤ 0.01) decreased by 52 and 60% at the midCHLORPYRIFOS FINAL

Page 60 of 74 and high doses, respectively. Mild changes in sper matogenic 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 not ed. 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 sper matogenic cells was also observed in these groups. Mild to severe vacuolation of the ep ithelium lining and congest ion 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, c ongestion of blood ve ssels, and alteration and degeneration of
muscle fibers were also obser ved 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 activit ies at 5 mg/kg/day were similar to
the control.

Chronic Toxicity/Carcinoge nicity (Rat, OCSPP 870.4300)

In a combined chronic toxicity/carcinogenicity study ( MRID 40952802 ), groups of 50 Fischer
344 rats/sex were fed diets containi ng chlorpyrifos (98.5% purity) at levels equivalent to doses of 0, 0.05, 0.1, 1, or 10 mg/kg/day for 104 weeks. Te n additional rats/sex/group were randomly allocated for a 12-rnonth sacrifice. Plasma and RBC ChE activity was assayed from 10rats/sex/dose level at 6, 12, 18, and 24 months. Brain ChE activity was determined from a halfbrain samples obtained at the scheduled 12-rn onth (10 rats/sex/dose) and 24-month (20 rats/sex/dose) sacrifices.

There were no treatment-related mortalities or clin ical 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 exceed ed 9%, and that overall body weight gains were decreased by approximately 10%. Periodic decr eases (p<0.05) were noted in the mid dose (1 mg/kg/day) males that also never exceeded 9%. Body weights were decr eased (p<0.05) in the high dose females during Days 56-231, but thes e decreases never exceeded 4%. Food consumption was unaffected by treatment. D ecreased 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 a nd relative adrenal weights were increased only at
the terminal sacrifice. No treatment-related ch anges were seen in the testes weights or ovaryCHLORPYRIFOS FINAL

Page 61 of 74 weights. No treatment-related histopathological lesions were seen in the testes, epididymides, and seminal vesicle, coagulating glands, pros tate, 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 wa s an exacerbation of th is vacuolation without obvious concomitant necrosis or cellular degenerati on. 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 male s at the mid and high dose as follows: 39-56% at 6 months; 71-87% at 12 m onths; 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 do se) 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 m onths; 14-88% (mid-low, mid, and high dose) at 18 months; and 60-82% at 24 mont hs. 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/da y. 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 se en 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 oncogeni c under the conditions of this study.CHLORPYRIFOS FINAL

Page 62 of 74 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 week s. Additional groups of 5 mice/sex were dosed as above for determination of plasma, RB C, 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 morta lity, differential white blood cell counts, organ weights, or gross pathology. Increased (p<0.05) incidences of excessi ve lachrymation were noted in the high dose males and fe males. Increased (p<0.05) ocular opacity was seen in the mid and high dose males and high dose females. Incr eased (p<0.05) hair lo ss 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 di et as a cause. Water consumption was consistently decreased throug hout 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 peribronc hial 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, t hyroid or pituitary glands. Ch lorpyrifos was not considered oncogenic under the cond itions of this study.

Plasma ChE was decreased (p<0.05) at all trea tment 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 th e parameters normally examined, plasma ChE activities were determined; RBC and brain ChE activity were not eval uated in this study.

There were no treatment related effects on mo rtality, clinical signs , organ weights, or histopathology. A significant decrease in body we ight gain was observed in high dose males during the first half of the study, and in high dose females dur ing the first three weeks.CHLORPYRIFOS FINAL

Page 63 of 74 However, body weight in exposed animals was similar to controls by Week 13. Food consumption in the high-dose animals was al so significantly increa sed during the time of increase body weight gain. He matological effects were obser ved 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 vol ume, increased urine pH, a higher specific gravity and a higher protein gradi ng, which appear to be treatment -related. No treatment-related changes were seen in the weights of the te stes 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-relate d differences were noted for clinical chemistry parameters, with the exception of ChE inhibi tion. Significant and dose-related plasma ChE inhibitions of 22, 37, and 72% were observ ed 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 inhi bited in the low dose group. However, the study author acknowledged the possibility thatthe ChE data for the 10 and 200 ppm female groups were accidently switched.

The LOAEL for plasma ChE inhibition is 0.5 pp m (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 ga ins 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 morta lity, clinical signs, food consumption, or organ weights. For the high dose males, body weights we re 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 th at these losses were due to palatabi lity 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 s lightly increased food consumption. It
was stated that PCV were decreas ed (p<0.05) in the high dose fema les, platelets were increased (p<0.05) in the mid and high dose males, and RB C 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 occu rred in a non-dose-dependent manner. A slightCHLORPYRIFOS FINAL

Page 64 of 74 increase in urine specific gravity was noted in the high dose females. No treatment-related changes were seen in the testes or ovary weight s. 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) vacuola tion in the adre nal 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, erythroc yte, and brain) at each time point measured in both sexes (val ues not include in review). At the interim sacrifice, animals of both sexes displayed decreases (p<0.05) in pl asma and RBC ChE activity at the mid low, midhigh, and high dose levels. Low dose females al so displayed reduced (p<0.05) levels. At terminal sacrifice, plasma ChE activity was d ecreased (p<0.05) at the mid low, mid-high and high dose in both sexes. RBC ChE activity was si gnificantly 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 T oxicity (Rat, OCSPP 870.3465)

In a subchronic nose-only inhalation study ( MRIDs 40013901 and 40166501 ), groups of Fischer 344 rats (10 rats/sex/con centration) 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 μ g/m 3

, respectively) 6 hours/day, 5 days/week for 13 w eeks. 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 ventil ation rate of 0.00715 m 3 /h (average for males and females).

