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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

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

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

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

.

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

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

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

CHEMICAL: DICHLOBENIL

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYDICHLOBENIL FINAL

ii TABLE OF CONTENTS

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

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

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

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

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

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

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

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9

1. Effects on Estrogen Pathway.......................................................9

2. Effects on Androgen Pathway....................................................14

C. Effects on Hypothalamic-Pituit ary-Thyroidal (HPT) Axis...........................19

IV. Committee’s Assessment of Weight of Evidence..........................................21

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................22

1. Tier 1 in vivo Assays..............................................................22

2. OSRI................................................................................22

B. Estrogen Pathway...........................................................................23

C. Androgen Pathway.........................................................................24

D. Thyroid Pathway...........................................................................24

E. Conclusions.................................................................................25

V. EDSP Tier 2 Testing Recommendations....................................................25

VI. References.......................................................................................26

LIST OF TABLES: Table 1. Tier 1 Assay for Dichlobenil.....................................................................8
Table 2. Estrogenic/Anti-Estroge nic Pathway for Dichlobenil.......................................11
Table 3. Androgenic/Anti-Androgenic Pathway for Dichlobenil.....................................16
Table 4. Thyroid Pathway for Dichlobenil..............................................................20

APPENDIX 1. EDSP Tier 1 Screening Assays.................................................................29

APPENDIX 2. Other Scientifically Relevant Information (OSRI).......................................40

APPENDIX 3. References Not Utilized in the Dichlobenil WoE Analysis.............................52DICHLOBENIL 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 AR 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 CAR 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

DER 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 )
ER 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 eneration
F2 Second filial g eneration
Fcd Fecundit yDICHLOBENIL FINAL

iv Abbreviation Terminology

FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y FQPA 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/M 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 Concentration

MTD Maximum Tolerated Dose
N Ne g ative
NE Not examined/evaluate d NF stage Nieuwkoo p and Faber’s Sta g in g AtlasDICHLOBENIL FINAL

v Abbreviation Terminology

NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level
NS Not Statisticall y Si g nificant
NR 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 )
PXR Pre g nane X rece p to r QC Quality Control

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel
SC Solvent Control

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

SEP Standard Evaluation Procedure
SD Standard Deviation or Sprague-Dawley

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

T4 Thyroxine (tetraiodothyronine)

TP Testosterone Pro p ionate
TR 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 OpeningDICHLOBENIL FINAL

vi Abbreviation Terminology

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

Page 1 of 52 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-pituitary thyr oid (HPT) axes. In addition to the available Tier 1 assay da ta, 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 dichlobenil interacts with E, A, or T hormone path ways, the number and type of effects induced, the magn itude and pattern of responses obs erved across studies, taxa, and
sexes were considered. Additi onally, the conditions under which effects occu r were considered, in particular, whether or not e ndocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On January 15, 2014, the Tier 1 Assay Weight of Evidence Review Comm ittee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination (OSCP) and Policy conducted a weight-of evidence (WOE) analysis of the potential intera ction of dichlobenil with the E, A or T hormone pathways. The T1WoERC conc lusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited OSRI for mammalian and wildlife studies ( 40 CFR Part 158 and literature).

For the estrogen pathway, there was no convi ncing evidence supporting po tential estrogenic or anti-estrogenic effects for dich lobenil. The Tier 1 ER binding, ERTA, and aromatase assays as well as the uterotrophic assay were negative, wher eas the results of the st eroidogenesis assay were equivocal (non-concentration dependent decreases in estradiol production) . Decreased uterine weights (wet and blotted) were noted in the female pubertal study, but there was no effect on uterine weights after adjustment for PND 21 body weights. In the FSTRA, reduced fecundity and changes in ovary histopathology ( ↑ oocyte atresia and a shift to mo re mature stages) were observed at the mid and high test concentration (0.21 and 2.1 mg/L). In the high treatment discoloration ( i.e ., lighter) in the fish was reporte d, which may be suggestive of stress. There were no effects on fertilization, GSI or VTG in this study. In the OSRI, estrogen-related findings in the absence of overt toxicity included a decrea se in uterus and ovary weight s in the Part 158 chronic dog study (which occurred in the presence of increased liver weights). Decreases in egg laying and hatchling success (survival and/or weight) were obser ved in the mallard duck and bobwhite quail reproduction study (decreases in male body we ight observed in the bobwhite quail study). Decreases in fecundity (fish and bi rds) in of themselves are limite d in their diagnostic specificity for the estrogen-pathway. A slight 4.2% decr ease in eggshell thinni ng was observed in the bobwhite quail study, however, the Tier 1 in vitro and in vivo uterotrophic assays were negative or equivocal (steroidogenesis, non-c oncentration decreases in estradiol productio n), and no effect onDICHLOBENIL FINAL

Page 2 of 52 eggshell thinning was observed in the mallard duck study with no or limited estrogen-related
effects observed in the other in vivo studies (sometimes observed in the presence of systemic toxicity).

For the androgen pathway, dichlobenil was a bind er in the Tier 1 AR binding assay and was equivocal in the steroidogenesis assay. No androgen-related eff ects were observed in the male pubertal assay and effects in the He rshberger assay occurred in the presence of overt toxicity. In the FSTRA dose-dependent re ductions in median male tubercle sc ores at all three dose levels (21, 14, and 12 in low, mid and high treatment, respectiv ely vs. 26 negative control); however, it is
noted that there were no other an drogen-related effects observed. It is noted that observation of discoloration in the fish was reported for the high treatment group. In the mice and rat OSRI studies, changes in androgen-rela ted endpoints occurred concomitantly with increases in liver weights. However, in hamsters, changes in pr ostate weights and histopa thology of the prostrate
and seminal vesicles occurred in the absence of over t toxicity or liver effect s. Effects on hatchling success (numbers and survival) was observed in both the bobwhite quail and mallard duck study, however, these endpoints are limited in informi ng potential androgen-related interactions.

The weight of evidence suggests that Dichlobenil may potentially interact with the androgen pathway based on: (i) positive AR binding; (ii) decreases in testosterone production in the steroidogenesis assay; (iii) tube rcle score reductions in FSTRA; and (iv) decreases in hamster testes and prostate weights and changes in pr ostate histopathology in the Part 158 studies.

There is no convincing evidence for a potential effect of dichlobe nil on the thyroid pathway. No thyroid-related effects were observed in the male or female pubertal assays. In the AMA, there was no effect when compared to solv ent control for the developmental endpoints. In the OSRI, there were no thyroid-related effects in the absence of overt toxicity ex cept for an increase in thyroid weight without any corresponding ch anges in thyroid histopathology in dogs in the chronic study.

For dichlobenil, the point of departure (POD) is 1 mg/kg/day used for human health risk assessment is based on liver toxicity (changes in liver enzymes, increased liver weights, and periportal hepatocytes hypertrophy ) in male dogs at 6 mg/kg/day (LOAEL) in a chronic toxicity study. This POD is used to derive the chronic Re ference Dose (RfD) for dietary exposure risk assessments. The POD of 3.0 mg/kg/day used for calculating Margin of Exposure (MOEs) for nondietary exposure risk assessments is base d on decreased pup body wei ght at weaning at 17.5 mg/kg/day (LOAEL) in a two-generation reprod uction study. Accordingly, the PODs (1 and 3 mg/kg/day) used for the overall risk assessments are 20 to 60-fold lower than the lowest dose (60 mg/kg/day) where a potentially androgen related effect was observed in the male pubertal assay. Consequently, the current RfD and MOEs are prot ective of potentially endo crine-related effects observed in the pubertal assay.

Based on weight of evidence considerations, mamm alian EDSP Tier 2 testing is not recommended for dichlobenil since additional testing will not impact the regulatory endpoints used for human
health risk assessments.DICHLOBENIL FINAL

Page 3 of 52 EDSP Tier 2, Medaka Extended One Generation Reproduction Test (MEOGRT) is recommended due to the potential interaction with the androge n pathway and the effects observed in the FSTRA assay. Currently, the chronic NOAEC endpoint for fish is <0.33 mg/L, based on growth effects at all concentrations tested in an early life-cycle study with rainbow trout. Effects on male tubercle
score in the FSTRA were observed at all concentrations tested ( ≥ 0.021 mg/L); additionally, fecundity was decreased at 0.21 and 2.1 mg/L. Th erefore, a Tier 2 MEOGRT is requested to provide data to understand potential effects of dichlobenil on fish reproduction and development.

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

.DICHLOBENIL FINAL

Page 4 of 52 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-pituitary thyr oid (HPT) axes. In addition to the available Tier 1 assay da ta, 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 a pesticide chemical inte racts with E, A, or T hormone pathways, the number and type of effects i nduced, the magnitude and pattern of responses observed across studies, taxa, and sexes we re considered. Additi onally, the conditions un der which effects occur were considered, in partic ular, whether or not endocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On January 15, 2014, the Tier 1 Assay Weight of Evidence Review Comm ittee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Offi ce of Science Coordination and Policy (OSCP) conducted a weight-of evidence (WOE) analysis of the potential intera ction of dichlobenil with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited OSRI for mammalian and wildlife studies ( 40 CFR Part 158 and literature).

Dichlobenil has a water solubi lity of 21.2 mg/L (at 25°C), a vapor pressure of 0.00101 mm Hg (at 25°C) and a partition coefficient (log K ow ) of 2.74. Dichlobenil dissipa tes in the environment principally by volatilization, although it may persis t under conditions that reduce the potential for volatilization. It is stable to hydrolysis, photodegradation in soil a nd metabolic degradative processes. Bioconcentration factors in fish are 32X, 63X and 110X for fillet, whole fish and viscera, respectively, with a high depuration rate (within 1 day of the 14 da y depuration period).

The available information considered in determin ing the potential interacti on of dichlobenil with the E, A, T pathways include submitted EDSP Tier 1 assays and other scientifically relevant information (OSRI) such as general toxicity studies and/or other published articles. These data are summarized in Sections III.A through III.C. An analysis of the data submitted to the agency, using the WoE approach outlined by the Agency (USEPA , 2011), is presented in Section IV. The EDSP
Tier 2 studies recommendations are presented in Section V.DICHLOBENIL FINAL

Page 5 of 52 II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

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

1.

