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

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## ~~~ 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: DIAZINON

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYDIAZINON FINAL

ii TABLE OF CONTENTS

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

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

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

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

B. Other Scientifically Rele vant Information (OSRI)......................................5

III. Weight of Evidence (WoE) Evaluation.......................................................5

A. EDSP Tier 1 Screening Assays.............................................................8

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9
1. Effects on Estrogen Pathway.....................................................9
2. Effects on Androgen Pathway..................................................15

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

IV. Committee’s Assessment of Weight of Evidence..........................................23

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................23

1. Tier 1 in vivo Assays.............................................................24

2. OSRI...............................................................................24

B. Estrogen Pathway...........................................................................25

C. Androgen Pathway.........................................................................26

D. Thyroid Pathway...........................................................................27

E. Conclusions.................................................................................27

V. EDSP Tier 2 Testing Recommendations....................................................27

VI. References.......................................................................................28

LIST OF TABLES: Table 1. Tier 1 Assay for Diazinon.....................................................................8
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Diazinon ......................................11
Table 3. Androgenic/Anti-Andr ogenic Pathway for Diazinon ...................................17
Table 4. Thyroid Pathway for Diazinon ............................................................22

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................33
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................44
APPENDIX 3. References Not Utilized in the Diazinon WoE Analysis..............................62DIAZINON 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 enerationDIAZINON 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 ConcentrationDIAZINON 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 Tier 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)DIAZINON 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-EvidenceDIAZINON FINAL

Page 1 of 63 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether diazinon interacts with E, A, or T hormone pathways, the number and type of effects induced, the magnitude and patt ern of responses observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, the dose(s) at which responses happen and whether overt toxicity was
present.

On March 12, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence analysis of the potential interaction of diazinon with the E, A or T signaling pathwa ys. 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).

Diazinon is a member of the organophosphate class of insecticides whose primary mode of toxic
action is cholinesterase inhibi tion leading to neurotoxicity ( e.g ., cholinergic signs).

There was no convincing evidence for diazinon to potentially interact w ith the estrogen pathway. Diazinon was not an ER agonist up to cytotoxi c concentrations in the estrogen receptor transactivation assay (ERTA) assay. The equivo cal responses in the estrogen receptor (ER) binding and aromatase assays occurred at concentrations near the li mit of solubility for the test compound and did not translate into effects in hi gher levels of biological organization in the
mammalian Tier 1 in vivo studies. No estrogen-related effect s were observed in the fish shortterm receptor assay (FSTRA) in the absence of overt toxicity, except for the decrease in vitellogenin (VTG) at the low dos e (which was not observed at the mid dose) for which there were no other complementary responses observe d. Decreases in the number of weight of offspring were observed in the Part 158 mallard duck study; however, no other treatment-related adverse reproductive effects were observed in this study or bobwhite quail reproduction study. In the existing Part 158 mammalian and fish st udies, the only estroge n-related effect was decreased fecundity in the developmental and reproduction studies. Decreases in fecundity in ofDIAZINON FINAL

Page 2 of 63 themselves are limited in their ability to inform about potential endocrin e-related effects. This effect is most likely attributab le to acetylcholine (AChE) i nhibition, as maternal systemic toxicity was sometimes present in treatment groups.

There is some evidence of an interaction wh en evaluating the androgen pathway responses
across all the considered studies. Diazinon showed potential interaction with the AR in the binding assay and decreased testosterone pr oduction in the steroidogenesis assay. In the Hershberger assay, diazinon treatment resulted in dose-related decreases in androgen-responsive tissue weights. However, no effects were found in the in vivo male pubertal assay. In the existing Part 158 mammalian studies, the only androgen-relate d effects were decreased fertility/fecundity in the developmental and reproduction studies. A dditionally, a slight (9.9%) decrease in testes weight was observed in the re production study. Although, there wa s a 1.9 day delay in PPS in the Part 158 developmental neurotoxicity study, this delay is at the upper ra nge (2 days) of what is considered normal biological variability. In the FSTRA, while th ere were two endpoints affected in the absence of overt toxicity (male GSI at 0.12 μg/L and gonadal histopathology at 0.011 and 0.12 μg/L), there were no other effects noted (i.e., fertilization success) and female body weight was reduced where these effects were observed (with signs of overt toxicity at the high dose) which may suggest that the fish in th is treatment were stressed. In addition, effects on growth and morphology were observed in chronic fish assays at concentrations of 3.2 and 4.8 μg/L; effects on AChE or neur ological symptoms were also observed at >0.47 μg/L in sheepshead minnow or at 2.4 μg/L in brook trout which also suggest that the fish in the
concentrations tested in the FSTRA may have been stressed.

Although these responses ar e consistent with the potential interaction with the A signaling pathway (via androgen antagonism and pertur bation of steroidogenesi s), there is some uncertainty around the observed responses. The in vitro effects were seen only at a high test
concentration that was near th e limit of solubility for the co mpound. In the FSTRA, there were only two endpoints affected in the absence of overt toxicity, and could be due to endocrine or
non-endocrine mechanisms. Effects in the Hershb erger assay were not observed in the male pubertal assay (rats with intact HPG) which wa s conducted at similar doses. Furthermore, while ChE activity or related clinical si gns were not measured in the Tier 1 assays and most of the Part 158 studies, based on information from the other mammalian toxicity studies which did measure ChE and the mode of action for diazinon, one w ould anticipate substantial ChE inhibition to occur at the doses where androgen pathway respons es were seen in these studies. Overall, the totality of the evidence does not support a potential interaction of diazinon interacting with the androgen pathway.

Based on an evaluation of the data, diazinon di d not demonstrate convincing evidence of a potential to interact with the thyroid pathway. The only obs erved response that could be attributed to a potential intera ction with the HPT axis was a slight non-dose responsive decrease ( ↓ 23% and 17% in the low and high dose, respectively) in serum T 4 levels observed in femaleDIAZINON FINAL

Page 3 of 63 pubertal rats. This decrease, however, was not as sociated with complementary alterations in thyroid stimulating hormone (TSH) levels or changes in thyroid weights, pituitary weights or thyroid histopathology in the male or female pubertal assays, excep t for a slight (10%) decrease in thyroid weight for males. In the amphibi an metamorphosis assay (AMA), there were no effects on developmental stage or effects on thyroid histopathology. Th e lack of thyroid pathway-related effects in the AMA and mammalia n Part 158 studies rein force the absence of thyroid interaction.

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

Page 4 of 63 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether diazinon interacts with E, A, or T hormone pathways, the number and type of effects induced, the magnitude of respons es, and the pattern of responses observed across studies, taxa and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whether or not endocrine-related responses occurred at does(s) that also resulted in general system ic toxicity or overt toxicity.

On March 12, 2014, the EDSP Tire 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence analysis of the potential interaction of diazinon with the estrogen (E), androgen (A) or thyroid (T) signaling pathways. The T1WoERC
conclusions from the WoE evaluation in this re port are presented by pathway (E, A and then T) beginning with the resu lts of the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Diazinon is a synthetic organophosph ate pesticide and its mode of action is based on altered
neurotransmission due to AChE inhibition. Diazinon has a low vapor pressure (9.0 × 10 -5 mm Hg at 25°C). Therefore, volatil ization is not expected to significantly contribute to the dissipation of diazinon in the environment. The compound has a lo w water solubility (0. 04 g/L at 20°C) and a moderate octanol/water partiti on coefficient (log Kow = 3.3 to 3.8). Diazinon is degraded by hydrolysis and photolysis. Diazi non has a whole body bioconcentra tion factor value in fish ranging from 3 to 200.

The available information considered in determ ining the potential intera ction of diazinon with the E, A and/or T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxi city studies and other pu blished articles. These data are summarized in Sections III.A through I II.C. An analysis of the data submitted to the Agency, using the WoE approach outlined by th e Agency (USEPA, 2011), is presented in
Section IV. The EDSP Tier 2 studies recommendations are presented in Section V.DIAZINON FINAL

Page 5 of 63 II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

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

1.

B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Orde r recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review T eam on diazinon (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any
additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies c onsidered but not util ized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

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

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

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

Page 6 of 63 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 organizati on from a molecular initiating ev ent, such as receptor binding ,through potential adverse effects in apical endpoints such as se xual development and fecundity at the whole organism level. The extent of expression of responses at higher levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide 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 bi ological organization? Finally, under what conditions did the responses occur including co nsideration of whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.DIAZINON FINAL

Page 7 of 63 This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting in July 2013 that evaluated scie ntific issues associ ated with the WoE evaluation of the EDSP Tier 1 screening proce ss. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All effects in endocrine se nsitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013). For these WoE analyses, overt toxicity wa s generally defined in accordance with EPA’s current approach as used by OPP in reviewi ng 40 CFR Part 158 studies for both human and ecological risk assessments. Specifically, in these an alyses, the effects that EPA considered to be potential evidence of overt toxicity included, but were not limited to: mo rtality; clinical signs such as tremors, ataxia and abnormal swimmi ng (fish and aquatic-pha se amphibians); and body weight decreases of ≥ 10% in mammals. Additionally, ot her effects including morphological ( e.g., organ weights/histopat hology), biochemical ( e.g., blood chemistry), and other clinical signs ( e.g., lethargy) were also considered when evaluating overt toxicity, especially if the effects were extreme. In some instances, one parameter ( i.e., death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

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

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

. These tables contain data that are considered scientifically and biol ogically relevant in regard to a treatment-relate d effect which supports a conclusion of whether a substance has the potential to interact with the E, A, or TDIAZINON FINAL

Page 8 of 63 pathway. Effects that occurred in th e presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not re ported in the table for E, A or T.

A. EDSP Tier 1 Screening Assays

The Tier 1 assays submitted in response to agen cy’s the test order for diazinon are shown below
in Table 1.

Table 1. Tier 1 Screening Assays for Diazinon.

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48616003)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48616004)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48670402)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48670403)

Aromatase Assay (Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48616002)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48616007)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48616006)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID No. 48616003)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID No. 48670401)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48616005)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 4861001)DIAZINON FINAL

Page 9 of 63 B. Effects on Hypothalamic-Pit uitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for diazinon to interact w ith the estrogen pathway is summarized in Table 2. The various targets of the estrogen pathway across the relevant Tier 1 a ssays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

The results for ER binding and aromatase inhi bition were considered equivocal and were potentially confounded by the solubility limit of diazinon. In a publishe d literature study, submitted as OSRI (Kojima et al ., 2005), diazinon was shown to be weakly estrogenic (an EC 10 of 4.6 x 10 -4 M) when the ER transactivation potential was evaluated in the BG1 (E-CALUX) test system; however, the chemical did not transactivate the ER in the EDSP Tier 1 ERTA assay when tested up to a cytotoxic concentration. Diazinon was also negative in non-guideline ERTA assay for estrogenic and anti-est rogenic activity in Chinese hamster ovary (CHO) cells (Kojima,

et al ., 2004). In the steroidogenesis assay, diazinon did not alter estradiol production.

Diazinon was negative in the Tier 1 uterotrophic assay and th e only estrogen-related endpoint altered in the female pubertal assay was a decrease in bodyweight at VO at the high dose.

In the FSTRA, there was a significant 94% decr ease in fecundity in the high-dose group (0.82 mg/L) with an 80% decrease in fertilization. Plas ma VTG concentrations were decreased in all female fish dose groups (not dose dependent; an d only significant at the low (0.011 mg/L) and high dose), and GSI was significantl y decreased 42% in the high-dose females. Also in the highdose treatment, female gonadal histopathology was affected with an incr eased incidence in the number of ovaries at Stage 0/1 w ith atrophy of perinuclear and early cortical follicles. However, significantly decreased body weight s in the female fish in the mid- and high-dose groups (18 and 23%, respectively) and overt morbidity (abnormal behavior and swimming) in the high-dose fish suggest that the effects obs erved, except for VTG in the low dose, may be due to overt toxicity.

In the existing Part 158 mammalian studies, the only treatment related effect was decreased fecundity in the developmental and reproduction studi es. This effect is most likely attributable to AChE inhibition, as maternal systemic toxi city was sometimes present in high-dose groups. In

vitro investigations with diazinon and porcine oocytes have s hown decreases in viability, in vitro
fertilization and maturation a nd morulae formation (Ducolomb et al. , 2009; Casa et al. , 2010).DIAZINON FINAL

Page 10 of 63 In the Part 158 avian reproduction studies, for th e mallard duck, a significant decrease in 14-day old offspring body weight and number of survivor s was reported at the high concentration. At this concentration, the male parental body weight gain was significantly decreased (130%), and effects on AChE were reported on both males and females at all doses tested. In the bobwhite quail reproduction study, no effect s on reproductive parameters we re reported but effects on
AChE were observed for both males and female s. Several fish reproduction studies were
available for diazinon. In a chronic fathead minnow study, hatch at the lowest concentration (3.2 μg/L) was reduced by 30%, however, parental fish did have an increased incidence of scoliosis compared to controls at this concentration. In the brook trout study, growth of the F1 generation
was reduced at all treatment le vels (0.55 to 9.6 μg/L); however signs of neurotoxicity were reported in the mid thru high treatment groups and increased incidents of scoliosis were reported at the two highest treatmen t groups. Finally, in the sheep shead minnow reproduction study, fecundity was reduced at all treatments (>0.47 μg /L) and brain AChE was also reduced at all
treatment levels. In Part 158 fish early life-stage toxicity studie s, growth was affected at all concentrations tested ( ≥ 92 μg/L) in the fathead minnow study; in the sheepshead minnow early life-stage study, growth was affected at ≥ 8.0 μg/L.DIAZINONFINALPage 11 of 63Table 2. Estrogenic/Anti-Estrogenic Pathway for DiazinonLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Diazinon1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian / Gonad Staging and Histo p atholo gy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48616003)EERTA(MRID 48616004)NSteroidogenesis(MRID 48670403)NAromatase(MRID 48616002)EUterotrophic(MRID 48616007)NCS(H)BW(↓15%)(H)Female Pubertal Rat(MRID 48670401)NNNNNNLW(L.H)NFSTRA(MRID 48616005)NENNNN↓63%(L)X4(H)DIAZINONFINALPage 12 of 63Table 2. Estrogenic/Anti-Estrogenic Pathway for DiazinonLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Diazinon1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian / Gonad Staging and Histo p atholo gy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRIERTAKojimaet al., 2004N(E-CALUX) ERTA,Kojima,et al., 2005PSubchronic toxicity(Rat; MRID 40815003)NNNN↑LW(H)NSubchronic toxicity(Dog; MRID40815004)NNNNNNDevelopmental toxicity(Rat; MRID 00153017)NR↓Fcd(H)NRNDevelopmental toxicity(Rabbit; MRIDs00079013, 00079017)NRNNRX(H)DevelopmentalNeurotoxicity (RatMRIDs 45842601,46195601)NRNNNRNTwo-generationreproduction(Rat; MRIDs 41158102and 41158101)NR N NP2:↓Frt(H) /↓Fcd(M, H)NRNChronic toxicity (Rat;MRID 41942002)NNNNNNDIAZINONFINALPage 13 of 63Table 2. Estrogenic/Anti-Estrogenic Pathway for DiazinonLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Diazinon1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight / GSI Ovarian / Gonad Staging and Histo p atholo gy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Chronic toxicity(Mouse; MRID00073372)NENENRNENENChronic toxicity (Dog;MRID 41942001)NNNNNX(MH, H)Avian reproduction(Duck; MRID41322901)NX(L-H)X (H)Avian reproduction(Quail; MRID41322902)NX(L-H)NChronic toxicityfreshwater fish(Fathead Minnow;MRID 40910904)NX(L-H)Chronic toxicityfreshwater fish (BrookTrout;MRID 40910904)NX(M-H)Chronic toxicityestuarine fish(Minnow; MRID40914801)↓Fcd:(L-H)X(L-H)N1 Key to responses: L=Low treatment, ML=Medium low treatment,M=Medium treatment, MH=Medium high treatment, H=High treatment.Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay.Changes in weight are absolute unless otherwise indicated. LW= Liver weight; Frt= Fertility; BW= Body weight; CS= Clinical signs2 The systemic toxicity in the Tier 1assays 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. ADIAZINONFINALPage 14 of 633. The overt toxicity in the Tier 1 assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For details4 Decreased (p<0.05) body weights in the female fish in the mid- and high-dose groups (18 and 23%, respectively) and overt morbidity (abnormal behavior andswimming) in the high-dose fish suggest that the high concentration are overtly toxic. While decreased body weight was reducedin the mid-dose, given thelack of other signs of toxicity, it was not considered to be overtly toxic in of itself, but does suggest that the fish may bestressed.E EquivocalP Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedDIAZINON FINAL

Page 15 of 63 2. Effects on Androgen Pathway

The potential for diazinon to interact w ith the androgen pathway is summarized in Table 3. The various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for androgeni c, anti-androgenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

Diazinon was classified as a binde r in the Tier 1 AR binding assa y. However it was negative in a non-guideline AR transcriptional activation (ARTA) assay evalua ting agonist and antagonist activity in CHO cells (Kojima et al ., 2004). Diazinon also sligh tly inhibited testosterone production in H295R cells in the Tier 1 steroidogenesis assay. In the in vivo Hershberger assay, diazinon showed a dose-dependent anti-androgenic re sponse in androgen-sensitive tissues (AST) weights at all three doses evaluated (15, 47 a nd 150 mg/kg/day). Seminal vesicle and ventral prostate weights were significantly decreased 29% and 26%, respectively at the low-dose, and
decreases at the intermediate dose were 37% fo r seminal vesicles, 43% for ventral prostate, and 21% for LABC. All five AST weights were decrea sed at the high-dose: 62% in seminal vesicles, 60% for ventral prostate, 44% for LABC, 48% fo r Cowper’s gland, and 13% for glans penis. However, when diazinon was tested in the male pubertal assay using animals with an intact HPG axis, no androgen-related effects were observed at doses up to 100 mg/kg/day.

