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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

# &

Health Effects Divison

# ::~:E£'~:::e 

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

Environmental

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

THR O UG H: Je ss Rowland

## ~ S

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

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

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

.

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

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

### EDSP: WEIGHT OF EVIDEN CE ANALYSIS OF POTENTIAL
INTERACTION WITH THE ESTROG EN, ANDROGEN OR THYROID
PATHWAYS

CHEMICAL: CHLOROTHALONIL

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYCHLOROTHALONIL FINAL

ii TABLE OF CONTENTS

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

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

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

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

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

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

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

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................8
1. Effects on Estrogen Pathway.....................................................8
2. Effects on Androgen Pathway..................................................13

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................17

IV. Committee’s Assessment of Weight of Evidence..........................................20

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................20

1. Tier 1 in vivo Assays.............................................................21

2. OSRI...............................................................................21

B. Estrogen Pathway...........................................................................21

C. Androgen Pathway.........................................................................22

D. Thyroid Pathway...........................................................................22

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

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

VI. References.......................................................................................24

LIST OF TABLES: Table 1. Tier 1 Assays for Chlorothalonil...........................................................7
Table 2. Estrogenic/Anti-Estrogeni c Pathway for Chlorothalonil ...............................9
Table 3. Androgenic/Anti-Androgenic Pathway for Chlorothalonil ...........................14
Table 4. Thyroid Pathway for Chlorothalonil ....................................................18

APPENDIX 1. EDSP Tier 1 Screening Assays .........................................................28
APPENDIX 2. Other Scientifically Relevant Information (OSRI)................................40

APPENDIX 3. References Not Utilized in the Chlorothaloni l WoE Analysis...................60CHLOROTHALONIL 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 enerationCHLOROTHALONIL 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 ConcentrationCHLOROTHALONIL FINAL

v Abbreviation Terminology

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

OPP Office of Pesticide Programs

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

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

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

T4 Thyroxine (tetraiodothyronine)CHLOROTHALONIL 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-EvidenceCHLOROTHALONIL FINAL

Page 1 of 61 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 chlorothalonil interacts with E, A, or T hormone pathways, the number and type of effects induced, the magnitude and pattern of respons es observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whethe r or not endocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On August 20 th , 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and th e Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence analysis of the potential interac tion of chlorothalonil with the E, A or T hormone pathways. The T1 WoERC 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).

There was no convincing evidence of potential inte raction with the estrogen pathway in either
the in vitro or in the in vivo Tier 1 assays. Chlorothalonil gave negative results for estrogen binding (ER) binding, ER α transcriptional activation assay ( ERTA), aromatase, and steroidogenesis. There was no increase in uterine weight in the uterotrophic assay. In the female pubertal assay, there were no estrogen-related effects observe d. In the fish short term reproduction assay (FSTRA), effects (decreased fecundity) was only in the presence of overt toxicity (decreased survival in males 63% and 88% in females). Th ere were estrogen-related effects were observed
in the mammalian Part 158 toxic ity studies in the absence of overt toxicity. Decreased egg production was seen in the Part 158 avian reproduction and fish lif e-cycle study in the absence of overt toxicity. However, these effects on this en dpoint alone is limited in diagnostic specificity for a potential interaction with the estrogen pathway. Furthermore there is a lack of redundancy across the assays.

There was no convincing evidence of potential inte raction with the androgen pathway either in
the in vitro or in the in vivo Tier 1 assays. Chlorothalonil was negative for androgen binding (AR) binding and steroidogenesis. In the Hershb erger assay, there were no changes in accessoryCHLOROTHALONIL FINAL

Page 2 of 61 sex tissue weights in the androgeni c or antiandrogenic portion of the assay. In the male pubertal assay and FSTRA, there were no androgen-rela ted effects observed. There were no androgenrelated effects in the mammalian or wildlife Part 158 studies in the absence of overt toxicity.

There was also no convincing evidence of intera ction with the thyroid pathway in mammals. While there was a lack of redundancy across the battery (i.e., no c onvincing evidence of interaction in mammals), the complementary ev idence of effects observed in the amphibian metamorphosis assay (AMA) (described below) indicate potential interact ion with the thyroid pathway. No thyroid-related effect s were seen in the male pubertal assay. In the female pubertal assay, there was an isolated finding of a 14% incr ease in T4 level at the low dose which was not complemented by any treatment-related changes in thyroid stimulating hormone (TSH) level, thyroid weight or histopathology. There were no thyroid related e ffects observed in the absence of overt toxicity in the mammalia n Part 158 toxicity st udies to suppor t a thyroid interaction in
mammals.

In the AMA study, there were complementarity of effects consistent with potential interaction with the thyroid pathway in amphibians, includin g: increased frequency a nd severity of thyroid gland hypertrophy and follicular ce ll hypertrophy and hyperplasia at the high dose. Furthermore, there was a significant decrease (25%) in 21-d ay normalized hind limb length (HLL) at the high
dose as well as an indication th at the developmental stage was de layed at the mid and high doses based on a slight, but significan t decrease in NF stage and dose-dependent decrease in the number of late stage frogs; no late stag e tadpoles were seen at the high dose.

Based on weight of evidence considerations, EDSP Tier 2 testing is not recommended for mammals or fish since there was no convincing evidence of potential interaction with the estrogen, androgen or thyroid pa thways in mammals or fish.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard
duck are available for chlorothalonil. In quail, effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from chlorothalonil exposure. Relative to the EDSP, additional testing is not recommended

As the results of the AMA suggest a potential interaction with th e thyroid pathway in amphibians at a concentration of 5.0 μg a.i. /L which is comparable to the current chronic endpoint value of 1.4 μg a.i./L for fish [which are surrogates fo r aquatic-phase amphibians; value from fathead minnow life-cycle study (MRID 00030391]an EDSP Ti er 2 Larval Amphibian Growth and Development Assay (LAGDA) is recommended.CHLOROTHALONIL FINAL

Page 3 of 61 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 chlorothalonil interacts w ith E, A, or T hormone pathways, the number and type of effects induced, the magnitude and pattern of respons es observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, whethe r or not endocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On August 20 th , 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and th e Office of Science Coordination and Policy (OSCP) conducted a WoE analysis of the potential in teraction of chlorothal onil with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) be ginning 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).

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitr ile) has a water solubility at 25°C of 0.8-0.96 mg/L. Chlorothalonil has an octa nol/water partition coefficient (log Kow) of 3.8. It has a vapor pressure of 5.72 × 10 -7 mm Hg at 25°C and a Henry’ s law constant of 2.6 × 10 -7 atm-m 3 /mol, and some degree of volatility from both soil and water (semi-volatile) could be anticipated

. S tudies
indicate chlorothalonil will tran sform primarily through aqueous photolysis in clear, shallow water, and is susceptible to microbial-mediated tr ansformation, and is slightly to hardly mobile in soil systems. Chlorothalonil is anticipated to be rapidly transformed in water/sediment systems under both aerobic and anaerobic condi tions. Field dissipation studies show that chlorothalonil dissipates ( e.g. , transformation or relocation) with half-lives less than 100 days; however, in a few field dissipa tion studies, chloroth alonil was observed in soil samples taken after one year.

The available information considered in determining the potential interaction of chlorothalonil with the E, A and/or T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxicity st udies and other published
articles. These data are summarized in Sections III.A through III.C. An analysis of the dataCHLOROTHALONIL FINAL

Page 4 of 61 submitted to the Agency, using the WoE appro ach outlined by the Agency (USEPA, 2011), is presented in Section IV. The ED SP Tier 2 studies recommendations are presented in Section V.

II. Sources of Scientific Data and Technical Information

A. EDSP T1 Screening Assays

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the agency’s test order 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 Screening assays were submitted as OSRI by the Te st Order recipients and/or the public. This included studies published in the open literatu re and/or data submitted to support pesticide registration (e.g., Part 158 guideli ne studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disr uptor Review Team on chlorothalonil (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any additional re levant information. Summarie s of the available OSRI are presented in Appendix 2. Additiona lly, literature/studies considered but not utilized for the WoE analysis are liste d 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 endoc rine-related processes (i.e., E, A, or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to 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 then characterize the main lines of evidence as well any conclu sions. Finally, the last step is to evaluate whether additional testing is needed based on the evidence and conclusions described
above.CHLOROTHALONIL FINAL

Page 5 of 61 As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, and 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

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

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

Page 6 of 61 toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.

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

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

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addressing th ese three main questions is fundamental to the WoE process and in determining whether addi tional data are needed. In addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) discussion of ke y studies; 3) description of inc onsistent or conflicting data; 4) overall strength of evidence supporting a conclusi on; and, 5) what, if a ny, additional data are needed and why. Assessing the need for additiona l data is based on a case-by-case analysis
which took all available toxi city data into account.CHLOROTHALONIL FINAL

Page 7 of 61 The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

A. EDSP T1 Screening Assays

The Tier 1 studies submitted in response to the agency’s test order for chlorothalonil are shown
below in Table 1

.

Table 1. Tier 1 Screening Assays for Chlorothalonil

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied (MRID No. 48615401)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48615402)

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied (MRID No. 48676301)

Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48669903)

Aromatase Assay (Human recombinant microsomes) OSCPP 890.1200 Requirement Satisfied (MRID No. 48615403)

Uterotrophic Assay (Rat) OSCPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48669904)

Hershberger Assay (Rat) OSCPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48669902)

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied (MRID No. 48699301)

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied (MRID No. 48699302)

Fish Short-term Reproduction Assay OSCPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48950801)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 49002801)CHLOROTHALONIL FINAL

Page 8 of 61 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential interactions of chlorothalonil with the estrogen pathway are summarized in Table
2. The various targets of the estrogen pathway acro ss the relevant Tier 1 assays are delineated so as to facilitate determination of potential for estr ogenic, anti-estrogenic, or HPG axis effects.
This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

In the Tier 1 in vitro studies, chlorothalonil was negative in the ER binding, ER TA, aromatase, and steroidogenesis assays. Furthermore in a li terature study considered as OSRI, Kojima et al. , 2004 reported negative results for ER transcriptional activation. Additionally there were no estrogenrelated effects in the in vivo uterotrophic or female pubertal assays. In the fish short-term reproduction assay (FSTRA), there was a 48% decr ease in fecundity at the highest tested concentration (9.9 μg a.i./L); however, survival wa s also decreased to 79% at this concentration.

In a Part 158 2-generation mammalian reprodu ction study, vaginal opening was delayed by 3.4 days at the high dose although body we ight at vaginal opening was co mparable to the control body weight. This finding was observed in the presence of both systemic (kidney and fore-stomach lesions were observed in parental animals and offspring of all generations) and overt (decreased parental and pup body weights) toxi city. In a developmental toxici ty study in rats, increased resorptions and post implantati on loss were observed at the hi gh dose. This finding was again observed in the presence of both sy stemic (clinical signs of toxicity ) and overt (parental mortality) toxicity. In addition, post im plantation loss and resorptions were not observed in a second developmental toxicity study in rats. No other es trogen-related effects were observed in the other Part 158 mammalian toxicity studies.

In an available Part 158 avian reproduction study on bobwhite quail, biologically relevant reductions in the number of eggs laid/hen at the high dose (624 mg/kg-di et) occurred in the absence of overt toxicity. In a set of reproduc tion studies with bobwhite quail and mallard ducks, effects on reproductive parameters in the presence of overt toxicity we re observed at 5,000 mg/kgdiet in the bobwhite and no reproductive effects were reported at concen trations of 10,000 mg/kgdiet in the mallard. There were no effects on mort ality or any reproductive parameters determined in two more avian reproductive toxicity studies (one each for mallard duck and bobwhite quail) up to and including the highest treatment concentrati on of 50 mg/kg-diet). In addition, reductions in egg production were also observed in a fathead minnow life-cycle toxicity study at 3.0 μg/L in the absence of overt toxicity. Chlorothalonil signif icantly reduced hatching success and survivability
of fathead minnows at concen trations at 6.5 μg/L (F1 reduced hatching success).CHLOROTHALONILFINALPage 9 of 61Table 2: Estrogenic/Anti-Estrogenic Pathway for ChlorothalonilLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-EstrogenicPathway for Chlorothalonil1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48615401)NERTA(MRID 48615402)NAromatase(MRID 48615403)NSteroidogenesis(MRID 48669903)NUterotrophic(MRID 48669904)NN NFemale Pubertal Rat(MRID 48699301)NNNNNNNX(H)4FSTRA(MRID 48950801)NENNNNNX(H)5OSRIER Binding (Kojima,etal., 2004)NTwo-generationreproduction (Rat; MRID41706201)NRNRNNNENX(L, M, H)X(M, H)Two-generationreproduction (Rat; MRID45710209)NRNNNN NNX(L, M, H)X(H)CHLOROTHALONILFINALPage 10 of 61Table 2: Estrogenic/Anti-Estrogenic Pathway for ChlorothalonilLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-EstrogenicPathway for Chlorothalonil1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Developmental toxicity(Rat; MRID 00130733)NRNX(H)X(H)Developmental toxicity(Rat; MRID 45710207)NRNX(H)X(H)Developmental toxicity(Rabbit; MRID 00047944)NRNNX(M, H)Developmental toxicity(Rabbit; MRID 45710208)NRNNNChronic toxicity/carcinogenicity (Rat;MRID 00146945)NRNNNRX(L, M, H)X(M, H)Chronic toxicity/carcinogenicity (Rat;MRID 45710212)NENNNX(L, ML, MH,H)NCarcinogenicity (Mouse;MRID 00127858)NENNNEX(L, M, H)NCarcinogenicity (Mouse;MRID 45710211)NNNNEX(ML, MH, H)NChronic toxicity (Dog;MRID 43653603)NE NE N NEX(H)X(M,H)Chronic toxicity (Dog;MRID 45710210)N N N NX(H)X(H)Subchronic toxicity (Rat;MRID 45710205)NNNNX(ML, MH, H)NSubchronic toxicity (Dog;MRID 45710206)NNNNX(M, H)X(H)CHLOROTHALONILFINALPage 11 of 61Table 2: Estrogenic/Anti-Estrogenic Pathway for ChlorothalonilLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-EstrogenicPathway for Chlorothalonil1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Avian reproduction(Quail;MRID 00044140NN NAvian reproduction(Quail; MRID 45710218)P6(H)N NAvian reproduction(Quail;MRID 40964104)NNX(M,H)Avian reproduction(Duck; MRID 00041441;40964102)NN NFish Life Cycle Toxicity(Fathead Minnow, MRID00030391)P7(M)NX(H)1.Key to responses: L=Low treatment, ML=Medium-low treatment,M=Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in thisassay. Changes in weight are absolute unless otherwise indicated.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 byan X in this column. For details see Section IV. A3.The overt toxicity in the Tier 1 assays are presented inthis column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column.For details see Section IV. A4. Decreased final body weight of 11% at the high dose.5. Survival was decreased to 88% for females atthe highest concentration (9.9 μg a.i./L).6. Reduced egg production.6. Reduction in egg production. F1 hatching success also reduced at mid-high 6.5 μg/L.P Positive findingsN Negative findings (invitro)/No effect (invivo)CHLOROTHALONILFINALPage 12 of 61NE Not examinedNR Not reportedCHLOROTHALONIL FINAL

Page 13 of 61 2. Effects on Androgen Pathway

The potential interactions of chlorothalonil with the androgen pathway are summarized in Table 3. The various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for androgenic, anti-androgenic, or HPG axis effects. This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reporte d in the table and not considered further in the WOE assessment.