B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Or ders, data believed 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 literature and/or data submitte d to support pesticide registration (e.g., Part 158 guidelin e studies). The Agency’s review of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team on dichlobenil (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any
additional relevant information. Summaries of the available OS RI are presented in Appendix 2. Summaries of the Tier 1 assays a nd the studies cited as other scie ntifically relevant information (OSRI) are presented in Appendix 1 and 2, respectiv ely. Additionally, literature/studies considered
but not utilized for the WoE anal ysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

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

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

Page 6 of 52 As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological ev ents including potential effects on receptor binding (estrogen and androgen agon ist and antagonist), steroidogene sis, and other effects on the HPG and HPT axes. OSRI may include published li terature studies as we ll as studies conducted under USEPA (often referred to as Part 158 data ) or OECD guidelines submitted in support of pesticide registrations. Each st udy is evaluated for scientific qu ality and relevance for informing interactions with the E, A, or T pathway. Add itionally, the consistency of the responses in the individual study is eval uated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a specific endpoint for discerning wh ether or not the chemical has interacted with the endocrine system, are consid ered. The collective resp onse of the individual endpoints, as well as the condi tions under which they were e xpressed, are considered when evaluating an overall indication 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 char acterization involves thr ee components. The first component is whether the data provide relevant , robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher-levels of bi ological organization? Finally, under what conditions dide the responses occur including consideration of whether the responses were observed in the presence of overt or systemic toxicity? The pr esence of overt and/or systemic t oxicity introduces uncertainty inDICHLOBENIL FINAL

Page 7 of 52 the ability to distinguish effect s specifically related to an endo crine-related effect from a nonendocrine toxic response.

This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific i ssues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, weight should be placed on signs of endocrine di sruption 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).

For these WoE analyses, overt toxicity was gene rally defined in accordance with EPA’s current approach as used by OPP in reviewing 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., organ weights, blood chemistry). The circumstances for which a dose/concentration was considered overtly toxic for a particular study are described in Section

IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a ch emical’s physical/chemical properties and its known
modes of action in its overall charac terization of a chemical’s potential to interact with the E, A or T pathway. Adequately addressing these three ma in components described above is fundamental to the WoE process and in determining whethe r additional 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 additional data is based on a case-by-case analysis which took all available toxicity data into account.DICHLOBENIL FINAL

Page 8 of 52 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 biologi cally relevant in regards 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 th e presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not re ported in the table for E, A or T.

A. EDSP T1 Screening Assays

The Tier 1 assays submitted in response to the ag ency’s test order for dichlobenil are shown below
in Table 1

.

Table 1: Tier 1 Screening Assays for Dichlobenil.

Tier 1 Assays Test Guideline Test Order Status

Estrogen Receptor (ER) Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied (MRID No. 48616104)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48616105)

Androgen Receptor (AR) Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied ( MRID No. 48616102 )

Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48616110)

Aromatase Assay (Human recombinant microsomes) OSCPP 890.1200 Requirement Satisfied (MRID No. 48616103)

Uterotrophic Assay (Rat) OSCPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48616111)

Hershberger Assay (Rat) OSCPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48616107)

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied (MRID No. 48616108)

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied (MRID No. 48616109 )

Fish Short-term Reproduction Assay OSCPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48616106)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 48616101)DICHLOBENIL FINAL

Page 9 of 52 B. Effects on Hypothalamic-Pit uitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The data utilized to assess the pot ential for dichlobenil to interact 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 determinat ion of potential fo r estrogenic, anti-estrogenic, or
HPG axis effects. This table also includes HPG- relevant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reported in the table and not considered fu rther in the WOE assessment.

Dichlobenil did not produc e responses indicative of estrogen agonism or antagonism in the ER binding assay, the ERTA assay, the aromatase assay, or the in vivo uterotrophic assay. However, the results of the Tier 1 steroidogene sis assay indicated that no changes in estradiol concentration were observed during Runs 1 and 2, but estradiol wa s decreased (p ≤ 0.05) in Run 3 at all dichlobenil concentrations except 100 and 0.1 μ M. Therefore, the results of the steriodogenesis assay were considered equivocal. In the female pubertal assa y, there was a dose-related decrease in mean absolute blotted uterus weight at doses of 20 mg/kg/day [( ↓ 12%; not significant [NS])] and 60 mg/kg/day (21%; p<0.05) and a decrease (NS) in m ean absolute wet uterus weight at 60 mg/kg/day ( ↓ 26%). Overt toxicity was not noted in the fema le pubertal assay; however, liver weights were increased by 18-19% at the high dose. In the FSTRA, fecundity was significantly (p<0.05) reduced by 53% and 90% at the 0.21 and 2.1 mg a.i./L tr eatment levels, respectiv ely, compared to the negative control. Also in the FS TRA, there was an increased incide nce and/or severity (minimal to moderate) of oocyte atresia and a sh ift to more mature stages in the ovaries in the 0.21 and 2.1 mg
a.i./L treatments. Fish at the hi gh dose in the FSTRA were reported to be discolored (i.e. lighter) compared to fish in other treatment groups includ ing controls whic h may suggest signs of stress in this treatment group, however, the incidence of th is effect was not reported.

In a Part 158 chronic toxicity study in dogs, absolute ( ↓ 50%) and relative ( ↓ 54%) ovarian weights and absolute ( ↓ 22%) and relative ( ↓ 24%) uterine weights were decrease d at the high dose. No overt toxicity was noted in this study, but both relative and absolute liver weights were increased by

≥ 18%. At the high dose (36 mg/kg/day), decreased uterine weights were accompanied by “early” maturation of the uterus (5/5). In the 2-generation reproduction toxicity study in rats, a decrease in implantations/dam (12.3 treated vs . 14.6 controls) was observed at 2000 ppm; however, this effect occurred in conjunction with overt toxicity based on decreased bod y weights. There were no other
effects observed in the 158 mammalian data.

In the Part 158 quail reproduction studies, decreases in egg laying were observed at the mid and highest dose tested (289 and 600 mg /kg-diet) as well as decreases in eggshell thinning (4.2%), number of hatchings and offspring weight at 600 ppm. Additionally, 14-day old survivors were
affected at all concentrations ( ≥ 146 mg/kg-diet). In this study, reductions in male body weight gain
were observed at all concentra tion (25-50% non-treatment responsiv e), however, no other clinical signs of toxicity were observe d. In the m allard duck reproducti on study, decreases in eggs laid,DICHLOBENIL FINAL

Page 10 of 52 number of hatchlings, and 14 day old survivors were also observed at 600 mg/kg-diet and signs of toxicity were not observed in the mallard duck study. In the Part 158 ea rly life-stage study with rainbow trout, effects on length were observed at ≥ 0.33 mg/L.DICHLOBENILFINALPage 11 of 52Table 2: Estrogenic/Anti-Estrogenic Pathway for DichlobenilLines of Evidence Indicating Potential Interaction with theEstrogenic/Anti-EstrogenicPathway for Dichlobenil1Study 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 AssaysER Binding(MRID 48616104)NERTA(MRID 48616105)NAromatase(MRID 48616103)NSteroidogenesis(MRID 48616110)EUterotrophic(MRID 48616111)NX(H)Female Pubertal Rat(MRID 48616108)↓21-26%(H)NNNNNLW(H)NFSTRA(MRID 48616106)NENP4(M,H)NFcd:↓53%,↓90%(M,H)NN5OSRISubchronic toxicity(Rats; MRID 00107106)NNNNX(M,H)X(H)DICHLOBENILFINALPage 12 of 52Lines of Evidence Indicating Potential Interaction with theEstrogenic/Anti-EstrogenicPathway for Dichlobenil1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Subchronic toxicity(Hamsters; MRID40600701)NNNNX(MH,H)X(H)Subchronic toxicity(Mice; MRID 48043302)NNNNX(M,MH,H)X(MH, H)Chronic toxicity(Dogs; MRID 43969701)↓78%(H)↓50%(H)P6(H)NX(M,H)NChronic toxicity /carcinogenicity(Rats; MRIDs 00147438,40401101)NNNNX(M,H)X (H)High-dose carcinogenicity(Hamsters; MRID42221201)NNNNX(L,M,H)X(L, M,H)Low-dose carcinogenicity(Hamsters; MRID41988301,42015101)NNNNX(H)X(H)Two-generationreproduction toxicity(Rats; MRID 41257303,42239101)NNNNX(M, H)Developmental toxicity(Rabbits; MRID41257302)NNX(H)DICHLOBENILFINALPage 13 of 52Lines of Evidence Indicating Potential Interaction with theEstrogenic/Anti-EstrogenicPathway for Dichlobenil1Study 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(Rats; MRID 00147437)NNX(M, H)Avian Reproduction(Quail; MRID 45280301)P7(L,M,H)NN8Avian Reproduction(Mallard; MRID45280302)P9(H)N N1 Key to responses: L=Low treatment, ML=Medium-low treatment,M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows (↓or↑) indicate the direction of a given response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. LW=Liver Weight 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 by an Xin this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Increased incidence and/or severity (minimal to moderate) ofoocyte atresia and a shift to moremature stages in the ovariesin the 0.21 and 2.1 mg a.i./Ltreatments.5. Fish were discolored (i.e. lighter) compared to fish in other treatment groups including controls, however, the incidence ofthis effect was not reported6. At the high dose (36 mg/kg/day), decreased uterine weights were accompanied by “early” maturation of the uterus (5/5).7. Decreases in egg laid at the mid and highest dose tested (289 and 600 mg/kg-diet) as well as decreases in eggshell thinning(4.2%), number of hatchingsand offspring weight at 600 ppm. Additionally, 14-day old survivors were affected at all concentrations (≥146 mg/kg-diet).8. Reductions in male body weight gainwere observed at all concentration (25-50%non-treatment responsive), however, no otherclinical signs of toxicitywere observed.9. Decreases in eggs laid, number of hatchlings and 14-d offspring survival at 600 mg/kg-diet.P Positive findingsN Negative findings (in vitro) / No effect (in vivo)E Equivocal findingsDICHLOBENIL FINAL

Page 14 of 52 2. Effects on A ndrogen Pathway

The data utilized to assess the pot ential for dichlobenil to interact with the androgen pathway are
summarized in Table 3. The various targets of th e androgen pathway across the relevant Tier 1 assays are delineated so as to facilitate determin ation of potential for andr ogenic, anti- androgenic,
or HPG axis effects. This table also includes HP G-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxi city are discussed in the text but are not reported in the table and not considered fu rther in the WOE assessment.