In the FSTRA, there was an 80% decrease in fe rtility at the high con centration (0.82 mg/L).
Plasma VTG concentrations in ma les were not affected but GSI was decreased (p<0.05) in the mid- and high-dose males by 29 and 31%, respectivel y. Male nuptial tubercle scores were also decreased at the high dose (29 in control vs. 7.8 at high dose). Histological examination indicated an increase in the proportion of spermatogonia in the testes at all diazinon exposures. However, the observed effects in the high treatment occurre d in the presence of overt toxicity based on
observations of abnormal behavi or and swimming (along with d ecreased female body weight).

In the Part 158 mammalian toxicity studies, in the developmental rat study, there was a decrease in the mean number of live fetuses at the high dose (11.9) compared to the controls (14.2). In the rat reproduction study, decreased ma ting and fertility were seen in the P2 animals at the high dose. In addition, absolute testes weights were decreased (9.9 %) in the P2 males; however, there no changes in relative weights or histopathology. In the developmental neurotoxicity study in rats, there was a 1.9 day delay in PPS. No othe r androgen-related effects were observed in the
other Part 158 mammalian studies.

In contrast, anti-androgenic effect s on reproductive parameters have been reported for male mice
and rats after diazinon treatment in two recent journal articles. In a non-guideline reproductive toxicity study focusing on effects in male mice, ElMazoudy and Attia (2012) observed doseDIAZINON FINAL

Page 16 of 63 dependent decreases in reproducti ve organ weights (testes, ep ididymides, prostate, seminal vesicles; ↓ 30 to 50%), as well as inconsistent al terations in testosterone and estradiol
concentrations at diazinon doses of 4.1 a nd 8.2 mg/kg/day (interme diate and high doses) administered to male mice for 38 days. Altered se minal parameters were also noted in the midand high-dose groups, including re duced sperm counts, increases in the number of abnormal sperm, and decreases in sperm motility and vi ability. After mating of treated males with untreated females, treatment-related decreases (p <0.05) were also noted in fertility, fecundity, and fetal body weights. Histological examinati on of male offspring showed increases in abnormal sperm and adverse effects on spermatogenesi s in the testes at th e intermediate and high dose. Although diazinon treatment resulted in significant effects on a number of endocrinerelated endpoints in male mice, it is noted the reproductive effect s occurred at doses which also resulted in overt (weight loss) as well as systemic toxicity (adverse clini cal signs of toxicity, and AChE inhibition).

In a non-guideline developmental study by Jayacha ndra and D’Souza (2013), female rats were treated with diazinon prior to and during mating (with untreated males), pregnancy, and weaning. Male pup body weights were initiall y decreased compared to contro l, but the weight differences were generally resolved by PND 21. Since male s from high-dose dams still had decreased body weights ( ↓ 10%) at PND 170, the high dose was considered to be overtly toxi c. Litter size was decreased in all diazinon-treated groups ( ↓ 12, 19, and 28%) compared to control. Testis weights were decreased (p<0.05 or 0.001) by 11 and 19% in the mid- and high-dose males, respectively, at PND 70, and by 14% in high-dose males at PND 170. Adverse histolog ical changes were noted in the testes and epididym ides of the mid- and high-dose males at puberty and adulthood. Changes in the testes were accompanied by chan ges in activities/concentr ations of selected enzymatic biomarkers isolated from the testes. Concentrations of testicular 3- β -hydroxysteroid dehydrogenase (3- β -HSD) were dose-dependently decreas ed (p<0.001) in the mid- and highdose groups at PND 70 and in all groups at PND 170. Activities of alkaline phosphatase, acid phosphatase and lactose dehydrogenase (LDH) were all increased in the high-dose group at PND 70 and 170. ChE activity was not assessed in th is study, however, the possibility that ChE inhibition may have a role in the observed effects cannot be ruled out.

There were no significant androge n-related endpoints in the avai lable chronic avian reproductive toxicity studies. In the brook trout study, growth of the F1 generation was reduced at all treatment levels; however signs of neurotoxicity were reported in the mid thru high treatment groups and increased incidents of scoliosis were reported at the two highest treatment groups.DIAZINONFINALPage 17 of 63Table 3. Androgenic/Anti-Androgenic Pathway for DiazinonLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Diazinon1Study Type / LiteratureCitation AR Binding Steroidogenesis Testosterone Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48670402)PSteroidogenesis(MRID 48670403)PHershberger(MRID 48616006)NESV:↓29/37/62%; VP:↓26/43/60%(L/M/H);LABC:21/44%(M/H);CG:↓48%,GP:↓13%(H)NE NMale Pubertal Rat(MRID 48670401)NNNNNNNNNNFSTRA(MRID 48616005)NE↓29%(M)↑%spermatogonia(L,M)NNNX4(H)DIAZINONFINALPage 18 of 63Table 3. Androgenic/Anti-Androgenic Pathway for DiazinonLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Diazinon1Study Type / LiteratureCitation AR Binding Steroidogenesis Testosterone Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRIARTA(Kojimaet al., 2004)NSubchronic toxicity(Rat; MRID 40815003)N N N N N NX(L-H)NSubchronic toxicity(Dog; MRID40815004)N N N N N NX(L-H)NDevelopmental toxicity(Rat; MRID 00153017)↓Fcd(H)NR NDevelopmental toxicity(Rabbit;MRID00079013 / 00079017)NNRX(H)DevelopmentalNeurotoxicity(Rat;MRIDs 45842601,46195601)N↑1.9 d(H)NR NDevelopmentalToxicity in Males(Rat; Jayachandra andD’Souza, 2013)P5(M)↓11%(M)P(M)NP(M)P6(L, M,H)X(H)Two-generationreproduction(Rat; MRIDs 41158102and 41158101)P2,↓9.9%(H)NP2:↓Frt(H) /↓Fcd(H)NR NDIAZINONFINALPage 19 of 63Table 3. Androgenic/Anti-Androgenic Pathway for DiazinonLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Diazinon1Study Type / LiteratureCitation AR Binding Steroidogenesis Testosterone Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Reproductive Toxicityin Males(Mouse; ElMazoudyand Attia, 2012)N N N NN NX(M,H)X(M, H)Chronic toxicity(Rat; MRID 41942002)N N N N N NX(L-H)NCarcinogenicity(Mouse;MRID 00073372)NENRNENRNENEN NChronic toxicity(Dog; MRID41942001)NNNNNNNX(MH, H)Avian reproduction(Duck;MRID 41322901)NX(L-H)X(H)Avian reproduction(Quail;MRID 41322902)NX(L-H)NChronic toxicityfreshwater fish(Minnow;MRID 40910904)NX(L-H)Chronic toxicityfreshwater fish(Trout;MRID 40910904)NX(M-H)Chronic toxicityestuarine fishNX(L-H)NDIAZINONFINALPage 20 of 63Table 3. Androgenic/Anti-Androgenic Pathway for DiazinonLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Diazinon1Study Type / LiteratureCitation AR Binding Steroidogenesis Testosterone Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3(Minnow;MRID 40914801)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. 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. Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP),Prostate (PR), Levator anibulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP) Frt= Fertility Fed=Fecundity2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1 assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4 Observations of abnormal behavior and swimming.5. Concentrations of 3-β-HSD were dose-dependently decreased (p<0.001) in the mid- and high-dose groups at PND 70 and in all groups at PND 170.6. Litter size was decreased in all diazinon-treated groups (↓12, 19, and 28%) compared to control.P Positive findingN Negative (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedDIAZINON FINAL

Page 21 of 63 C. Effects on Hypothalamic-Pitui tary-Thyroidal (HPT) Axis

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

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

Decreases ( ↓ 23% and 17%) in serum T 4 levels were observed in fema le pubertal rats treated with diazinon at 50 and 100 mg/kg bw/da y, respectively. A decrease ( ↓ 15%) in T 4 was also seen in the male pubertal assay at 50 mg/kg bw/day, but not at 100 mg/kg bw/day. The depression in T4 was
not associated with alterations in TSH levels . Decreased (p<0.05) thyroid weights of 10 and 11%, respectively were seen in the male pubertal at 50 and 100 mg/kg/day, respectively. In the AMA, there were no histological effects on the thyroid or changes in developmental stage. A decrease in the Day 21 normalized hind-limb length (HLL) value ( ↓ 12%) at the intermediate dose (with an identical, but NS, decrease at th e high dose) was observed. In addition decreased (p<0.05) body weight (19%) was seen in the hi gh-concentration (0.82 mg a.i./L) animals on Day

21.

There were also no changes in thyroid weights, pituitary weights or thyr oid histopathology in the
mammalian Part 158 studies evaluated.DIAZINON FINAL

Page 22 of 63 Table 4. Thyroid Pathway for Diazinon

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

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

EDSP Tier 1 Assays

Female Pubertal Rat (MRID 48670401) N N ↓ 23, 17% (L,H) N N N  LW (L,H) N

Male Pubertal Rat (MRID 48670401) ↓ 10, 11% (L,H) N ↓ 15% (L) N N N N N

AMA (Frog; MRID 48616001) N HLL: ↓ 12% Day 21

(M) BW: ↓ 19% (H) N

OSRI Subchronic toxicity (Rat; MRID 40815003) N N N X (H) N

Subchronic toxicity (Dog; MRID 40815004) N N N X (L-H) N

Chronic toxicity (Rat; MRID 41942002) N N NE NE N X (L-H) N

Carcinogenicity (Mouse; MRID 00073372) NE NR NE NE NE N N

Chronic toxicity (Dog; MRID 41942001) N N NE NE N N X (MH, 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 re sponse. A shaded cell indicates that is parameter is not routinely evaluated or is not ap plicable in this assay. Changes in weight are absolute unless otherwise indicated. LW= Liver weight 2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 3. The overt toxicity in the Tier 1 a ssays 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 P Positive findings N Negative findings NR Not reported NE Not examinedDIAZINON FINAL

Page 23 of 63 IV. Committees Assessment of Weight of Evidence

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

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

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

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

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

Page 24 of 63 1. Tier 1 in vivo Assays

The only T1 assay that showed overt toxicity was the high dose (250 mg/kg/day) group in the uterotrophic assay (15% BW decr ease). Cholinesterase (ChE) activ ity was not measured in any of the Tier 1 assays. Although clinical signs s uggestive of ChE inhibiti on were not observed in
the Tier 1 mammalian in vivo assays, tremors and other signs of cholinergic toxicity can be missed in studies that are not spec ifically designed to detect neur otoxicity. It is plausible that signs of cholinergic toxicity occurred in mamma lian in vivo assays, but were not noted during the standard observation periods asso ciated with these studies.

In the FSTRA, diazinon exposure was associated with a decrease in body weight of female fish at 0.12 and 0.82 mg/L ( ↓ 18% and 23%, respectively). Ov ert toxicity, in cluding abnormal behavior and swimming, was also observed in fish exposed to the high dose. However, no significant effect on su rvival was observed.

2. OSRI

In a combined chronic dietary toxicity/carcinoge nicity study in rats, male and female Sprague Dawley rats were fed diets containing Diazinon at 0, 0.004, 0.06, 5 and 10 mg/kg/day for males and 0, 0.005, 0.07, 6 and 12 mg/kg/day for females for 98 weeks. For plasma cholinesterase inhibition, the NOAEL is 0.005 mg/kg/day and the LOAEL is 0.06 mg/kg/day based on ChE activity. For RBC and brain AChE inhibition, th e NOAEL is 0.07 mg/kg/day and the LOAEL is 6 mg/kg/day.

In the development neurotoxicity study, the NOA EL for maternal cholinesterase inhibition was 0.026 mg/kg/day. The LOAEL was 2.36 mg/bw/day ba sed decreases in plasma, RBC and brain cholinesterase activities. For offspring syst emic toxicity, the NOAEL was 2.36 mg/kg/day; the LOAEL of 24.2 mg/kg/day based reduced body we ight, reduced body weight gains and delayed sexual maturation in males and females. For offspring cholinesterase in hibition, the NOAEL was 0.026 mg/kg/day. The LOAEL was 2.36 mg/kg/da y based on decreased plasma and RBC
cholinesterase activ ities both sexes.

In a comparative cholinesterase assay (CCA), blood and brain cholin esterase activity in male and female adult and neonatal/juvenile rats were examined following both acute and repeated exposures. In the acute study adult rats were ad ministered single doses of 0, 0.3, 3.0, 30, or 300 mg/kg bw in corn oil and PND 11 and PND 21 p ups were administered single doses of 0, 0.3, 3.0, 10, or 100 mg/kg bw by gavage. In the repeat CCA dose study, adult rats were administered diazinon at 0, 0.03, 0.3, 10, or 100 mg/kg bw/day in se ven consecutive daily doses. Ten male and 10 female pups received doses of 0, 0.03, 0.3, 3, or 30 mg/kg bw/day from PND 11 to 17. Following acute dosing, clinical si gns of toxicity included tremor s in 4/8 adult females at 30 mg/kg, in all adults at 300 mg/kg, in 2/8 fema le PND 11 pups at 10 mg/kg, and in 7/8 male andDIAZINON FINAL

Page 25 of 63 6/8 male PND 11 pups at 100 mg/kg. Acute or re peated exposure to diazinon resulted in statistically and biologically significant decreases (>0.05) in the cholinesterase activity in plasma, RBC, and brain of young adults animal s, and PND 11, PND 17, and PND 21 pups. In adults, effects were noted at 3 mg/kg bw in bot h sexes following single dose exposures and at 0.3 mg/kg bw/day in females after repeated exposur es. Following single doses to pups, effects were observed at 3 mg/kg bw in both sexes of PND 11 pups and in bot h sexes of PND 21 pups. After repeated exposures, effects were noted in PND 17 pups at 0.3 mg/kg bw/day in both sexes. Acute doses of 0.3 mg/kg bw and repeated doses of 0.03 mg/kg bw caused no significant effects.

Clinical signs, particularly tremors, consistent with cholinergic toxicity were observed in some studies, but not all. It is importa nt to note that clini cal signs may only last for a short period of time and in repeating dosing studies may not occu r every day. The most robust source of data on tremors can be found in the acute CCA study wher e tremors are seen at 10 mg/kg in PND11 pups and 30 mg/kg in adults. In the mallard study, a si gnificant decrease in male body weight gain of 130% at the high dose was reported. In the Ch E portion of the study, decreased serum ChE
concentrations were observed at all treatment le vels, while decreased brain ChE concentrations
were observed at the low and high treatment levels.

In the bobwhite quail study, no signs of overt toxicity were obser ved. In the ChE portion of the study, decreased serum ChE concentrations were observed in both male and females at all treatment levels, while decreased brain ChE concen trations were only observed for females at all
treatment levels.

Fathead minnows exposed to 3.2 ug/L from 5 days after hatch through spawning had an increased incidence of scoliosis compared to the control.

Brook trout exposed to ≥ 4.8 ug/L began developing scoliosis and lordosis within a few weeks of exposure and growth was substantially inhib ited during the first 3 months. Neurological symptoms were observed at concentrations ≥ 2.4 ug/L but were rarely observed after 4 or 5 months of exposure. Exposure of mature trout for 6 to 8 months to concentrations ranging from 0.55 to 9.6 ug/L resulted in equally reduced growth rates fo r their progeny.