Chlorothalonil was classified as negative in the in vitro AR binding and steroidogenesis assays. Furthermore, in a literature study considered as OSRI, Kojima et al. (2004) reported negative results for an AR transcriptional activation.

Additionally, the mammalian in vivo assays (Hershberger and Male P ubertal) were also negative for all of the androgen-sensitive parameters evalua ted. In addition, no effects on androgen-related endpoints were noted in the FSTRA.

In the mammalian Part 158 data, the only finding related to the androgen pathway was a delay in preputial separation (PPS) of 3.2 days at the high dose in a 2-generation reproduction study in rats. The body weight at PPS was comparable to the control body weight. The finding of delayed PPS was observed in the presence of both systemic (kidne y and fore-stomach lesions in parental animals) and overt (decreased body weight in F1 males and F1/2 pups) t oxicity. There were no androgen
related effects in the wildlife Part 158 data.CHLOROTHALONILFINALPage 14 of 61Table 3: Androgenic/Anti-Androgenic Pathway for ChlorothalonilLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Chlorothalonil1Study Type/Literature Citation AR Binding/ARTA Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48676301)NSteroidogenesis(MRID 48669903)NHershberger(MRID 48669902)NNN NMale Pubertal Rat(MRID 48699302)NNNNNNNNNNFSTRA(MRID 48950801)NENNNNNX(H)4OSRIAR Binding (Kojima,2004)NTwo-Generationreproduction (Rat;MRID 41706201)NRNENNENNENEX(L, M, H)X(H)Two-Generationreproduction (Rat;MRID 45710209)NENNNNNNN NX (L, M, H)X(H)Chronic toxicity /Carcinogenicity (Rat;MRID 00146945)NNNRNNRNRX(L, M, H)X(M,H)Chronic toxicity /Carcinogenicity (Rat;MRID 45710212)NNNENRNNEX(ML, MH,H)NCHLOROTHALONILFINALPage 15 of 61Table 3: Androgenic/Anti-Androgenic Pathway for ChlorothalonilLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Chlorothalonil1Study Type/Literature Citation AR Binding/ARTA Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Carcinogenicity(Mouse; MRID00127858)NNNENENENEX(L,M,H)X(H)Carcinogenicity(Mouse; MRID45710211)NNNENNENEX(L,ML,MH, H)NChronic toxicity (DogMRID 43653603)NNNNNENEX(H)X(H)Chronic toxicity (DogMRID 45710210)NNNNNNX(H)X(H)Subchronic toxicity(Rat; MRID45710205)NNNENNNX(L,ML,MH,H)NSubchronic toxicity(Dog; MRID45710206)NNNENNNX(M,H)X(H)Avian reproduction(Quail;MRID 00044140NNNAvian reproduction(Quail;MRID 00044140NNX(M,H)Avian reproduction(Duck; MRID00041441)NNNAvian reproduction(Quail; MRID45710218)NN NCHLOROTHALONILFINALPage 16 of 61Table 3: Androgenic/Anti-Androgenic Pathway for ChlorothalonilLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Chlorothalonil1Study Type/Literature Citation AR Binding/ARTA Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Fish Life CycleToxicity (FatheadMinnow, MRID00030391)NNX(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 response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight areabsolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate(PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP).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 inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For detailssee Section IV. A4. Survival of males was decreased to 63% at highest concentration (9.9 μg a.i./L).P Positive findingsN Negative findings (invitro)/No effect (invivo)NE Not examinedNR Not reportedCHLOROTHALONIL FINAL

Page 17 of 61 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

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

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

There were no effects of chloroth alonil noted on serum TSH levels, p ituitary or thyroid weights, or thyroid histopathology in either the male pubertal or female pube rtal assays. Although treatmentrelated increases in serum T 4 levels were noted in the female pubertal assay, these changes were not supported by changes in TSH levels or in th yroid weight and histology. In the AMA, no asynchronous development was observed, but medi an NF developmental stage was delayed slightly at the high concentration on Day 7 (53.5 vs . 54), and at the mid and high concentrations on Day 21 (59 and 58.5 vs. 59.5 in control). Additi onally, there was an observed dose dependent decrease in the number of late stage frogs with increasing chlorothalonil concentration (control (15.5%), low (15%), mid (8.8%), and high (0%) treatment groups). In addition, thyroid gland hypertrophy and follicular cell hypertrophy and hyperplasia were observed at the highest concentration (5 μg/L), along w ith a decrease in normalized HLL on Day 21. Decreases in body weights ( ↓ 42, 23%) and reductions in SVL ( ↓ 18, 7%) were also observed at the high concentration on Days 7 and 21.

In the Part 158 chronic rat study, the transient increase in adjusted thyroid weight (19%) seen in females at the high dose was determined to be bi ologically not significant si nce it was seen only at week 52 (not week 104) and was not seen in male s at any dose or time period. Also, the increase was seen in the presence of st omach irritation and inflammation a nd nephrotoxicity. In the chronic dog study, increased thyroid weight was observed at the highest dose tested but this was in the presence of both systemic (macroscopic and microscopic effects on the stomach and hypertrophy
of cells in the zona fasciculata of the adrenal glands) and overt (decreased body weight in males) toxicity.CHLOROTHALONIL FINAL

Page 18 of 61 Table 4. Thyroid Pathway for Chlorothalonil.

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

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage (±) orasynchronous or HLLGrowth(BW, SVL)Systemic Toxicity2Overt Toxicity Observed3

Male Pubertal Rat (MRID 48699302) N N N N N N N

Female Pubertal Ra t (MRID 48699301) N N ↑ 14% (L) N N N X 4 (H)

AMA (MRID 49002801) P 5 (H) P 6 (M, H) Days 7 and 21:
BW: ↓ 42, 23%; SVL: ↓ 18, 7% (H) N

OSRI

Two-Generation reproduction (Rat; MRID 45710209) NE N N X (L, M, H) X (H)

Chronic toxicity/ Carcinogenicity (Rat; MRID 00146945) NR N NR X (L, M, H) X (M,H)

Chronic toxicity/ Carcinogenicity (Rat; MRID 45710212) N N N X (L, ML, MH, H) N

Carcinogenicity (Mouse; MRID 00127858) NE N NE X (L, M, H) X (H)

Carcinogenicity (Mouse; MRID 45710211) NE N NE X (L, ML, MH, H) N

Chronic toxicity (Dog, MRID 43653603) N N N X (H) X (M,H)

Chronic toxicity (Dog, MRID 45710210) N N N X (H) X (H)

Subchronic toxicity (Rat; MRID 45710205) N N N X (L, ML, MH, H) N

Subchronic toxicity (Dog; MRID 45710206) N N N X (M, H) X (H)CHLOROTHALONIL FINAL

Page 19 of 61 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 applicable in this assay.
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 assa ys are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A
4. Decreased final body weight of 11% at the high dose. 5. Thyroid gland hypertrophy, and follicular cell hypertro phy and hyperplasia were noted at the high concentration. 6. Normalized HLL decreased by 25% on Day 21 at the high concentration; NF developmental stage was delayed slightly on Day 7 in the high concentration, and at the mid and high concentrations on Day 2. Also there was an observed dose dependent decrease in the number of late st age frogs with increasing chlo rothalonil concentration. No asynchronous development. P Positive findings N Negative findings NE Not examined NR Not reportedCHLOROTHALONIL FINAL

Page 20 of 61 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of chlorothalonil to interact with endoc rine related processes ( i.e., E, A, or T hormonal pathways) as well as recommendations regarding Tier 2 testing. The re sults of the Tier 1 assays are considered, along with other scien tifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available peer-r eviewed studies). WoE analysis in the context of the EDSP follows the Agency’s guidance (USEPA 2011) and is conducted on a case-by-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 cons istency, coherence, and interrelationships among the measured endpoints within and across studies. Th e available findings from standard toxicology studies on the substance may contribut e to the WoE evaluation in helpi ng elucidate if effects seen in the Tier 1 assay are related to pe rturbations of the endocrine system per se or alternatively a sequelae of systemic effects. Endocrine m odes of action may elicit a number of phenotypic consequences other than those ev aluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt to xicity may result in uncertainty as to whether or not the responses are mediat ed through an endocrine pathwa y, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also considered in
this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of
evidence evaluation of the results of the Tire 1 assays stated that “In summary, the Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in the presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. second ary effects related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. Generally, one parameter (i .e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concen tration to be considered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were ge nerally less severe forms of toxicity (e.g., changes in orga n weights or blood chemistry).CHLOROTHALONIL FINAL

Page 21 of 61 1. Tier 1 in vivo Assays

No signs of overt or systemic toxicity were noted in the Hershberger Uterotrophic, or male pubertal assays at dose levels up to 1000 mg/kg/day. Overt toxicity was observed at the highest dose tested (175 mg/kg) in the female pubertal as say. In the FSTRA, survival was reduced to 79% at the highest test concentr ation (males 63%, females 88%).

2. OSRI

Systemic and/or overt toxicity observed in nearly all the mammalian toxicity studies were changes in body weight, and histopathological changes in the gastro-intestinal tract a nd kidneys. In the two 2-generation reproduction studies, kidney and fore stomach lesions were observed at all dose levels. In the development toxicity studies, syst emic toxicity was eviden t based on reductions in body weight or body weight gains with increases in mortality at the highest doses. Nephrotoxicity and gastro-intestinal irritation and inflammation were also seen in the two chronic rat studies; these were observed both macro- and microscopicall y. In two mouse carci nogenicity studies, histological changes were noted in the kidneys and stomach. In two chronic dog studies, macroand microscopic lesions were reported in the stom ach with concomitant re duced body weight gain and mortality at the high dose. The subchronic rat studies demonstrated reductions in body weight, stomach lesions and effects on kidneys. In the dog subchronic study, reduc tion in body weight and body weight gain were seen, along with effects on the liver.

No systemic or overt toxicity was seen in the qua il avian reproductive stud y at fed levels up to 642 ppm. In the minnow life-cycle st udy, overt toxicity wa s noted at the highest test concentration (16.0 μg/L), based on reductions in survival.

B. Estrogen Pathway

There was no convincing evidence of potential intera ction with the estrogen pathway in either the

in vitro or in the in vivo Tier 1 assays. Chlorothalonil was negative for ER binding, ERTA, and steroidogenesis. There was no increase in uterine we ight in the uterotrophic assay. In the female pubertal assay, there were no estrogen-related effects observed. In the FSTRA, there was a significant decrease (48%) in fecundity in the pres ence of overt toxicity (decreased survival in males 63% and 88% in females); no other effects were noted. There were estrogen-related effects were observed in the mammalian Part 158 toxicity studies in the absence of overt toxicity. Decreased egg production was seen in the Part 158 avian reproduction and fish life-cycle study in the absence of overt toxicity. Howe ver, these effects on this endpoint alone is limited in diagnostic specificity for a potential interaction with the es trogen pathway. Furthermore there is a lack of redundancy across the assays.CHLOROTHALONIL FINAL

Page 22 of 61 C. Androgen Pathway

There was no convincing evidence of potential intera ction with the androgen pathway in either the

in vitro or in the in vivo Tier 1 assays. Chlorothalonil was negative for AR binding and steroidogenesis. In the Hershberger assay, there were no changes in accessory sex tissue weights in the androgenic or antiandrogeni c portion of the assay. In the male pubertal assay and FSTRA, there were no androgen-related effects observed. There were no androgen-re lated effects in the
mammalian or wildlife Part 158 studies in the absence of overt toxicity.

D. Thyroid Pathway

There was also no convincing evidence of interact ion with the thyroid path way in mammals. While there was a lack of redundancy acr oss the battery (i.e., no convinci ng evidence of interaction in mammals), the complementary evidence of eff ects observed in the amphibian metamorphosis assay (AMA) (described below) indicate potenti al interaction with th e thyroid pathway. No thyroid-related effects were seen in the male pubertal assay. In the female pubertal assay, there was an isolated finding of a 14% increase in T4 leve l at the low dose which was not complemented by any treatment-related changes in thyroid stimul ating hormone (TSH) level, thyroid weight or histopathology. There were no thyroid related effect s observed in the absence of overt toxicity in the mammalian Part 158 toxicity studies to support a thyroid interaction in mammals. In the AMA study, there were complementarity of effects consiste nt with potential intera ction with the thyroid pathway in amphibians, including: increased freq uency and severity of thyroid gland hypertrophy and follicular cell hypertrophy and hyperplasia at the high dose. Furthermore, there was a significant decrease (25%) in 21 -day normalized hind limb length (H LL) at the high dose as well as an indication that the developmental stage was delayed at the mid and high doses based on a slight, but significant decrease in NF stage and dose-dependent decrease in the number of late stage frogs; no late stage tadpoles were seen at the high dose.

E. Conclusions

There was no convincing evidence of potential inte raction with the estrogen pathway or androgen pathway in mammals and wildlife. There was also no convincing evidence of interaction with the thyroid pathway in mammals. While there was a lack of redundancy acros s the battery (i.e., no convincing evidence of interaction in mammals), the complementary evidence of effects observed in the AMA indicate potential interaction with the thyroid pathway.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations, EDSP Tier 2 testing is not recommended for mammals or fish since there was no convincing evid ence of potential interac tion with the estrogen, androgen or thyroid pathwa ys in mammals or fish.CHLOROTHALONIL FINAL

Page 23 of 61 For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard duck are available for chlorothalonil. In quail, eff ects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from chlorothalonil exposure. Relative to the EDSP, additional testing is not recommended

As the results of the AMA suggest a potential interaction with th e thyroid pathway in amphibians at a concentration of 5.0 μg a.i./L which is comparable to the cu rrent chronic endpoint value of 1.4 μg a.i./L for fish [which are surrogates for a quatic-phase amphibians; value from fathead minnow life-cycle study (MRID 00030391]an EDSP Tier 2 La rval Amphibian Growth and Development Assay (LAGDA) is recommended.CHLOROTHALONIL FINAL

Page 24 of 61 VI. References

Cary, George A., 1980. A Chronic Study on the Fa thead Minnow (Pimephales promelas) with Technical Chlorothalonil. Unpublished report prepared by EG & G, Bionomics for Diamond Shamrock Corporation. Received 2/19/80. MRID 00030391. Unpublished.

Diamond Shamrock Corp. (1983) A Chronic Dietary Study in Mice wi th Technical Chlorothalonil: Document No. 108-5TX-79-0102-004. (submitted by Diam ond Shamrock Agricultural Chemicals, Cleveland, OH; CDL:071541- E; 071542; 071543; 071544; 071545; 071546), April 21, 1983. MRID 00127858. Unpublished.

Diamond Shamrock Corp. (1983) A Teratology St udy in Rats with Technical Chlorothalonil: Document No. 517- STX-82-0011-003. (submitte d by Diamond Shamrock Agricultural Chemicals, Cleveland, OH; CDL:251069-A), July 7, 1983. MRID 00130733. Unpublished.

Huntingdon Research Centre, Inc. (1993) Chlorothalonil: A study of the effect on pregnancy of the rat. Huntingdon Research Centre, Inc. Laboratory Study No.: VCM 22/930637. MRID 45710207. Unpublished.

Huntingdon Research Centre, Inc. (1994) Chlorothal onil: A study of the effect on pregnancy of the rabbit Huntingdon Research Centre, Inc. Laboratory Study No.: VCM 22/930638. MRID 45710208. Unpublished.

Huntingdon Research Centre, Inc. (1995) Chloro thalonil: A study of the effect on reproductive function of two generations in the rat. Huntingdon Research Centre, Inc. Laboratory Study No. VCM 211942505. MRID 45710209. Unpublished.