Dichlobenil was a binder in the AR assay, and gave equivocal results in the steroidogenesis assay based on a non-dose-de pendent decrease in test osterone levels in two of three runs. In the Hershberger assay decreased seminal vesicle, ventral prostate, LABC, and Cowper’s gland weights at the high dose of 180 mg/kg/day; howev er the effects occurred in conjunction with overt toxicity ( ≥ 10% decrease in body weight). At the mid dose (60 mg/kg/day), a decrease in seminal vesicle weight was observed; however, th is dose was considered to be negative since only one of the five androgen-responsive tissue weights was decreased (a positive response requires changes in two or more of the five AST weights). Additiona lly, liver weights were increased (32-40%) at the mid and high dose. In the FSTRA, dose-de pendent reductions in
median male tubercle scores were observed at all three dose levels (21, 14 and 12 in low, mid and high-concentration vs. 26 in negative control). There were no androgen-related effects observed in the male pubertal assay.

In the Part 158 studies, testes (with epididym ides) weights were decreas ed 12-13% at 117 and 277 mg/kg/day, and pigmented macroph ages and dilated seminiferous tubules were observed at 277 mg/kg/day in a carcinogenicity study in hamste rs. In another carcinogenicity study evaluating lower doses in hamsters, reduced secretion of th e prostate and seminal ve sicles was noted at 9.4 and 46 mg/kg/day. In a subchronic study in hams ters, testes weights were decreased 33% and seminal vesicle weights were d ecreased 31%, both at 263 mg/kg/ day; prostate weights were decreased 19-36% at 16, 79, and 263 mg/kg/day. In addition, a nd small testes, decreased number of spermatocytes, prostatic mine ralization, tubular degeneration in the testes, small epididymides, and slight hemorrhagic area in the epididymid es were noted at 263 mg/kg/day. In a chronic toxicity study in dogs, testes weights were increa sed by 33% at the high dose (36 mg/kg/day) and there were dose-dep endent decreases ( ↓ 14-37%) in prostate weights, which attained significance (p<0.05) at the high dose. Finally, in a subchr onic study in mice, absolu te and relative testes weights were increased 7-13% at 19, 95, and 473 mg/kg/day. Overt toxicity was noted in each of these studies at the high dose and three of these st udies reported increases ( ≥ 13%) in liver weights. No androgen-related effects were seen in the remaining Part 158 studies.

In the Part 158 avian reproduction studies, e ffects on offspring survival were observed ≥ 146

m g/kg-diet and number of hatchlin gs/offspring weight at 600 mg/k g-diet in the bobwhite quail study; decreases in number of hatchlings/offspring survival at 600 mg/kg-diet was observed in the mallard duck study . In the bobwhite quail study, reductions in male body weight gain wereDICHLOBENIL FINAL

Page 15 of 52 observed at all concentration ( 25-50% non-treatment responsive), how ever, no other c linical signs of toxicity were observed.DICHLOBENILFINALPage 16 of 52Table 3: Androgenic/Anti-Androgenic Pathway for DichlobenilLines of Evidence Indicating Potential Interaction with theAndrogenic/Anti-AndrogenicPathway for Dichlobenil1Study Type/ LiteratureCitation AR Binding Steroidogenesis Testosterone Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity 2 Overt Toxicity Observed 3EDSP Tier 1 AssaysAR Binding(MRID 48616102)PSteroidogenesis(MRID 48616110)PHershberger(MRID 48616107)N↑LW(M,H)↓≥10%BW(H)Male Pubertal Rat(MRID 48616109)NNNNNNNNLW(H)NFSTRA(MRID 48616106)NNNTuberclescore:↓(L,M, H)NN4OSRISubchronic toxicity(Rats; MRID 00107106)NNNNNNX(M,H)X(H)Subchronic toxicity(Hamsters; MRID40600701)NNNNNProstate:↓19 -36%;(ML,MH)X(ML,MH,H)X(H)DICHLOBENILFINALPage 17 of 52Lines of Evidence Indicating Potential Interaction with theAndrogenic/Anti-AndrogenicPathway for Dichlobenil1Study Type/ LiteratureCitation AR Binding Steroidogenesis Testosterone Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity 2 Overt Toxicity Observed 3Subchronic toxicity(Mice; MRID48043302)↑7-13%(ML,MH)NNNNNX(ML,M,H)X(H)Chronic toxicity(Dogs; MRID43969701)NNNNNNX(M,H)X(H)Chronic toxicity /carcinogenicity(Rats; MRIDs00147438, 40401101)NNNNNNX(M,H)X(H)High-dosecarcinogenicity(Hamsters; MRID42221201)NNNNNNX(L, M, H)X(L, M, H)Low-dosecarcinogenicity(Hamsters; MRID41988301, 42015101)NP(MH)NNNNX(MH, H)X(H)Two-generationreproduction toxicity(Rats; MRID 41257303,42239101)NNNNNNX(M, H)DICHLOBENILFINALPage 18 of 52Lines of Evidence Indicating Potential Interaction with theAndrogenic/Anti-AndrogenicPathway for Dichlobenil1Study Type/ LiteratureCitation AR Binding Steroidogenesis Testosterone Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity 2 Overt Toxicity Observed 3Avian Reproduction(Quail; MRID45280301)P5(L,M,H)NN6Avian Reproduction(Mallard; MRID45280302)P7(H)NN1. 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.LW= Liver Weight. Changes in weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV),Ventral prostate (VP), Dorsal prostate (DP), Levator ani- bulbocavernosus (LABC), Epididymides (EP).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 Xin this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Fish were discolored (i.e. lighter) compared to fish in other treatment groups including controls, however, the incidence ofthis effect was not reported5. Decreases in number of hatchlings/offspring weight at 600 mg/kg-diet. Additionally, 14-day old survivors were affected at all concentrations (≥146mg/kg-diet).6. Reductions in male body weight gainwere observed at all concentration (25-50%non-treatment responsive), however, no otherclinical signs of toxicitywere observed.7. Decreases in number of hatchlings and 14-d offspring survival at 600 mg/kg-dietP Positive findingsN Negative findings (in vitro) / No effect (in vivo)DICHLOBENIL FINAL

Page 19 of 52 C. Effects on Hypothalamic-Pituitary-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 detect changes in the HPT axis, i.e ., the female and male (rat) pubertal assays, and the AMA (frog).

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

No effects on thyroid weights, hormones or histol ogy were noted in the ma le pubertal assay. In the female pubertal assay, serum TSH le vels were increased by 18-22% (20 and 60 mg/kg/day, respectively), but these changes did not attain statistical significance and were not accompanied by any changes in thyroid weig ht or histology. Substantial increases in liver weights were also noted at the high dose in both the male ( ↑ 35-36%) and female ( ↑ 18- 19%) pubertal assays. In the AMA, the 21-day normalized HLL was increased by 7-11% at all concentrations (0.02, 0.2, a nd 2.0 mg/L, respectively), and a lower incidence and severity of follicular cell hypertrophy was noted at 2.0 mg/L compared to the negative control. However there were no significant differences for the 21-day normalized HLL endpoint, when treatment groups were compar ed to the solvent control.

In a Part 158 chronic toxicity study in dogs (MRID 00067649), increased absolute and relative thyroid weights were observed in both se xes. No other thyroi d-related effects were reported in the Part 158 studies.DICHLOBENIL FINAL

Page 20 of 52 Table 4: Thyroid Pathway for Dichlobenil

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

Study Type / Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stageor asynchronous &NHLLGrowth (BW,SVL)SystemicToxicityObserved2Overt SystemicToxicity3

EDSP Tier 1 Assays

Male Pubertal (Rat) N N N N N N N ↑ LW (H) N

Female Pubertal (Rat) N N N N N N N ↑ LW (H) N

AMA (Frog) N Norm. HLL: ↑ 7- 11% (21- d; L, M, H) 4 N N

OSRI

Subchronic toxicity (Rats; MRID 00107106) N N X (M,H) X (H)

Subchronic toxicity (Hamsters; MRID 40600701) N N X (ML, MH, H) X (H)

Subchronic toxicity (Mice; MRID 48043302) N N X (ML, MH, H) X (H)

Chronic toxicity (Dog; MRID 00067649) P (H) N X (H) N

Chronic toxicity (Dogs; MRID 43969701) ↑ 33- 56% (L, M; females ) N X (M, H) X (H)

Co m b ined chronic t oxicity / carcinogenicity ( Ra t s ; MRIDs 00147438

, N N N X (M,H) X (H)
High-dose carcinogenicity (Hamsters; MRID 42221201 ) N N N X (L,M,H) X (L,M,H)
Low-dose carcinogenicity (Hamsters; MRID 41988301

, 42015101 ) N N N X (MH,H) X (H)DICHLOBENIL FINAL

Page 21 of 52 1. Key to responses: L=Low treatment, ML=Med ium-low treatment, M=Medium treatment, MH=Mediumhigh 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. LW = Liver Weight. 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 by an X in this column. For details see Section IV. A 3. The overt toxicity in the Tier 1 assays are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 4. No effect on HLL was observed when treatment groups were compared to solvent control. P Positive finding N Negative finding

IV. Committee’s Assess ment of Weight of Evidence

This section of the document will describe the weight of evidence (WoE) determination on the potential of dichlobenil to interact with endocrine related processes ( i.e., E, A, or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated analysis of those lines of evidence. The WoE evaluation includes considerations of biologi cal plausibility of the findings from the different lines of evidence by examining the consistency, coherence, and interrelationships among the measured endpoints within and across studies. The availabl e findings from standard toxicology studies on the substance may contribute to the WoE evaluation in helping elucidate if effects seen in the Tier 1 assay are related to pertur bations of the endocrine system per se or alternatively a sequelae of systemic effects. Endocrine mode s of action may evoke a number of phenotypic consequences other than those ev aluated in the Tier 1 assays.

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

Page 22 of 52 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 below. In addition to the endocrine-related effects described above
for the in vivo Tier 1 assays and OSRI, other eff ects observed which may be considered overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. However, in other instances, mo re than one parameter was needed to determine overt toxicity. Effects which were considered to be signs of systemic toxicity were generally less severe forms of toxicity (e .g., organ weights, blood chemistry).

1. Tier 1 in vivo Assays

In the uterotrophic assay, the high dose (180 mg/kg/day) caused significant body weight losses (8-9%) on Days 3 and 4. Overt toxicity was not observed in the female pubertal assay; however, liver weights (18-19%) and cholesterol (18%) were in creased at the high dose (60 mg/kg/day). In the Hershberger assay at th e high dose (180 mg/kg/da y), overt toxicity, manifested as decreased body weights (>9-11%) and body weight gains in both the androgenic (>44%) and anti-androgenic (>40%) of the assay. Although overt t oxicity was not identified in the male pubertal assay, liver weights were increased by >35 – 36%, however no clinical chemistry changes were observed.