Sheepshead minnows exposed to >0.47 ug/L ha d no signs of overt t oxicity. Brian AChE
inhibition was observed in fish at all concentrations after 7 days.

B. Estrogen Pathway

There was no convincing evidence for diazinon to potentially interact w ith the estrogen pathway. Diazinon was not an ER agonist up to cytotoxi c concentrations in the estrogen receptor transactivation assay (ERTA) assay. The equivo cal responses in the estrogen receptor (ER) binding and aromatase assays occurred at concentrations near the li mit of solubility for the testDIAZINON FINAL

Page 26 of 63 compound and did not translate into effects in hi gher levels of biological organization in the
mammalian Tier 1 in vivo studies. No estrogen-related effect s were observed in the fish shortterm receptor assay (FSTRA) in the absence of overt toxicity, except for the decrease in vitellogenin (VTG) at the low dos e (which was not observed at the mid dose) for which there were no other complementary responses observe d. Decreases in the number of weight of offspring were observed in the Part 158 mallard duck study; however, no other treatment-related adverse reproductive effects were observed in this study or bobwhite quail reproduction study. In the existing Part 158 mammalian and fish st udies, the only estroge n-related effect was decreased fecundity in the developmental and reproduction studies. Decreases in fecundity in of themselves are limited in their ability to inform about potential endocrin e-related effects. This effect is most likely attributab le to acetylcholine (AChE) i nhibition, as maternal systemic toxicity was sometimes present in treatment groups.

C. Androgen Pathway

There is some evidence of an interaction wh en evaluating the androgen pathway responses
across all the considered studies. Diazinon showed potential interaction with the AR in the binding assay and decreased testosterone pr oduction in the steroidogenesis assay. In the Hershberger assay, diazinon treatment resulted in dose-related decreases in androgen-responsive tissue weights. However, no effects were found in the in vivo male pubertal assay. In the existing Part 158 mammalian studies, the only androgen-relate d effects were decreased fertility/fecundity in the developmental and reproduction studies. A dditionally, a slight (9.9%) decrease in testes weight was observed in the repr oduction study. Although th ere was a 1.9 day delay in PPS in the Part 158 developmental neurotoxicity study, this delay is at the uppe r range (2 days) of what is considered normal biological variability. In the FSTRA, while there were two endpoints affected
in the absence of overt toxici ty (male GSI at 0.12 μg/L and gonadal histopathology at 0.011 and 0.12 μg/L), there were no other effects noted (i.e ., fertilization success) and female body weight was reduced where these effects were observed (with signs of overt toxicity at the high dose) which may suggest that the fish in this treatment were stressed. In addition, effects on growth and morphology were observed in chronic fish as says at concentrations of 3.2 and 4.8 μg/L; effects on AChE or neurological symptoms were also observed at >0.47 μg/L in sheepshead minnow or at 2.4 μg/L in brook trout which also suggest that the fish in the concentrations tested in the FSTRA may have been stressed.

Although these responses ar e consistent with the potential interaction with the A signaling pathway (via androgen antagonism and pertur bation of steroidogenesi s), there is some uncertainty around the observed responses. The in vitro effects were seen only at a high test
concentration that was near th e limit of solubility for the co mpound. In the FSTRA, there were only two endpoints affected in the absence of overt toxicity, and could be due to endocrine or
non-endocrine mechanisms. Effects in the Hershb erger assay were not observed in the maleDIAZINON FINAL

Page 27 of 63 pubertal assay (rats with intact HPG) which wa s conducted at similar doses. Furthermore, while ChE activity or related clinical si gns were not measured in the Tier 1 assays and most of the Part 158 studies, based on information from the other mammalian toxicity studies which did measure ChE and the mode of action for diazinon, one w ould anticipate substantial ChE inhibition to occur at the doses where androgen pathway respons es were seen in these studies. Overall, the totality of the evidence does not support a potential interaction of diazinon interacting with the androgen pathway.

D. Thyroid Pathway

Based on an evaluation of the data, diazinon di d not demonstrate convincing evidence of a potential to interact with the thyroid pathway. The only obs erved response that could be attributed to a potential intera ction with the HPT axis was a slight non-dose responsive decrease ( ↓ 23% and 17% in the low and high dose, respectively) in serum T 4 levels observed in female pubertal rats. This decrease, however, was not asso ciated with complementary alterations in TSH levels or changes in thyroid weights, pituitary weights, or thyroid hist opathology in the male or female pubertal assays, except for a slight (10%) decrease in thyr oid weight for males. In the AMA, there were no effects on developmental st age or effects on thyr oid histopathology. The lack of thyroid pathway-related effects in th e AMA and mammalian Part 158 studies reinforce the absence of thyr oid interaction.

E. Conclusions

Based on weight of evidence considerations, there was no convincing evidence of potential interaction with the estrogen, a ndrogen or thyroid pathways.

V. EDSP Tier 2 Testing Recommendations

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

Page 28 of 63 VI. References

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Barnes, T., Hazelette, J. and Arthur, A. (1988) . Diazinon (MG-8): Pilot 6-week Oral Feeding Study in Rats: Laboratory Study Number: 88076 (MIN 872348). Unpublished study prepared by Ciba-Geigy Corporation., July 20, 1988. (MRID 40815003).

Casas, E., Bonilla, E., Ducolomb, Y., and Beta ncourt, M. (2010). Di fferential effects of herbicides atrazine and fenoxaprop-ethyl, and in secticides diazinon and malathion, on viability and maturation of porcine oocytes in vitro. Toxicol in Vitro , 24 (2010) 224–230.

Ciba-Geigy (1981). A Teratology Study of Diazinon in New Zealand White Rabbits. Study No. 281005. Unpublished study prepared by Ciba-Gei gy Corporation, Research Department, Pharmaceuticals Division. July 28, 1981. (MRID 00079013).

Davis, J. (2011). The Hershberger Bi oassay (OPPTS 890.1400); Diazinon. Unpublished study prepared by Integrated Laboratory Systems, Inc., Durham, NC. Labor atory Project Study ID: C193-200, November 2, 2011. (MRID 48616006).

Davis, J. and I. Lea (2011). The Uter otrophic Assay (OPPTS 890.1600). Unpublished study prepared by Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Study Number: C193-100, Project Number: R28280, September 6, 2011. (MRID 48616007).

Davis, J.P. (2012). Pubertal Development and Th yroid Function in Intact Juvenile/Peripubertal Female and Male Rats: Diazinon. Unpublished study prepared by Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study No.: C193-300, January 25, 2012. (MRID 48670401).

Ducolomb, Y., Casas, E., Valdez, A., González, G., Altamirano-Lozano, M., and Betancourt, M. (2009). In vitro effect of malathion a nd diazinon on oocytes fertilization and embryo development in porcine. Cell Biol Toxicol 25 (6) p. 623–633.

ElMazoudy, R.H. and Attia, A.A. (2012). E ndocrine-disrupting and cytotoxic potential of anticholinesterase insecticide, diazinon in reproductive toxicity of male mice. Journal of
Hazardous Materials , 209–210 (2012) 111–120.

Giknis, M.L.A

. (1989). Diazinon Technical: A Two Ge neration Reproductive Study in Albino Rats: Project ID MIN 852218. Unpublished study prepared by Ciba-Geigy Corporation, Research Department, Pharmaceu ticals Division. February 9, 1989. (MRID 41158101).DIAZINON FINAL

Page 29 of 63 Goodman, L.R., Hansen, D. J., Coppage, D.L., M oore, J.C., and Matthews, E. (1979). Diazinon: Chronic Toxicity to, and Brain Acetylcholinesterase Inhibition in, the Sheephead Minnow, Cyprinodon variegatus . Transactions of the Ameri can Fisheries Society 108: 479–488. (MRID 40914801)

Harris, S.B. et al ., (1981). A Teratology Study of Di azinon (CAS Number 333-41-5) in New Zealand White Rabbits. Unpublished study prepar ed by Science Applications, Inc. LaJolla, Calif. Study No.: 281995. July 23, 1981. (MRID 00079017).

Infurna, R.N. (1985). A Teratology Study (S egment II) in Albino Rats. Report No. 52-83. Unpublished study prepared by Ciba-Geigy Corpor ation, Research Department, Pharmaceuticals
Division

. April 19, 1985. (MRID 00153017 ).

Jayachandra, S. and D’Souza

, U.J.A. (2013). Pre - and postnatal toxicity of diazinon induces
d isruption of spermatogenetic cell line evidenced by increased testicular marker enzymes
activities in rat offspring. Journal of Environmental Pathology, Toxicology, and Oncology

, 32 (1) p .73 –90.

Kirchner, F.R., McCormick

, G

. C

. , and Arthur

, A.T. (1991). Diazinon (MG- 8): One/Two Year Oral Toxicity Study in Rats: Lab Project Number: 882018. Unpublished study prepared by Ciba-Geigy Corporation, Research Departme nt, Pharmaceuticals Division. June 13, 1991. (MRID 41942002).

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

Kojima, M

. , Fukunaga

, K.

, Sasaki

, M.

, Nakamura, M

., Tsuji, M. and Nishiyam a , T. (2005). Evaluation of estrogenic activit ies of pesticides using an in vitro reporter gene assay.
Int J Environ Health Res 15:271–280. (MRID 48074203).

Lee, M. R. (2011). Diazinon Technical — Am phibian Metamorphosis Assay with African Clawed Frog (Xenopus laevis) Following O PPTS Test Guideline 890.1100 and OECD Test Guideline 231. Unpublished study prepared by Sm ithers Viscient, 790 Main Street, Wareham, MA 02571-1037. Study No. 11742.6161. October 25, 2011. (MRID 48616001).

Mandella, R.C. (2003). Diazinon: A Developm ental Neurotoxicity Study in Rats. OPPTS 870.6300. Unpublished study prepared by Huntingdon Life Sciences, Study No. 01-4532. September 18, 2003. (MRID 46105601 ).DIAZINON FINAL

Page 30 of 63 Marselas, G. (1989). Diazinon: A One-Gene ration Reproduction Study with the Northern Bobwhite ( Colinus Virginianus ): Lab Project Number: 108/292. Unpublished study prepared by Wildlife International Ltd. (MRID 41322902).

Marselas, G. (1989). A one-generation reproduction study with the mallard duck ( Anas platyrhyncos ) using parental reproduction. U npublished study conducted by Wildlife International, Easton, MD for Ciba-Geigy Corporation, Greensboro, NC. November 30, 1989. (MRID No. 41322901).

National Toxicology Program (1979). Bioassa y of Diazinon for Possible Carcinogenicity. Carcinogenesis Testing Program, Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health . Report No. TRT-137; NIH 79-1392. (MRID 00073372).

Parker, S.P. (2003) Relative sensitivity of ne onatal/juvenile and young adult CD rats after an acute exposure to diazinon. Center for Life Sciences and Toxicology, RTI International, P.O. Box 12194, Research Triangle Park, NC 27709- 2194. Project. No. 08882.4. December 23, 2003. MRID 46166301. Unpublished

Parker, S.P. (2003) Comparative sensitivity of ne onatal/juvenile and young adult CD rats after repeated exposure to diazinon. Center for Life Sciences and Toxicology, RTI International, P.O. Box 12194, Research Triangle Park, NC 27709-2194. Project. No. 08882.005. December 23, 2003. MRID 46166302. Unpublished

Plunkett, L., Gordon, E., Sharpe, R.M (2012) Diazinon: A weight of evidence evaluation of the U.S. EPA’s Endocrine Disruption Screening Pr ogram Tier 1 Screening Assays. Makhteshim Chemical Works, Ltd. Project Number 839 12/5. MRID 49013901

Raab, D., Yoreneff, M., Giknis, M., and Yau, E. (1989a). Supplemental to the Two Generation Reproduction Study: a One-generation (Two Litte r) Reproduction in Rats. Laboratory Study No. MIN 842294. Unpublished study prepared by Ci ba-Geigy Corporation. January 25, 1989. (MRID 41158102).

Raab, D., Yoreneff, M., Giknis, M., and Ya u, E. (1989b). Two Generation Reproduction Study in Albino Rats. Laboratory Study No. 852218. Unpublished study prepared by Ciba-Geigy Corporation. February 9, 1989. (MRID 41158101).

Rudzki, M.W

., McCormick, G.C., and Arthur, A.T

. (1991). Diazinon (MG-8): 52-Week Oral Toxicity Study in Dogs: Lab Project Number: 882014. Unpublishe d study prepared by CibaGeigy. June 14, 1991. (MRID 41942001).DIAZINON FINAL

Page 31 of 63 Singh, A.R. (1988). Diazinon (M G-8): 90-Day Oral Toxicity Study in Rats. Project ID 882011. Unpublished study prepared by Ciba-Geigy Corpor ation, Research Department, Pharmaceuticals Division. August 4, 1988. (MRID 40815003).

Sousa, J. (1997). Diazinon—Chroni c Toxicity to Sheepshead Minnow, ( Cyprinodon variegatus ), Under Flow-Through Conditions: (Final Report): Lab Project Number: 97-2-6887. Unpublished study prepared by Novartis Crop Protection, Inc. (MRID 44244802).

Surprenant, D. C. (1988). The Toxicity of Diazinon Technical to Fathead Minnow ( Pimephales promelas ) Embryos and Larvae. Report No. 88-5-2702. Submitted by Ciba-Geigy Corporation

. (MRID 40782301).

USEPA. (2005). Interim Reregistration Elig ibility Decision (IRED) for Diazinon. LIST A. CASE 0238. May, 2004. EPA 738-R-04-006, Office of Pollution Prevention and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC.

USEPA. (2010). Diazinon: Report of the Endocrine Disruptor Review Te am — Test Order #: EDSP-057801-36. August 5, 2010. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

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

Wagner, H. (2012). Diazinon: steroidogenesis (human cell line — H295R). Unpublished study prepared by CeeTox, Inc., Kalamazoo, MI. La boratory Study No.: 9141V-100358STER, January 25, 2012. (MRID 48670403).

Wilga, P. (2011). Diazinon: Human Recombinant Aromatase Assay. Unpublished study prepared by CeeTox, Kalamazoo, MI. Study No. 9142V-100358AROM, June 20, 2011. (MRID 48616002).

Willoughby, J.A. (2012). Diazinon: a ndrogen receptor binding assay us ing rat prostate cytosol. Unpublished study prepared by CeeTox Inc., Kalamazoo, MI. Laborat ory Study No.: 9141V100358ARB, January 25, 2012. (MRID 48670402)

Willoughby, J.A., (2011). Diazinon: Estrogen Recep tor Binding (Rat Uterine Cytosol). Unpublished study prepared by CeeTox, Kalama zoo, MI. Laboratory report number 9141V100358ERB, November 1, 2011. (MRID 48616003).DIAZINON FINAL

Page 32 of 63 Willoughby, J.A. (2011). Diazinon: Estrogen Recepto r Transcriptional Ac tivation (Human Cell Line (HeLa-9903)). Unpublished study prepar ed by CeeTox, Kalamazoo, MI. Study No. 9141V100358ERTA, study completion date November 1, 2011. (MRID 48616004).

York, D. (2011). Diazinon — Short-term Reproduction Assay with Fathead Minnow ( Pimephales promelas ). Unpublished study performed by Smithers Viscient (formerly Springborn Smithers Laboratories), Wareham, Ma ssachusetts. Laboratory Project ID No.: 11742.6162. Submitted by Makhteshim Chemical Works, Ltd., Raleigh, NC. (MRID 48616005).DIAZINON FINAL

Page 33 of 63 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100)

The 21-day assay (MRID No. 4861001) of diazinon (98.7% purity) on amphibian metamorphosis of African clawed frogs Xenopus laevis was conducted under fl ow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 tadpoles per treatment) were exposed to a negative control and nominal diazinon conc entrations of 0.01, 0.1, and 1.0 mg a.i./L. Test
chemical measured concentrations were <0.0000374 (<LOQ; control), 0.0076, 0.082 and 0.820 mg a.i./L. The test system was maintained at 21 to 23 °C and a pH of 7.9 to 8.2.