Huntingdon Research Centre, Inc. (1995) Chloro thalonil: Potential tumorigenic effects in prolonged dietary administration in mice. Huntingdon Research Centre, Inc. Laboratory Study No.: VCM 16/943065. MRID 45710211. Unpublished.

Huntingdon Research Centre, Inc. (1995) Chlorothalonil: Toxicity to dogs by repeated dietary administration for 52 weeks. Huntingdon Resear ch Centre, Inc. Laboratory Study No.: VCM 14/943124. MRID 45710210. Unpublished.

Huntingdon Research Centre, Inc. (1996) Chloro thalonil: Potential tumorigenic effects in prolonged dietary administration in rats. Huntingdon Research Cent re, Inc. Laboratory Study No.: VCM 15/943286. MRID 45710212. Unpublished.

International Research and Development Corpor ation. (1976) Teratology Study in Rabbits: IRDC No. 293-032a. (submitted by Diamond Shamrock Ag ricultural Chemicals, Cleveland, OH; CDL:095783-K). MRID 00047944. Unpublished.CHLOROTHALONIL FINAL

Page 25 of 61 Kojima, H., E. Katsura, S. Takeuchi, K. Niiy ama, and K. Kobayashi. (2004) Screening for estrogen and androgen receptor act ivities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. Environmental Health Perspectives 112:524-531.

Lee, M.R. (2012) Chlorothalonil- Amphibian Metamorphosis Assay with African Clawed Frog ( Xenopus laevis ). Smithers Viscient, Wareham, MA . Laboratory report number: 1781.6774, November 29, 2012. MRID 49002801. Unpublished.

Lucas, F.; Benz, G. (1990) A Two Generation Reproduction Study in Rats with Technical Chlorothalonil. Ricerca, Inc., and Experiment al Pathology Labs, Inc. Lab Project Number: 87- 0121: 1722-87-0121-TX-003. MRID 41706201. Unpublished.

Mizns, M., Laveglia, J (1994) A chronic (12 mont h) oral toxicity study in dogs with technical chlorothalonil. Pharmaco LSR Inc. and Experime ntal Pathology Labs, Inc. Lab Project Number: 92-135: 92-0457: 5211-92-0457-TX-003. MRID 43653603. Unpublished.

Redgrave, V. (1993) Chlorothalonil: Bobwhite Quail Dietary Reproduction and Tolerance Studies: Lab Project Number: VCM 11/930496. Unpublishe d study prepared by Huntingdon Research Centre Ltd. 314 p. MRID 45710218.

Sawhney Coder, P. (2012) Chlorothalonil – Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Female Rats. WIL Resear ch Laboratories, LLC, Ashland, OH. Laboratory Study No.: WIL-639143, March 21, 2012. MRID 48699301. Unpublished.

Sawhney Coder, P. (2012) Chlorothalonil – Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Male Rats. WIL Resear ch Laboratories, LLC, Ashland, OH. Laboratory Study No.: WIL-639142, March 21, 2012. MRID 48699302. Unpublished.

Sawhney Coder, P. (2012) Chlorothalonil – He rshberger Bioassay in Orchidoepidiymectomized Rats Final Report. WIL Research Laboratories, LLC, Ashland, Ohio. Laboratory Project ID: WIL639141, January 26, 2012. MRID 48669902. Unpublished.

Sawhney Coder, P. (2012) Chlorothalonil – Uter otrophic Assay in Ovariectomized Rats Final Report. WIL Research Laboratories, LLC, As hland, Ohio. Laborator y Project ID: WIL-639140, January 26, 2012. MRID 48669904. Unpublished.

SDS Biotech Corp. (1985) A Tumorigenicity Study of Technical Chlorothalonil in Rats:. SDS Biotech Corp. Document No. 099-STX- 80-0234-008. MRID 00146945. Unpublished.

Spencer-Briggs, D. (1994) Chlorothalonil: Toxicity to rats by dietary administration for 13 weeks. Huntington Research Centre Ltd. La b Project Number: VCM/9/90339. MRID 45710205. Unpublished.CHLOROTHALONIL FINAL

Page 26 of 61 Spencer-Briggs, D. (1994) Chlorothalonil: Toxicity to dogs by dietary administration for 13 weeks. Huntington Research Centre Ltd. La b Project Number: VCM/12/92413. MRID 45710206. Unpublished.

USEPA (2010) CHLOROTHALONIL - Report of the Endoc rine Disruptor Review Team - Test Order #: EDSP-081901-113 to EDSP-081901-118. December 8, 2010. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, D.C.

USEPA. (2011) Endocrine Disruptor Screeni ng Program – Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. September 14, 2011. Office of Chemical Safety and Pollution Preven tion. U.S. Environmental Protection Agency, Washington, DC.

USEPA (2013) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting held July 30 - August 1, 2013 on Weight-of-Ev idence: Evaluating Resu lts of EDSP Tier 1 Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

Wagner, H. (2012) Chlorothalonil - H295R Ster oidogenesis Assay. CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: R9047V-100331STER, Fe bruary 1, 2012. MRID 48669903. Unpublished.

Wilga, P.C. (2011) Chlorothalonil - Human Recombinant Aromatase Assay. CeeTox, Inc., Kalamazoo, MI. Study No. :9047V- 100331AROM, November 30, 2011. MRID 48615403. Unpublished.

Willoughby, J.A., (2011). Chlorothalonil - Estrogen r eceptor binding (rat uterine cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9047V-100331ERB, November 30, 2011. MRID 48615401. Unpublished.

Willoughby, J.A. (2011) Chlorothalonil - Estrogen r eceptor transcriptional activation (Human Cell Line (HeLa-9903)) final report. CeeTox, Inc., Kalamazoo, MI. Laborat ory Report No.: 9047V100331ERTA, November, 30 2011. MRID 48615402. Unpublished.

Willoughby, J.A. (2012) Chlorothalonil: Androgen Receptor Binding (Rat Prostate Cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Project ID No.: 9047V-100331ARB, February 2, 2012. MRID 48676301. Unpublished.

Willoughby, J.A. (2012) Supplemental Information - Laboratory Proficiency Data for ERTA assays and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. CeeTox, Inc., Kalamazoo, MI. July 10, 2011. MRID 48843501. Unpublished.

York, D.O. (2012) Chlorothalonil – Fish Shor t-Term Reproduction Assa y with Fathead Minnow ( Pimephales promelas ). Smithers Viscient, Wareham, MA. Laboratory report number: 1781.6775.CHLOROTHALONIL FINAL

Page 27 of 61 Study submitted by Syngenta Crop Protection, LLC, Greensboro, NC, October 1, 2012. MRID 48950801. Unpublished.CHLOROTHALONIL FINAL

Page 28 of 61 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100)

The 21-day assay (MRID 49002801) of chloro thalonil (99.0% purity) on amphibian metamorphosis of African clawed frog (X enopus laevis) was conducted under flow-through conditions. Amphibian larvae at developmental Nieuwkoop-Faber Stage (NF) 51 (80 larvae per group) were exposed to nominal concentra tions of 0 (negative control), 0.000090, 0.00060, and 0.0040 mg a.i./L. The reviewer-calculated time-we ighted average (TWA) concentrations were <0.000027 (<LOQ; control), 0.000093, 0.00061, 0.0050 mg a.i. /L. The test system was maintained at 22 to 24°C and a pH of 7.4 to 8.1.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. Mortality was observed in the control (2.5%), mid (3.75%), and high (2.5%) treatment groups; no deaths occurred in the low-dose treatment group. Spinal deformities were
observed in the control and a ll treatment groups; 18%, 19%, 25%, and 3% in the 0 (negative control), low, mid, and high treatment groups, re spectively, and were not considered treatmentrelated.

There was a slight but significant delay of median NF developmen tal stage after 7 days (53.5 vs. 54 in control) in the high treatment group, and on Day 21, again, slight but significant (p<0.05) delays were noted in the mid and high treatment groups (58.5 and 59 in mid and high-treatment vs. 59.5 in control). Additionally, it is noted that la te stage tadpoles (NF>60 ) were observed in the control (15.5%), low (15%), and mid (8.8%) treatment groups, but the high treatment group did not contain any late stage tadpol es, further suggesting a development delay. Late stage tadpoles (NF>60) were excluded from statis tical analysis of the growth pa rameters (HLL, normalized HLL, wet weight, and SVL). No asynchronous developm ent was observed. Chlorothalonil had no effect on Day 7 normalized hind-limb length (HLL) at any tr eatment level relative to the negative control (p>0.05), whereas Day 21 HLL was significantly (p >0.05) reduced by 24.5% in the high treatment group. Significant decreases in body weights ( ↓ 42, 23%) and reductions in snout-vent length (SVL) ( ↓ 18, 7%) were also observed at the high con centration on Days 7 and 21. Thyroid gland histopathology observations incl uded increased frequency and severity of thyroid gland hypertrophy, and/or follicular cell hypertrophy and hype rplasia in the control and treatment groups. Treatment-related thyroid gland histopathology effects were observed at the high treatment group (thyroid gland hypertrophy and follicular cell hypertrophy and hyperplasia).

Androgen Receptor (AR) Bi nding Assay (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID # 48676301) ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to examine the ability of chlorothalonil toCHLOROTHALONIL FINAL

Page 29 of 61 bind to the AR receptor. Competitive binding e xperiments were performed with chlorothalonil (99.0% a.i., Batch# 617642). The competitive binding assay was conducted to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (logarithmic increase from 10

− 11 to 10

− 3 M) of chlorothalonil. DMSO was used as the solvent vehicle at a final assay concentrat ion of approximately 3.2%. A total of three runs were performed, and each run included dexameth asone as a weak positiv e control, and R1881 as the ligand reference standard.

A saturation binding experiment was conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and wa s functioning with appropriate affinity for the radiolabeled reference androgen (R1881) prior to routinely conducting AR competitive binding experiments. Saturation binding data were not or iginally 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 preparations, evaluated for binding activity at different dates, were used for each of the three competitive binding runs, so data from the three saturation binding experi ments were compiled. For the first cytosolic preparation evaluated on March 30, 2 011, 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 cytoso l that was prepared. For th e second cytosolic preparation evaluated 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 si ngle batch of prostate cytosol that was prepared. For the third cyto solic preparation evaluated 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 for the three experiments were generally below the range reported in the EPA validation program (0.685 to 1.57 nM), alth ough two individual values were within the range and one value was above the range. C onfidence 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.

In the competitive binding experiment, precipitati on of chlorothalonil was visually observed at the highest tested concentration (10

− 3 M) in the first run. Theref ore, the highest concentration
used for data evaluation in Run 1 was 10

− 4 M, and the concentration range in Runs 2 and 3 was

10

− 11 to 10

− 4 M. Specific binding was >75% ( i.e . <25% displacement of radioligand) at soluble
concentrations ≤ 10

− 4 M in all three runs. An IC 50 and relative binding affinity (RBA) could not
be calculated for chlorothalonil.

The estimated average log IC 50 was

− 9.3 M for R1881 and

− 4.4 M for the weak positive control (dexamethasone). Compared to R1881, the mean relative binding affinity (RBA) for dexamethasone was 0.0022%. Confidence in these numbers is high for dexamethasone due toCHLOROTHALONIL FINAL

Page 30 of 61 the generally small variation, but there was greater than a 1-log difference for the IC 50 of R1881 in the second run compared to Runs 1 and 3. Howe ver, this variance did not appear to affect the study results as all perf ormance criteria were met in the three runs.

Based on the results from the three runs, chloro thalonil is classified as a Non-Binder in the Androgen Receptor Binding Assay.

Aromatase Assay (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48615403) , chlorothalonil (99% a.i., Lot # 617642) in dimethyl sulfoxide (DMSO) at logarithmic concentrations from 10 –10 to 10

− 3 M was
incubated with human recombinant aromat ase and tritiated androstenedione ([1 β

3 H(N)]-androst4-ene-3,17-dione; [ 3 H]-ASDN) for 15 minutes at 37°C to asse ss the potential of chlorothalonil 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 (L SC). Three independent runs were conducted,
and each run included a full activ ity control, a background 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 ) with 3 repetitions per concentration.

Aromatase activity in the fu ll activity controls ranged from 0.166 to 0.293 nmol·mgprotein

− 1 ·min

− 1 for the three test runs, with a m ean and standard deviation of 0.218 ± 0.050 nmol·mg-protein

− 1 ·min

− 1 . Activity in the background controls ranged from 0.47% to 0.80% and averaged 0.67% of the full activ ity controls. The response of the full activity controls and background controls was acceptable for each run.

Results for the positive control were generally within the recommended ra nges for the top of the curve, bottom curve, Hill slope, log IC 50 , and coefficient of varia tion for replicates of each concentration within runs. Also, the coefficien ts of variations (%CVs) for replicates of each
concentration of chlorothalonil within a run were generally within the 15% guideline. For 4-OH ASDN, the estimated log IC 50 averaged -7.17, and the slope was -0.94.

For chlorothalonil, arom atase activity averaged 0.205 ± 0.052 nmol·mg-protein

− 1 ·min

− 1 at the
lowest tested concentration of 10 -10 M and 0.194 ± 0.045 nmol·mg-protein -1 ·min

− 1 at the highest successfully tested concentration of 10

− 4 M (highest non-precipitating dose in all 3 runs). Precipitation was observed in Run 1 at 10

− 3 M and in Run 2 at 10 -3.5 M. The data for
chlorothalonil were not suitable for modeling. The average lowest portion of the response curve
across runs was >75% activity (88.75% at 10 –4 M chlorothalonil).CHLOROTHALONIL FINAL

Page 31 of 61 Based on the data from the average response curve, chlorothalonil is classifi ed as a Non-inhibitor of aromatase activity in this assay.

Estrogen Receptor (ER) Bi nding Assay (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID# 48615401), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiments. A competitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-17 β

estradiol (1 nM) in the presence of increasi ng concentrations of chlorothalonil (logarithmic
increase from 10

− 10 to 10

− 3 M or 10

− 11 to 10

− 4 M). DMSO was used as the vehicle at a final concentration of 2%. The assay included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17 β -estradiol as the na tural ligand reference
material.

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID# 48843501) from the performing laboratory were submitted following a request by the Agency. Data from two separate saturation binding assays were reported due to the use of two separate cyto sol preparations during this ER assay for
chlorothalonil. For the firs t batch of cytosol evaluate d on the September 27, 2010, the K d for [ 3 H]-17 β -estradiol was 0.081±0.006 nM, and the estimated B max was 52.70±1.05 fmol/100 μg protein. The second batch evaluated on July 10, 2011 resulted in a K d for [ 3 H]-17 β -estradiol of 0.303±0.090 nM, and the estimated B max was 33.59±0.70 fmol/100 μg protein. The K d for each run was within the expected range of 0.03 to 1.5 nM . Confidence in these numbers is high due to the goodness of fit and the small variation among runs.

The reference compounds performed as expected in the assay. Octyltriet hoxysilane had no effect on binding, and 17 β -estradiol and 19-norethindrone i nhibited binding at the expected concentrations. The mean log IC 50 was

− 5.6 M for 19-norethindrone and

− 9.2 M for

17 β -estradiol. The relative binding affinity (RBA) was 0.026% for 19-norethindrone when compared to the natural ligand.

In the competitive binding experiment, chloro thalonil had no effect on specific binding at
concentrations of ≤ 10 -4 M in Runs 1 and 2, and ≤ 10 -5 in Run 3. Precip itation occurred at 10

− 3 M in Runs 1 and 2, and at 10

− 4 M in Run 3; therefore, data from these chlorothalonil concentrations
were not used. Chlorothalonil displaced <25% of the radiolabeled estradiol at the highest concentrations successfully tested (10

− 4 or 10

− 5 M) in the three independent runs.