Discoloration [(i.e., lighter, more yellowish color compared to the fish in the controls and other treatment levels (incidence rate not reported)] was observed in the FSTRA in the high treatment

group.

2. OSRI

The major target organs for dichlobenil-induced toxicity are the liver and the nose in mice and rats and the male reproductive syst em in the hamsters. In a 90-day study with mice, absolute (7- 13%) and relative (7-11%) testes weight were increased at >19 mg/kg/day. In a 90 day oral toxicity study in rats, inflammation and necros is were observed in the liver of males at 45 mg/kg/day, and increased liver weight and liver histopathology (swellin g and vacuolation of hepatocytes) were observed in females at 135 mg/ kg/day. In a 90 day oral toxicity study with hamsters, when administered in the diet at 395 mg/kg/day, there was an excessive body weight loss (>13-25%) in both sexes afte r 2 weeks of treatment. Therefor e, the dose was reduced to 263 mg/kg/day from weeks 3 until week 13. Mean body weight in males at the high dose remained decreased (>21%) at the end of treatment and mean food cons umption was decreased (>11%) after lowering the dose. In males at the high dose, there was increase in absolute liver weights (>13%) and enlarged liver at ne cropsy; histopathology revealed calculi in the gall bladder and hepatocytes in the liver. Mean absolute pr ostate weight was reduced (>19%) 16 mg/kg/day
while mean absolute seminal vesicle and testis weights were reduced at 16 mg/kg/day (>16%)DICHLOBENIL FINAL

Page 23 of 52 and at 263 mg/kg/day (33%). Histopathology revealed a marked decrease in the number of spermatocytes at the high dose. Marginal to m oderate prostatic mineralization was seen at 16 mg/kg/day. Marginal to marked tubular degeneration in the testes was seen at the high dose. In females, increased liver weight, enlarged liver and swollen hepatocytes were seen at 79 mg/kg/day. In the chronic toxicity study in dogs, lower mean relative body weights (12-16%) were seen throughout the study in males only at 36 mg/kg/day. At dos es >6 mg/kg/day, both sexes of dogs exhibited increases in several hepatic enzymes and increased liver weights, males revealed periportal hypertrophy of the liver. Male dogs also exhibited dose-dependent decreases in absolute weight of the prostate, increases in absolute testes weight at 36 mg/kg/day and increases in relative testes weight at 6 and 36 mg/kg/day. None of these weight increases were corroborated with histopathologi cal lesions in these organs . In the combined chronic toxicity/carcinogenicity study with rats, at the high dose, (173 mg/kg/day), there were significant decreases in body weight in males (33%) and females (34%), changes in multiple hepatic enzymes, increases in absolute and relative liver weights, enlarged livers at necropsy, and histopathological lesions of the liver at 18.9 mg/kg/day In a carcinogenicity study with hamsters, at 51 mg/kg/day, there were decrease s in body weight, enlarged liver, increases in absolute and relative liver weights, histopathol ogical lesions changes, and increases in liver

tumors

In the Part 158 bobwhite quail reproduction study , reductions in male body weight gain were observed at all concentration (25-50% non-treat ment responsive), however, no other clinical signs of toxicity were observed. No clinical si gns of toxicity were observed in the mallard duck reproduction study.

B. Estrogen Pathway

There was no convincing evidence supporting potent ial estrogenic or anti-estrogenic effects for dichlobenil. The Tier 1 ER binding, ERTA, and aromatase assays as well as the uterotrophic assay were negative, whereas the results of the steroidogene sis assay were equivocal (nonconcentration dependent decreases in estradiol production). Decreased uterine weights (wet and blotted) were noted in the female pubertal study, but there was no effect on uterine weights after adjustment for PND 21 body weights. In the FSTRA, reduced fecundity and changes in ovary histopathology ( ↑ oocyte atresia and a shift to more mature stages) were observed at the mid and high test concentration (0.21 and 2.1 mg/L). In the high treatment discoloration ( i.e

., lighter) in the fish was reporte d, which may be suggestive of stress. There were no effects on fertilization, GSI or VTG in this study. In th e OSRI, estrogen-related findings in the absence of overt toxicity included a decrease in uter us and ovary weights in the Part 158 chronic dog study (which occurred in the presence of increa sed liver weights). Decreases in egg laying and hatchling success (survival and/or weight) we re observed in the mallard duck and bobwhite quail reproduction study (decreases in male body weight observed in the bobwhite quail study).DICHLOBENIL FINAL

Page 24 of 52 Decreases in fecundity (fish and birds) in of themselves are limited in their diagnostic specificity for the estrogen-pathway. A slight 4.2% decrease in eggshell thinning was observed in the bobwhite quail study, however, the Tier 1 in vitro and in vivo uterotrophic assays were negative or equivocal (steroidoge nesis, non-concentration decreas es in estradiol production), and no effect on eggshell thinning was observed in the mallard duck study with no or limited estrogen-related effects observed in the other in vivo studies (sometimes observed in the presence of systemic toxicity).

C. Androgen Pathway

Dichlobenil was a binder in the Tier 1 AR binding assay and was equivocal in the steroidogenesis assay. No androgen-related eff ects were observed in the male pubertal assay and effects in the Hershberger assay occurred in the presence of overt t oxicity. In the FSTRA dose-dependent reductions in medi an male tubercle scores at a ll three dose levels (21, 14, and 12 in low, mid and high treatment, respectively vs . 26 negative control); however, it is noted that there were no other androgen-related effect s observed. It is noted that observation of discoloration in the fish was reported for the high treatment group. In the mice and rat OSRI studies, changes in androgen-rela ted endpoints occurred concomitantly with increases in liver weights. However, in hamsters, changes in prostate weights a nd histopathology of the prostrate and seminal vesicles occu rred in the absence of overt toxi city or liver effects. Effects on hatchling success (numbers and survival) was observed in both the bobwhite quail and mallard duck study, however, these endpoints ar e limited in informing potential androgenrelated interactions.

The weight of evidence suggests that Dichlobenil may potentially interact with the androgen pathway based on: (i) positive AR binding; (ii) decreases in testosterone production in the steroidogenesis assay; (iii) tube rcle score reductions in FSTRA; and (iv) decreases in hamster testes and prostate weights and changes in pr ostate histopathology in the Part 158 studies.

D. Thyroid Pathway

There is no convincing evidence fo r a potential effect of dichl obenil on the thyroid pathway. No thyroid-related effects were observed in the male or female pubertal assays. In the AMA, there was no effect when compared to solvent control for the developmen tal endpoints. In the OSRI, there were no thyroid-rela ted effects in the absence of overt toxicity except for an increase in thyroid weight w ithout any corresponding changes in thyroid histopathology in dogs in the chronic study.DICHLOBENIL FINAL

Page 25 of 52 E. Conclusions

There is no convincing evidence for interact ion with the estrogen pathway. The observed estrogen-related effects in the absence of overt toxicity occurred in isolation without pathway concordance or redundancy. However, there is ev idence for potential for interaction with the androgen pathway. Dichlobenil did not show c onvincing evidence for a potential interaction with the thyroid pathway.

V. EDSP Tier 2 Testing Recommendations

For dichlobenil, the point of departure (POD) is 1 mg/kg/day used for human health risk assessment is based on liver toxicity (changes in liver enzymes, increased liver weights, and periportal hepatocytes hypertrophy ) in male dogs at 6 mg/kg/day (LOAEL) in a chronic toxicity study. This POD is used to derive the chronic Re ference Dose (RfD) for dietary exposure risk assessments. The POD of 3.0 mg/kg/day used fo r calculating Margin of Exposure (MOEs) for non-dietary exposure risk assessments is base d on decreased pup body we ight at weaning at 17.5 mg/kg/day (LOAEL) in a two-generation reproduction study. Accordingly, the PODs (1 and 3 mg/kg/day) used for the overall risk asse ssments are 20 to 60-fold lower than the lowest dose (60 mg/kg/day) where a potentially androge n related effect was observed in the male pubertal assay. Consequently, the current Rf D and MOEs are protective of potentially
endocrine-related effects obser ved in the pubertal assay.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is NOT recommended for dichlobenil since additional testing will not impact the regulatory endpoints
used for human health risk assessments.

EDSP Tier 2, Medaka Extended One Gene ration Reproduction Test (MEOGRT) is recommended due to the potential interaction with the androgen pathway and the effects observed in the FSTRA assay. Currently, the ch ronic NOAEC endpoint for fish is <0.33 mg/L, based on growth effects at all concentrations te sted in an early life-cycle study with rainbow
trout. Effects on male tubercle score in the FST RA were observed at all concentrations tested ( ≥ 0.021 mg/L); additionally, fecundity was decrease d at 0.21 and 2.1 mg/L. Therefore, a Tier 2 MEOGRT is requested to provide data to understa nd potential effects of dichlobenil on fish reproduction and development.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for dichlobenil. In both sp ecies, effects on reproduc tive parameters were observed. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating pot ential reproductive effects to birds from dichlobenil exposure. Relative to EDSP, additional testing is not recommended.DICHLOBENIL FINAL

Page 26 of 52 VI. References

Barker L. (1989). Dichlobenil: oral (gavage) teratology study in the rabbit (experiment II), repeat study. Unpublished study prepared by Hazleton UK (England). Report No. 56645/21/89. (MRID 41257302).

Barker, L. (1992). Dichlobenil: 2-generation oral (dietary admi nistration) reproduction study in the rat; Addendum 1 – Pathology report. Unpublished study prepared by Hazleton UK (England). Report No. PD R 5798-65/18 Addendum 1, January 1992. (MRID 42239101).

Chambers, PR, Hornby, SB, Gibson, WA, et al . (1992). Dichlobenil: potential tumorigenic effects in prolonged dietary ad ministration to hamsters. Un published study prepared by Huntingdon Research Centre, Ltd. (England ). Report No. PDR 511/9178, February 10, 1992. (MRID 42221201).

Chambers, PR, Brown, R, Gibson, WA, et al . (1992). Background tumor incidence in hamsters (Background data study). Unpublished study prep ared by Huntingdon Research Centre, Ltd. (England). Report No. PDR 540/920478, September 22, 1992. (MRID 42563601).

Cooke, A (1977). Effects of fiel d applications of the herbicid es diquat and dichlobenil on amphibians. Published. Envir. Pollut. 12 : 43-50.

Dijkstra, A. (1988). Chronic feeding study on dichlobenil in the rat (Duphar Report No. 56645/41/84): Addendum 2. (MRID 40823801).