Unless otherwise indicated, all effects are re ported based on comparison to the negative (FETAX) control. Two mortalitie s each (3.3%) were observed in the control and low treatment group and one mortality each (1.7%) were obser ved in the mid and high treatment groups. Curvature of the spine was observed in the cont rols (7/58; 12%), and low (10/58; 17%), mid (17/59; 29%), and high (7/59; 12%) treatment gr oups. “Several” tadpoles were reported to be pale and “small” throughout the exposure, but the number of tadpoles affect ed and the associated treatment levels were not reported. No other clinical signs were reported.

Diazinon did not significantly aff ect Day 7 normalized (for snout-vent length) hind-limb length (HLL) in any treatment group. Reductions in nor malized HLL at Day 21 in the mid and high treatment levels were similar, based on comparison to the negative control, but the effect was only statistically significant at the mid treatment level (p=0.04 5). No significant change of median NF developmental stage was observed at Day 7 or Day 21 at any treatment level. Asynchronous development was not observed in any of the treatments. Body weight was significantly reduced on Day 21 at the high treatment level only (19%; p=0.01); no other effects on snout-vent length (SVL) or body weight were observed. Although mild follicular cell hypertrophy and hyperplasia were reported across all treatment groups (10 to 32%), there was no treatment-related pattern. Incidents of increased or decreased follicular lumen area were sporadic (5 to 10%), but also were not treatment-related. Late stag e tadpoles (NF Stage >60) were excluded from analyses of continuous endpoi nts (SVL, body weight, and normalized HLL).

Androgen Receptor (AR) Bindi ng Assay; (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48670402), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment, which measured the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (10

− 10 to 10

− 3 M) of diazinon (98.7% a.i., Batch# 00896074). Dimethyl sulfoxide (DMSO) was used as the solv ent vehicle at a final assay concentration of approximately 3.2%. A total of three independent runs were performed, and each run included dexamethasone as a weak positive control, a nd R1881 as the ligand reference standard.DIAZINON FINAL

Page 34 of 63 Prior to conducting the competitive binding expe riment, saturation binding experiments were conducted to demonstrate that AR from the rat cy tosol preparations was present in adequate numbers and functioning with the appropriate affi nity for the radiolabeled ligand. Saturation binding data were not originally provided in the study report; however , summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency. Separate cytosolic pr eparations were used for each of the three competitive binding runs, so data from three satu ration binding experiments were evaluated. For the cytosolic preparation conducted on Marc h 30, 2011, the mean dissociation constant (K d ) for [ 3 H]-R1881 was 0.614±0.065 nM, and the estimated B max was 1.080±0.109 fmol/100 μg protein for the single batch of prostate cytosol that was prepared. For the cytosolic preparation conducted on July 23, 2011, the K d for [ 3 H]-R1881 was 1.080 ± 0.690 nM, and the estimated

B max was 1.071 fmol/100 μg protein (±0.167) for the single batch of prostate cytosol that was prepared. For the cytosolic preparation conducted on September 24, 2011, the dissociation constant (K d ) for [ 3 H]-R1881 was 0.613 ± 0.041 nM, and the estimated B max was 0.817 fmol/100 μg protein (±0.049) for the single batch of prosta te cytosol that was prepared. The mean and
individual K d values were generally below the range reported in the EPA validation program (0.685 to 1.57 nM), although two individual values were within the range and one value was above the range. Confidence in these numbers is generally high according to the goodness of fit (R 2 = 0.880-0.992) and the small variation in R 2 and K d estimates among runs (although greater variability was observed in the Ju ly 23, 2011 saturation binding data).

In the competitive binding experiment, precipitat ion of diazinon was visu ally observed at the highest tested concentration (10

− 3 M) in the first run; therefore, the data at this concentration for this run were not evaluated. No precipita tion was observed at concentrations up to 10

− 3 M in
Runs 2 and 3.

In all three runs, specific binding of [ 3 H]-R1881 was 93.5-100.5% at diaz inon concentrations of

10

− 10 to 10

− 5 M, and specific binding declined to 71.9-80.5% at 10

− 4 M and to 36.7-41.6% at

10

− 3 M (Runs 2 and 3). Based on the average binding curve for diazinon, the estimated log IC 50 for diazinon was

− 3.6 M, the relative binding affinity (RBA) was 0.00017%, and the Hill slope was approximately 1.00.

The estimated average log IC 50 s for R1881 (

− 9.3 M) and the weak positive control, dexamethasone (

− 4.4 M), were within the expected ranges, and the mean RBA for
dexamethasone was 0.0022%. The solvent control responses indicated no drift in the study assay, and the performance cr iteria were generally met.

Based on the results from the th ree runs, diazinon is classifi ed as a Binder in the Androgen Receptor Binding Assay.DIAZINON FINAL

Page 35 of 63 Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48616002), diazinon (98.7% w/w, Batch # 00896074) in dimethyl sulfoxide (DMSO) was incubated with human recombinant aromatase and tritiated androstenedione ([1 β

3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]-ASDN) at concentrations of 0, 10

− 3 , 10

− 4 , 10

− 5 , 10

− 6 , 10

− 7 , 10

− 8 , 10

− 9 , or 10

− 10 M for 15 minutes at 37°C to assess the potential of diazinon 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 to 10

− 5 M) using a known inhibitor (4-hydr oxyandrostenedione; 4-OH ASDN), and the test chemical series (10

− 10 to 10

− 3 M) with three repeti tions per concentration.

Aromatase activity in the fu ll activity controls ranged from 0.230 to 0.347 nmol·mgprotein

− 1 ·min

− 1 for the four test runs, with a m ean and standard deviation of 0.285±0.037 nmol·mg-protein

− 1 ·min

− 1 . Activity in the background controls ranged from 0.44% to 0.53% and averaged 0.48% of the full activ ity controls. The response of the full activity controls and background controls was acceptable.

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.257±0.048 nmol·mg-protein

− 1 ·min

− 1 at the lowest tested concentration (10

− 10 M) and 0.002±0.001 nmol·mgprotein

− 1 ·min

− 1 at the highest tested concentration (10

− 5 M). These results were generally within the recommended ranges for the top of the curve, bottom curve, hill slope, log IC 50 , and coefficient of variation for replicates of each concentration.

For diazinon, aromatase activity averaged 0.261±0.033 nmol·mg-protein

− 1 ·min

− 1 at the lowest
tested concentration of 10 -10 M and 0.115±0.041 nmol·mg-protein -1 ·min

− 1 at the highest tested
concentration of 10

− 3 M. Although precipitation was noted at 10

− 3 M, the percent activity was decreased at this concentration compared to 10 –4 M in all four runs. The goodness of fit assessed by a least squares fit with 4- variable nonlinear regression improves greatly when the 10

− 3 M concentration was included, based on increased R 2 values. No data were presented detailing the solubility of the test compound at any other concentrations between 10 –3 and 10 –4 M.

Excluding Run 1, which showed no inhibi tion for diazinon, the estimated log IC 50 for diazinon ranged from –4.93 to –3.90 M and averaged –4.55 M compared to 4-OH ASDN, which averaged -7.22 M. The averag e slope of the con centration response curve was –5.29 for diazinon and –1.03 for 4-OH ASDN. Confidence in the mean log IC 50 and the hill slope for the positive control is high due to the small variation (CV <15%).DIAZINON FINAL

Page 36 of 63 Aromatase activity was inhibited at the high concentration (10 -3 M) by more than 50%, but the results were cofounded by evidence of insolubility at this concentrati on. The next lowest
concentration inhibited aromatas e activity by approximately 34% (66% activity). There is no indication in the study repor t that 1⁄2 log concentrations of diaz inon were tested. Therefore at the highest soluble concentrati on of diazinon evaluated (10 -4 M), the average lowest portion of the response curve fell between 50 and 75% activ ity, indicating an e quivocal response.

Based on the data from the average response curv e, diazinon is classified as Equivocal for aromatase inhibition in this assay.

Estrogen Receptor (ER) Bindi ng Assay; (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48616003), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiment s. A saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was functioning with appropr iate affinity for the radiolabeled reference estrogen prior to conducting ER competitive binding experiments. The competitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of increasing concentrat ions of diazinon (logar ithmic increase from 10 -10

to 10 -4 M). DMSO was used as a vehicle at a fina l concentration of 4% . The assay included 19-norethindrone as a weak positive control, oc tyltriethoxysilane as a negative control, and 17 β

estradiol as the natural ligand reference material.

Saturation binding data were not provided in the study report; however , summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency.

For the competitive binding experiment, an IC 50 could not be calculated fo r diazinon as it did not The mean log IC 50 was -8.9 M for 17 β -estradiol and -5.3 M for the weak positive control, 19- norethindrone. The mean RBA and log RBA for 19-norethindrone were 0.027% and -1.56%, respectively.

The performance criteria were met. The curves fitted to the Hill equation for the reference material showed that increasing concentrations of unlabeled 17 β -estradiol displaced [ 3 H]-17 β

estradiol in a manner consistent with one-site binding, as indicated by a hill slope of -1.1 and -1.0
for the first two runs. For th e third run, the Hill Slope wa s outside the recommended range,

at -1.6.

Diazinon was tested over a concentration range that fully defined the top of the curve. The maximum percent binding , 111.3%, 111.8% and 100.4% for the first, second and third runs,DIAZINON FINAL

Page 37 of 63 respectively, was within 25 percen tage points of the value for th e lowest concentration of the estradiol standard, 92.8%, 105.0% and 96.7%, respectively. In the in dividual competitive binding runs, diazinon was interactive in one run and not interactive in th e other two runs. Based on the results from the three runs, diazinon is classifi ed as Equivocal in the Estrogen Receptor Binding Assay.

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

In an estrogen receptor transcriptional activ ation assay (MRID 48616004) cultured cells were exposed to diazinon (98.7% a.i. Lo t# 00896074) at concentrations of 10 -11 to 10 -3 mg/mL in DMSO (0.1%) for approximately 24 hours. Experi ments were performed in 96-well plates and each diazinon concentration was tested in six wells/plate. Cells were exposed to test agents for

24±2 hr to induce reporter gene prod ucts. Luciferase expression in response to estrogen receptor activation was measured upon addition of luciferase substrate and detection with a luminometer.

Diazinon was tested up to the limit of cytot oxicity, which occurred at concentrations ≥ 10 -4 M.
The RPC Max was 8.8%, and the associated PC Max was 10 -5 M for the first run, and the RPC Max was 5.3%, and the associated PC Max was 10 -6 M for the second run. Performance criteria were met; log PC 50 and log PC 10 for 17 α -methyltestosterone were lower than the acceptable values,
but this indicates increased se nsitivity of the cells to weak agonists. Because the RPC Max < PC 10 in both assay runs, diazinon was considered ne gative for estrogen receptor transcriptional activation in this test system.

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

The 21-day short-term reproduction assay (MRID No. 48616005) of technical grade diazinon (98.7% active ingredient; a.i.) with fathead minnows ( Pimephales promelas) was conducted under flow-through conditions. Adult fish, 16 spawning groups (2 males and 4 females in each

group: ca . 5 months old) were exposed to diazinon at nominal concentrations of 0 (negative control) 0.01 (low), 0.1 (mid) and 1.0 (high) mg a.i./L; mean-measured concentrations were <level of quantification (LOQ <0.00083), 0.011, 0.120, and 0.820 mg a.i./L. The test system was
maintained at 24 to 25 o C and a pH of 6.0 to 7.9.

Overall mean survival values were 100, 96, 92, and 92% in the control, low, mid, and high treatment groups, respectively; no significant e ffects on survival were observed (p>0.05). Mortality was limited to female fish in each tr eatment. However, there was a statistically significant reduction (p<0.05) in body weight of female fish at the mid and high treatment levels, and overt toxicity, including a bnormal behavior and swimming, wa s observed in fish (male and female) exposed at the high concentration leve l. Based on the reductions in body weight andDIAZINON FINAL

Page 38 of 63 behavior, the mid and high exposur e levels were considered over tly toxic to females while the high exposure level was consider ed overtly toxic to males.

In the control replicates, spaw ning occurred at least every 4 da ys in only two of the four replicates and fecundity in the control was 11 e ggs/female/day/replicate (fecundity ranged from 6.2-16 in controls, and in only one of the four replicates was ≥ 15 eggs/female/day validity criterion). Fertilization success in the control group averaged 91% (fer tilization ranged 83-95%) and was less than the 95% validity criterion. Di azinon significantly (p<0 .05) reduced fecundity by 94% and fertility by 80% relative to th e control at the high treatment level.

Plasma vitellogenin (VTG) was signi ficantly reduced relative to cont rol in female fish at the low and high treatment levels (p<0.05) by 63% and 71 %, respectively. Male pl asma VTG levels were not significantly different from the control and did not exhibit a monotonic trend (p>0.05).

A significant 29% and 31% reduction (p<0.05, relati ve to control) was observed in gonadosomatic index (GSI) in male fish at the mid a nd high treatment levels, as well as a significant 42% reduction for GSI in female fish (p<0.05, relati ve to control) at th e high treatment level. Nuptial tubercle scores were al so significantly reduced (p<0.05; 73% compared to control) in male fish at the high treatment level; no tuberc les were observed for females. Effects on gonadal histopathology were observed in female fish at the high treatment level and included an increased incidence of stage 0/1 ovaries with atrophy of perinucleolar and early cortical follicles. Male fish displayed greater incidence and se verity (ranging from minimal to moderate) of increased percentage of spermatogonia at all di azinon treatment levels; a testis-ova was observed
in one male at the mid concentration.

Hershberger Assay; (OCSPP 890.1400)

The Hershberger Assay consists of androgenic a nd anti-androgenic components. To screen for potential androgenic activity, diazinon (98.7% a.i., Batch # 00896074) in corn oil was administered daily via oral gava ge to ten 56- to 57-day old, castr ated male Sprague Dawley rats at dose levels of 0 (vehicle), 47, or 150 mg/kg/day. An androgenic positive control group consisted of 10 castrated rats exposed to 0.4 mg/kg/day testosterone propionate (TP) by subcutaneous (s.c.) injection for 5 days, th en TP + corn oil by gavage for 5 days.

To screen for potential anti-a ndrogenic activity diazinon (98.7% a. i., lot # 00896074) in corn oil was administered daily via oral gavage to ten 56- to 57-day old, castrated male Sprague Dawley rats at dose levels of 15, 47, or 150 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by subcutaneous in jection. The anti-andr ogen vehicle control consisted of 10 castrated rats expo sed to 0.4 mg/kg/day of TP (s.c.) for 5 days, then TP + corn oil by gavage for 5 days. An anti-androgenic positiv e control group consisted of 10 castrated rats exposed to 0.4 mg/kg/day TP and 3.0 mg/kg/ day flutamide (FT) in corn oil.DIAZINON FINAL

Page 39 of 63 For both components of the assay, the animal s were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration to determine weights of the five androgen-dependent tissues.

One animal from the TP and corn oil control (positive control for a ndrogen agonist, vehicle control for anti-androgen) was found dead on da y 10, and one animal from the 150 mg/kg/day group of the anti-androgen assay was found moribund prior to dosing. The remaining animals
survived until scheduled termination.

In the androgen agonist assay, no clinical signs of toxicity we re observed in any of the dose groups. In the anti-androgen assay, clinical signs included one 47 mg /kg/day rat with throat scab or lesion, and shoulder scab throughout the study. For one 150 mg/kg rat, clinical signs included fur discoloration (neck/face) a nd soft feces on day 3, thin on da ys 5 and 6, and hunched and/or thin on days 8-10. No clinical signs were observed in the 15 mg/kg/day group.

In the androgen agonist assay, body weight s in the 47 and 150 mg/kg/day groups were comparable to controls throughout the duration of the assay. At 150 mg/kg/day, overall (days 1-11) body weights gains were decreased (p< 0.05) by 64%. Animals dosed with TP had increased overall body weight gains (p<0.05) of 52%. There were no st atistically significant increases in any of the accessory sex organs of animals dosed w ith diazinon. Animals in the positive control TP group had accessory sex organ weight increases (p<0.05) as follows: 1649% in seminal vesicles; 1210% in ventral prostate ; 247% in levator ani- bulbocavernosus (LABC); 669% in Cowper’s gland; and 88% in glans penis. The performance criteri a indicated that this assay was performing as expected.