Based on the results from the three runs, chlorothalonil is classified as Not Interactive in the Estrogen Receptor Binding Assay.CHLOROTHALONIL FINAL

Page 32 of 61 ER α Transcriptional Activatio n (ERTA) Assay (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48615402), hER α -HeLa-9903
cells cultured in vitro were exposed to chlorothalon il (99.0% a.i., Batch # 617642) at logarithmically increasing concentrations from 10

− 11 to 10

− 4 M in cell culture media containing 0.1% DMSO for 24 hours in two independent test s. The experiments were performed using 96- well plates and each chlorothalonil concentration wa s tested in 6 replicates/plate. Cells were exposed to the test agent for 24±2 hours to induce reporter (luciferase) gene products. Luciferase expression in response to activation of the es trogen receptor by chlorothalonil was measured using a proprietary lu ciferase assay.

Chlorothalonil was tested up to the limit of solubility (10 –4 M); however, chlorothalonil was cytotoxic at concentrations ≥ 10 –6 M in the first run and at ≥ 10 –5 M in the second run. Cytotoxicity was also observ ed in the first run for 17 β -estradiol (10 –13

, 10 –8 and 10 –9 M),

17 α -estradiol (10 –6 M) and corticosterone (10 –4 M) and in the second run for corticosterone (10 -4 M). The study also included supplemental data evaluating the response of chlorothalonil at each concentration (2 replicates/concen tration) in the presence of the hER α antagonist ICI 182,780 (4-hydroxytamoxifen, 1 μM) to identify non-sp ecific luciferase induction. Performance criteria for reference chemicals were generally no t met, especially in the first run. However in most cases, values were lower than validated ra nges and suggested increa sed sensitivity of the assay to weak ligands. In the abse nce of cytotoxicity, the mean RPC Max was 3.0% for the first run and 7.6% for the second run, and the associated PC Max was 10 –7 M for the first run and 10 –5.5 M for the second run. In run 2, a higher RPC Max was achieved (12.5% at 10 -5 M) in the presence of cytotoxicity. However, both RPC Max were within the variability range for each assay.

Because the RPC Max < PC 10 in at least two assay runs, chloro thalonil was considered negative for estrogen receptor transcriptional activation in this test system. The data seems to indicate that
chlorothalonil is not an ER α agonist under the conditions of this study; however, confidence in the results is lessened due to the excess cytoto xicity observed with the reference chemicals.

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350)

The 21-day short-term reproduction assay (M RID 48950801) of chlorothalonil with fathead minnow (Pimephales promelas) was conducted u nder flow-through conditions. Groups of adult (ca. 6 months old) fish (16 spawning groups of 2 males and 4 females per replicate tank with 4 replicates per test concentra tion) were exposed to chloroth alonil (99.0% purity) at nominal concentrations of 0 (neg ative control), 0.000070, 0.00070, and 0.0070 mg a.i./L. The reviewercalculated time-weighted average (TWA) concentr ations were below the limit of concentrationCHLOROTHALONIL FINAL

Page 33 of 61 (<LOQ; <0.000028 mg a.i./L, control), 0.000074, 0.00077, and 0.0099 mg a.i./L). The test system was maintained at 24 to 26°C and a pH of 6.8 to 7.9.

Survival at test termination was 100% in the co ntrol and 96% in both th e low and mid treatment groups. Overall survival was decr eased (p<0.05) to 79% at the hi gh concentration (males - 63%; females - 88%), compared to the control. At test termination, a single bloa ted female was observed in the high treatment group. Chlorothalonil exposure was associated with an increase (4%; p<0.05) in male length at the high concentration, but wet weight was not significan tly different between the control and treatment groups.

Spawning in controls occurred at least every three days and the m ean fecundity in the control was 24.7 eggs/female/day/replicate (range: 19.8-27.8); fert ilization success in controls was 98%. At the high concentration, fecundity was significantly decreased (p< 0.01) by 48%, and fertility was significantly increased (p<0.05) al though the difference was minor (99% versus 98% for control). There were no significant diffe rences in plasma vitellogenin (VTG) or gonado-somatic index (GSI) between control and treate d fish for both males and female s. Similarly, nuptial tubercle score was not significantly differe nt between control and treated males; no nuptial tubercles were
detected in females.

Histopathological examinations showed increased tes ticular generation in male fish in the control and low and mid treatment groups. For females, mi nimal to moderate incr eased oocyte atresia was
noted in the control and all trea tment groups, though moderate cases were restricted to the mid and high concentrations. Oocyte atresia was associated with minimal to mild multifocal granulomatous inflammation 53% of the time (9 of the 17 cases); protozoal spor es (presumptive microsporidia) were observed in two fish. Minimal egg debris in the oviduct was found in two females each in the control and high concentration gr oups. Minimal granulomatous in flammation was observed in the control and all treatment groups and ranged in prevalence from 7 to 29% with the highest occurrence in the high treatment group. Overall, the study author reported that there were no apparent treatment-related patter ns in histopathological findings fo r males or females and that the findings were considered b ackground or incidental.

Hershberger Assay (OCSPP 890.1400)

In a Hershberger Assay (MRID 48669902) screen ing for androgenic activity, chlorothalonil (99.0% a.i., Batch # 617642) in aqueous 1% car boxymethylcellulose (CMC) was administered daily via oral gavage to groups of six 54- to 56-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 300 or 1000 mg/kg/day (limit dose). An androgenic positive control group consisted of six castrated rats exposed to the vehicle (1% CMC by oral gavage) plus 0.2 mg/kg/day of testosterone propionate (T P) by subcutaneous (s.c.) injection.CHLOROTHALONIL FINAL

Page 34 of 61 To screen for potential anti-a ndrogenic activity, chlorothalonil in 1% CMC was administered daily via oral gavage to groups of six 54- to 56-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300 or 1000 mg/kg/ day (limit dose) in conjunction with a daily dose of reference androgen TP at 0.2 mg/kg/day by s.c. injection. The anti-androgenic positive control group consisted of six cas trated rats exposed to 3 mg/kg/ day flutamide (FT) in corn oil via oral gavage and 0.2 mg/kg/da y TP (s.c. injection). The positive control animals for the androgen agonist assay served as th e anti-androgenic negative controls.

For both components of the assa y, body weights and food consump tion were determined daily. The animals were dosed for 10 consecutive days a nd terminated approximately 24 hours after the final dose administration. At necropsy, the fi ve androgen-dependent ti ssues and liver were collected and weighed.

One animal in the androgen agonist assay 1000 mg/kg/day group was euthanized in extremis on Day 4 following a body weight loss of 33.1 g and essentially no food consumed on Days 3-4.
The death of this animal was not considered to be test substance related, and all other rats in the study survived until scheduled sa crifice. Clinical findings not ed for surviving rats in the chlorothalonil-treated groups were limited to soft stool, which infrequently occurred (one animal from the 1000 mg/kg/day chlo rothalonil group and one anim al from the 1000 mg/kg/day chlorothalonil + TP group).

In the androgen agonist assa y, body weights throughout the study and overall body weight gains in the chlorothalonil treated gr oups were comparable to the ve hicle controls. Body weights throughout the study in the positive control (vehic le + TP) group were similar to the vehicle controls, but overall body weight gains were incr eased (p<0.05) by 19% compared to the vehicle controls. Food consumption was unaffected by the administration of chloro thalonil or TP. There were no treatment-related effects in any of th e accessory sex organs of chlorothalonil-dosed animals. Animals in the positive control (vehic le + TP) group had accessory sex organ weight increases (p<0.05) as follows: 351% in seminal vesicles; 628% in ventral prostate; 191% in levator ani-bulbocavernosus (LABC); 451% in Co wper’s glands; and 60% in glans penis.

In the anti-androgen assay, body we ights throughout the study a nd overall body weight gains in the chlorothalonil treated groups we re comparable to the negative (vehicle + TP) controls. Food consumption was unaffected by the administration of chlorothalonil (+ TP). However, the overall mean food consumption in the positive control (FT +TP) group was decreased (p<0.05) by 9% compared to the negative controls; no corresponding effects on mean body weight gains
were noted. There were no trea tment-related decreases in accessory sex organ weights for all treatment groups in the anti-androgen assay. Animals in the positive control (FT + TP) group had accessory sex organ weight decreases (p<0.05) as follows: 70% in seminal vesicles; 77% in ventral prostate; 45% in LABC; 70% in Cowp er’s glands; and 28% in glans penis.CHLOROTHALONIL FINAL

Page 35 of 61 The high dose of chlorothalonil (1000 mg/kg/day) used in the assays represents the maximum dose level recommended in the guidelines. Altho ugh one animal was euthanized in extremis on Day 4, this was not considered to be treatment related, and no extreme signs of toxicity were observed in the surviving animals. Liver weig hts in the chlorothalonil treated groups were comparable to the vehicle/negative controls. The performance criteria indicated that the assays were performing as expected.

Statistically significant changes were not seen in two or more of the five androgen sensitive tissue weights. Chlorothalonil was negative for androgenicity and anti-a ndrogenicity in the Hershberger assay.

Female Pubertal Assay (OCSPP 890.1450)

In a Female Pubertal Assay (MRID# 48699301) , 15 Sprague-Dawley rats/dose group were treated daily via oral gavage with chlorothal onil (99.0% a.i., Batch #: 617642) in aqueous 1.0% carboxymethylcellulose at doses of 0, 90 or 175 mg/kg/day from post-natal day (PND) 22 to 42 or 43. The animals were examined for vagina l opening (VO) daily beginning on PND 22, and age and body weight at day of attainment were recorded. Following sacrifice on PND 42 or 43,
blood was collected for clinical chemis try analyses, including thyroxine (T 4 ) and thyroid stimulating hormone (TSH) which were analyz ed using electrochemiluminescent immunoassays or radioimmunoassays, respectivel y. Liver, kidney, pituitary, adrenals, thyroid, ovaries, and uterus were weighed and examined macroscopica lly. The thyroid, ovaries , uterus, and kidneys were examined microscopically.

No treatment-related effect was observed on surviv al, clinical signs, age of attainment of VO, body weight at VO, or gross pathology. No signi ficant differences were observed in liver, thyroid, uterus, or ovary weight s of the treated groups compared to controls. No treatmentrelated effect was noted on cycle status at necropsy, mean age at first estrus, mean cycle length, percent cycling, or percent regularly cycling. No treatment-related lesions were observed on the thyroid, ovary, uterus, or kidney.

At 175 mg/kg/day, the final body weight wa s decreased by 11% (p<0.01), and overall bodyweight gain was decreased (p<0.05) by 15%. Mean body weights were decreased (p<0.05) by 10-16% during PND 41-43. Mean body weight gains were generally comparable to the control group from PND 22-40. However, a mean body weight loss (-3.2 g) was noted in this group compared to controls (6.5 g) during PND 40-41, followed by lower mean body weight gains during PND 41-42 ( ↓ 61%) and 42-43 ( ↓ 32%). Mean food consumption (g/cage/day) was generally similar to the c ontrol group during PND 22-40, butdecreased mean cage foodCHLOROTHALONIL FINAL

Page 36 of 61 consumption was noted during PND 40-43 (corre sponding to lower body weight gains). Body weight, body weight gain, and mean food consumption were similar to controls at 90 mg/kg/day.

Also at 175 mg/kg/day, organ-to -body weight ratios were incr eased (p<0.01) by 26% in the adrenal gland, 19% in kidney, and 15% in the pituitary. Additionally, increased (p<0.01) kidney-to-body weight ratio was increased by 10% in the 90 mg/kg/day group. However, adjusted (and unadjusted) values in the treated groups did not differ statistically from controls. As there were no significant ch anges in unadjusted and adjusted organ weights, the slight changes in relative organ we ights are not considered to be biologically significant.

Serum TSH levels in the tr eated groups were simila r to controls. Serum T 4 was increased (p<0.05) by 14% at 90 mg/kg/day and by 27% at 175 mg/kg/day. The changes in T 4 levels were not corroborated by changes in TSH levels, thyr oid gland weight, or gross or histological changes in the thyroid.

A gas-filled stomach and intestine was noted in one 175 mg/kg/day females, but no other gross findings were observed. This findi ng was considered incidental.

The doses tested were adequate based on the observed decrease s in body weight and body weight gain at the high dose.

Male Pubertal (OCSPP 890.1500)

In a Male Pubertal Assay (MRID# 48699302), 15 Sprague-Dawley rats/dose group were treated daily via oral gavage with chlorothalonil (99.0% a.i., Batch #: 617642) in aqueous 1.0% carboxymethylcellulose at doses of 0, 90 or 175 mg/kg/day from post-natal day (PND) 22 to 53 or 54. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and age and body weight at day of attainment were recorded. Following sacrifice on PND 53 or 54,
blood was collected for clinical chemistry analyses, including selected hormones. Total serum

testosterone a nd thyroxine (T 4 ) levels were determined us ing electrochemiluminescent immunoassays, and thyroid stimulating hormone (TSH) levels were determined using a radioimmunoassay. Liver, adrenals, pituit ary, and urogenital orga n were weighed, and microscopic examinations were performed on the testes, epididymides , thyroid, and kidneys.

No treatment-related effects were observed on mortality, clinical signs, body weights, body weight gain, food consumption, organ weights, or gross pathology. Age at incomplete PPS in the treated groups was similar to controls. Thyroid hormone levels, colloid area and follicular cell height scores were similar in the treated groups to the co ntrol group. Serum testosterone levels were similar in the treated groups to th e control group. No treatme nt-related findings were noted in the epididymis, testis, and kidneys.CHLOROTHALONIL FINAL

Page 37 of 61 At 175 mg/kg/day, age at PPS was decreased (p <0.05) by 1.7 days (43.7 days vs. 45.4 days). When a second analysis was performed, adjusting th e day of attainment for those males with 3 or more days of incomplete separation (persistent threads), there were no statistically significant differences between the 175 mg/ kg/day group (43.2 days) and the control group (41.8 days). The significant effect on complete PPS for the 175 mg /kg/day group was the result of a greater number of control animals with incomplete se paration (persistent thr eads). Unadjusted and adjusted body weight at PPS was decreased (p<0.05) by 11%. The unadjusted mean body weight at PPS (203.8 g) was similar to the mini mum mean value in the WIL historical control data (203.7 g) and is not consid ered biologically significant.

In the kidneys, unadjusted, adjusted, and rela tive (to body) organ weights were increased (p<0.05) at 90 mg/kg/day by 10-12% , and relative weight was in creased (p<0.001) by 10% at 175 mg/kg/day. Relative (to body) liver wei ghts were increased (p<0.05) by 8% at 90mg/kg/day and by 7% at 175 mg/kg/day. These effects were considered minor and without dosedependency, and adjusted organ weights were similar to controls in the high dose group. Furthermore, no additional evid ence of toxicity was observed on the kidney and liver in this study.

Although there was no evidence of an adverse effect in this study, the high dose was considered adequate based on decreased terminal body weig hts of 11% in a 14-Day range finding study (MRID 48699303; see Appendix) and the 11% d ecrease in body weight observed in the concurrent female pubertal study (MRID 48699301). Mean terminal body weight at the high dose in this study (male pubertal) was decrea sed by only 3% compared to controls; no explanation was provided for this discrepa ncy with the other stated results.