Hodge, HC. (1961). Ninety day feeding study of casoron in rats. Unpublished study prepared by Dept. of Pharmacology, Univ. of Rocheste r School of Medicine and Dentistry, Nov. 25, 1961. (MRID 00107106).

Hooks, WN, Cary, RJ, Colman, K, et al. (1991). Dichlobenil: pot ential tumorigenic effects in prolonged dietary administration to hamsters . Unpublished study prepared by Huntingdon Research Centre, Ltd. (England). Re port No. PDR 478/90177, May 3, 1991. (MRID 41988301).

Hooks, WN, Cary, RJ, Colman, K, et al. (1991). Dichlobenil: pot ential tumorigenic effects in prolonged dietary administration to hamsters (S upplemental data). Unpublished study prepared by Huntingdon Research Centre, Ltd. (E ngland). Report No. PDR 478/91486, June 27, 1991. (MRID 42015101).

Inoue, H, and Enomoto, M. (1983). Chronic fe eding study on dichlobenil in the rat. Unpublished study prepared by Biosafety Research Center, Foods, Drugs, and Pesticides (Japan). Report No. 56645/41/84, November 30, 1983. (MRID 00147438).DICHLOBENIL FINAL

Page 27 of 52 Kemp, A., DeHaan, M, and Dawe s, R (1987). A 13-week (die tary) toxicity study with
dichlobenil in male and female mice. Unpublished study prepared by Duphar B.V. Project ID DT 87/01, April 1987. (MRID 48043302).

Kemp, A, and Dawes, R. (1987). Chronic feeding study on dichlobe nil in the rat: Addendum to Duphar Report No. 56645/41/84. (MRID 40401101).

Kemp, A, de Haan, M, and Jager, FC. (1987). A 13-week toxicity study with dichlobenil in male and female hamster followed by a 4-week recovery period. Unpublished study prepared by Duphar B.V., Dept. of Toxicology (Nethe rlands). Laboratory study number 56645/04/87, June 10, 1987. (MRID 40600701).

Koëter, HB. (1984). Oral embryotoxicity/terat ogenicity study with dichlobenil in rats. Unpublished study prepared by Netherlands Orga nization for Applied Sc ientific Research. Report No. V 84.026/231045, January 1984. (MRID 00147437).

Leeming, NM. (1989). Dichlobenil: 2-generation oral (dietary administration) reproduction study in the rat. Unpublished study prepared by Hazleton UK (England). Report No. PDR 5798-65/18, August 15, 1989. (MRID 41257303).

Meerts, I.A.T.M. (2011). Dichlobenil Tec hnical: Estrogenic Receptor Transcriptional Activation Assay (Human Cell Line (HeLa-9903)). Unpublished study prepared by NOTOX B.V, Hertogenbosch, The Netherlands. La boratory Report No.: 495782, December 9, 2011. (MRID 48616105).

Pickersgill, N. (1995). Dichlobe nil — 52 week oral (cap sule) toxicity study in the Beagle dog. Unpublished study prepared by Pharmakon Europe (France). Study No. 65/510, December 20, 1995. (MRID 43969701).

McVey, E. A (2012) Weight of evidence summary of Tier 1 and OSRI data from dichlobenil Technical. Chemtura Corporation. Middl ebury, CT. NOTOX Project 498767. MRID No.
48670501.

Til, HP, Feron, VJ, Huismans, JW, et al . (1969). Chronic (Two-year) Toxicity Study with Dichlobenin (H133) in Beagle Dogs. Unpublished study prepared by Thompson-Hayward Chemical Co. Report No. R2813. (MRID 00067649).

USEPA. (2010). Di chlobenil — Report of the OPP Endocrine Disrupt or Review Team; Test Order #: EDSP-027401-120. December 7, 2010. Offi ce of Chemical Safety and Pollution Prevention. U.S. Environmental Pr otection Agency, Washington, DC.

USEPA. (2011). Endocrine Disruptor Scre ening Program — Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agen cy, Washington, DC.DICHLOBENIL FINAL

Page 28 of 52 USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envi ronmental Protection Agency, Washington, DCDICHLOBENIL FINAL

Page 29 of 52 APPENDIX 1. EDSP T1 Screening Assays

Amphibian Metamorphosis Ass ay (Frog); OSCPP 890.1100; OECD 231

The 21-day assay (MRID No. 48616101) of Dichlobenil (99.3% purity) on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) st age 51 (80 per control and treatme nt group) were exposed to a negative control and a solvent control (0.1 mL/L; acet one) and nominal dichlobe nil concentrations of 0.02, 0.2, and 2.0 mg a.i./L. Mean-measured concen trations were <0.001 (<LOQ; negative and solvent), 0.019, 0.19, and 1.9 mg a.i./L. The test system was maintained at 21.0 to 22.5°C and a pH of

7.3 to 8.2.

No mortalities were observed in the solvent contro l, or low or mid treatment groups. One mortality each was recorded in the negative control and high treatment group. Clinical signs ( i.e. , behavioral and other sublethal effects) including curved spine were observed in tadpoles in the solvent control and 0.019 and 0.19 mg a.i./L treatment groups. This effect did not appear to be related to the test substance.

No significant acceleration or dela y of body weight, median NF deve lopmental stage, snout-to-vent length (SVL), hind-limb length (HLL), or norma lized (for SVL) HLL was observed at Day 7 compared to the negative control. Furthe r, no asynchronous development was observed.

At Day 21, >20% of amphibians were at NF stage >60. When excluding organisms with NF stages greater than 60, dichlobenil signif icantly increased Day 21 normalized HLL at all test concentrations (7-10% greater than negative control for non-late st age amphibians, p<0.05). No significant treatment effects were detected for Day 21 median developm ental stage, body weight, or SVL for non-late stage amphibians compared to the negative control. Ad ditionally, no significant effects on development or growth were observed when comparing treatment a nd negative control late-sta ge amphibians (NF>60). Normalized HLL at Day 21 was also significantly greater (1 0%, p<0.05) in the solvent control than in the negative control, as was NF development stage.

There was a lower incidence and severity of fo llicular cell hypertrophy in the high treatment group compared to the negative or solvent control. No treatment-related effects were observed for thyroid gland hypertrophy or atrophy, or follicular cell hype rplasia, and there were no differences in the amount or quality of follicular colloids.

AR Binding Assay (rat pros tate cytosol); OSCPP 890.1150

In an androgen receptor (AR) binding assay (MRI D 48616102), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a saturation binding experiment and a competitive binding experiment. The saturation bi nding experiment was conducted to demonstrateDICHLOBENIL FINAL

Page 30 of 52 that the AR in the rat prostate cytosol was pres ent in reasonable numbers and was functioning with appropriate affinity for the radio-labeled reference androge n (R1881) prior to conducting the competitive binding experiments. The competiti ve binding assay was conducted to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (10

− 10 to 10

− 3 M) of dichlobenil (99.3%, Lot# AM9/K12A 222). Dimethyl sulfoxide (DMSO) was used as the solvent vehicle at a final assay concentrati on of 3.3%. A total of three independent runs were performed, and the assay included dexamethasone as a weak positive control, and R1881 as the ligand
reference standard in each run.

Only one run was considered acceptable in the satu ration binding experiment, but the estimates for the dissociation constant (K d ), B max , and specific binding generally met the acceptance criteria, and the Scatchard plot was linear (r 2 = 0.994). For the successful run, the dissociation constant (K d ) was 0.65 nM, and the B max was 2.58 fmol/100 μ g protein. The specific bindi ng reached a plateau, and nonspecific binding was less than 20% of total binding (4.2%).

In the competitive binding experiment, dichlobe nil reduced the mean specific binding of the radiolabeled ligand in a concentr ation dependent manner in all thr ee runs. The maximum reduction in [ 3 H]-R1881 binding occurred at th e highest concentration of dichlobenil tested (10

− 3 M), with specific binding being reduced to 40-46%. Speci fic binding was also reduced at 10

− 4 M (52-56%), but not at
concentrations ≤ 10

− 5 M (86-103% binding). Dichl obenil is classified as a binder in the AR assay as specific [ 3 H]-R1881 binding to the AR was reduced to <50% at the highest concentration tested in all
three runs.

The estimated mean log IC 50 was

− 3.85 M for dichlobenil,

− 8.73 M for R1881 and

− 4.58 M for dexamethasone (weak positive control). Compared to R1881, the mean Relative Binding Affinity (RBA) was 0.0013% for dichlobenil and 0.0071% for de xamethasone. All performance criteria were met except that the ligand deplet ion was greater (~18%) than the ≤ 10-15% criteria, and the bottom percent binding for R1881 was low in one of the runs.

Although only one acceptable run was conducted for the saturati on binding assay, and the K d was low for that run, the androgen receptor was present in a reasonable concentration and functioning with appropriate affinity for the ligand in the competit ive binding assays based on the performance criteria
and estimated IC 50 s for R1881 and the weak positive control. It is unlikely that information from additional saturation binding runs would ch ange the interpretati on of this study.

Based on the results from the three r uns, dichlobenil is classified as a Binder in the Androgen Receptor Binding Assay.DICHLOBENIL FINAL

Page 31 of 52 Aromatase Assay (Human recombinant microsomes); OSCPP 890.1200

In an in vitro aromatase (CYP 19) assay (MRID 48616103), dichlobenil (99.3%, Lot # AM9K12A222)
was incubated with human recombinant arom atase and tritiated androstenedione (1- β [ 3 H(N)]-androst4-ene-3,17-dione; [ 3 H]ASDN) in ethanol at l og concentrations of 10 -10 to 10 -3 M for 15 minutes to assess the potential of dichlobeni l to inhibit aromatase activity.

Aromatase activity was determined by measuring the am ount of tritiated water pr oduced at the end of a
15-minute incubation for each concentration of chemi cal. Tritiated water was quantified with liquid scintillation counting (LSC). Thr ee runs were conducted, and each run included a full activity control, a background activity control, a positive control series (10

− 10 to 10

− 5 M) using a known inhibitor (4- hydroxyandrostenedione; 4-OH ASDN), and a dichlobenil series (10

− 10 to 10

− 3 M) with three repetitions per concentration.

Aromatase activity in the full activity controls ranged from 0.4156 to 0.7358 nmol·mg-protein

− 1 ·min

− 1for the three test runs, with a mean and standard deviation of 0.5750 ± 0.1208 nmol·mgprotein

− 1 ·min

− 1 . The response of each full act ivity control within a run was between 90 to 110% of the mean. Activity in the background controls ranged from 0.06% to 0.39% and averaged 0.21% of the full activity controls. For the positive control substance (4-OH ASDN), aromatase activity results were within the recommended ranges for the performance criteria. The estimated log IC 50 for 4-OH ASDN averaged

− 7.34 M and the Hill slope was

− 0.94.