There were no treatment-relate d effects on body weight, clinical signs, urinalysis, hematology, clinical chemistry, gross or microscopic path ology, or plasma, red blood cell or brain ChE activities. Although female rats of all treatme nt 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 obs ervations were not considered treatmentrelated due to a lack of dose-res ponse, 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, epididymid es, and seminal vesicle, prostate, uterus, ovaries, oviducts, vagina, cervix, mammar y, coagulating, adrenal, thyroid or pituitary glands.

No LOAEL was identified in this study. Theref ore, the NOAEL for systemic toxicity and ChE inhibition exceeds 20 ppb or 0.082 mg/kg/day.CHLORPYRIFOS FINAL

Page 65 of 74 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 contai ning 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 he n 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 n ecropsy; 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 debeake d. A reviewer of this study suggested that the high number of eggshell cracks a nd mortalities at 25 ppm resu lted from substandard housing conditions (crowding). However, there were no apparent eff ects 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 reproduc tion 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 th e onset of egg laying (calculated by reviewer), and then during a 12 week egg laying period. The total duration of the st udy 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 producti on 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 indicat ed 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 sign ificant 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 repo rted (egg weights, cracked orCHLORPYRIFOS FINAL

Page 66 of 74 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 obse rved 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 cont aining chlorpyrifos at 0, 25, or 125 ppm. The birds were fed the diets prior to the onset of egg laying (dura tion of treatment not provided), and then during egg laying (duration of treatment not provide d). The total duration of the study was not provided. The birds were housed with two male a nd 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 mortal ities occurred in either of these groups. Ducks fed 125 ppm exhibited locomotor dysfunc tion during Week 5. Sy mptoms 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 feat her condition and were significantly thinner than controls. Food consumption was mu ch 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 drak e 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. N ecropsy 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 le vel, 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 si gnificant decreases in egg weight (14%) and eggshell thickness (9%) at 125 mg/kg-diet.

In another avian reprodu ction toxicity study ( MRID 42144901 ), groups of 42 Mallard ducks (Anas platyrhyncos) were fed diets containing ch lorpyrifos 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 12CHLORPYRIFOS FINAL

Page 67 of 74 week egg laying period. The tota l duration of the study was 22 weeks. The birds were housed with two male and five females pe r pen, with six pens per dose group.

One bird died during the acclimation period and wa s replaced. Adult mortality during the preegg laying period (Weeks 1-10) was as follows: 1 c ontrol 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 th e birds were replaced during the pre-egg laying peri od and no replacements were ma de 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, th ere was a significant reduction in body weight in the highest treatment group during th e pre-egg production period (Days 1-70).

When compared to the control group, food c onsumption in the 30 and 90/60 ppm groups was significantly greater during the egg production period. During We ek 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 comp ared to the control group, there were no significant differences in cracked or broken eggs, egg we ights, egg shell thickness, infertile eggs, or early or late embryonic deat hs at any concentration. When compared to the control group, there was a significantly higher pr oportion 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 differe nces in offspring bodyweight am ong groups for weight at hatch or at 14 days of age. No abnormalities were de tected 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 concentratio ns of 0 (negative and solvent controls), 63, 125, 250, 500, or 1000 ng/L under flow-through conditi ons for 238 days. Mean measured concentrations of chlorpyrifos we re 82.6, 143.8, 300.1, 568.3, and 1093.2 ng/L. At study initiation, the embryos were approximately 24 h pos t-fertilization. On Day 25, the larvae were randomly culled to 25 larvae/aquaria. Spawning s ubstrates were placed in the aquaria on Day 80, after which time the fish were observed fo r secondary sex characteristics and breeding behavior. On Day 105, the grow-out section of each aquarium was partitioned into fourCHLORPYRIFOS FINAL

Page 68 of 74 spawning cells, each containing a single spawning substrate. One male and two females were placed in each of four spawning cel ls 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 fr om each spawning unit. The viability of at least three sets of embryos for 70% of the sp awning sets were evalua ted. Observations of reproductive effects continued until 88% of the wate r 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, a nd assigned to hatching cups using the same procedure as that followed for P embryos. Ei ghty F1 embryos, 40 from a single spawning cell for each replicate, were hatched, and growth a nd survival observed until 28 days post-day-tomean-hatch of the controls. At termination, the length (to the n earest 1 mm) and weight (to the nearest 0.01 g) of all surviv ing fish were recorded.