Steroidogenesis (OCSPP 890.1550)

In a steroidogenesis assay (MRID# 48669903), H295R cells cultured in vitro in 24-well plates
were incubated with chlorothalonil (99.0% purity, Batch # 617642) at concentrations of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 μM for 48 hours in triplicat e for four independent experiments. Dimethyl sulfoxide (DMSO) was used as a ve hicle, at a final c oncentration of 0.05%. Precipitation of the test compound was observed microscopically at 100 μM in Runs 2 and 4. Cytotoxicity greater than 20% was also obs erved at 10 and 100 μ M chlorothalonil in all three valid runs; therefore, the results from these wells were not evalua ted statistically. Run 1 was not analyzed because a forskolin concentration on the quality control (QC) pl ate was incorrect (3.33

μ M was used instead of 1 μ M).

Testosterone and estradiol levels were measured using HPLC-MS/MS with internal standards. A QC plate was run concurrently with each independ ent run of a test chemi cal plate to demonstrateCHLOROTHALONIL FINAL

Page 38 of 61 that the assay responded properly to positive contro l agents at two concentration levels; positive
controls included the known inhi bitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production.

Guideline acceptability recommendations and requirements were met, including adequate production of testosterone and estradiol, acceptabl e reproducibility (low % CV), and appropriate
induction and inhibition with positive controls.

Chlorothalonil treatment to H295R cells did not affect testosterone or estradiol production. Inhibition (p ≤ 0.05) of testosterone production was obser ved in Run 4 at 0.01 μM (0.89-fold) and Run 3 at 1 μM (0.82-fold). Induction (p ≤ 0.05) of estradiol production was noted in Run 2 at 1 μM (1.09-fold). However, these changes were minor and not reproducible in this study. All
other values of testosterone and estradiol in the treated cells were similar to the solvent control

group.

Based on hormone responses in three independent runs, chlorothalonil trea tment did not result in statistically significant and repr oducible alterations in testoste rone or estradiol production.

Uterotrophic (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48669904) conducted to screen for potential estrogenic activity, chlorothalonil (99.0% a. i., Batch# 617642) in aqueous 1% carboxymethylcellu lose (CMC) was administered daily via oral gavage to groups of six ovariectomized Sprague Dawley rats at dose levels of 0 (vehicle), 300 or 1000 (limit dose) mg/kg/day on post-natal days (PND) 65-67 or 66- 68. A positive control group was tr eated with a daily dose of 17 α -ethynyl estradiol (EE) in corn oil at 0.3 mg/kg/day by oral gavage. Body we ights and food consumption were determined daily. All animals were terminated and necropsied on PND 68 or 69 approximately 24 hours
after the final dose administration to dete rmine wet and blotted uterine weights.

All animals survived until scheduled terminati on and no significant clinical observations were noted in any of the animals during the study. Body weights throughout the duration of the study and overall (Day 1-4) body weight gains in the ch lorothalonil-dosed groups were comparable to the vehicle controls. Food consumption was unaff ected by the administrati on of chlorothalonil. The overall average food consumption was decr eased (37%) in the positive control (EE) group and corresponded to the decrease in overall body weight gain; body weights for the EE group were decreased (p ≤ 0.01) by 9% on Day 4, and overall body weight gain was decreased (p ≤ 0.001) by 208% in comparison with the vehicle controls.CHLOROTHALONIL FINAL

Page 39 of 61 Uterine weights in the chloroth alonil treated groups (300 and 1000 mg/kg/day) were comparable to the vehicle controls. Absolute wet and blotted uterus weights for the positive control (EE) group were increased (p ≤ 0.01) by 752% and 221%, resp ectively, as expected.

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

Page 40 of 61 APPENDIX 2. Other Scientifical ly Relevant Information (OSRI)

Published literature and Part 158 ge neral toxicity studies considered relevant for determining the potential interaction of chlorothalonil with the en docrine system are summarized below. Given the breadth of toxicity data availa ble on chlorothalonil, the only Part 158 studies discussed in this appendix are those that fulfill current guideline requi rements and are directly relevant to assessing potential endocrine effects (i.e., multiple-gener ation rodent reproductive studies, development studies, chronic toxicity studies, etc.).

ER and AR Transcriptional Acti vation Assays (ERTA & ARTA)

In a published, non-Guideline study ( Kojima et al ., 2004) , examined 200 pesticides, including chlorothalonil (>95-100% purity), for their potential to inte ract with two human ER subtypes (hER α and hER β ) and one human androgen receptor (hAR) in transactivation assays with Chinese hamster ovary cells (CHO-K1 cells). The human ER (hER α ) and AR (hAR) expression vectors (pcDNAER α and pZeoSV2AR, respectively) were cons tructed as described previously (Kojima et
al. , 2003). The hER β expression vector was co nstructed by cloning the hER β cDNA with reverse transcriptase-polymerase chain reaction from hu man placental RNA. The sequence was verified and inserted into the mammalian expre ssion vector pcDNA3.1Zeo(-) creating pcDNAER β . The construction of the estrogen or androgen responsive elements containing reporter plasmid (pGL3- tkERE and pIND-ARE, respectively) was described previously (Kojima et al. , 2003). pRL-SV40 containing the Renilla luciferase gene was used as an inte rnal control for transfection efficiency.

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. Flutolanil was tested over a concentration range from

10

− 8 to 10

− 5 M, and the responses were compared to the activity of 17 β -estradiol (E 2 , >97% purity)

or 5 α -dihydrotestosterone (DHT, 95% purity) 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 along w ith the test compound, re spectively. Agonistic activities were evaluated by the relative activity e xpressed 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. Antagonist ic activities were evaluated similarly by the relative activity expressed as 20% relative inhibito ry concentration (RIC 20 ), the concentration of the compound showing 20% inhibition of the activity of E 2 or DHT against ER α , ER β , and AR, respectively. The range of concentrations tested was ≈ 10 -8 to 10 -5 M; all pesticides
were tested at concentrations ≤ 10 -5 M to avoid cytotoxicity. Wh en the activity of the test compound was higher than the REC 20 the pesticide was judged to be positive for activity. When the activity of the test co mpound was higher than the RIC 20 the pesticide was judged to be positive for inhibitory activity.CHLOROTHALONIL FINAL

Page 41 of 61 Chlorothalonil was negative for agonist or antagonist activity against ER α and ER β , and negative for agonist or antagonist activity against AR in this transcri ptional activation test system.

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a two-generation repr oduction toxicity study ( MRID 41706201, 1990 ), chlorothalonil (98.1% purity) was administered to 35 Spra gue Dawley rats/sex/dose via the diet at dose levels equivalent to 0, 38, 115, and 234 mg/kg/day during the initial pre-mating period of 10 or 14 weeks (P or F 1 generations, respectively); with exposure continuing in dams through gestation, lactation and weaning. Both the P and F 1 generation rats were mated twice to produce the F 1a and F 1b litters and

F 2a and F 2b litters respectively. The parent animal s were rested for two weeks between mating periods. The standard reproductive study paramete rs were evaluated. Or gan weights were not
measured.

There were no treatment related effects on mortalit y, food consumption, or clinical signs in P or F 1 adult animals. In the males, body weights were decreased (p<0.01, ↓ 7-11%) in the 234 mg/kg/day group during all interv als in the P and F 1 generations. In the females, there were significant (p ≤ 0.05) lower group mean weights ( ↓ 5-8%) in the 234 mg/kg/day group during the 10 weeks prior to mating (F 1a ). During gestation for F 1a , group mean body weights were decreased by 5-7% and mean body weight gains were essentially the same for all 4 groups (117-119 g). For the F 1a lactation period (3 weeks), group mean gains were -1, -3, 13, and 23 g in the control, 38, 115, and 234 mg/kg/day groups, respectively, so that the group mean final lactation body weights for all 4 groups was essentially the same (307-317 g). Gr oup mean body weight gains during the 2 week rest period between F 1a and F 1b were decreased (p<0.01) by 7-8% at 234 mg/kg/day compared to
the controls.

During the 14 weeks pre-mating for F 2a , group mean body weights were decreased (p<0.05) by 6- 10%, 4-9%, and 6-13% in the 38, 115, and 234 mg /kg/day groups, respectively, compared to controls. Body weight gains for this period were 209, 190, 186, and 178 g in the control, 38, 115, and 234 mg/kg/day groups, respectively. During the F 2a gestation period, body weights were decreased (p<0.01) by 9-14% in th e 234 mg/kg/day group; however, the all treated groups gained 118-121 g (vs 116 g control). During the F 2a lactation period, body we ights were decreased (p<0.01) by 6-9% in the 234 mg/kg/day animals. The control, 38, and 115 mg/kg/day groups lost 3-10 g; whereas, the 234 mg/kg/day group gained 12 g. During the rest period, body weights remained decreased (p<0.01) in the high-dose group ( ↓ 10-11%) compared to controls, and all treated groups gained 2 g each (vs 6 g control). Although there were significantly (p<0.01) lower body weights in the 234 mg/kg/day group ( ↓ 9-13%) during the F2b gestat ion period compared to controls, the weight gained during this 3 week period was 117-124 g for all treated groups (vs 116 g control). The 234 mg/kg/day group gained 8 g during lactation compared with losses of 16, 12 and 4 g in the control, 38, and 115 mg/kg/day groups.CHLOROTHALONIL FINAL

Page 42 of 61 No treatment-related effects were observed in the reproductive parameters (male or female fertility indices, gestation index, gesta tion length, live birth index, vi ability index, lactation index, parturition, or fetal sex ratio). No treatment-rela ted changes were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, uterus, or vagina at any dose in either generation.

The NOAEL for parental toxicity was not attain ed. The LOAEL for parental toxicity was 38 mg/kg/day based on kidney and forestomach lesi ons. The LOAEL for reproductive effects was 234 mg/kg/day based on decreased neonatal body weights on LD 21. The NOAEL for reproductive effects was 115 mg/kg/day.

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a two-generation repr oduction toxicity study ( MRID 45710209, 1995 ), chlorothalonil (99.16% purity) was administered continuously in th e diet to Crl: CD®BR VF/Plus rats (32 animals/sex/dose) at dose levels of 0, 400, 1200, or 3000 ppm (equivalent to 0/0, 30.8/34.3, 92.5/106.0, and 247.5/270.0 mg/kg/day [M/F]). The P and F 1 parents were dosed for 10 weeks and 12 weeks, respectively, before th ey were mated to produce the F 1 and F 2 litters. The F 1 pups were weaned on postnatal day (PND) 21, and 28 pups/sex/g roup (1 pup/sex/litter as nearly as possible) were randomly selected as parents of the F 2 generation.

In the parental animals, no tr eatment-related effects were obser ved on survival, or food or water consumption. In the 3000 ppm P animals, body we ight gains during Week s 0-1 were decreased, and food consumption for Week 1 was decrease d. Body weight gains were decreased during Week 1-10 in the males in this generation, but cumulative food consumption was comparable for Weeks 2-10 in both sexes in this generation. In the 3000 ppm F 1 males, body weights were decreased throughout treatment (Weeks 4-26), resu lting in decreased body weight gains during pre-mating (Weeks 4-16), that continued to termin ation (Weeks 17-26). Similarly in the 3000 ppm

F 1 females, body weights were decreased during pr emating (Weeks 4-16), resulting in decreased body weight gains during this period . In the males, food consumption was not affected, while food consumption was slightly decreased during Week 1 in the females.
Treatment-related effects were observed on th e kidney at 1200 and 3000 ppm. Adjusted (to terminal body weight) kidney weights were increased in both sexes in both generations. Enlarged kidney was observed in the P and F 1 males compared to controls , and the following microscopic pathological findings were noted: i) trace to modera te dilated/basophilic cort ical tubules containing colloidal material in the P males compar ed to controls; ii) trace to moderate hyperplasia/hypertrophy of the inner co rtical tubules in both sexes in both generations compared to controls; and iii) trace to minimal dystrophic minera lization at the corticomedullary junction in the P females compared to controls. Bright yell ow/orange urinary staini ng and wetter than normal feces on the cage tray paper unde r the cages of the P and F 1 parents; and cage sawdust yellow stained and wet during lacta tion phase in the P and F 1 females was observed.CHLOROTHALONIL FINAL

Page 43 of 61 In the 400 ppm and above P and F 1 animals, the forestomach was observed to be thickened and roughened, compared to controls, with depressions in the epithelial aspect compared to controls. At 1200 ppm and above, white areas in the forest omach were noted in the P animals, and the limiting ridge of the forestomach was prominent in the F 1 animals both compared to controls. In the 400 ppm and above P and F 1 males and females, trace to moderate hyperplasia and hyperkeratosis of the non-glandula r epithelium of the stomach was obs erved compared to controls. At 3000 ppm, minimal focal ulceration of the no n-glandular epithelium of the stomach was observed in the P females, and trace to moderate erosion of the non-glan dular epithelium of the
stomach was noted in the F 1 females, both compared to contro ls. Increases were observed in relative kidney weights ( ↑ 9-13%) in the 400 ppm P and F 1 males. The kidney was enlarged in 3 males in the P generation and in 7 males in the F 1 generation. Microsc opically, there was an increased incidence of trace to minimal dystrophic mineralization at the co rticomedullary junction of the kidneys in the P females (34%) when compared to controls (13%).

No treatment-related changes in absolute or relative weights were seen for parental animals in the testes, epididymides, prostate (weighted with seminal vesicle and coagulating gland), ovaries, pituitary or adrenal glands of the parental animals. In addition, No treatment-related histopathological changes were seen in the testes, epididymides, seminal vesicle, prostate, coagulating gland, ovaries, uterus , cervix, vagina, mammary, thyr oid, adrenal and the pituitary glands at any dose in either generation.

The LOAEL for parental toxicity was 400 ppm (equivalent to 30.8/34.3 mg/kg/day male/female), based on macroscopic and microscopic pathological findings in the stomach- including: thickened, roughened, and white areas in forestomach with depressions in the epithelial aspect, and hyperplasia and hyperkeratosis of non-glandular epithelium in the st omach, and kidney effects, i.e., enlargement, relative weight increases and dystr ophic mineralization at the corticomedullary junction. The NOAEL was not established.

In the offspring, no treatment-related effects were observed on post-implan tation survival, live birth, viability, or lactation indice s, on the sex ratio, clinical si gns, gross pathology, or microscopic pathology.

At 3000 ppm, pup body weights were d ecreased during PND 12-21 in the F 1 and F 2 pups, resulting in decreased overall (PND 0-21) pup body weight gains. Litter weights were decreased in the F 1 pups during PND 16-21 and in the F 2 pups during PND 8-21, resulting in decreased overall litter weight gains. Additionally, ba lano-preputial skinfold cleavage was delayed and vaginal opening was delayed compared to controls. However, since no differences were observed in the subsequent mating and reproductive performance of these animals, these effects were considered equivocal and were attributed to the reduction in body weight gain observed during lactation. ThisCHLOROTHALONIL FINAL

Page 44 of 61 assertion is supported by the comparable body weight at attainment of cr iterion in all treatment

groups.

At ≥ 400 ppm, a dose-dependent increase in the incidence of thickeni ng and/or roughening of the forestomach was observed in both generations, and increased incidence and severity of trace to moderate hyperplasia and hyperkeratosis of the non-glandular epith elium of the stomach and trace to marked sub-epithelial edema in the non-glandular region of the stomach were observed compared to controls. Additionally, in the F 1 group, trace to moderate inflammatory cells in the non-glandular region of the stomach was noted compared to controls.

The LOAEL for offspring toxicity was 400 ppm (equivalent to 30.8/34.3 mg /kg/day male/female), based on thickening and/or roughening of the fore stomach with depressions in the epithelial aspect, and hyperplasia and hyperkeratosis of th e non-glandular epithelium of the stomach. The
NOAEL was not established.