Aromatase activity at the lowest a nd highest successfully tested c oncentrations of dichlobenil (10 -10

and 10

− 4 M) were 99.6% and 87.5% of control. The highe st tested concentrati on of dichlobenil (10

− 3 M) showed precipitation during the 15-minute incubation period; theref ore, the results of the highest test concentration were not used for interpretation.

Based on the data from the average response curve, dichlobenil is classified as a Non-inhibitor of aromatase activity in this assay. There is no indicati on in the study report that half-log concentrations (i.e., 10 -3.5 M) were tested for solubility as recommended by the Test Guideline. At concentrations up

to 10 -4 M, dichlobenil would be classifi ed as a non-inhibitor. However, as there is no indication in the study report that 10 –3.5 M was evaluated for solubility, there is less confidence in the non-inhibitor
classification. It should be noted that based on the sh ape of the response cu rve, if a soluble 10 –3.5 M concentration had been tested, it is possible that the lowest portion of the curve would fall within the 50-75% activity range resulti ng in an equivocal classifi cation for this chemical.DICHLOBENIL FINAL

Page 32 of 52 ER Binding Assay (Rat ut erine cytosol); OSCPP 890.1250

In an estrogen receptor (ER) binding assay (M RID 48616104), uterine cytosol from Sprague Dawley
rats was used as the source of ER to conduct satu ration binding and competitiv e binding experiments. The saturation binding experiment was conducted to dem onstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was functioni ng with appropriate affin ity for the radiolabeled reference estrogen prior to routinely conducti ng ER competitive binding experiments. The competitive binding experiment was conducted to m easure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of increasing conc entrations (10 -10 to 10 -3 M) of dichlobenil (99.3% a.i., Batch #AM9K12822). DMSO was used as the solvent vehicle at a final concentration of 2%. Three independent runs we re performed, and each run included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17 β -estradiol as the natural ligand
reference material.

For the saturation binding experiment, the appropriat e protein concentration fo r the saturation binding assay was determined to be 40 μg/tube, as it bound 30.2% of the [ 3 H]-17 β -estradiol. The mean K d for [ 3 H]-17 β -estradiol was 0.12 nM and the mean B max was 57.19 fmol/100 μ g protein for the prepared rat uterine cytosol. The data produced c onsistent and linear Scatchard plots (r 2 >0.98). In the competitive binding experiments, the reference standard (E2), the weak positive contro l (19-nonethindrone), and the negative control (octyltriethoxy silane) each exhibited the expected responses. The estimated mean log IC 50 s were

− 9.08 and

− 5.87 M for E2 and 19-norethindrone, respectively, and the relative binding affinity (RBA) was 0.062% for 19-norethindrone , compared to the natural ligand.

Precipitation of dichlobenil was not observed at any concentration. In all three runs, dichlobenil showed minimal displacement (93-104% binding) of radiolabeled estradiol at dichlobenil concentrations up to 10

− 3 M; therefore an IC 50 and RBA could not be determined for dichlobenil.

Based on the results from the three competitive runs, di chlobenil is classified as Not Interactive in the Estrogen Receptor Binding Assay.

ER α Transcriptional Activatio n Assay (Human cell line He La 9903); OSCPP 890.1300; OECD

455

In an estrogen receptor transcript ional activation assay (MRID 48616105), hER α -HeLa-9903 cells
cultured in vitro were exposed to dichlobenil (99.3% a. i., Batch # AM9K128222) at logarithmically increasing concentrations from 1 × 10 -9 to 2.5 × 10 -4 M in DMSO (0.1%). The experiments were performed using 96-well plates and each dichlobe nil concentration was tested in triplicate (3 wells/plate). Cells were exposed to the test agent for 20-24 hours to determine if reporter (luciferase) gene products were induced. Luciferase expression in response to activation of the estrogen receptorDICHLOBENIL FINAL

Page 33 of 52 was measured upon addition of a luciferase substrate and detection with a luminometer with acceptable sensitivity. A total of four inde pendent runs of the assay were c onducted. Laboratory proficiency was demonstrated by testing the 10 chemical s listed in the test guideline.

Dichlobenil was tested up to the limit of solubility, 2.5 × 10 -4 M. The mean RPC Max for dichlobenil was 0.9% to 5.1%, and the associated PC Max was 10 -8 or 10 -6 M. Performance criteria were met for

17 β -estradiol, 17 α -estradiol and cor ticosterone. For 17 α - methyltestosterone, the Log PC 50 was not calculable for Run 1, Run 2 and Run 4, and the Log PC 10 was higher than the expected range for all four Runs. The responsiveness of the cel ls to the very weak positive control 17 α -methyltestosterone was lower than the expected values, indicating a decreased sensitivity of the assay to very weak agonists. Although the conditions of this assay we re not optimal to detect very weak activity, dichlobenil responses were similar to those of the negative control co rticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to reach a maximum of 34-51% PC. Dichlobenil was only able to reach a maximum of 0.9-5.1% PC when tested up to the highest concentration possible based on cy totoxicity. Because the RPC Max < PC 10 in both assay runs, dichlobenil was considered negative for estrogen recepto r transcriptional activation in this test system.

Fish Short-term Reproduction Assay; OSC PP 890.1350; OECD 229

The 21-day short-term reproduction assay (MRID N o. 48616106) of dichlobenil with fathead minnows ( Pimephales promelas) was conducted under flow-through conditi ons. Adult fish, 20 spawning groups (2 males and 4 females in each group; 6-7 months old) were exposed to dichlobenil (99.3% purity) at nominal concentrations of 0 (negative control) , 0 (solvent control; 0.1 mL/L acetone), 0.021, 0.20, and 2.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.001; both controls), 0.021, 0.21, and 2.1 mg a.i./L. The test system was maintained at 24.3 to 26.2 o C and a pH of 7.3 to 7.9. Unless otherwise indicated, the treatment groups were comp ared to the negative control.

There were no statistically signifi cant differences (p>0.05) in mean male or female body weights or lengths at any test leve l compared to the control. Overall m ean survival values were 100, 96, 100, 100 and 92% in the negative control, solvent control, and low, mid, and high treatment levels, respectively. Fish exposed to the high treatment level were observed to have a lighter, more yellowish color compared to the fish in the contro ls and other treatment levels; howev er, the incidence of this effect was not reported. No other changes in be havior or appearance were noted.

Spawning frequency in the negative and solvent contro l was at least every 4 days in 3 and 2 of the 4 replicates, respectively; the mean fecundity in the negative and so lvent controls was 12.1 (range 6.6- 15.8) and 6.5 (range 4.3-11.6) eggs/female/day/rep licate. Fecundity was significantly (p<0.05; Jonckheere-Terpstra) reduced by 53% and 90% at th e mid and high treatment levels, respectively, compared to the negative control. The study author di d not provide fertility da ta, but reported fertility was “more than 99.9% in all test groups” and fertility of eggs from control animals was at least 95%.DICHLOBENIL FINAL

Page 34 of 52 Median male tubercle score show ed a statistically significant reduc tion from 26 in the negative control to 21, 14, and 12 in the low, mid and high treat ment groups, respectively (p<0.05; JonckheereTerpstra). No tubercles were observed for females, except for one female with one tubercle in the low treatment group which was reported not to be treatme nt related. There were no significant differences in male or female gonadal stage index (GSI) or vitellogenin (VTG).

In males, no treatment-related hist opathological gonadal findings were reported. In females, there was an increased incidence and/or severity (minimal to moderate) of oocyte atre sia and a shift to more mature stages in the ovaries in the mid and high treatment groups.

Sex steroids (testosterone and 17 β -estradiol) were not measured. No dorsal nape pad measurements or other dorsal nape pad observ ations were reported.

While not statistically significant (p=0.078), the fecundity in the solv ent control group was reduced by 46% compared to the negative control. However, large variability was observed between the preexposure and exposure fecundity rate for both the ne gative and solvent contro l replicates, suggesting this difference may not be solvent related. A dditionally, this was the only endpoint for which a notable difference between the negative and solvent control was observed.

Hershberger Assay (Ra t); OSCPP 890.1400; OECD 441

In a Hershberger assay (MRID 48616107) screening fo r androgenic activity, dichlobenil (99.3% a.i., Lot# AM9K12A222) in 1.0% gum tragacanth was admini stered daily via oral gavage to groups of 55- day old, castrated male Sprague Dawley rats (6/g roup) at dose levels of 0 (vehicle), 20, 60, or 180 mg/kg/day. An androgenic positive control group c onsisting of six castrate d rats exposed to 0.2 mg/kg/day testosterone propionate (TP) by subcutaneous (s.c.) in jection was also included as a positive control.

To screen for potential anti-androgenic activity, dichlobenil in 1.0% gum tragacanth was also administered daily via oral gavage to 55-day old, castrated male Spra gue Dawley rats (6/group) at dose levels of 20, 60, or 180 mg/kg/day in conjunction w ith a daily dose of reference androgen TP at 0.2 mg/kg/day by s.c. injection. The vehicle control groups consisted of six castrated male rats dosed daily with TP (0.2 mg/kg/day) by s.c. injection in corn oil, and the anti-androgenic positive control group consisted of six castrated male rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg/day in conjugation with a daily s.c. dose of TP (0.2 mg/kg/day).DICHLOBENIL FINAL

Page 35 of 52 For both components of the assay, the animals were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administra tion to determine weights of liver, kidneys and the five androgen-dependent tissues.

All animals survived until scheduled termination. There were no trea tment-related clinical signs of toxicity.

In the androgen agonist assa y, body weights and overall body we ight gains for the 20 and 60 mg/kg/day treatment groups were comparable to controls throughout the assa y. At 180 mg/kg/day, body weights were decreased (p<0.05) by 9-10% on Days 2-7, and 9. The overall (Days 0-10) body weight gains for 180 mg/kg/day were decreased (p<0.05) by 44%. Live r weights were not affected in the low-dose and positive contro l groups, but were increased (p<0.05) in the 60 mg/kg/day ( ↑ 24%) and 180 mg/kg/day ( ↑ 32%) groups. Kidney weights were unaffect ed, except in the TP group, in which kidney weights increased (p<0.05) by 8%. There were no significant changes in weight of the accessory sex organs for dichlobenil-dosed animals. Animals in the TP group (positive control) had increases (p<0.05) in accessory se x organ weights as follows: 339% in seminal vesicles, 638% in ventral prostate, 165% in levato r ani-bulbocavernosus (LABC), 513% in Cowper’s gland, and 72% in glans penis. All %CV values were less th an the maximum recomme nded guideline values.