In the anti-androgen assay, there were no effects on body weights or body weight gains in the 15 and 47 mg/kg/day group, or the positive contro l group. At 150 mg/kg/da y, overall body weight gains were decreased (p<0.05) by 42%. Statistically significant decreases in two or more of the target accessory sex organ weights were obs erved in animals dosed with diazinon. At 15 mg/kg/day, the seminal vesicles weights were decreased 29% (p<0.05) and ventral prostate weight was decreased 26% (p<0.05). At 47 mg/ kg/day, accessory sex or gan weight decreases (p<0.05) were noted as follows: 37% in seminal vesicles; 43% in ventra l prostate and 21% in LABC. At 150 mg/kg/day, accessory sex organ weight decreases (p<0.05) were noted as follows: 62% in seminal vesicles; 60% in vent ral prostate; 44% in LABC; 48% in Cowper’s gland; and 13% in glans penis. Animals dosed with TP + FT had decreased (p<0.05) accessory sex organ weights as follows: 86% in seminal vesicles; 77% in ventral prostate; 45% in LABC; 69% in Cowper’s gland; and 20% in glans penis. The %CV fo r mean LABC weight of the 150 mg/kg/day group (24%) exceeded the maximum allowable %CV (20%). All other %CV values were less than the maximum permissible values.DIAZINON FINAL

Page 40 of 63 The high-dose level selected for this study (150 mg/kg/da y) was considered to be adequate based on previous studies showing reduced body weight and body wei ght gain after dosing at 200 mg/kg/day.

Diazinon was negative for androge nic activity in the Hershbergerassay as no statistically significant increases in organ weights were seen in two or more of the five androgen sensitive tissues in the androgen agonist as say. However, diazinon was posit ive for anti-androgenicity in the Hershberger assay as statistically significant d ecreases in organ weights were seen in two or more of the five androgen sensitive ti ssues in the androgen antagonist assay.

Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48670401), 16 Sprague-Dawley (Crl:CD ® [SD] IGS) female rats/dose group were treated daily by oral ga vage with diazinon (95.9% a.i.; batch # 00896074)
in corn oil at doses of 0 (vehicle), 50 or 100 mg/k g/day from post-natal day (PND) 22-42/43. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainment was recorded. Be ginning on the day of complete VO, vaginal smears
were examined for evaluation of the estrous cycle. After sacrifice on PND 42/43, serum thyroxine (T 4 ), and thyroid stimulating hormone (T SH) concentrations were analyzed by radioimmunoassay (RIA). Adrenal, liver, thyroid, pituitary, a nd urogenital organ weights were recorded, and microscopic examinations of the uterus, ovaries, thyroid, and kidney were
conducted.

No treatment-related effects were observed on mort ality, clinical signs, age at VO, mean age at first estrus, estrous cycle status, clinical chemistry, or gross or microscopic pathology.

The majority of females (11/16) lost weight from PND 21 to 22 in the high-dose group, but gained weight for the remainder of the st udy. Body weights ranged from 90.5 to 96.2% of control ( ↓ 4 to 10%) during treatment (PND 22-42), suggesting that 100 mg/kg/day was an adequate high dose. Additionally, at this dos e, body weight at VO was decreased (p<0.05) by 10%, with a decreasing (p<0.05) trend across both dose groups.

Relative (to body) liver weights we re increased (p<0.05) by 7 and 11% in the low- and high-dose animals, respectively. Additiona lly, an increasing (p<0.05) trend in unadjusted adrenal weights was observed ( ↑ 3 to 8%). R elative kidney weights were increa sed (p<0.05) by 7%, and relative adrenal weights were increased by 13%, in the high-dose group. These increased relative organ weights correlated with slight decreases in final body weights and slight in creases in unadjusted organ weights.DIAZINON FINAL

Page 41 of 63 Serum T 4 concentrations were decreased (p<0.05) by 17 and 23% in the treatment groups. However, serum TSH concentrations were similar to control, and there were no effects observed on thyroid weights, or thyroid follicul ar cell colloid area or cell height.

In the high-dose group, mean estrous cycle lengt h was increased (p<0.05) from 3.7 days to 4.6 days, and the percent regularly cycling was decreased (not significant [NS]) from 92% to 69%. As mean age at first estrus and estrous cycle status were unaffected, th e biological significance of these findings in the context of female pubertal development is not clear. The doses tested were determined to be adequate.

Male Pubertal A ssay; (OCSPP 890.1500)

In a male pubertal assay (2012, MRID 48670401), 16 Sprague-Dawley (Crl:CD ® [SD] IGS) male rats/dose group were treated daily via gavage with diazinon (95.9% a.i.; batch # 00896074) in
corn oil at doses of 0 (vehicle), 50 or 100 mg/ kg/day from post-natal day (PND) 23-53/54. Animals were examined for preputial separation (PPS) daily beginning on PND 30 and weight at day of attainment was recorded. Following s acrifice on PND 53/54, total serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were analyzed by radioimmunoassay (RIA). Adrenal, liver, thyroid, pituitary, a nd urogenital organ weights were recorded, and microscopic examinations of th e testis, epididymis, thyroid, and kidney were performed.

No treatment-related effects were observed on mortality, clinical signs, body weights, body weight gains, age or weight at PPS, organ weights (except thyroid) , clinical chemistry parameters, gross pathology, or microscopic examin ation of the thyroid, te stes, epididymides, or kidney. Thyroid weights were decreased by 10 an d 11%, respectively, in the low- and high-dose groups, with a decreasing (p<0.05) tre nd across both dose groups. Serum T 4 concentration was decreased (p<0.05) by 15%, and serum TSH concen tration were increased not significant [NS] by 35% in the low-dose group; however, these parame ters were similar to control values in the high-dose group. Serum testosterone concentrations were unaffected by treatment. The minimal changes observed in the t hyroid hormone levels (T 4 and TSH) were not considered to be toxicologically significant due to the minimal magnitude and lack of dose response.

Although no treatment-related effects were noted on pubertal development, the thyroid, or signs of systemic toxicity, the dosing levels utilized in the current study are considered adequate. Even though acetylcholinesterase (AChE) levels were not determined in this study, previous studies are available showing that treatment with diazinon at thes e dose levels results in AChE
inhibition.DIAZINON FINAL

Page 42 of 63 Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48670403), H295R cells cultured in vitro in 24-well plates
were incubated with diazinon

, (98.7% purity, Batch # 00896074) at log concentrations from 0.0001 to 100 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as a vehicle,
at a final concentration of 0.05%.

Testosterone and estradiol concentrations we re measured by HPLC/MS-MS using positive ion multiple reaction monitoring. Three independent experiments were performed. A Quality Control (QC) plate was run concur rently with each independent r un of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels; positive controls include d the known inhibitor (p rochloraz) and indu cer (forskolin) of estradiol and testosterone production. Because of precipitation in two runs and cytotoxicity in the third, the highest suitable concentration of diazinon in Runs 1 through 3 was 10 μM.

Guideline acceptability recommendations and requirements were generally met, including lack of cytotoxicity, adequate producti on of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with positive controls. Although the betweenplate %CVs for estradiol were slightly above the recommended Guideline limit of 30% for each set, these data are considered acceptable.

Statistically significant decreases in testoster one production were observe d after incubation with 10 μM diazinon in all three runs. Statistically significant increases were also observed at several
lower concentrations in Runs 2 and/or 3, but these changes were not reproducible across all three test runs. Statistically significant decreases in estradiol production were observed in Run 1 at concentrations of 0.0001 to 0.1 μM, and a significan t increase was observed in the same run at the 10 μM concentration.

Based on the hormone responses in each of th e three independent runs, diazinon treatment resulted in statistically signi ficant and reproducible decreases in testosterone production, but
diazinon treatment did not result in statistically significant an d reproducible alterations in estradiol production.

Uterotrophic Assay; (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48616007) conducted to screen for potential estrogenic activity, diazinon (98.7% a.i., Lot # 00896074) in corn oil wa s administered daily via oral gavage to 56- day old ovariectomized female Sprague-Dawley rats (8/group) at dose levels of 0 (vehicle), 78 or 250 mg/kg/day. The study also included a posi tive control group of eight ovariectomized females dosed via oral gavage with 17 α -ethynyl estradiol (EE) at 0.1 mg/kg/day. Animals wereDIAZINON FINAL

Page 43 of 63 dosed for three consecutive days and necropsie d approximately 24 hours after the final dose to
determine wet and blo tted uterine weights.

All animals survived until scheduled termination. One rat administered 78 mg/kg exhibited mild lethargy prior to dosing on Day 3. Rats admini stered 250 mg/kg/day exhibited the following
adverse clinical effects: two rats displayed nasal discharge, thinness and fur discoloration on Day 2; on Day 3 those same two rats plus three additional rats (total of five) displayed head and scapula fur discoloration, lethargy and hunched postu re; these same five rats displayed head and scapula fur discoloratio n prior to euthanasia.

Body weights were decreased by 15% (p<0.05) in the 250 mg/kg/day group compared to controls. Body weight gains we re decreased (p<0.05) by 62% in the 78 mg/kg/day group and the 250 mg/kg/day animals actually lo st weight during the overall study (-18.1 g) compared to controls (18.5 g). These fi ndings indicate that the dose le vels tested were adequate.

Uterine weights in the diazinon tr eated groups were comparable to the vehicle controls. The mean blotted uterine weight in the control gr oup was 0.031% of the mean terminal body weight, which meets the Guideline requirement (<0.04%). Absolute wet and blotted uterus weights for the positive control group (0.1 mg/kg/day EE) were increased (p<0.05) by 254% and 162%, respectively. These increased uterine weights were in the expected range.

No statistically significant changes were seen in uterine weights in this assay. Diazinon was negative in the uterotrophic assay.DIAZINON FINAL

Page 44 of 63 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 studies consid ered relevant for dete rmining the potential
interaction of diazinon with the endocrine system are summarized below. Given the breadth of toxicity data available on diazi non, the only Part 158 studies discu ssed in this appe ndix are those that fulfill current guideline requirements and are directly re levant to assessing potential endocrine effects ( i.e. , multiple-generation rodent reproduc tive studies, development studies, chronic toxicity studies, etc.).

ER Transcriptional Activation

In a published, ( Kojima et al ., 2004) , the potential of 200 chemicals (including diazinon; purity 95-100%) to interact with two human ER subtypes (hER α and hER β ) and one human androgen receptor (hAR) in transactivati on assays with Chinese hamster ovary cells (CHO-K1 cells) was investigated. For detection of hER α or hER β activity, cells were tr ansfected with pcDNAER α or pcDNAER β , pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were transfected with pZeoSV2AR, pIND-ARE, and pRL-SV40. Flut olanil was tested over a concentration range
from 10

− 8 to 10

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

− 11 or 10

− 10 M E 2 or 10

− 10 M DHT was added to the cell cultures al ong with the test compound, respectively. Agonistic activities were evaluated by the relative activity ex pressed as 20% relative effective concentration (REC 20 ), the concentration of the compound de monstrating 20% of the activity of

10

− 10 M E 2 , 10

− 9 M E 2 , or 10

− 9 M DHT for ER α , ER β , and AR, respectively. Antagonistic activities were evaluated similarly by the relative activity expressed as 20 % relative inhibitory concentration (RIC 20 ), the concentration of the compound showing 20% in hibition of the activity
of E 2 or DHT against ER α , ER β , and AR, respectively. Diazi non did not show agonist or antagonist activity for ER α , ER β , or AR in this study.

In a published study by Kojima et al . (2005) , the response of an in vitro ER–related reporter gene assay was investigated af ter exposure to thirty two pest icides either alone or in combination. The source of the test compounds was not reported; however diazinon had a reported purity of >96%. The test compounds were prepared in DMSO at 10

− 2 M. The ECALUX Assay (Xenobiotic Detection Systems, In c., 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. Estr ogen-responsive concat emers (repetitive DNA sequences), inserted directly a bove the promoter, confer estrog en responsiveness to the viral promoter and reporter gene. The tr ansfected cells are made stable by an antibiotic resistance gene resulting in the cell line us ed in the assay (BG1Luc4E 2 ). Compounds that interact with estrogenresponsive DNA are identified by the luciferase gene response and quantified in terms of Relative Light Units (RLU).DIAZINON FINAL

Page 45 of 63 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. The reported EC 10 for diazinon in this assay was 4.6 x 10 -4 M.

90-Day (Subchronic) Oral T oxicity in Rodents (Rat)

In a subchronic oral toxicity study ( MRID 40815003 ), male and female SD rats were administered diazinon in both pilot (6 weeks) an d 90-day (13 weeks) studies. In the pilot study, males and females (10/sex) were administer ed diazinon (MG-8, 87.7% purity, Lot/Batch #FL872049) at nominal concentrations of 0, 0.5, 2, 100, and 1000/2000/4000 ppm in the diet for 42 days [analytical concentrations of 0.04/0.05, 0.2/0.2, 8.4/9.4, and (105.1/152.7/236.6)/ (114.9/194.2/284.9) mg/kg/day (M/F)]. The high dose was increased from 1000 to 2000 ppm on Day 15, and increased again from 2000 to 4000 ppm on Day 36. Soft feces, and decreased body weight gain and food consumption, were observed in the high-dose animals and considered to be
related to treatment. Dose-related inhibition of serum AChE activity occu rred in all groups, but inhibitions in the low-dose group (0.5 ppm) were very small (4 to 9%; NS). Significant inhibition (p ≤ 0.01) occurred in males ≥ 100 ppm and in females ≥ 2 ppm. RBC AChE activity was inhibited in the two highest dose groups of both sexes, and brain AChE was inhibited in males (p ≤ 0.01) in the high-dose group and females (p ≤ 0.05 and 0.01, respectively) in the two highest dose groups. Histopathology was not conducted and there was no mortality. The NOEL was 0.5 ppm, and the LEL was 2.0 ppm based on inhibition of serum AChE activity.

In the main study, males and females (15/sex) we re administered diazinon (MG-8, 87.7% purity, Lot/Batch #FL-872049) at nominal concentrations of 0, 0.5, 5, 250, and 2500 ppm in the diet for 90 days [analytical concentrations of 0.03/ 0.04, 0.3/0.4, 15/19, and 168/212 mg/kg/day (M/F)]. Clinical signs observed in the high-dose an imals included soft feces, hypersensitivity, and aggressiveness; no mortality wa s observed. Body weight gain was decreased (generally p ≤ 0.01) in high-dose males through Week 6 and female s through Week 7, with <10% decreases (NS) through study termination. Decreased food consum ption was observed in the high-dose animals for Week 1 (males) and Weeks 1 and 2 (females) only. Dose-re lated inhibition (p ≤ 0.01) of serum and RBC AChE activity occurred in all female dose groups, except inhibition in the low-dose group (0.5 ppm) were very small ( ↓ 4 to 12%; NS). Dose-related inhibition (p ≤ 0.01) of serum AChE activity occurred in males with the ex ception of the low-dose gr oup, and inhibition of RBC AChE activity was similar be tween the two highest dose groups . Brain AChE was inhibitedDIAZINON FINAL

Page 46 of 63 in males (p ≤ 0.01) in the high-dose group and females (p ≤ 0.01) in the two hi ghest dose groups. No organ weight or histopatholog ical effects were reported for adrenals, epididymides, ovaries, pituitary, prostate, testes, t hyroid, and uterus. Increased ( ↑ 18%; p ≤ 0.05) liver weight in the highdose females and an increased incidence of he patocellular hypertrophy in 3/15 (250 ppm) and 13/15 (2500 ppm; p ≤ 0.001) in females was also observed. The NOEL was 0.5 ppm (serum) and 5 ppm (RBC and brain) for males, and 0.5 ppm (serum and RBC) and 5 ppm (brain) for females based on inhibition of AChE activity.

90-Day (Subchronic) Oral Toxi city in Non-Rodents (Dog)

In a subchronic oral toxicity study ( MRID 40815004 ), male and female Beagle dogs were administered diazinon in both pilot (4 weeks) an d 90-day (13 weeks) studies. In the pilot study, males and females (4/sex) were administer ed diazinon (MG-8, 87.7% purity, Lot/Batch #FL-872049) at nominal concentrations of 0, 0.5, 2, 20, and 500 ppm in the diet for 28 days [analytical concentrations of 0.020/0.023, 0.073/0.082, 0.80/0.75, and 14.68/15.99 mg/kg/day (M/F)]. Emesis, and decreased body weight gain and food consumption, were observed in the high-dose animals and considered to be rela ted to treatment. Dose-related inhibition (p ≤ 0.01) of serum AChE activity occurred in all female groups, and al l males except the 0.5 ppm group. RBC and brain AChE activity was inhibited (p ≤ 0.01) in the high-dose gr oups of both sexes. Histopathology was not conducted and there was no mortality. The esse ntial NOEL was 0.5 ppm in males and <0.5 ppm in females based on inhibition of serum AChE activity. The accompanying LEL values (not reported) would be 2 and 0.5 ppm in males and females, respectively.