No treatment-related effects were observed on estrous cycle length or periodicity, pre-coital interval, duration of gestation, or on ma ting, gestation, or pa rturition indices.

The LOAEL for reproductive performance was not observed. The NOAEL for reproductive performance was 3000 ppm (equivalent to 247.5/270.0 mg/kg/day males/females).

Developmental Toxicity (Rat, OCSPP 870.3700)

In an oral developmental toxicity study ( MRID 00130733, 1983 ), chlorothalonil (98% purity) in 0.5% methylcellulose was administered daily by oral gavage at a dose volume of 10 mL/kg bw to 25 female Sprague-Dawley rats/group at dose le vels of 0, 25, 100, or 400 mg/kg/day on gestation days (GD) 6 through 15. All surviving dams were killed on GD 20; their fetuses were removed by
cesarean section and examined.

There were no treatment-related effects on mo rtality, clinical signs , body weight gain, food consumption, or food efficiency observed in the 25 or 100 mg/kg/day groups. In the 400 mg/kg/day group, there were 3 mort alities; however, the cause of death was not determined. Clinical signs of toxicity in this group included: mucoid, loose or white discoloration of the feces; brown material around the nose and mouth; matting of the urogenital fur; s light increase in the
number of animals with hair lo ss; and 2 animals with a red vagi nal discharge occurring between GD 12 and 18. During the dosing period, body wei ght gains were decreased (p<0.01) by 52%, food consumption was decreased by 29% and food efficiency was decreased by 33% compared to
the control.

There were no effects of treatmen t on gross pathology at any dose.CHLOROTHALONIL FINAL

Page 45 of 61 The maternal LOAEL was 400 mg/kg/day based on mortality, clinical signs, decreased body weight gains, reduced food cons umption, and lower food efficiency during the dosing period. The maternal NOAEL was 100 mg/kg/day.

No treatment-related changes were seen in pregnancy rate, number of corpora lutea, implantation sites, implantations/dam, late resorptions, number of live fetuses/litter, fetal sex ratio, or soft tissue abnormalities at any dose. Increases in total reso rptions and resorptions/dam were observed in the 400 mg/kg/day dose group with a related increase in post-implantation loss (13.4% treated vs. 5.2% controls). No effects of treatment were observed on fetal growth. There were no treatmentrelated external, visceral, or skel etal malformations or variations.

The developmental LOAEL was 400 mg/kg/day base d on an increase in to tal resorptions and resorptions/dam with a related increase in post-implantation loss. The developmental NOAEL was 100 mg/kg/day.

Developmental Toxicity (Rat, OCSPP 870.3700)

In an oral developmental toxicity study ( MRID 45710207, 1993 ), chlorothalonil (99.15% purity) in 1.0% methylcellulose was administered daily by gavage at a dose volume of 15 mL/kg bw to 25 female (Crl: CD®[SD] BR VAF/Plus) rats/group at dose levels of 0, 80, 200, or 500 mg/kg/day on GD 6 through 15. All dams were killed on GD 20; their fetuses were removed by cesarean section
and examined.

There were no effects of treatment on gross pathology.

At 500 mg/kg/day, one dam was f ound dead on GD 9. This animal exhibited noisy respiration prior to dosing on the previous day, and post-mort em examination revealed red/brown staining of the perinasal and perioral regions, enlarged cervical lymph nodes, firm lungs with patchy congestion, and gaseous disten sion of the gastrointestinal tract. Also at the above dose level, two
dams were killed for humane reasons on GD 12. These dams exhibited loss of body tone, noisy/irregular respira tion, piloerection, and brown perinasal staining immediately prior to death, as well as lethargy, distended abdomen, and ye llow stained urogenital region. Post-mortem examination revealed enlarged cervical lymph nodes and severe gaseous distension of the gastrointestinal tract in both animals; one anim al was also found to have a roughened forestomach. All other animals survived to study termination. Wet feces were noted on GD 7 or 8 to GD 16, post-dosing salivation was observed in 6/22 da ms on GD 11/12, and noisy/irregular respiration
was noted in 2/22 dams on GD 10-16 and 13-19. Body weight gains were decreased (p ≤ 0.05) during treatment on GD 6-8 and 6-14 (decreased 19- 41%). Body weight gains continued to be decreased (p ≤ 0.05) to termination (GD 6-20; decreased 9%) and for the overall (GD 2-20) study (decreased 7%; not significant [NS]). Food consumption was decreased (p ≤ 0.05) during GD 6-7 (decreased 13%), and water consumption was increased (p ≤ 0.05) during GD 8-9, 12-13, 14-15, and 18-19 (increased 21-33%).CHLOROTHALONIL FINAL

Page 46 of 61 The maternal LOAEL was 500 mg/kg/day, based on incr eased mortality, clinical signs of toxicity, decreased body weight gains and food consum ption, and increased water consumption. The maternal NOAEL was 200 mg/kg/day.

There were no abortions, premature de liveries, or dead fetuses. No effects of treatment were noted on numbers of litters, live fetuses, resorptions (ear ly, late, or complete litter), or on sex ratio or post-implantation losses. No effects of treatment were observed on fetal growth. There were no treatment-related external, visceral, or skeletal malformatio ns or variations.

The developmental LOAEL was not observed. Th e developmental NOAEL was the highest dose of 500 mg/kg/day.

Developmental Toxicity (Rabbit, OCSPP 870.3700)

In an oral developmental toxicity study ( MRID 00047944, 1976 ), chlorothalonil ( ≥ 99% purity) was administered daily by gavage (vehicle and do se volume were not reported) to 10 female Dutch Belted rabbits/group at dose levels of 0, 1, 2.5, or 5.0 mg/kg/day on gestati on days (GD) 6 through 18. All dams were killed on GD 28; their fetuse s were removed by cesarean section and examined.

No significant adverse effects were evident at 1.0 mg/kg/day. Dose dependent maternal toxicity
was evident at ≥ 2.5 mg/kg/day in the form of one death and one abort at 2.5 mg/kg/day and 2 deaths and four aborts at 5 mg/kg/day.

The maternal LOAEL was 2.5 mg/kg/day, based on mortality and abortion. The maternal NOAEL is 1.0 mg/kg/day.

No treatment-related changes were seen in pregnancy rate, number of corpora lutea, implantation sites, implantations/dam, early or late resorptions, number of live fetuses/litter, fetal sex ratio, or soft tissue abnormalities at any dose.

The developmental LOAEL was not observed. Th e developmental NOAEL was 5.0 mg/kg/day.

Developmental Toxicity (Rabbit, OCSPP 870.3700)

In an oral developmental toxicity study ( MRID 45710208, 1994 ), chlorothalonil (99.15% purity) in 1.0% (w/v) aqueous methylcellulose was admini stered daily by gavage at a dose volume of 5 mL/kg bw to 16 female New Zealand White ra bbits/group at dose levels of 0, 5, 10 or 20 mg/kg/day on gestation days (GD) 6 through 18. All does were killed on GD 29; their fetuses were removed by cesarean section and examined.

No effects of treatment were observed on mort ality, clinical signs, body weights, body weight gains, food consumption, or gross pathology.CHLOROTHALONIL FINAL

Page 47 of 61 At 5 mg/kg/day, one doe died prior to dosing on GD 8. One 10 mg/kg/day female aborted her litter on GD 29; this animal exhib ited cold ears during dosing, and anorexia/few feces, cold ears, red material/blood on tray paper, and no feces pos t-dosing. One 20 mg/kg/da y aborted her litter on GD 25-26; this animal exhibited anorexia/few feces prior to dosing, anorexia /few feces, cold ears, and no feces during dosing, and anorexia/few f eces, cold ears, dark eyes, no feces, fouled anogenital region, nasal exudate, soft/liquid feces, and thin appearance post-dosing. It was stated that similar findings were not observed in a prel iminary developmental toxicity study in rabbits,
and in view of the low incidence of animals aborti ng prior to terminal sacrifice, these findings are
considered to be incidental to treatment.

The maternal LOAEL was not observed. The maternal NOAEL was 20 mg/kg/day.

No effects of treatment were noted on numbers of litters, live fetuses, resorptions (early, late, or complete litter), or post-implantation losses. Fetal growth was una ffected by treatment.

There were no treatment-related external , visceral, or skeletal malformations.

At 20 mg/kg/day, increased (p ≤ 0.05) incidence of thirteen ribs , a variant, was observed (75.0% fetuses; 100% litters) compared to concurrent controls (49.2 % fetuses; 93.3% litters), and reduced sternebrae, a variant, wa s observed (17.8% fetuses; 76.9% litters) compared to concurrent controls (8.3% fetuses; 40.0% litters). In the absence of historical control data, these findings are
considered treatment-related.

The developmental LOAEL was 20 mg/kg/day base d on increased incidence of 13th ribs and reduced sternum. The developmental NOAEL was 10 mg/kg/day.

Chronic Toxicity/Carcinoge nicity (Rat, OCSPP 870.4300)

In a combined chronic toxicity/carcinogenicity study ( MRID 00146945, 1985 ), groups of 60
Fischer 344 rats/sex were fed diet s containing chlorothaloni l (98.1% purity) at le vels equivalent to doses of 0, 40, 80, or 175 mg/kg/day for 116 (males) or 129 (females) weeks.

Survival was comparable in all groups, both sexes, for the first 24 months. Continuation on study decreased survival in the 178 mg/kg/day males re sulting in all males sacrificed at 27 months.
Females were terminated on schedule at 30 mont hs. The major cage-side clinical observation included dark yellow urine in the 175 mg/kg/ day males and females from Weeks 27-91. An increased brown, red staining around the anogenital region of the ≥ 80 mg/kg/day females was also observed. There was a significant body weight decrease ( ↓ 10-29%) in both sexes at 175 mg/kg/day throughout study, as well as a 5-12% body weight decrease in both sexes at 80 mg/kg/day. There was no body weight reducti on in either sex at 40 mg/kg/day. Food consumption was unaffected, except for an increase in high dose animals, generally towards the last half of the study.CHLOROTHALONIL FINAL

Page 48 of 61 No treatment-related changes were se en in absolute or relative we ights of the testes, ovaries or adrenal glands at any dose level. No treatment-related histopathol ogical lesions were seen in the testes, epididymides, seminal vesi cle, prostate, ovaries, uterus, thyroid, adrenal and the pituitary glands at any dose level.

Parameters measured which were compound related and associated with the effects on the kidneys
included increased BUN and serum creatinine in 175 mg/kg/day males and females, decreased serum albumin and serum glucose in 175 mg/kg/da y males and females, increased urine volume and decreased specific gravity in all treated ma les throughout the study, and in all treated females initially (first year), but in 175 mg/kg/day fe males only, after the first year. Relative kidney weights were significantly increased in all treated males and in the ≥ 80 mg/kg/day females. Relative liver weight was affected in the same groups, being significantly in creased in all treated males, and the ≥ 80 mg/kg/day females. Gross necropsy of ' all animals demonstrated a compound related effect on the kidneys and stomach. In all treated male groups and the 175 mg/kg/day female group there were kidney masses and/or nodul es as well as increased granularity of' the surface of' the kidneys (the latter ob served in all dose groups). Ther e were increased incidences of'
erosions and ulcerations in the non-glandular stomach of all treate d rats as well as a significant
increase in discoloration of' th e mucosa in 175 mg/kg/day males.

Histologically there was evidence of compound rela ted effects on the kidneys, esophagus, stomach and duodenum. Non-neoplastic changes in the ki dney included: chronic glomerulonephritis which increased in severity in a dose -related manner in all groups; dose related increase in cortical tubular hyperplasia in all dosed ra ts; increased incidence of' tubular cysts in all dosed rats; and
increased incidence in dosed male s only of' hyperplasia of the papi llary/pelvic epithelium. Other changes included increased hyperpla sia/hyperkeratosis of' the squa mous mucosa of the esophagus (all dose groups); increased mucosal hype rtrophy of the duodenum (all dose groups); hyperplasia/hyperkeratosis of the parathyroid (all dosed male and 175 mg/kg/day females, considered a secondary lesion as a result of severe chronic renal disease); increased hyperplasia/hyperkeratosis of the squamous mucosa of all dose groups; in creased incidences of foci of necrosis or ulcers in the glandular st omach of all dose groups; increased incidence of suppurative prostatitis in all male dose groups (considered associ ated with treatm ent related renal lesions); complete involu tion of the thymus was increased in 175 mg/kg/day males and all female dose groups. Interesting inverse do se related changes included: ch ronic interstitial prostatitis (increased in control and 40 mg/ kg/day males); increased inciden ce of medullary tumors of the adrenal (control and 40 mg/kg/day females); increased incidences of ' osteosclerosis of the femur and sternum (control females); and an increased incidence of basophilic cell focus/foci of the liver (control females, a common finding in aging Fischer 344 rats).

Neoplastic changes associated with treatme nt were observed in kidneys and stomach (forestomach). Tubular adenomas and carcinomas , aneplastic renal carcinomas and transitional
cell carcinomas were observed in th e kidney of treated rats only, be ing statistically significant inCHLOROTHALONIL FINAL

Page 49 of 61 all treated rats except the 40 mg/kg/day females. There was also a possible decrease in time to tumor in 175 mg/kg/day rats for renal adenomas and carcinomas. There was no evidence however,
that the occurrence of' co rtical tubular hyperplasia or tubular cysts predisposed animals to such tumors since only 1/11 tumor bearings low dose rats al so had a tubular cyst. This was also true at the higher doses as well.

Papillomas and carcinomas of' the squamous mucosa or the stomach were present in treated rats only, but statistically significan t in the 175 mg/kg/day females only. There was no evidence that hyperplasia or hyperkeratosis predis posed the animals to such tumors as the degree or severity or hyperplasia/hyperkeratosis varied from slight to marked in these animals, and was equally prevalent in non-tumor bearing rats, although the seve rity of response increased in a dose related

manner.

The LOAEL was 40 mg/kg/day based on renal adenomas and carcinomas in both sexes. The NOAEL was <40 mg/kg/day.

Chronic Toxicity/Carcinoge nicity (Rat, OCSPP 870.4300)

In a combined chronic toxicity/carcinogenicity study ( MRID 45710212, 1996 ), Chlorothalonil (99.28% purity) was administered in the diet for 2 years to 50 Crl:CD (SD) BR rats/sex/dose at doses of 0, 15, 60, 240, and 1200 ppm; these dietary levels were equivalent to 0, 0.7, 2.7, 10.6 and 54 mg/kg/day in males and 0, 0.9, 3.3, 13.9 and 70 mg/kg/day in females). Additionally, 20
rats/sex/dose were treated simila rly and sacrificed at 12 months.

No treatment-related effects were observed on mortality, food and water consumption, food efficiency, ophthalmoscopic examinations, hema tology, and non-protein thiol concentration in
liver.

At 1200 ppm, an increased incidence of generalized yellow staining of the fur was observed in the interim and terminal groups (66-100% treated vs 0% controls). This si gn was observed generally
from Week 16 in males and Week 19 in females. Body weight gain was decreased (p<0.05) by 15- 17% in the females during Weeks 0-1, 13-26, and 26-52; however, overall (Weeks 0-104) body weight gain was similar to controls, i ndicating recovery during the second year.