In the anti-androgen assay, body weights and overal l body weight gains for the 20 and 60 mg/kg/day treatment groups were comparable to controls throughout the assay. At 180 mg/kg/day, body weights were decreased (p<0.05) by 9-11% on Days 1-10. The overall (Days 0-10) body weight gains for 180 mg/kg/day were decreased (p<0.05) by 40%. For the positive control group (TP + FT), overall body weight gains were decreased (p<0.05) by 13%. No changes were noted in liver weight for any dose group and kidneys were only affected in the positiv e control (TP + FT) group, in which kidney weights significantly decreased ( ↓ 10%).

Statistically significant decreases in 4 of the 5 targ et accessory sex organ wei ghts were observed at the high dose (180 mg/kg/day). The following decrease s (p<0.05) in accessory sex organ weights were noted at 180 mg/kg/day: 36% in seminal vesicles, 35% in ventral pr ostate, 27% in LABC, and 49% in Cowper’s gland. In addition, a decrease (21%; p< 0.05) in seminal vesicle weight was observed at 60 mg/kg/day. Animals dosed with TP + FT (positiv e control) had decreased (p<0.05) accessory sex organ weights as follows: 73% in seminal vesicles , 75% in ventral prostate, 48% in LABC, 71% in Cowper’s gland, and 20% in glans penis. The maximum guideline-specified %CV was exceeded for the glans penis (20% vs. 17%) in the TP +FT (pos itive control) group. All other %CV values were
less than the maximum recommended values.

Dichlobenil was negative for androgenic activity in th e Hershberger assay as no statistically significant increases in organ weights were se en in two or more of the five androgen sensitive tissues in the androgen agonist assay. Dichlobenil was positive for anti-androgenicity in the Hershberger assay asDICHLOBENIL FINAL

Page 36 of 52 statistically significant decreases in organ weights we re seen in four of the five androgen sensitive tissues in the androgen antagonist assa y. However, bodyweights were decreased ≥ 10% at the dose that resulted in the changes in androgen-sensitive tissue weights indicating overt toxicity.

Pubertal Female Ass ay (Rat); OSCPP 890.1450

In a Female Pubertal Assay (MRID 48616108), 15 Spra gue Dawley rats/dose group were treated daily via oral gavage (5 mL/kg) with dichlobenil (99.3 % a.i., lot/batch #: AM9K12A222) in aqueous 1.0% gum tragacanth at doses of 0 (vehicle), 20, or 60 mg/kg/ day from post-natal day (PND) 22 to 42. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainment was recorded. Blood samples fo r chemical chemistry analyses were taken following sacrifice on PND 42. Total serum thyroxine (T 4 ) and thyroid stimulating hor mone (TSH) levels were determined using electrochemiluminescent immunoa ssays and radioimmunoassays. The weights of adrenals, kidneys, liver, pituitar y, thyroid, and urogenital organs were recorded and microscopic examination of the ovaries, uterus, thyroi d, liver, and kidneys were performed.

There were no dichlobenil-relate d effects observed on mortality, cl inical signs of toxicity, body weights, body weight gains, age and weight at attainment of VO, mean age at first vaginal estrus, mean cycle length, or percent cycling a nd regularly cycling. Organ weights were similar between the control and dichlobenil treated groups with the exceptions of liver and uterus weights. Unadjusted, adjusted and related (to body) liver weight was increased (p<0.05) by 18–19% at 60 mg/kg/day, and relative liver weight was increased by 9% at 20 mg/kg/day. Blotted uterus weight was decreased by 12% (NS) and 21% (p<0.05) at 20 and 60 mg/ kg/day, respectively, and wet uterus weight was decreased by 26% (NS) at 60 mg/kg/day. However, no hi stological changes were noted in the liver, uterus or ovaries.

Serum T4 and TSH levels were altered in a dose dependent manner, but the changes did not attain significance. At 20 and 60 mg/kg/day, T 4 levels were decreased by 4% and 7%, respectively, and TSH levels were increased by 18 and 22%, respectively. The changes in thyroid hormone levels were not corroborated by changes in t hyroid weight or histology.

The dose levels tested were determined to be adeq uate based on the dose dependent changes were seen in the uterus and the thyroid in this assay, and the results of the dose range-finding study.

Pubertal Male Ass ay (Rat); OSCPP 890.1500

In a Male Pubertal Assay (MRID 48616109), 15 Spra gue-Dawley (Crl:CD(SD)) rats/dose group were treated daily via oral gavage (5 mL/kg) with di chlobenil (99.3% a.i., Lot #: AM9K12A222) in aqueous 1.0% gum tragacanth at doses of 0, 20, or 60 mg/ kg/day from post-natal day (PND) 23 to 53. The animals were examined for preputial separati on (PPS) daily beginning on PND 30, and age and bodyDICHLOBENIL FINAL

Page 37 of 52 weight at day of attainment was recorded. Fo llowing sacrifice on PND 53, to tal serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were analyzed using electrochemiluminescent immunoassays and radioimmunoassays. The weights of adrenals, liver, pituitary, thyroid, and urogenital or gans were recorded and microscopic examinations of the testes, epididymides, thyroid, liver, and kidneys were performed.

No adverse treatment-related eff ects were observed in mortality, clinical signs of toxicity, body weight, body weight gain, age or weight at PPS, clinical chemistr y, gross pathology, or microscopic examination of the testes, epididymides, thyroid, liver, or kidney.

The age and weight at attainment of PPS at all doses was within the acceptable range of the performance criteria. The unadjusted values for organ weights and hormone levels in the control group were within the acceptable range of the performance criteria prov ided in the Guideline with the exception of a minor departure in the thyroid weight of 13.44 mg comp ared to Guideline criterion of 14-26 mg. All thyroid glands examined in all dose groups were considered within normal limits of
control tissues.

Increased (p ≤ 0.05) unadjusted, adjusted (covariate PND 23 body weight), and organ-to-body weight ratio liver ( ↑ 35–36%) and kidney ( ↑ 9–10%) weights were observed at 60 mg/kg/day. Increased (p ≤ 0.05) organ-to-body wei ght ratio liver ( ↑ 16%) and kidney ( ↑ 7%) weights were observed at 20 mg/kg/day. Increased (p ≤ 0.05) adjusted adrenal weights were also noted at 60 mg/kg/day ( ↑ 13%). Except for the liver weights at 60 mg/kg/day, these di fferences were considered minor. Furthermore, in the absence of corroborating evidence of hepatot oxicity from gross and microscopic examination of the liver and clinical chemistry, the increased weight was considered an adaptive response.

No biologically significant effect was noted on clinical chemistr y parameters in the treated groups; however several clinical chemistry parameters in the 60 mg/kg/day group differed statistically (p ≤ 0.05) from controls.

Although no treatment-related effects were noted on pubertal development, th e thyroid, or signs of systemic toxicity, the dosing levels utilized in th e current study are considered appropriate based on
the results of the dos e range-finding study.

Steroidogenesis Assay (Human cel l line H295R); OS CPP 890.1550; OECD 456

In a steroidogenesis assay (M RID 48616110), H295R cells cultured in vitro in 24-well plates were
incubated with dichlobenil (99.3% a.i., Batch # AM9K12A222) at se rial log concentra tions of 100 to 0.0001 μM for 48 hours in triplicate for four independent experiments. Dimethyl sulfoxide (DMSO) was used as a vehicle, at a final concentration of 0.1%.DICHLOBENIL FINAL

Page 38 of 52 Testosterone and estradiol levels were measured using ELISA. A Quality Control (QC) plate was run concurrently with each independent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents at tw o concentration levels. The positive controls included a known inhibitor (proch loraz) and inducer (forskolin) of estradiol and testosterone production.

Guideline acceptability recommendations and requir ements were generally met, including adequate production of testosterone and estradiol, acceptabl e reproducibility (low %CV), and appropriate induction and inhibition with positiv e controls. Precipita tion of the test compound was not reported, and cytotoxicity was not observed.

Treatment-related effect from dichlobenil observe d on testosterone or estr adiol levels were not consistent across all runs. In Run 1, there were si gnificant reductions in test osterone levels at 0.0001, 0.001, 0.01, 1 and 100 μM of dichlobenil, with the grea test reduction (0.71-fold ) occurring at 0.01 μM. During Run 2, decreased (p ≤ 0.05) testosterone levels were also noted at 0.0001 and 0.01 μM. However, no reductions in testosterone were noted in Run 3. No changes in estradiol concentration were observed during Runs 1 and 2, but estradiol was decreased (p ≤ 0.05) in Run 3 at all dichlobenil concentrations except 100 and 0.1 μM.

Due to the inconsistencies and variability in the hor mone alterations across the three runs, the results for dichlobenil in this assay were considered to be equivocal.

Uterotrophic Assay (Rat)

In a Uterotrophic Assay (MRID 48616111) conducted to screen for potential estrogenic activity, dichlobenil (99.3% a.i., Lot #AM9K12A222) in 1.0% gum tragacanth in deionized water was administered daily via oral gavage to groups of si x ovariectomized Sprague Dawl ey rats at dose levels of 0 (vehicle), 20, 60 or 180 mg/kg/day on post-nata l days (PND) 63-65. A positive control group was
treated with a da ily dose of 17 α -ethynyl estradiol (EE) at 0.003 mg/kg/day by subcutaneous (s.c.) injection. Body weights were determined daily. All animals were terminated and necropsied on PND 66 approximately 24 hours after th e final dose administration to de termine wet and blotted uterine weights.

All animals survived until scheduled termination and no clinical findings were observed in animals dosed with dichlobenil at any leve l. Body weights were decreased (p<0.05) by 8% on Day 3 and 9% on Day 4 (termination) at 180 mg/kg/day compared to controls, resulting in a decrease (p<0.001) of 129% in overall body weight gain. Body weights in the 20 and 60 mg/kg/day dose groups were comparable to controls throughout the duration of the assay. Uterine weights in the dichlobenil treated groups were comparable to the vehicle controls.DICHLOBENIL FINAL

Page 39 of 52 The body weights for the positive control (EE) group were decreas ed (p<0.05) by 8% on Day 4 and overall body weight gain for the EE animals was decreased (p<0.001) by 104% in compared with the controls. Absolute wet and blotted uterus weig hts for the positive control group were increased (p<0.001) by 766% and 234%, respectively, as expected.