In the main study, males and females (4/sex) were administered di azinon (MG-8, 87.7% purity, Lot/Batch #FL-872049) at nominal concentrations of 0, 0.1, 0.5, 150, and 300 ppm in the diet for 90 days [analytical concentr ations of 0.0034/0.0037, 0.020/0.021, 5.9/5.6, and 10.9/11.6 mg/kg/day (M/F)]. Clinical signs observed in two high-dose males and one high-middose female included emesis (with or without food), bloody feces and/or diarrhea; no mortality was observed. Body weight gain was decreased (NS) in high-dose males ( ↓ 34%) and in highmid- and high-dose females ( ↓ 34 and 45%, respectively). Food consumption was not affected in treated animals. Dose-related inhibition (p ≤ 0.01) of serum and RBC AChE activity occurred in males and females in the high-mid- and high-dos e groups. No inhibition was observed in females in the low dose group; inhibition (generally NS ) was observed in low-dose males and low-middose males and females. Brain AChE was inhibited (p ≤ 0.01) in males and females in the highmid- and high-dose groups. No organ weight or histopathological eff ects were reported for adrenals, epididymides, liver, ovaries, pituitary, prostate, testes, thyroid, uterus, and vagina; pancreatic acini atrophy was noted in a si ngle high-dose male. The NOEL was 0.1 ppm (NS
inhibition in males and no inhibi tion in females) and the LEL was 0.5 ppm (significant inhibition in males and NS inhibition in females) based on effects on serum AChE activity.DIAZINON FINAL

Page 47 of 63 Prenatal Development in Rodents (Rat)

In a rat teratology study ( MRID 00153017 ), randomized groups of 27 assumed pregnant COBS CDT (SD) (BR) rats were administered di azinon (97.4% purity, Lot/B atch FL #821568) at 0 (vehicle only), 10, 20, and l00 mg/kg/day by intubation during GDs 6 to 15. All dams survived until sacrifice on GD 20. Food-consumption was significantly reduced on GDs 6 to 9, and body weight was reduced on all measurement days (GDs 6, 10, 14, 18, and 20) in high-dose females. These finding were considered compound-related. Similarly, body weight gain in the high-dose females was -11 ± 2 g during GDs 6 to10 of gesta tion; body weight gain in the other dose groups was similar to control (14 ± 2 g). Body weight gain during GDs 10 to 14 in high-dose females was greater compared to contro l values indicating recovery; ho wever, overall body weight gain in high-dose females was reduced (significance not reported) compared to control and is
considered treatment-related.

Comparable incidences among groups of cor pora lutea and implantations were observed. Resorptions were decreased (sig nificance not reported) in the mid-dose group (0.4) and increased in the high-dose group (1.8) compared to control (1.0), and the mean number of live fetuses was decreased in the high-dose group (11.9) compared to control (14.2). Pre-implantation and postimplantation loss, and fetal sex ratio, variations were also considered incidental to treatment. Male and female fetal weights were increased (significance not reported) in the high-dose group in comparison to controls; this was considered rela ted to treatment, but not a sign of fetotoxicity. Three single instances of external malformati on were observed at the high-dose. Since the
malformations were not related morphologically, and considered s econdary to maternal toxicity, they were not considered related to treatment. Fetotoxicity (mottled liver) and the incidence of rudimentary 14th ribs at the high-dose were within normal or historical ranges for control data.

The rat teratology study is acceptable as Guidelin e data. Diazinon was not teratogenic at doses up to 100 mg/kg. The maternal toxicity LOEL is 100 mg/kg based on decreased food consumption, body weight, and body weight gain. The NOEL is 20 mg/kg/day. The LOEL for developmental toxicity is 100 mg/kg/day based on the marginal increase in rudimentary ribs. The NOEL is 20 mg/kg/day.

Prenatal Development in Non-Rodents (Rabbit)

In a dose range-finding rabbit teratology study ( MRID 00079013 ), randomized groups of 6 assumed-pregnant New Zealand White Rabbits were administered diazinon (purity and Lot/Batch not reported) at 0 (vehicle only) , 7, 25, 100, 200, 300, and 400 mg/kg/day by gavage during GDs 6 to 18. Significant maternal mort ality was observed at 100 (16.7%), 200 (83.3%), 300 (33.3%), and 400 (50.0%) mg/kg/day prior to sacrifice on GD 30. Because doses

≥ 200 mg/kg/day resulted in substantial matern al toxicity, these treatment groups wereDIAZINON FINAL

Page 48 of 63 discontinued. Neither embryotoxicity nor frank external malformations were seen as a result of diazinon exposure.

In the main rabbit develo pmental toxicity study ( MRID 00079017 ), randomized groups of 6 pregnant New Zealand White Rabbits were admi nistered diazinon (89.2% purity, Lot/Batch not reported) at 0 (vehicle only), 7, 25, and 100 mg /kg/day by gavage during GDs 6 to 18. Mortality was biologically significant at the high dose (40.9% death; p<0.07); no mortality was observed in the two lower dose groups. C linical symptoms including tremors and convulsions and body weight gain decreases. Gastroin testinal hemorrhages and erosions were noted at necropsy. There were no significant differences among the groups regarding the mean number of implantation sites, and proportions of live, de ad, or resorbed fetuses per litter. Similarly, fetal weights and fetal sex ratio were similar in control and treatment groups.

Various skeletal variations were observed among treated and control litters, but no significant difference in incidence was determined. There were no indications of prenatal toxicity/teratogenicity at any of the doses tested. Because 100 mg/kg/day diazinon approached the maternal multiple-dose LD 50 (40.9% mortality) with no signs of fetotoxicity, it is inferred that maternal rabbits were more sensitive to the test article than embryos or fetuses. These findings demonstrated that diazi non was not fetotoxic or terat ogenic in rabbits at doses of

≤ 100 mg/kg/day. The LOEL for maternal toxici ty is 100 mg/kg/day based on decreased body weight, tremors, convulsions and deaths. The NO EL for maternal toxicity is 25 mg/kg/day. No compound-related effects on the fetuses were evid ent. The LOEL for developmental toxicity is >100 mg/kg/day. The NOEL for developmental toxicity is ≥ 100 mg/kg/day.

Developmental Neurotoxicity in Rodents (Rat)

In a range-finding developm ental neurotoxicity study ( MRID 45842601 ), diazinon (94.2% purity, Lot/Batch #9896144) was ad ministered to 10 female SD CD rats/dose in the diet at concentrations of 0, 0.1, 0.5, 50, or 300 ppm fr om GD 6 to PND 21. The average daily test article intake during the study was 0, 0.0125, 0.063, 6.56, and 38.06 mg/kg/day. A Functional Operational Battery (FOB) was performed on 5 dams/dose on GDs 7, 14, and 20, and on PNDs 1, 4, 7, 14, and 20. On PND 4, litters were culled to yield five males and five females (as closely as possible). A FOB was also conducted on eight pups/sex/group on PND 4, 7, 11, 14, 17, and
20. Blood and brain AChE activities were measur ed in eight pups/sex/group on PND 4 and 21, in two dams/group on PND 5 or 6, and in eight dams/group on PND 21.

All dams survived to scheduled sacrifice. During gestation, no eff ects on body weight or body weight gain were noted. Duri ng lactation, body weight gain by the high-dose dams was decreased by 25 to 80% of cont rol. No differences in food c onsumption were observed during gestation and lactation. Maternal necropsy was unremarkable. The maternal systemic LOAEL is 300 ppm (38.06 mg/kg/day) based on clinical si gns of toxicity, and the NOAEL is 50 ppm (6.56 mg/kg/day).DIAZINON FINAL

Page 49 of 63 In the main developmental neurotoxicity study (MRID 46195601, 2003), di azinon (92.9% purity, Lot/Batch #9896144) was administered to 27 female Crl:CD(SD)BR IGS rats/dose in the diet at concentrations of 0, 0.30, 30 or 300 ppm from GD 6 through PND 21. The average daily intake of diazinon was 0, 0.026, 2.36, or 24.2 mg/kg/da y during gestation and 0, 0.039, 4.06, or 39 mg/kg/day overall (GD 6 to PND 21). A FOB was performed on 10 dams/dose on GDs 13 and 20, and on PNDs 7, 14, and 20. On PND 4, litters were culled to yield five males and five females (as closely as possible). Offspring representing at least 20 litters/dose were allocated for detailed clinical observations (F OB), assessment of motor activity, assessment of auditory startle response habituation, assessment of auditory startle pre-pulse i nhibition, assessment of learning and memory, and neuropathology at study termina tion (Day 60 of age). On PND 21, the whole brain was collected from 10 pups/sex/dietary level for micropathologic examination and morphometric analysis. Pup sexual maturation was assessed by age at VO for females and at completion of PPS for males. Plasma, RBC, and br ain AChE activities were measured in dams on PND 21, and in selected pups on PNDs 4 and 21.

In dams, no treatment-related e ffects on mortality, clinical si gns, body weight, FOB parameters, or necropsy findings were noted. There was a slig ht decrease in body weight gain (38%; NS) and food consumption (10%; p ≤ 0.01) in the high-dose dams during lactation. This is supported by a dose-dependent inhibition of AChE in all three compartments. Enzyme activity was inhibited in the mid- and high-dose groups by 67 and 92.6% , respectively, in plasma; by 87.3 and 100%, respectively, in RBC; and by 24.8 and 86.8%, respec tively, in brain. No inhibition was observed
in low-dose dams.

For maternal systemic toxicity, the NOAEL is 300 ppm (24.2 mg/kg/da y), the highest dose
tested. A LOAEL was not established. For ma ternal AChE inhibition, the NOAEL is 0.3 ppm (0.026 mg/kg/day). The LOAEL is 30 ppm (2.36 mg/kg/day) based on decreases in plasma, RBC, and brain AChE activities.

No treatment-related effects were noted on body weight or body wei ght gain for pups in the lowor mid-dose groups. At the high dose, absolute body weights of male and female pups were decreased (p ≤ 0.05 or 0.01) compared to control beginning on PND 7 for males and PND 4 for females. Correspondingly, high-dose pups had re duced body weight gain compared with the controls at all intervals throughout lactat ion. In addition, offspring had decreased (p ≤ 0.05 or 0.01) body weight through PND 60 for males (m id- and high-dose) and PND 42 for females (high-dose only). Intermittent decreases in body weight gain were observed in males and females through-out the post-weaning period. Males a nd female pups from the high-dose group had delays (p ≤ 0.01) in PPS (1.9 days after c ontrol) or VO (1.3 days afte r control), respectively. However, mean body weight at attainment of PPS or VO was similar between the treated and control groups for males and females.DIAZINON FINAL

Page 50 of 63 In 4-day old high-dose male and female pups, AChE activity was inhibited (p ≤ 0.01) in plasma by 50.1 and 48.4%, respectively, in RBC by 41.3 a nd 37.8%, respectively, and in brain by 16.7 and 13.4%, respectively. On PND 21, dose-related i nhibition of AChE activ ity was observed in male and female pups from the mid- and high-dose litters. Plasma enzyme activity was inhibited (p ≤ 0.01) in the mid- and high-dose offspring by 34.4 and 68.2%, respectively, in males and by 18.6 (p ≤ 0.05) and 51.2%, respectively, in females. In 21-day old high-dose male and female pups, AChE activity was inhibited (p ≤ 0.01) in RBC by 58.3% and 53.8%, respectively, and in brain by 44.2 and 28.6% i nhibition, respectively.

For offspring systemic toxicity, the NOAEL is 30 ppm (2.36 mg/kg/day). The LOAEL is 300 ppm (24.2 mg/kg/day) based on reduced body weight and body weight gain and delayed
sexual maturation in males and females. For offspring AChE inhibition, the NOAEL is 0.30 ppm (0.026 mg/kg/day). The LOAEL is 30 ppm (2.36 mg/kg/day) based on plasma and RBC AChE
activities in both sexes.

Comparative Cholinesterase Assay

In a special neurotoxicity study ( MRIDs 46166301 and 46166302 ), diazinon (92.9% a.i., batch/lot # 9896144) was administered to groups of Crl:CD® (SD) IGS BR rats by gavage. In the acute study, treatment groups consisted of eight adult rats/sex administered single doses of 0, 0.3, 3.0, 30, or 300 mg/kg bw in Mazola® corn oil. Eight PND 11 pups/sex and eight PND 21 pups/sex (one/sex/litter) r eceived single doses of 0, 0.3, 3.0, 10, or 100 mg/kg by gavage. In the repeat dose study, eight male and eight fema le adult Crl:CD® (SD) IGS BR rats were administered diazinon at 0, 0.03, 0.3, 10, or 100 mg/ kg/day in seven consecutive daily doses. Ten male and 10 female pups received doses of 0, 0.03, 0.3, 3, or 30 mg/kg/day from PND 11 to 17. The primary purpose of this study was to de termine the effect of diazinon on blood and brain cholinesterase activity in male and female adu lt and neonatal/juvenile rats following both acute and repeated exposure.

The test material did not did not increase mo rtality or affect body weight. Following acute dosing, clinical signs of toxicity included tremors in 4/8 adult females at 30 mg/kg, in all adults at 300 mg/kg, in 2/8 female PND 11 pups at 10 mg/kg, and in 7/8 male and 6/8 male PND 11 pups at 100 mg/kg. A high incidence of tremor s was noted for all dose groups of PND 17 pups following repeated dosing. Additional clinical signs following acute dosing included rustcolored fur in adults at doses  3 mg/kg or  0.3 mg/kg following single or repeated doses, respectively; lethargy in 2/8 adult males at 300 mg/kg and in one male and female PND 11 pup at 100 mg/kg; and ataxia in 2/8 male and 2/ 8 female PND11 pups at 100 mg/kg. Increased salivation was seen in adults after repeated dosing with 10 mg/kg/day (1/8 males) or 100 mg/kg/day (2/8 males, 3/8 females). Re peated exposure of PND 17 males to 3 or 30 mg/kg/day resulted in decreased absolute brain weight (5% or 9%, respectively).DIAZINON FINAL

Page 51 of 63 Acute or repeated exposure to diazinon resulted in statistically and bi ologically significant
decreases in the cholinestera se activity in plasma, red bl ood cell (RBC), and brain of young adults animals, and PND 11, PND 17, and PND 21 pups. In adults, effects were noted at 3 mg/kg in both sexes following single dose exposur es and at 0.3 mg/kg/day in females after repeated exposures. Following single doses to pups, effects were observed at 3 mg/kg in both sexes of PND 11 pups and in both sexes of PND 21 pups. After repeated exposures, effects were noted in PND 17 pups at 0.3 mg/kg/day in both se xes. Acute doses of 0.3 mg/kg and repeated doses of 0.03 mg/kg caused no significant effects.

For acute exposure, the overall LOAEL for cholines terase inhibition in adults is 3 mg/kg based on plasma; the adult NOAEL is 0.3 mg/kg.

For acute exposure, the overall LOAEL for c holinesterase inhibiti on in PND 11 and PND 21 pups is 3 mg/kg based on plasma; the pup NOAEL is 0.3 mg/kg.

For repeated exposure, the overall LOAEL for c holinesterase inhibition in adults is 0.3 mg/kg/day based on plasma; the NOAEL is 0.03 mg/kg/day.

For repeated exposure, the overall LOAEL for c holinesterase inhibition in PND 17 pups is 0.3 mg/kg/day based on plasma; the NOAEL is 0.03 mg/kg/day.

Developmental Toxicity in Male Rodents (Rat)

In a published non-guideline developmental toxicity study examining the effects of diazinon on reproductive development in male rats (Jayachan dra and D’Souza, 2013), groups of six female SD rats (10 to 12 weeks old) were administer ed diazinon (purity 98.6%) vi a gavage at 0 (normal saline), 10, 15, or 30 mg/kg/day prior to and during mating, pregnancy, and lactation. Mating was conducted with undosed males after 14 days of treatment. Male offspring were assessed on PND 70 (pubertal stage) or 170 (a dult development) for histologica l changes in the testes and epididymides and for changes in selected enzy me activities in the testes and changes in
concentrations of 3- β -hydroxysteroid dehydrogenase (3- β -HSD). AChE activity was not
assessed.