At 1200 ppm, nephrotoxicity was evident. Urinar y protein concentration was increased (p<0.05) by 137-279% in the males at Weeks 52, 78, and 104. Adjusted (for terminal body weight) kidney weights were increased (p<0.05) in the males at Weeks 52 and 104 ( ↑ 21-28%). Necropsy of the males, revealed uniform cortical scarring of th e kidneys at the interim (15% treated vs 0% controls) and terminal (44% trea ted vs 14% controls) sacrifices. Kidney enlargement in the males (44% vs 20%) was also observed at the terminal necropsy. At the te rminal sacrifice, there was an increased incidence of minimal to marked progre ssive glomerulonephrosis in the males (70% vs 52%) and the females (40% vs 26%).CHLOROTHALONIL FINAL

Page 50 of 61 At necropsy following the interim s acrifice, the forestomach in the ≥ 240 ppm groups was thickened, roughened, and white. Trace to moderate epithelial hyperplasia and hyperkeratosis in the non-glandular region and in the limiting ridge of the stomach was observed in these animals. At necropsy following the terminal sacrifice, the forestomach had depressions in the ≥ 60 ppm groups; and was thickened, roughened, and white in the ≥ 240 ppm groups; and limiting ridge thickened or prominent in the 1200 ppm males. Th ere was an increased incidence in the following lesions in these animals: (i) ep ithelial hyperplasia and hyperkeratos is in the nonglandular region of the stomach in all treated animals; (ii) epithelial hyperplasia and hyperkeratosis in the limiting ridge of the stomach in ≥ 240 ppm males or the 1200 ppm females; (iii) stomach ulceration in the nonglandular region in the ≥ 60 ppm females; and (iv) submucosal fibrosis and inflammatory cells in the nonglandular region of the stomach in ≥ 60 ppm females.

No treatment-related changes were seen in absolute or relative weights of the testes, ovaries, adrenal and pituitary glands at any dose level. At Week 52, adjusted thyr oid weight was increased (p<0.05) by 19% in females at the high dose; howev er, no difference in thyroid weight was seen at Week 104, or in males at any dose or interval.

No treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, uterus , vagina, mammary, adrenal or pituitary glands at any dose level. In male rats, there was a non-statis tically increase in the incidences of thyroid C-cell adenomas at 2.7 mg/kg/day (2%), 10.6 mg/kg/day (2%) and 54 mg /kg/day (4%) compared to control males (0% ). Thyroid C-cell carcinomas were increased only at the 10.6 mg/ kg/day (4%) compared to control males (2%). There were no corrobor ative non-neoplastic lesions or changes in the absolute or relative weight of the thyroid in males. It is important to note that C-cell tumors arise from parafollicular cells that secr ete calcitonin and not involved in thyroid hormone regulation. No
treatment-related lesions were seen in females.

The LOAEL was 15 ppm (0.9 mg/kg/day) in females based on an increased in the incidence and severity of epithelial hyperplas ia, hyperkeratosis and ulceration of the nonglandular region of the stomach females, and 60 ppm (2.7 mg /kg/day) based on an increase in the incidence and severity of epithelial hyperplasia and hyperk eratosis of the nonglandular region of the stomach in males. The NOAEL was <15 ppm (0.9 mg/kg/day) in fe males and 15 ppm (0.7 mg/kg/day) in males.

Carcinogenicity (Mouse, OCSPP 870.4200)

In a carcinogenicity study ( MRID 00127858, 1983 ), groups of 60 CD-1 mice/sex were fed diets containing chlorothalonil (97.7% purity) at 0, 750, 1500, or 3000 ppm for approximately 24 months, equivalent to doses of approxim ately 113, 225, and 450 mg/kg/day, respectively.CHLOROTHALONIL FINAL

Page 51 of 61 No effects of treatment were observed on clinical signs, body weights, or food consumption. The
number of mortalities was increa sed in the 450 mg/kg/day males (39/ 60 treated vs 29/60 controls). Mortality in all other groups was similar to controls.

At 450 mg/kg/day, decreased hem oglobin, hematocrit and red cell values were reported in the
males at 24 months and in the females at 12 and 24 months. Hyperplastic bone marrow was reported in all treatment group males and females. Hyperplasia of the splenic red pulp was noted in the male treatment groups. Hemosiderosi s was not a prominent finding in this study.

Relative spleen weights were repor ted in the high-dose females; however, no significant pathology was reported. Spleen enlargement occurred in the ≥ 225 mg/kg/day males. Ovarian weight ratios were decreased in all treatment groups; however, no histopathological findings were associated
with this. The same was true for the relative testes weights in the 450 mg/kg/day males. Kidney weights were significantly increas ed (p<0.01) in all treatment groups . This finding was considered
to be treatment-related. No treat ment-related changes were seen in absolute or relative weight of adrenal glands at any dose level. No treatment-related histopathol ogical lesions were seen in the testes, seminal vesicle, prostate, preputial gla nd, ovaries, uterus, mammary , thyroid, adrenal, and pituitary glands at any dose level.

Compound related effects in the kidney were descri bed as renal enlargement, discoloration, surface irregularities, pelvic dilation, cysts, nodules and ma sses in all treatment groups.

In the stomach, the incidence and severity of hype rplasia and hyperkeratos is of the esophageal squamous mucosa in treated males and females was significant and was co nsidered to be doserelated. This was not seen control animals. Th ere was a significant increase in gastric squamous cell tumors in the 450 mg/kg/day females but this was not dose related. Glandular epithelial tumors were present in the treated groups but not in statistically significant numbers.

In the kidney, chronic glomerulonephritis was se en in all groups, but the incidence was not significantly different among them , although it was higher in the 450 mg/kg/day males. Increased tubular degeneration was noted in the ≥ 225 mg/kg/day males and in the 450 mg/kg/day females. Increased incidences of cortical cysts were s een in all treated males and in the 450 mg/kg/day
females.

Adenomas and carcinomas of the cortical tubules were increased in all treated males but not in females. The only neoplasm seen in the females was one renal hemangiosarcoma in a 113 mg/kg/day female.

The LOAEL was 113 mg/kg/day based on increased in cidence of adenomas and carcinomas of the
cortical tubules in the kidneys of males. The NOAEL was not established.CHLOROTHALONIL FINAL

Page 52 of 61 Carcinogenicity (Mouse, OCSPP 870.4200)

In a carcinogenicity study ( MRID 45710211, 1995 ), chlorothalonil (99.28% purity) was administered in the diet to Crl:CD(SD)BR mice (50/sex/dose) at concen trations of 0, 15, 60, 240, or 960 ppm for up to 18 months. These dietary c oncentrations were equivalent to 0, 1.9, 7.8, 30.4 and 130 mg/kg/day in males and 0, 2.5, 9.9, 40.6 and 157 mg/kg/day in females.

No treatment-related effects were observed on mo rtality, clinical signs, body weights, body weight gains, food consumption, food utilization, or hematology. No treatment-related changes were seen in absolute or relative weights of the testes, uterus or ovaries at any dose level. No treatmentrelated histopathological lesions we re seen in the testes, epididym ides, seminal vesicle, prostate, ovaries, uterus, vagina, mammary glands, thyroid, or pituitary glands at any dose level.

Minimal to moderate epith elial hyperplasia of the nonglandular region of the stomach in all treated male groups (23-42 vs 10 control) and epithelial hyperplasia (limiting ridge) of the stomach in all treated male groups (13-32 vs 3 control) was observed. Minimal to moderate epithelial hyperplasia and of the nonglandular regi on of the stomach was observed in ≥ 60 ppm females (27- 42 vs 9 control). Folding of the epithelium of the nonglandular region of the stomach was
observed in the ≥ 60 ppm males (16-18 vs 3 control). Hype rkeratosis of the nonglandular region of
the stomach was observed in the ≥ 240 ppm males (39-45 vs 23 control) and ≥ 240 ppm females (45-47 vs 26 control). At ≥ 60 ppm in the kidney, increased (p ≤ 0.05) incidences (# affected/50 in treated vs controls) of the follo wing histological lesions were observed in males: (i) minimal to moderate basophilic cortical tubules vs minimal in the controls ( 23-32 vs 12 control); (ii) cystic atrophic glomerulus with hypertro phic parietal epithelium (13-22 vs 4 control); and (iii) foci of mineralization in the cortex (17-19 vs 9 control; not statistically significant at 240 ppm). The increase in this spectrum of lesions indicates a treatment-related effect that is considered adverse because it suggests an acceleration of the natura l, age-related degenera tion of the kidney. Urinalysis or PAS staining (eva luating the integrity of the ba sement membrane) could have provided further information regarding the im pact of these lesions on renal function.

At 960 ppm, eosinophilic droplets in keratin was observed in the stomach (7 vs 0) of males; and an increased incidence of enlarged kidneys were obse rved in males, and in uniform cortical scarring of the kidneys in both sexes. Additionally, the adjusted kidney weight was increased (p ≤ 0.01) in both sexes. In the 960 ppm males, increased incidences of sinus histiocytosis and erythrophagocytosis were observed (p ≤ 0.05) in the mesenteric lym ph node. Focal epithelial hyperplasia of the nonglandular re gion of the stomach (7 vs 0) and papillomatous hyperplasia (nonglandular region and limiting ridge) was observed in the 960 ppm females (5 vs 0, each region).

The LOAEL was 15 ppm (equivalent to 1.9 mg/kg/da y) in males, based on epithelial hyperplasia of the nonglandular region of the stomach and epithelial hyperplasia (limiting ridge) of theCHLOROTHALONIL FINAL

Page 53 of 61 stomach. The NOAEL was < 15 ppm (equivalent to 1.9 mg/kg/day) in males. The LOAEL was 60 ppm (equivalent to 9.9 mg/kg/ day) in females based on epithelial hyperplasia and of the nonglandular region of the stomach. The NOAEL wa s 15 ppm (equivalent to 2.5 mg/kg/day) in
females.

Chronic Toxicity (Dog, OCSPP 870.4100)

In a chronic toxicity study ( MRID 43653603, 1994 ), chlorothalonil (98.3% purity) was administered orally by gelatin capsules to Beag le dogs (5/sex/group) at doses of 0, 15, 150 or 500 mg/kg/day for 52 weeks.

There were no effects on mortal ity, clinical signs, food cons umption, ophthalmology, hematology, urinalysis, or macroscopic pathology. No tr eatment-related effects were observed on organ weights, including absolute or relative testes weight at any dose level. No treatment-related histopathological lesions were observed in the testes, ep ididymides, prostate , ovaries, uterus, mammary, thyroid, adrenal and pituit ary glands at any dose level.

There were decreases in body weight gains in males and females at 500 mg/kg/day. Effects on clinical chemistry were limited to decreased alanine aminotransferase levels ( ↓ 90%) in all dosed animals compared with controls as well as with pretreatment levels.

The LOAEL was 500 mg/kg/day based on decrease d body weight gains in both sexes. The NOAEL was 150 mg/kg/day.

Chronic Toxicity (Dog, OCSPP 870.4100)

In a chronic oral toxicity study ( MRID 45710210, 1995 ), 4 beagle dogs/sex/dose were given chlorothalonil (99.28% pu rity) in the diet at nominal concentrations of 0, 160, 1280, or 10,240 ppm for 52 weeks. These dietary concentrations were equivalent to doses of 0, 5.10, 43.26 and 374 mg/kg/day in males and 0, 5.92, 45.30 and 354 mg/kg/day in females.

There were no effects of treatment on ophthalmos copy, hematology, or urinalysis. No treatmentrelated changes were seen in absolute or relati ve weights of the testes, epididymides, prostate, ovaries, uterus, thyroid (except in males), adrenal or pituitary glands at any dose level. No treatment-related histopathological lesions were seen in the testes , epididymides, prostate, ovaries, uterus, vagina, mammary, thyroid, and p ituitary glands at any dose level.

Vasodilatation of the gums and/or ears was noted in all of the 10,240 ppm males and females compared to 0 controls, with males affected fo r 33-84 days and females affected for 30-191 days. With the exception of a single female, this effect tapered off after 26 weeks.

One 10,240 ppm female (#370) was killed for humane reasons on the first day of Week 31, and the
death was considered to be treatm ent-related. The following were noted in this animal prior toCHLOROTHALONIL FINAL

Page 54 of 61 death: vasodilatation of the gums later followe d by paleness of gums; decreased body weight, food consumption, red cell indices, and albumin; reticu locytosis; and increased total globulin. Postmortem examination revealed microscopic change s in the heart, spleen, bone marrow, stomach, liver, and kidneys.

At 10,240 ppm, body weights were d ecreased (decreased 4-11%) in the males beginning at Week 2, resulting in significantly decreased (p<=0.05) cu mulative body weight gains beginning at Week 8 (decreased 36-47%) and an overa ll (Weeks 0-52) body weight gain decrease of 44% compared to controls. In the females at this dose, cumulative body weight gains were decreased (p ≤ 0.01) early in treatment, with decreases of 50% compared to controls at Week 4 a nd decreases of 38% at Week 8. However, overall body weight gains were comparable to controls.

At 10,240 ppm, weekly food consumption was decreased during the first six months of the study in the males and generally throughout treatment in the females, with greater decreases in the females (decreased ≤ 18% compared to controls) than in the males (decreased ≤ 8%). Average food consumption for the overall study was decreased in both sexes by 2% compared to controls, with the difference attaining significance (p ≤ 0.05) in the females.

Cholesterol was increased (increased 7-48%) in the ≥ 1280 ppm males throughout treatment. Total protein was decreased (decreased 8-14%; p ≤ 0.05) at Weeks 39 and 52 in the ≥ 1280 ppm males and in the 10,240 ppm females. Albumin was decreased (decreased 7-20%; p ≤ 0.05) at 10,240 ppm in both sexes throughout treatment. Alkaline phosp hatase was increased (increased 39-68%) throughout treatment in the 10,240 ppm males.

Alanine aminotransferase (ALT) was severely decreased (p ≤ 0.01) in all treated groups of both sexes, with average values of <1 to 3 mU/mL in the treated groups compared to 18-27 mU/mL in
the controls. It was stated that normal ALT leve ls were attained when a pre-incubation stage was incorporated into the assay using pyridoxal-5'- phosphate (IFCC reagent) and that the initial decreases were attributed to a decrease in pyridox al-5'-phosphate, a cofactor necessary for the full expression of ALT in addition to the metabolism of cysteine conjugates of β -lyase. The reduction in ALT activity was therefore consid ered not to be an indication of toxicity but a consequence of pyridoxal-5'-phosphate depletion by β -lyase during the metabolism of chlorothalonil. However, no data were provided for the levels measured from the assay incorporating this pre-incubation stage with pyridoxal-5'-phosphate.

Non-protein thiol concen tration was increased (p ≤ 0.05) compared to controls in the: (i) liver and kidneys of the 10,240 ppm males (increas ed 30-40%); (ii) kidneys in the ≥ 160 ppm females (increased 30-80%); (iii) liver in the >=1280 ppm females (increas ed 60%); and (iv) stomach in the 10,240 ppm females (increased 20%). It was stat ed that these increases are likely due to the conjugation of chlorothalonil with glutathione in these organs.CHLOROTHALONIL FINAL

Page 55 of 61 At ≥ 1280 ppm, absolute liver weights in the males and absolute and adjusted liver weights in the females were increased (increased 9-21%), with the adjusted va lues in the females attaining significance. Increased absolute (increased 27%) and adjusted (increased 47%; p<=0.05) thyroid weights were noted in the 10,240 ppm males. However, there were no macroscopic or microscopic findings to corrobor ate an effect of treatment in either of these organs.

In males, an increased incidence of minimal hyper trophy of the cells of the zona fasciculata of the
adrenals was seen at the low (2/4) and mid (2/4) dose group dogs compared to controls males (114). All high dose males (4/4) ha d moderate hypertrophy of the ce lls of the zona fasciculata.
Adrenal lesions were not seen in females.