There was no significant difference in the percenta ge 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 differe nce was not evident on Day 5. On Days 12, 19, and 25, a significant increase in mortality was obs erved 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 wh en 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 we ights of the non-breeders at 568 ng/L and in the water control when compared to the solvent c ontrol. Here again there was no dose-related response and these observations are considered s purious. After 216 days of exposure, there was no significant difference in grow th (length and weight) of the breeders when compared to the
solvent control. Sublethal effects were dem onstrated 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 da ta for the acetone cont rols was consistent
with the remainder of the treatment levels and there was no statistically significant treatmentrelated effects on any of the re productive parameters analyzed.

For the F1 generation, there was no significant di fference in growth, percentage hatched, or percentage normal at hatch between the contro ls and treatments. Mortality at 1093 ng/L was statistically higher on Days 8, 11, 14, 19, 22, 27, and 32.CHLORPYRIFOS FINAL

Page 69 of 74 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 reducti ons 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 concentr ations produced reduced weight gain relative to controls. When chlorpyrifos was detected in test fish, bioconcentra tion 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 concentr ation that caused no signi ficant effect and the lowest concentration that caused a statistically si gnificant 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.CHLORPYRIFOS FINAL

Page 70 of 74 APPENDIX 3: References Not Utilized in the Chlorpyrifos WoE Analysis

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

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

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

Abbott, D, Padmanabhan, V, and Dumesic, D (2 006) Contributions of androgen and estrogen to fetal programming of ovarian dysfunction. Reprod. Biol. Endocrinol. 4 : 17.

Akhtar, N., Srivastava, M., and Raizada, R. (2 009) Assessment of chlo rpyrifos 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 th e United States population. Environ. Res. 99 : 314-326.CHLORPYRIFOS FINAL

Page 71 of 74 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 th e pubertal male assay in rats (WA 4-15). WIL Research Laboratories, LLC, Ashland, OH. Labor atory Study No. WIL-431005, December 13, 2005.

Breslin, W, Liberacki, A, Dittenber, D, et al . (1996) Evaluation of the developmental and reproductive toxicity of chlo rpyrifos in the rat. Fundam. Appl. Toxicol. 29 : 119-130.

Buratti, F, De Angelis, G, Ricceri, L, et al . (2011) Fetal and neonatal e xposure 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 chlo rpyrifos interferes with the responses to 17 β -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 activit y 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 e ffects 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, Farabollin i,F, Dessi-Fulgheri, F, et al . (2008) Environmental-like esposure to low levels of estrogen affects sexual beha vior 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- pyridino l, 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. (19 84) Method for early life- stage toxicity test using three atherinid fishes and results with chlorpyrifos. MRID 00154718. Published: AquaticCHLORPYRIFOS FINAL

Page 72 of 74 Toxicology and Hazard Assessment: Seventh Sym posium, ASTM STP 854, R.D. Cardwell, R. Purdy, and R.C. Bahner, Eds., American Societ y for Testing and Materials, Philadelphia, 1985, pp 145- 154.

Gray, L, Ostby, J, Furr, J, et al . (2001) Effects of environmen tal antiandrogens on reproductive development in experimental animals. Human Reprod. Update 7 (3) : 248-264.

Haviland, J., Butz, D., and Porter, W. (2010) Lo ng-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 develo pment and thyroid function in juvenile male rats. Huntingdon Life Sciences, East Mill stone, 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 toxicolo gical and ecotoxicological data. Reg. Toxicol. Pharmacol. 25 : 1-5.

Lund, T, Munson, D, Haldy, M, et al . (2004) Equol is a novel anti-a ndrogen that inhibits prostate growth and hormone feedback. Biol. Reprod. 70 : 1188-1195.

Mandal, T., and Das, N. (2011) Correlation of tes ticular toxicity and oxid ative 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 acu te 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) Eval uation 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 Sera m, P (2008) Effects of biological and technical
factors on brain and muscle cho linesterases in Nile tilapia, Oreochromis niloticus : implications for biomonitoring neurotoxic contaminations. Arch. Environ. Contam. Toxicol. 54 : 309-317.CHLORPYRIFOS FINAL

Page 73 of 74 Ricceri, L, Venerosi, A, Capone, F, et al . (2006) Developmental neurotoxicity of organophosphorous pesticides: fetal and neonatal e xposure 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 antia ndrogens 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 insec ticide 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 delaye d 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 di sruption contribute to the developmental neurotoxic ity 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 a ssessment 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 m echanis ms of action are involved in chlorpyrifos effects in estrogen-dependent or -independent breast cance r 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 chlorpyr ifos and piperophos. J. Steroid. Biochem. Mol. Biol. 120(1) : 22-29.CHLORPYRIFOS FINAL

Page 74 of 74 York, R (2006) Interlaboratory validation of th e pubertal male assay. CR-DDS Argus Division, Horsham, PA. Laboratory Project ID RTP00002, January 3, 2006.