Litter size was decreased in all diazinon-treated groups ( ↓ 12, 19 and 28%) compared to the control, and male pup body weight deficits were noted in all dose groups; however, differences
in the low- and mid-dose animals were reso lved by PND 14 and 21, respectively. By PND 21, male pup body weight was decreased (p<0.05) by 9% at the high dose. By puberty (PND 70), body weights were similar among on all dose groups , but testis weights were decreased (p<0.05 or 0.001) by 11 and 19% in the mid- and high- dose males, respectively. By adulthood (PND 170), body weight and testis wei ght were decreased (p<0.001 and 0.01, respectively) in highdose males by 10 and 14%, respectively. Sperm countwas also decreased (p<0.001) in the highdose group at puberty compared to control.DIAZINON FINAL

Page 52 of 63 Histological changes were noted in the testes an d epididymides of the mid- and high-dose males. At puberty, testes showed evidence of sloughing of the seminiferous epithelium with an increase in the interstitial spa ce. The numbers of spermatozoa were fe wer, and some tubules did not have any spermatozoa at the high dose. In epididym is, there was an evidence of sloughing of the epithelium and distortion of the shape of the tubules , with an increase in th e interstitial space. Similar findings were noted in the mid- and high-dose groups at PND 170.

The changes noted in testes and epididymides were accompanied by changes in
activities/concentrations of selected enzymatic biomarkers isolated from the testes.
Concentrations of 3- β -HSD, which is an enzyme involved in steroidogenesis, were decreased (p<0.001) by 29 and 42% in the mid- and hi gh-dose groups at puberty (PND 70) and by 25, 44 and 52% in the low-, mid-, and high-dose groups by PND 170. Activities of alkaline phosphatase (p<0.01 or <0.001) and acid phosphatase (p<0.05 or <0.01) were increased in the high-dose group at PND 70 by 87 and 29%, respectively, a nd at PND 170 by 48 and 21%. The activity of LDH was also increased (p<0.01 or <0.001) in the high dose group by 23% at PND 70 and 170. The study authors suggest that the increases in the activities of these three enzymes are indicative of the degenerative changes being obse rved in the testis at the high dose.

Two-Generation Reproduction in Rodents (Rat)

In an initial Multiple -Generation Reproduction study in the rat ( MRID 41158102 ), diazinon (94.9% purity; Lot/Batch #FL841650) was admini stered at target doses of 0, 10, 100, and 1000 ppm in the diet to SD rats (Crl:COBS CD(SD) Br for one generation (two litters; abbreviated study). Actual exposure ranges were 0, 0.49 to 0.99, 4.88 to 9.75, and 49.64 to 93.92 mg/kg/day and 0, 0.60 to 0.96, 6.14 to 9.60, and 65.87 to 89.48 mg/kg/day in F0 males and females, respectively.

A NOEL of 100 ppm was determined in this study, based on decreases in the male and female fertility indexes, male mating indices, pregna ncy index, and the number of litters per group; the duration of gestation also was increased. Only a single generation (with two litters) was bred, and the study was terminated early due to poor survival of the F1b pups. Data indicating a possible effect on the number of litters in the low- and mid-dose groups was considered equivocal. Additional data was requested to resolve these issues (effects on litter size; number of litters; relative ovary weights; weight gain; reproductive indi ces; pup weight at birth; and pup survival). This study is cl assified as supplementary.

In a follow-up Multiple (Two)-Generati on Reproduction study in the rat ( MRID 41158101 ), diazinon (94.9% purity; Lot/Batch #FL841650) was administered at target doses of 0, 10, 100, and 500 ppm in the diet to SD rats (Crl:COBS CD(SD)Br for two generations. Mean exposures (first and second generation) were 0, 0.67, 6.69, and 35.15 mg/kg/day and 0, 0.7, 7.63, and 41.43 mg/kg/day in F0 (P1) males and females, respecti vely. The P1 rats were fed the diet for 10 weeks prior to mating, through gestation and lactation of the F1 litter, and up to three weeks afterDIAZINON FINAL

Page 53 of 63 weaning. The F1 litters were weaned at PND 28. P2 rats (selected from F1 pups) were treated for 11 weeks after weaning prior to mating, and the F2 pups and P2 adults were sacrificed after weaning.

Three females died in the first parental group (p ossibly treatment-related and associated with dystocia) and two died in the second group (unrel ated to treatment); three and four females, respectively in each parental high-dose group ha d tremors. Effects on body weight gain were observed in both parental groups, but observed decreases were transient or NS in the P1 animals. In the second parental group (P 2), decreased (p<0.05) body weight gain occurred in mid- and high-dose males ( ↓ 6 and 9%, respectively); decreases body weight gain for females in was generally NS (sometimes due to high variability). No treatment-related changes were observed in absolute or relative weights of ovaries in eith er parental group, and no histopathological lesions
were seen in the ovaries . Absolute testis weight was decreased ( ↓ 9.9%; p<0.05) in P2, but relative testis weight was comparable to control, and no treatment-related histopathological
lesions were seen in the test es in either parental group.

Precoital interval, gestation inde x, and the number of viable litters were not affected for either parental group. Male and female mating and fertility indices were slightly decreased (significance not reported) for both parental grou ps. Mean duration of ge station was increased by approximately one day in the high-dose females; a similar increase was observed with both sets
of litters from the init ial one-generation study.

The viable mean litter size and total number of vi able pups were decreased for both litters (NS, except p<0.05 for second litter size ), and pup weight was slightly decreased (NS). Pup sex ratio was higher (p<0.05) in favor of males in the fi rst group, but not the second. Pup survival was decreased (p<0.05) for high-dose pups in both bree dings (overall, LDs 0 to 4, and LDs 4 to 21). Mean pup body weight was decreased (generally p< 0.05) from LD 4 (pre-cull) through LD 21 in the high-dose animals of both sexes. No definitive treatment-related histopathological findings were observed in pups.

The NOAEL for parental toxicity is 10 ppm (0.67 and 0.77 mg/kg/day; M and F) and the LOAEL is 100 ppm (6.69 and 7.63 mg /kg/day; M and F), based on decreased parental weight gain (P2 males, with transitory decreases for P2 females). No decrease in ovary weight was observed in the two-generati on reproduction study, although absolute testis weight was decreased ( ↓ 9.9%) in P2 males. The NOAEL for reproductive toxicity is 100 ppm (6.69 and 7.63 mg/kg/day; M and F), and the LOAEL is 500 ppm (35.15 and 41.43 mg /kg/day; M and F), based on decreased mating index an d fertility in the P2 parents and an increase in gestation length (approximately 1 day). The NOAEL for offspring toxicity is 10 ppm (0.67 and 0.77 mg/kg/day; M and F), and the LOAEL is 100 ppm (6.69 and 7.63 mg /kg/day; M and F), based on decreased pup body weight gain and survival.DIAZINON FINAL

Page 54 of 63 Reproductive Toxicity in Male Rodents (Mouse)

In a published non-guideline study examining the repr oductive toxicity of diazinon in male mice (ElMazoudy and Attia, 2012), 12-week old CD-1 (ICR) male mice (25/dose group) were administered diazinon (purity 98%) by gavage at 0 (distilled water), 2.0, 4.1, and 8.2 mg/kg/day for 38 days. After 28 days of dosing, the treated ma les were each paired with an untreated female for ten days (with continued dos ing of males) to assess effect s on mating and fertility. Males were terminated after the mating period and fe males were terminated on GD 20. The males were examined daily during dosing for clinical signs and mortality. Data collected from males included: body weight, reproducti ve organ weights, functional seminal parameters, plasma hormone concentrations (LH, FSH, estradiol, testosterone and prol actin), plasma AChE concentrations, sperm morphol ogy, and histology of the testes and epididymis. Data on reproductive outcomes were collected from the females at sacrifice.

All animals in each dose group survived until the end of the experimental period, and no significant effects were noted for any of the eval uated parameters for low-dose males. Males in the mid- and high-dose groups exhibited clinical signs for cholinergic toxicity throughout the study. Mean final body weight was reduced (p<0 .05) in mid- and high-dose males by 26 and 29%, respectively, compared to control; and males in these groups actually lost weight during the study (-4.9 and -5.6 g, respectively) . In addition, plasma AChE was reduced (p<0.05) by 37 and 24% at the mid- and high-dose. Plasma concentra tions of testosterone, es tradiol, and prolactin were increased (p<0.05) by 90, 84 and 96%, respectiv ely at the mid-dose, but testosterone and prolactin were decreased (p<0.05) by 63 and 11% at the high-dose. Plasma levels of LH and FSH were decreased (p<0.05) at both the mid-dose ( ↓ 45% and ↓ 56%) and high-dose ( ↓ 49% and ↓ 69%). Reproductive organ weights were decrease d (p<0.05) in a dose-dependent manner at the intermediate and high doses. Absolute weights for the mid- and high-dose groups were decreased by 30 and 35% for the testes, 50% for the epidid ymides and prostate, and 32 and 94% of the seminal vesicles. Relative organ weights were also decreased for the epididymides ( ↓ 33%) and prostate ( ↓ 30%). The mating and fertility indices we re decreased (p<0.05) by 12 to 14% and 40%, respectively, at both the intermediate and high dose. In addition, sperm counts and morphology was adversely affected at the intermediate and high dose, with sperm counts being reduced by 38 and 67% in the ep ididymides, and 39 and 68% in the testes. There were dosedependent decreases in sperm motility and viabilit y and increases in the percentage of abnormal sperm. With regard to the e ffects on reproductive outcomes, the number of pregnant females and litters were reduced (p<0.05) by 24% (NS) and 64% at the intermediate and high dose, and fetal weights were reduced (p<0.05) by 14 and 26%. Also at the high dose, the number of live fetuses/litter was reduced (p<0.05) by 30%, and the percentages of early a nd late resorptions and post-implantation loss were increased by 179, 265 and 252%, respectively. Sex ratio was not affected by treatment. Histological examinations showed increased in abnormal sperm and a dose-dependent effect on spermatogenesis in th e testes, including the loss, derangement, andDIAZINON FINAL

Page 55 of 63 sloughing of the germ cells, the vacuolization of germ cell cy toplasm, and disruption of spermatogenic cells.

The LOAEL for the study is 4.2 mg/kg/day based on adverse clinical signs, reduced plasma AChE concentrations, decreased body weights, and adverse effects on male reproductive organs and parameters. The NOAEL is 2.0 mg/kg/day. The u tility of this study for assessing potential
endocrine effects of diazinon is limited because the effects noted on hormone concentrations, and reproductive parameters and outcomes for male mice, occurred at doses that also showed signs of overt toxicity (weight loss, adverse clin ical signs of toxici ty, and reduced AChE concentrations).

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat)

In a chronic one/two-year oral toxicity study in rats ( MRID 41942002 ), diazinon (MG-8, 87.7% purity, Lot/Batch #872049) was admini stered as 0 (negative contro l and vehicle control groups), 0.1, 1.5, 125, or 250 ppm in the diet to Crl: VAF/Plus CD (SD) Br rats (10/sex in the 52-week dose groups and 20/sex in the 98-week dose groups ) for 52 or 98 weeks; a separate group of high-dose, interim-sacrifice animals (52 w eeks) included a 4-week recovery period.

No clinical signs of toxicity co uld be attributed to administration of the test material. Survival was comparable among the control and the treated groups, but due to higher mortality in the low-dose group, the study was terminated at Week 97. No adverse treatment-related effects were noted, but body weight and food consumption valu es were increased re lative to the negative
control. Mean compound intake was 0.004, 0.06, 5, or 10 mg/kg/day for males and 0.005, 0.07, 6, and 12 mg/kg/day for females in the low-, mi d-, high-mid-, or high-dose groups. Dose-related inhibition (p ≤ 0.01 or 0.05) of serum AChE activity occurr ed in males and females in the mid-, high-mid- and high-dose groups, with no inhibi tion was observed in the low-dose group. Brain and RBC AChE was generally inhibited (p ≤ 0.01) in males and females in the high-mid- and high-dose groups only. Organ we ights, gross pathology, an d neoplastic/non-neoplastic microscopic pathology were unrem arkable (no treatment-related findings). Organs examined included adrenals, epididymides, liver, ovaries, pituitary, prosta te, seminal vesicles, testes, thyroid, uterus, and vagina. No evidence of carci nogenicity was observed in the present study at diazinon exposures of ≤ 250 ppm. A NOEL of 0.1 pp m is determined in this chronic study, with a LEL of 1.5 ppm based on decreased serum AChE activity (with serum, RBC, and brain AChE activity decreased at ≥ 125 ppm. The systemic NOEL was >250 ppm.

Carcinogenicity in Rodents (Mouse)

In an NCI oncogenicity study in mice ( MRID 00073372 ), diazinon (98% purity, Lot/Batch #741308) was administered as 0, 100, or 200 ppm in the diet to B6C3F1 mice (25/sex in the control group and 50/sex-treate d in the treated groups) for 103 weeks, followed by a 2-week observation period. No trend in dose-related mo rtality was observed through Week 76. NoDIAZINON FINAL

Page 56 of 63 treatment-related effects on body weight were seen in males, with slig htly lower weights in females for the final 20 weeks of the study. No hematology, clinical chemistry, or urinalysis determinations; no organ weights; and no correla tion of macroscopic a nd microscopic pathology
for individual animals were included. No evidence of carcinogeni city was observed at diazinon exposures of ≤ 200 ppm. This study is cons idered core minimum.

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

In a chronic one-year oral toxicity study in dogs ( MRID 41942001 ), diazinon (MG-8, 87.7% purity, Lot/Batch #872049) was ad ministered as 0, 0.1, 0.5, 150, or 300/225 ppm in the diet to Beagle dogs (4/sex) for 52 weeks. The high dose (300 ppm) was decreased to 225 ppm after Week 14 due to insufficient body weight gain in the high-dose animals.

One high-dose male was sacrificed and one mid-dose female died due to gastrointestinal
infections in the first two w eeks of the study (animals were replaced). One high-dose male displayed treatment-related dehydration a nd emaciation. Body weight gain and food consumption values were generally low or d ecreased relative to control throughout the study. The high dose was reduced after Week 14 due to the lack of body wei ght gain. Mean compound intake was 0.0032, 0.015, 4.7, or 7.7 mg/kg/day for males and 0.0037, 0.020, 4.5, and 9.2 mg/kg/day for females in the low-, mid-, high-mid-, or high-dose groups. Dose-related inhibition (p ≤ 0.01) of serum AChE activity occurred in males in the high-mid- and high-dose groups, with generally NS d ecreases in the mid-dose gr oup. Dose-related inhibition (p ≤ 0.01 or 0.05) of serum AChE activity generally occurred in females in all dose groups through Day 268, with inhibition (p ≤ 0.01) only in the high-mid- and high- dose groups at study termination. RBC AChE was generally inhibited (p ≤ 0.01) in males and females in the high-mid- and high-dose groups only. Brain AChE was inhibited in the high-mid- and high-dose males (NS) and females (p ≤ 0.05 and 0.01, respectively). No e ndocrine-related organ weight changes were observed at study termination. Organs examined included ad renals, epididymides, liver, ovaries, pituitary, prostate, testes, thyroid, uterus , and vagina. Gross and microsc opic pathology were unremarkable (no treatment-related findings). For AChE inhibition, the NOEL is 0.1 ppm and the LEL is 0.5 ppm based on decreased serum AC hE activity. For systemic toxi city, the NOEL is 0.5 ppm and the LEL is 150 ppm based on decreased food consum ption in males and females, and decreased body weight and body weight gain in males.

In Vitro Porcine Oocyte Viability

In a published non-guideline study (Casas et al. ( 2010), the effect of diazinon exposure in vitro on porcine oocyte viability and in vitro matu ration was examined. Diazinon was tested at concentrations of 0 (deionized water), 25, 50, and 100 μM and incubated for 44 hours at 38.5°C. Oocyte viability was decreased with a calcula ted LC50 of 72 μM; oocyte maturation was also decreased with an IM50 of 22 μM (approximate ly 30% of the LC50). The mechanism(s) for toxicity was not investigated.DIAZINON FINAL

Page 57 of 63 In another published no n-guideline study (Ducolomb et al ., (2009), the effect of diazinon exposure in vitro on porcine oocyte viability, and in vitro fertilization and development, was
examined. Diazinon was tested at concentratio ns of 0 (deionized water), 50, 100, and 500 μM after maturation (incubation for 44 hours at 38.5°C). Oocyte viability was not significantly decreased (LC 50 not calculable), although in vitro fertilization of oocytes was decreased (FI 50 502 μM). Similarly, early embryo development af ter 96 hours was not significantly affected (DI 50 not calculable), but progression to mo rulae formation after 144 hours had an MI 50 of 344 μM. The mechanism(s) for toxicity was not investigated, although th e potential role of
AChE inhibition was discussed.