The Pathology Working Group concluded that cha nges were limited to the dogs fed at 10,240 ppm, with the exception of a s light increase in brown pigment in corti cal tubules of the kidney in dogs at 1,240 ppm. All treatment-related cha nges were minor in severity considering the magnitude of the dose and length of exposure. They consisted of a small increase in the in cidence and severity of brown pigment in the tubular epithel ium of the renal cortex, a slight increase in severity (but not in incidence) of intraepith elial nuclear pyknosis in the mucosal epithelium of the antrum of the stomach, and a very slight increase in the inciden ce and severity of hypertrophy of the cells in the zona fasciculata of the adrenal glands. Changes c onsidered to be the result of treatment were not
observed in the stomach or adrenals of ma le and female dogs receiving 1,280 or 160 ppm
chlorothalonil in the diet for 52 weeks.

The LOAEL was 10,240 ppm (equivalent to 374/354 mg/kg/day in M/F) based on death in a female, decreased body weight gain and food consumption, macroscopic and microscopic pathological findings in the stomach including: th ickened appearance of the stomach, often with a catarrhal adhesion to the mucosal surface, and in tra-epithelial nuclear pyknosis in the mucosal epithelium of the antrum of the stomach and a ve ry slight hypertrophy of the cells in the zona fasciculata of the adrenal glands. The NOA EL was 1280 ppm (equivalent to 43.3/45.3 mg/kg/day in M/F).

Subchronic Toxicity (Rat, OCSPP 870.3100)

In a subchronic oral toxicity study ( MRID 45710205, 1994 ), 10 Crl:CD (SD) BR rats/sex/dose were exposed to Chlorothalonil ( 99.18% purity) in the diet at nominal con centrations of 0, 30, 60, 300, or 1500 ppm (equivalent to 0/0, 2.3/2.7, 4.7/5.5, 23.6/28.8, and 117/130 mg/kg/day in males/females) for up to 3 months.

No treatment-related adverse effects were obser ved on mortality, clinical signs, body weight, food or water consumption, food effi ciency, ophthalmoscopic examination, hematology, or clinical chemistry. No treatment-related changes were seen in absolute or relative weights of the testes, ovaries, uterus, thyroid, adrenal and pituitary glands. No trea tment-related histopathologicalCHLOROTHALONIL FINAL

Page 56 of 61 lesions were seen in the testes, epididymides, seminal vesicle, prostate ovaries, uterus, cervix, vagina, mammary, thyroid, adre nal and pituitary glands.

Body weight gain was decreased (p<0.05) by 27-36% in the 1500 ppm group during the first week
of treatment which was attributed to a palatability pr oblem. The forestomach was roughened in all
animals in the ≥ 300 ppm groups. The forestomach was also thickened in the 1500 ppm males (10 treated vs 0 controls) and females (4 treated vs 0 controls). The incidences of minimal to moderate epithelial hyperplasia and hyperk eratosis observed at the limit ing ridge and the non-glandular region of the forestomach were increased in the ≥ 300 ppm groups (7-10/10 treated vs 0/10 controls). The incidence of minimal to mode rate hyperplasia of the duodenum was increased in the 1500 ppm group (3/10 each treated vs 0/10 contro ls). The incidence of minimal epithelial hyperplasia and hyperkeratosis observed at the li miting region of the forestomach was also increased in the 30 and 60 ppm males a nd the 60 ppm females (4-5/10 treated).

The LOAEL was 30 ppm (equivalent to 2.3/2.7 mg/ kg/day [M/F]) based on minimal epithelial hyperplasia and hyperkeratosis observed at the li miting ridge and/or non-glandular region of the
forestomach in males and females. The NOAEL was < 30 ppm (equivalent to < 2.3/2.7 mg/kg/day [M/F]).

Subchronic Toxicity (Dog; OCSPP 870.3100)

In a subchronic oral study ( MRID 45710206, 1994 ), chlorothalonil (99.18% purity) was administered to 4 beagle dogs/sex/dose in the diet at nominal concentrations of 0, 160, 1600, or 16,000 ppm for13 weeks. These dietary levels were equivalent to 0, 5.6, 56.5 and 597 mg/kg/day in males and 0, 6.1, 61.0 and 570 mg/kg/day in females.

There were no treatment-related effects on survival, hematology, ophthalmoscopy, or gross pathology. No treatment-related cha nges were seen in absolute or relative weights of the testes, prostate, ovaries, uterus, thyroid or pituitary glands. No treatme nt-related histopat hological lesions were seen in the testes, epididymides, prostate , ovaries, uterus, mamma ry, vagina, thyroid and pituitary glands.

At 16,000 ppm, body weights were d ecreased throughout treatment in both sexes, resulting in a decrease of 167-188% (p ≤ 0.01) in overall body weight gains. Weekly food consumption was decreased by 3-29% in the females throughout treat ment, resulting in decreased mean overall food consumption of 14%.

At ≥ 1600 ppm, in the liver, minimal foci of necr otic hepatocytes with inflammatory cell infiltration was observed in the males (1-2/4 vs. 0 controls). Also, in the liver at ≥ 1600 ppm, minimal parenchymal foci of inflammatory cells wa s noted in both sexes (1-3/4). Adjusted liver weights were increased by 23-25% (p ≤ 0.05) in the females at these doses. Albumin was decreasedCHLOROTHALONIL FINAL

Page 57 of 61 (p<=0.05) by 5% in both sexes at 1600 ppm at Week 13 and additionally by 13-21% at 16,000 ppm at Weeks 6 and 13.

At ≥ 1600 ppm, trace to minimal brown pigment was obser ved (vs 0 controls) in the epithelium of
the cortical tubules of the kidneys in both sexes (1 to 3 of 4 dogs). Thiol concentration was decreased prior to alkali hydr olysis in the males at this dose (decreased 64-100%; p ≤ 0.05). Additionally at 16,000 ppm, urinalysis showed in creased protein in the females (incr. 91%; p ≤ 0.01), and thioether c oncentration (per μ mole creatinine) was increased in the males for the 0-8 hour urine sample (increased 69%; p ≤ 0.01).

In the adrenals at 16,000 ppm, increased width of the zona glomerulosa was noted in the females (3 of 4 dogs vs 0 controls), and absolute adre nal weights were increas ed (increased 21%; p ≤ 0.05)
in the males.

The LOAEL for this study was 1600 ppm (equiva lent to 56.5/61.0 mg/kg/da y in M/F) based on minimal foci of necrotic hepatocytes with infl ammatory cell infiltration in males, and minimal parenchymal foci of inflammatory cells in bot h sexes. The NOAEL was 160 ppm (equivalent to 5.6/6.1 mg/kg/day).

Avian Reproductive Toxicity (OCSPP 850.2300)

In an avian reproduction study with the mallard duck ( Anas platyrhynchos ), ( MRID 00041441

1976 ), birds of unspecified age were administered chlorothalonil (99.6% pur ity) in the feed at nominal concentration of 0, 5, or 50 mg/kg-diet. Five pens were us ed at each test level with 2 males and 5 females per pen. There were no si gnificant effects (p>0.05) to any reproductive parameters examined in the study up to and includ ing the 50 ppm concentra tion.. It was not noted whether any mortality occu rred in the study.

In an avian reproduction study with the mallard duck ( Anas platyrhynchos ), ( MRID 4096402,

1988 ), birds were administered ch lorothalonil (98.3%) in the feed at nominal concentration of 1000, 5000 and 10000 mg/kg-diet. There were no significant effects (p>0.05) to any reproductive parameters examined in the study up to and incl uding the 10000 ppm concentration. No clinical signs were reported the study.

In an avian reproduction st udy with the bobwhite quail ( Colinus virginianus) ( MRID 00041440,

1976 ), birds of unspecified age were administered chlorothalonil (99.6% pur ity) in the feed at nominal concentration of 0, 5, or 50 mg/kg-diet. Twelve pens were used, with 1 male and 2 females per pen. There was no mo rtality or effect on any reproductive parameter tested up to and including the 50 ppm concentration.

In an avian reproduction st udy with the bobwhite quail ( Colinus virginianus) , ( MRID 4096404,

1988 ), birds were administered ch lo rothalonil (98.3%) in the feed at nominal concentration ofCHLOROTHALONIL FINAL

Page 58 of 61 1000, 5000 and 10000 mg/kg-diet. Signi ficant effects on reproductive parameters and clinical signs of toxicity were repor ted at 5000 and 10000 ppm.

In another avian reproduction study ( MRID 45710218, 1993 ), 6-month old Bobwhite quail were administered chlorothalonil (99.18% purity) in the feed at nomina l concentrations of 0, 40, 160, or 640 mg/kg diet for 22 weeks (10 weeks prior to th e start of egg production and 12 weeks of egg production). Mean-measured concentrations were 1.5 (<LOD, control), 41, 153, and 624 ppm, respectively. Each treatment group was comprised of 24 mating pairs, with one pair per pen. A progressive photo period was em ployed in this study: 7 hours/day through Week 8 and 17 hours/day until the end of the study. Temperatur e was maintained at 23-27°C throughout the study and humidity was maintained at ca 55%. Individu al adult body weights were recorded at Week 0, 2, 4, 6, 8 and 22, and body weight changes were determined for each interim period. Food consumption in each replicate was measured week ly throughout the pre-treatment and treatment periods of the adult phase. I ndividual chick body weights were r ecorded after hatching and on Day 14 after hatching; egg shell th ickness was measured every tw o weeks during egg production.
Adult birds and chicks were observed rout inely for mortality and clinical signs. There was no reported treatment-related effect on adult mortality. There were 1, 2, and 5 deaths during the study in the low-, medi um-, and high-dose groups, respectively, but the deaths were generally attributed to pen-mate aggression. Adult body weight change s and food consumption during the treatment period were generally similar in all groups, and there was no evidence of a
treatment-related effect. No treatment-related effects were observed on egg quality, fertility, embryonic development, hatchabili ty, or survival of hatchlings . Although not statisticallysignificant, a treatment-related re duction in the number of eggs laid/hen was observed at 640 ppm,
which also resulted in a reduction of the number of 14-day old survivors/hen. The number of eggs laid/hen were in the control, low-, mi d- and high-dose groups were 62.0, 62.4, 68.9, and 51.0 respectively, and the resulting number of 14- day old survivors/hen were 37.2, 42.8, 42.2, and 30.4, respectively. These findings are c onsidered to be biologically signi ficant. Therefore, reproductive LOAEC is 624 ppm based on decreased e gg production, and the NOAEC is 153 ppm.

Life-cycle Toxicity in Freshwater Fish (OPPTS 850.1500)
! In a life-cycle toxicity study ( MRID 00030391, 1980 ). Fathead minnows ( Pimphales promelas ) were exposed to chlorothalonil (96% purity) under flow-t hrough conditions, beginning as embryos (F 0 ) through to spawning and hatching of the subsequent generation (F 1 ). Mean-measure concentrations of chlorothalonil were 0 (c ontrol, <LOD), 0.6, 1.4, 3.0, 6.5 and 16 μg a.i./L. The following parameters were asse ssed: hatching success of F 0 generation eggs, survival of F 0 generation fry at 35 and 64 days, number of spawns of F 0 generation fish, number of F 1 eggs per spawn of F 0 fish, hatching success of F 1 generation fry at 34 days.

Chlorothalonil significantly reduced hatching su ccess and survivability of fathead minnows at concentrations as low as 6.5 ppb, with most deaths occurred before Day 35 at all concentrations.CHLOROTHALONIL FINAL

Page 59 of 61 The hatching success of F 1 generation eggs spawned from exposed F 0 parents at the 6.5 ppb test level was significantly less than the control in the F 1 generation, less than the F 0 generation eggs at the 6.5 ppb test level, and less than the F 1 generation eggs exposed to 6.5 ppb chlorothalonil but spawned from unexposed F 0 parents. The growth of fish exposed to chlorothalonil was not reduced significantly compared to the c ontrols. Although reductions in the number of eggs per spawn and hatchability of F 1 eggs only attained significance at 6.5 μg/L , a decrease in the total number of spawns and eggs was observed in the 3.0 μ g/L compared to the control. The number of eggs per female in the negative control was 494 whereas it was 103 in the 3.0 μ g/L group; however, because there is inherent variability in egg pr oduction and the use of only two replicates, this decrease (79% difference) was not significant. Ho wever, because this decrease is thought to be biologically significant and tr eatment-related, the LOAEC is 3.0 μg/L and the NOAEC is 1.4 μ g/L where the number of eggs per female was 459.

! !CHLOROTHALONIL FINAL

Page 60 of 61 APPENDIX 3: References Not Utilized in the Chlorothalonil 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 Chlorothalonil but we re not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the compound of interest wa s not used in the study; 3) the test material was not adequately describe d; 4) a formulated end- use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only da ta on invertebrates.

Andersen, H.R., A.M. Vinggaard, T.H. Rasmussen, I.M. Gjermandsen, and E.C. BonefeldJorgensen. (2002) Effects of currently used pestic ides in assays for estr ogenicity, androgenicity, and aromatase activity in vitro

. Toxicology and Applied Pharmacology 179:1-12.

Diamond Shamrock Corp. (1982b) A Chronic Dietary Study in Mi ce with DS-3701: Document No. 098-STX-78- 0024-001. (submitted by Diamond Shamrock Agricultural Chemicals, Cleveland, OH; CDL: 0 71531-A; 071532; 071533; 071534; 071548; 071530), April 21, 1983. MRID 00127848. Unpublished.CHLOROTHALONIL FINAL

Page 61 of 61 Diamond Shamrock Corp. (1981) A 90-day Toxicity Study of Technical Chlorothalonil in Rats: Document No. 099-5TX-80-0200-006. (submitted by Diam ond Shamrock Agricultural Chemicals, Cleveland, OH; CDL:071535- E; 071536), April 21, 1983. MRID 00127850. Unpublished.

Hastings, T.F.; Stemmer, K.L. (1975) 120-Day Di etary Toxicity Study--Rats: DAC-3701: Project No. 24-051. Final report. Bio/Tox Research Labo ratories, Inc., submitted by Diamond Shamrock Agricultural Chemicals, Cleveland, OH; CDL:095783-A). MRID 00047936. Unpublished.

Nishihara, T., J. Nishikawa, T. Kanayama, F. Da keyama, K. Saito, M. Imagawa, S. Takatori, T. Kitagawa, S. Hori, and H. Utsumi. (2000) Estr ogenic activities of 517 chemicals by yeast twohybrid assay. Journal of Health Science 46:282-298.

Petit, F. P. Le Go, J.-P. Cravedi, Y. Valota ire, and F. Pakdel. (1997). Two complementary bioassays for screening the estrogenic potency of xenobiotics: Recombinant yeast for trout estrogen receptor and trout hepatocyte cultures. Journal of Molecular Endocrinology 19:321-335.

Serrone, D.M. and F.C. Killeen, Jr. (1990) A 90-da y oral toxicity study in dogs with 3-carbamyl2,4,5 trichlorobenzoic acid. Biodynamics . Document No.: 3080-88-0082-TX-003. MRID 41564805.

Soto, A.M., C. Sonnenschein, K.L. Chung, M.F. Fe rnandez, N. Olea, and F.O. Serrano. (1995) The ESCREEN assay as a tool to id entify estrogens: An update on estr ogenic environmental pollutants. Environmental Health Perspectives 103 (suppl. 7):113-122.

Yu, S., Wages, M. R., Cobb, G. P., and Maul , J. D. (2013). Effects of chlorothalonil on development and growth of amphibian embryos and larvae. Environ Pollut . 181:329-334.