Avian Reproduction (OCSPP 850.2300)

In an avian reproduction study (MRID 41322901), 25-week old Mallard ducks ( Anas platyrhynchos ) were dosed with diazinon in the feed at concentrations of 5, 10 or 20 ppm for up
to 20 weeks. Mean measured diazinon concen trations in the diet were <0.05, 4.02, 8.30, and 16.33 ppm. Each dose group consisted of 20 ducks/se x, with a single drake and hen being paired in 20 pens/dose group. The photoperiod was eight hours of light per day during the first 8 weeks (pre egg-laying), decreased to 7 hours of light per day during Weeks 1 and 2, and was increased to 17 hours of light per day for the remainder of the study. All adult birds were observed daily for mortality, signs of toxicity, and abnormal behavior. Body weights we re recorded at study initiation, Week 2, and at study termination. Daily egg production was recorded, and parental incubation was employed instead of artificial incubation. The s econd and fourth eggs laid (as available) were selected fo r egg weight, and eggshell thic kness was determined. Feed consumption was measured every week. The stu dy room temperature was maintained at 18.8 ± 2.9°C and 80% relative humidity. Hatchlings were housed with the hen until 14 days of age and fed non-treated diets; surviving ha tchlings were weighed on Day 14.

In the ChE segment of the study housing and hus bandry were identical to the reproductive segment. Blood samples were collected from the a dult birds (4/sex/dose) on Day 7, at the start of egg production, at approximately mid-incubation, at hatch, and at 14 days post-hatch. Brain samples were collected at the end of the adult exposure period. Serum and brain samples were refrigerated or frozen, respect ively, immediately after collecti on, and were analyzed for ChE activity.

No adult mortalities occurred in any test group, a nd no overt signs of toxicity or clinical signs were observed. No effects were seen at any te st dose on female body weight or average feed consumption in males and females; it was noted th at feed consumption was highly variable. Male body weight gain was decreased (p<0.05) in the high-dose group ( ↓ 130%; lost weight). There were no apparent treatment-related effects upon e gg weight, shell thickne ss (0.36 to 0.38 mm), or shell strength at any of the concentrations tested.DIAZINON FINAL

Page 58 of 63 In general, no reproductive impairment (p>0.05) was seen at any dose. Eggs laid per hen and days in production were increased (as oppose to decrease) (p<0.05) in the high-dose females which is treatment-related. A >2-fold increase (N S) in eggs cracked per hen was noted in the high-dose group. The mean weight and number of 14-day old survivors was decreased (p<0.05; ↓ 33 and 41%, respectively) in the high-dose treatm ent. Similar reductions in serum and brain
AChE concentrations were observed in male and female Mallard ducks. Decreased (p<0.05) serum AChE concentrations were gene rally observed in the low-dose group ( ↓ 33 to 61%), and were observed consistently in the mid- ( ↓ 49 to 74%) and high-dose ( ↓ 62 to 84%) groups. Decreased (p<0.05) brain AChE concentratio ns were observed in the low-dose males ( ↓ 53%), and the high-dose males and females ( ↓ 70 and 68%, respectively). Ba sed on the reduction in 14- day hatching survival, the LOAEL for reprodu ctive effects is 16.33 ppm, and the NOAEL is 8.3 ppm. The NOEL for AChE inhibition is not determined (<4.02 ppm, low dose), and the LOAEL is 4.02 ppm.

In another avian reproduction study (MRID 41322902, 1989 ) , 6-month old Northern Quail ( Colinus virgnianus ) were dosed with diazinon in the feed at concentrations of 10, 20 or 40 ppm for up to 20 weeks. Mean measured diazinon co ncentrations in the diet were <0.05, 8.3, 16.3, and 32.0 ppm. Each dose group consisted of 32 quails , with a single cock and hen paired in 16 pens/dose group. The photoperiod was eight hours of light per day during the acclimation period (pre egg-laying) and the first tw o weeks on study, and was increased to 17 hours of light per day for the remainder of the study. Body weights were recorded at study initiation, Week 2, and at study termination. Samples were randomly sele cted for eggshell strength and thickness measurements. Feed consumption was measured every week. Eggs were collected daily, marked, cleaned, and stored at 10.3°C and 75% relative humidity. All eggs were candled to detect cracks prior to incubation and then measured for viab ility on Day 11 of incubation. On Day 21, embryo survival was determined and eggs were placed in a hatcher. On Day 25 or 26, all hatchlings, unhatched eggs, and eggshells were removed and the average body weight of hatchlings determined. Hatchlings were housed in brooder pens until 14-days old, and were weighed on Day 14.

In the AChE segment of the study housing and husbandry were identical to the reproductive segment. Blood samples were collected from the a dult birds (4/sex/dose) on Day 7, at the start of egg production, Week 4 of egg production, and at Day 14 post-hatch. Brain samples were collected at the end of the adult-exposure peri od. Serum and brain samples were refrigerated or frozen, respectively, immediately after collect ion, and were analyzed for AChE activity.

No mortality occurred during the reproductive portion of the study, a nd behavioral effects
observed were considered unrelated to treatmen t. Adult body weights and feed consumption in the treated groups were comparable to the cont rol group (high variability in feed consumption was noted due to wastage). There was no treatmen t-related effect on the prevalence of cracked eggs, and mean eggshell thickness in treatment groups was similar to control, except for a slightDIAZINON FINAL

Page 59 of 63 decrease ( ↓ 5%; p<0.05) at the high dose, wh ich was not considered to be biologically significant. Decreases (p<0.05) in eggs laid, viable embr yos, and live 3-week embryos (per hen) were observed in the mid-dose group were due to decreas ed output in a few hens, and were not doseresponsive. All other reproducti ve parameters, including 14-day old survivor weights and
numbers were similar to control values. Similar reductions in serum AChE concentrations were observed in male and female Northern qua il. Decreased (p<0.05 or 0.01) serum AChE concentrations were generally observed in the low-dose group ( ↓ 37 to 56%), and were observed consistently in the mid- ( ↓ 58 to 74%) and high-dose ( ↓ 83 to 88%) groups. Decreased (p<0.01)
brain AChE concentrations were observed in all treated females ( ↓ 35 to 40% across all doses);
no effect was noted in males. A LOAEL for re productive effects is no t determined; the NOAEL is 32 ppm (high dose). The NOEL for AChE inhibition is not de termined (<8.3 ppm, low dose), and the LOAEL is 8.3 ppm.

Fish Early Life-Stage Test (Freshwat er and Estuarine/Marine) (OPPTS 850.1400)

In a freshwater fish early life-stage test ( MRID 40782301 ), Fathead minnow ( P. promelas ) embryos were exposed to diazi non (87.7% purity, Lot/Batch #872049) at nominal concentrations of 0 (negative and solvent control), 0. 094, 0.19, 0.38, 0.75, and 1.5 mg/L. Achieved exposure concentrations were 0.092, 0.17, 0.38, 0.76, and 1.60 mg/L for the treated groups. At the end of the 28-day exposure, both length and wet weight were decreased (p< 0.05 or <0.01) at all concentrations. From the low- to high-dos e groups, body length was decreased by 4, 10, 23, 32 and 46%, respectively, compared to the contro l, and wet weight was decreased by 14, 20, 38, 58 and 79%, respectively. This study was scientif ically sound; however, it does not fulfill the Guideline requirements for a fish early life-stage test since the NOAEC value of diazinon for P. promelas could not be determined due to the adve rse effects on growth observed at all dose concentrations. Egg hatching and la rval survival were not affected by any test concentrations of
diazinon.

In an estuarine fish early life-stage test ( MRID 44244802 ), Sheepshead minnow, (Cyprinodon variegatus) embryos were exposed to diazinon (87.3% pur ity, Lot/Batch not reported) at nominal concentrations of 0 (negative and solven t control), 3.8, 7.5, 15, 30, and 60 μg/L. Achieved exposure concentrations were 4.3, 8.0, 13, 28, and 56 μg/L for the treated groups. This study is scientifically sound and fulfills the guideline requirements for an estuarine fish early life-stage test. After the 28-day exposure, both body length and weight (wet and dry) were decreased (p<0.05) in the mid-low, mid-, mid-high and high- dose groups compared to control. For the four highest dose groups, body length was decreased by 4, 9, 11 and 21%, respectively. Wet weights were decreased by 8, 21, 21 and 42%, respectivel y, and dry weights were decreased by 23, 31, 30 and 54%. Based on the most sensitive endpoints (length, dry weight, wet weight), the NOEC
and LOEC were 4.3 and 8.0 ppb (μg/L), respectively.DIAZINON FINAL

Page 60 of 63 Chronic Toxicity in Fish (Freshwater and Estuarine/Marine)

In two chronic toxicity studies on freshwater fish ( MRID 40910904 ), fathead minnows ( P. promelas ) and brook trout ( Salvelinus fontinalis ) were exposed to diazinon (92.5% purity) continuously under flow-through conditions. In th e main fathead minnow study, 5-day old fish (50 fish/dose) were exposed for up to 274 days to diazinon at concentrations of 0, 3.2, 6.9, 13.5,
28.8 and 60.3 μ g/L. Survival and growth were assessed on Days 31, 64, 97 and 167. At Day 167, each test group was thinned to 15 fish to assess spawning. Larvae from the initial hatches were exposed for 30 days, and subsequent hatche s were exposed for 60 days. Fathead minnows exposed to the lowest concentration tested ( 3.2 ug/L) from 5 days af ter hatch through spawning had an increased (p<0.05) incidence of scoliosi s compared to control, and the hatch of their progeny was reduced by 30% at this concen tration. The LOAEC for this study is 3.2 μ g/L, and a
NOAEC was not observed.

In the trout study, yearling brook trout (12 fish/dose) were exposed to diazinon at 0, 0.55, 1.1, 2.4, 4.8, and 9.6 μ g/L. Growth (length and weight) were assessed in the adult fish on Days 91 and 173, and then each treatment group was thinned to 6 fish (2 males, 4 females) to assess spawning (egg production and hatcha bility). Larval surv ival and growth were assessed at 30, 60, 90 and 122 days post-hatch. Year ling Brook trout exposed to ≥ 4.8 μ g/L diazinon began developing scoliosis and lordosis within a few weeks of exposure, and growth was substantially inhibited during the first 3 months at ≥ 4.8 μ g/L. Neurological symptoms were evident in brook

trout at ≥ 2.4 ug/L early in the tests, but were rarely observed after 4 or 5 months of exposure. Exposure of mature trout for 6 to 8 months to concentrations ranging from 0.55 to 9.6 μ g/L resulted in equally reduced gr owth rates for their progeny. Tran sfer of progeny between test
concentrations indicated that e ffects noted for the progeny from lo wer test concentrations were the result of parental exposure alone and not the exposure of the progeny after fertilization. The LOAEC for this study is 0.55 μ g/L based on reduced larval growth, and a NOAEC was not
observed.

In an estuarine/marine fish chronic toxicity study ( MRID 40914801 ), sheepshead minnows ( Cyprinodon variegatus ) were exposed under flow-through cond itions to diazinon (92.6% purity) at nominal concentrations of 0, 0.63, 1.25, 2.5, 5.0 and 10.0 μ g/L to diazinon for 108 days, followed by a 32-day depuration period. Treatments included a solvent wa ter control, and the mean measured concentrations of diazinon were 0.47, 0.98, 1.8, 3.5 and 6.5 μ g/L. Wild-caught juvenile sheephead minnows (110/treatment ) were initially e xposed in a single aquarium/treatment. Initial analyses of a random sample of fish indicated that diazinon concentrations were nondetectable prior to st udy initiation. After atta ining sexual maturity, groups of three females and two males were pl aced into separate glass spawning chambers within each aquarium. Fish were observed for sig ns of toxicity and mortality throughout the study. Selected fish from each aquarium we re sampled on exposure days 1, 4, 7, 14, 29, 43, 85 and 108, and depuration days 8, 19 and 32 for analysis of diazinon concentrations in tissues andDIAZINON FINAL

Page 61 of 63 brain AChE activity. Five spawning trials were conducted during th e study, 3 during the exposure phase and 2 during the depuration phase. During these tr ials, egg production (egg/female/day) was monitored, embryos and hatched fish were observed daily for mortality and clinical toxicity, and body le ngths of fry were determined after 28 days of exposure. In addition in the third spawning trial, effects on progeny based on prior exposure of adults was investigated by comparing (1) embryos from treated parents raised at the same concentration as the parents; (2) control embryos ra ised at the five test concentr ations; and (3) embryos from the five dose groups raised under control conditions.

No treatment-related mortality was observed in the fish at any te sted concentration, and transient signs of toxicity were only observed during ha ndling. Brain AChE activities were decreased in all treated groups with the occurrence and magnitude of the decreases being dose-dependent. At the low dose (0.47 μ g/L), AChE activities were decreased by 17 to 40% on 4 out of 8 sampling days, whereas at the high dose (6.5 μ g/L), AChE activities were d ecreased by 61 to 78% on 8 out of 8 sampling days. During the depuration phase , brain AChE activity at the high dose was comparable to the control value by Day 19.

Diazinon treatment decreased p<0.05) egg production ( ↓ 31 to 55%) in all treatment groups compared to control. In addition, during the de puration phase of the st udy (Days 23 to 31), egg production in the 3.5 μ g/L group remained lower (ca. ↓ 40%; p<0.05) compared to control, but was not significantly different from fish continuously exposed at 3.5 μ g/L, even though tissue concentrations of diazinon, and br ain AChE activities, were comparable to control. These data
indicate that the effect of diazinon on egg production can exte nd well beyond exposure and after
AChE activities have returned to normal. Although diazinon reduced egg production, there was no effect on fertility or on survival and grow th of fry at diazinon concentrations up to 6.5 μ g/L, and there were no significant eff ects on fry survival or growth based on prior exposure of the adults. Based on the effects on egg production, the LOAEC is <0.47 μ g/L and a NOAEC was not
observed.DIAZINON FINAL

Page 62 of 63 APPENDIX 3: References Not Utilized in the Diazinon 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 Diazinon but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the com pound of interest was not us ed in the study; 3) the test material was not adequately described; 4) a formulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only da ta on invertebrates.

Leong, C.T., D'Souza, U.J., Iqbal, M., and Mustap ha, Z.A. (2013) Lipid peroxidation and decline in antioxidant status as one of the toxicity measures of diazinon in the testis. Redox report :
communications in free radi cal research. 18(4):155-164.

Manabe, M., Kanda, S., Fukunaga

, K. , Tsubura, A., and Nishiyama, T. (2006). Evaluation of the estrogenic activities of some pesticides and thei r combinations using MtT/Se cell proliferation

assay

. Int J Hyg Environ Health 209 : 413–421. (MRID 48125701).

Oh, Y.J., Jung, Y.J., Kang, J.-W

., and Yoo, Y.S. (2007). Inves tigation of the estrogenic activities of pesticides fro m Pal-dang reservoir by in vitro assay. Sci. Total Environ . 388:8–15.DIAZINON FINAL

Page 63 of 63 Okamura, A., Kamijima, M., Oh tani, K., Yamanoshita, O., Naka mura, D., Ito, Y., Miyata, M., Ueyama, J., Suzuki, T., Imai, R., Takagi, K., and Nakajima, T. (2009) Broken sperm, cytoplasmic droplets and reduced sperm motility are principal markers of decreased sperm quality due to organophosphorus pesticides in rats. J Occup Health . 51(6):478-487.

Oksay, T., Naz ı ro ğ lu, M., Ergün, O., Do ğ an, S., Özatik, O., Arma ğ an, A., Özorak, A., and Çelik, O. (2013) N-acetyl cysteine attenuates diazinon exposure-induced oxidative stress in rat testis. Andrologia . 45(3):171-177.

Relyea, R.A. (2009) A cocktail of contamin ants: how mixtures of pesticides at low concentrations affect aquatic communities. Oecologia . 159(2):363-376.

Sarabia, L., Maurer, I., and Bu stos-Obregón, E. (2009) Melatonin prevents damage elicited by the organophosphorous pesticide diazinon on the mouse testis. Ecotoxicol Environ Saf

. 72(3):938-942.

Wang, D., Kamijima, M., Okamura, A., Ito, Y., Yanagiba, Y., Jia, X. F., Naito, H., Ueyama, J., and Nakajima, T. (2012). Evidence for diazinon-re lated inhibition of cis-permethrin metabolism and its effects on reproductive t oxicity in adult male mice. Reprod Toxicol . 34(4):489-497.