The highest dose tested (180 mg/kg/da y) was determined to be adequate based on the decrease in body weight gain (129%) at this dose.

No statistically significant changes were seen in ut erine weight in this assa y. Dichlobenil was negative in the uterotrophic assay.DICHLOBENIL FINAL

Page 40 of 52 APPENDIX 2. Other Scientifica lly Relevant Informat ion (OSRI) Subchronic Oral Subchronic

Oral Toxicity Study (Rat)

In a 90-day oral toxicity study ( MRID 00107106 ), dichlobenil technical (90.5% a.i.) was
administered in the diet to al bino rats (12/sex/ dose) at daily dose leve ls of 0, 100, 1000, or 3000 ppm (equivalent to 0, 4.5, 45, or 135 mg/kg/day; adjusted for purity) for 3 months. An additional 6 males were treated with 10,000 ppm (453 mg/kg/day) for 3 months. Five (of 6) males treated with 10,000 ppm dichlobenil died by Week 7 of the study (causes undetermined). Mortality, due either to respiratory infection or “other cau ses”, was evenly distributed acro ss males in all other groups (2/12, 2/12, 1/12, and 2/12 at 0, 100, 1000, and 3000 ppm, resp ectively); 2/12 females died on study at 1000 ppm only. Mean body weights were decreased in males ( ≥ 14%) by the end of the study at ≥ 3000 ppm. There were no treatment-re lated effects on urinary protein or glucose up to 3000 ppm. In addition, no adverse effects were ob served on hematological parameters . Mean absolute liver weights were increased in males (39%) and females (52%) at 3000 ppm. The effect wa s dose-dependent and is considered adverse since swelling and vacuolation of the hepatocytes with or without necrosis was
observed in all rats/sex at ≥ 3000 ppm. An inflammatory, necrot ic focus was also observed in the liver of 1 male at 1000 ppm, while vacuolar swelling was observed in a second animal at the same
dose.

No treatment-related changes were observed in abso lute or relative testes we ight, and no histological lesions were noted in the testes, ovaries, thyroid and adrenal glands.

The LOAEL is 1000 ppm (45 mg/kg/da y) in males, based on hepatocytic inflammation and necrosis; and 3000 ppm (135 mg/kg/day) in females, based on increased liver weight and liver histopathology (swelling and vacuolation of hepatocytes). The NOAEL is 100 (4.5 mg/kg/ day) in males and 1000 ppm (45 mg/kg/day) in females.

Subchronic Oral Toxicity Study (Hamster)

In a 90-day oral toxic ity study (MRID 40600701), dichlobenil technical (98.5% a.i., batch # FUX 0100000) was administered to 10 Bio F1 Alexander hamsters/sex/dose in the diet at dose levels of 41, 209, 1289, or 7500/4648 ppm (adjusted due to evaporation; eq uivalent to 3, 16 , 79, or 395/263 mg/kg/day). Twenty animals/sex we re tested as a control group. Ten hamsters/sex from the control and high dose groups were kept on study for an additiona l 4-week recovery period during
which the animals received diet w ithout dichlobenil. Due to exce ssive weight loss in both sexes after 2 weeks (13%-25%) at 7500 ppm, all high- dose an imals received 4648 ppm dichlobenil from
Week 3 until Week 13.

There were no treatment-related effe cts on survival, and no ad verse clinical signs or ophthalmological changes were observed. Body weight gains in hi gh-dose males did not rebound to control levels until Week 10; however, mean body weights in high-dose males remained decreased (21%) at the end ofDICHLOBENIL FINAL

Page 41 of 52 treatment. Mean food consumption was decreased ( ≥ 11%) in high-dose males after dose lowering.
Mean serum cholesterol and phospholipids were increased by 55% each (p<0.01) in females at 4648 ppm. Concentrations of these cl inical chemistry parameters, body weight gain, and food consumption
returned to control leve ls during the recovery pe riod. Other changes in he matological or clinical chemistry parameters were slight and not considered toxicologically significant. Mean absolute liver weights were increased by ≥ 13% (p<0.01) in males at ≥ 1289 ppm and by 16% (p<0.01), 34% (p<0.01), and 85% (p<0.01) in females at 209, 1289, and 4648 ppm, re spectively. Mean absolute prostate weight was reduced by ≥ 19% (p<0.05) at ≥ 209 ppm, while mean absolute seminal vesicle and testicular weights we re reduced by 16% at ≥ 209 ppm and 33% (p< 0.01) at 4648 ppm, respectively. Statistically significant changes in other mean absolute and/or relative organ weights (e.g., kidney, spleen, brain, uterus ) were observed at the highest dos e tested; however, in the absence of histopathological correlates, these changes we re not considered toxico logically significant.

At necropsy, livers of 5/10 (p<0.01) males were enlarged at 4648 ppm, wh ile 5/10 or 10/10 (p<0.01) females had enlarged livers at 1289 or 4648 ppm, respectively. Rough surface of the liver was also noted for 3/10 and 8/10 (p<0.01) fe males at 1289 or 4648 ppm, respectiv ely. Black grit was found in the gallbladders of 2/10 (p<0.01) fe males at 4648 ppm at the end of tr eatment. Control incidences for each of these hepatobiliary observa tions were 0/20. At 4648 ppm, sm all testis was ob served in 3/9 males (p<0.01), while small epididym is was seen in 2/9 males (p< 0.01); the control incidences for each of these observations was 0/20. Slight hemorrhag ic area in the epididym is and seminal vesicle was also observed in 1/9 males (v s. 0/20 controls) at 4648 ppm. Bile granules in the gall bladder
were observed in 2/10 male s and 3/10 females in the high-dos e recovery group (vs. 0/10 and 1/10, respectively, in controls).

Histopathological observation reveal ed a marked decrease in the num ber of spermatocytes at the end of treatment in 5/9 high-dose males (vs. 1/20 in controls). Marginal to moderate prostatic
mineralization was observed in 3 or 4 (of 9 or 10) males at ≥ 209 ppm after 13 weeks (vs. 1/20 in controls). Marginal to marked tubular degeneration in the testes was observed in 7/9 (p<0.01) males at 4648 ppm (vs. 2/20 in controls). Calculi in the gall bladder were observed at the end of treatment in 7/10 males (p<0.01) and 10/10 (p<0.01) females at 4648 ppm (vs. 0/ 20 in each control group). Swollen hepatocytes were noted in 2/9 males at 4648 ppm and in 2/ 10 females at 209 (marginal), 4/10 females at 1289 (marginal to s light; p<0.01), and 4/10 females at 4648 ppm (marginal to moderate; p<0.01). Control incidences for swollen hepatocytes were 1/20 for males and 0/20 for females.

The L OAEL for males is 209 ppm (16 mg/kg/day), ba sed on decreased weight and mineralization of the prostate and decreased mean absolute seminal vesicle and test icular weights; and 1289 ppm (79 mg/kg/day) for females, based on increased liver weight, enlarged liver (with rough surface), and swollen hepatocytes. The NOAEL fo r males is 41 ppm (3 mg/kg/day) and 209 ppm (16 mg/kg/day)
for females.

Subchronic Oral Toxicity Study (Mouse)DICHLOBENIL FINAL

Page 42 of 52 In a 90-day oral toxicity study ( MRID 48043302 ), dichlobenil was administered to 22-day old male and female CD-1 mice (number not specified) in the diet at 0, 25, 125, 625, or 3125 ppm ( 0, 4, 19, 95, or 473 mg/kg/day). Adverse effect s in the liver (specific effects were not reported) were observed in female mice at the 95 mg/kg/day. Absolute (7 -13%) and relative (7 -11%) testes weights were
increased at ≥ 19 mg/kg/day. No treatment-related histopath ological lesions were seen in the testes, epididymides, seminal vesicle, pros tate, ovaries, uterus, vagina, thyr oid, adrenal or pituitary glands.

Chronic Toxicity (Dog)

In a non-guideline chronic toxicity study ( MRID 00067649 ), groups of beagle dogs (4/sex/dose) were given dichlobenil in the diet for 2 years at dos ing levels of 0, 20, 50, or 350 ppm (0, 0.5, 1.25, or 8.75 mg/kg/day). The NOEL for systemic toxicity was 1.25 mg/kg/day. The LOEL for systemic toxicity was 8.75 mg/kg/day based on (1) an increase in absolute and relative liver and thyroid weights in both sexes; (2) an increase in serum alanine aminotrans ferase in females, and serum alkaline phosphatase in both sexes; (3) an increase in liver enzyme glucose-6- phosph atase and glucose-6-phosphatase dehydrogenase activity in both se xes; and (4) leukocytic infiltra tion and fibrinoid degeneration around the central hepatic veins of both sexes.

Chronic Toxicity (Dog)

In a chronic toxicity study ( MRID 43969701 ), dichlobenil (98.8% a.i.; Batch FUX 011000/FUN 87E08A) was administered by capsule to 5 beagle dogs per sex at doses of 0, 1, 6, or 36 mg/kg/day for 52 weeks.

All animals survived to the scheduled termination date. No treatment-related clinical signs of toxicity were observed in any dogs, and no compound-re lated effects were noted on food or water consumption, ophthalmoscopic exam ination, urinalysis, or gross pathology. Lower mean body weights (12-16%) and body weight gains (36-44%; p<0.05) were obs erved throughout the study in males at 36 mg/kg/day (relative to controls), wh ereas no biologically significant changes in body weight or body weight gain were observed in females.

Increased serum cholesterol, triglyceride, phos pholipid, and al kaline phosphatase levels were
observed in males and females at ≥ 6 mg/kg/day. Gamma-glutamyltransferase was also increased in males only. Increased ( ≥ 18%) absolute and relative liver wei ghts were observed in mid- and highdose males and females. This effect corresponded to periportal hepatocytic hypertrophy in males at

≥ 6 mg/kg/day and at 36 mg/kg/day in females.

Absolute thyroid weights were si gnificantly (p <0.01) increased at the low (34%), mid (33%) and high (50%) dose groups for fe males and relative thyroid weight was increased (63%) at the high dose. For males, both absolute (56%) and relative (84%) th yroid weights were increas ed at the high dose. However, no thyroid lesions were seen in either sex at any dose level.

In the males, there was a dose-depe ndent decrease in absolute weight of the prostate glands with theDICHLOBENIL FINAL

Page 43 of 52 decrease reaching statistical significance (p <0.05 ) at the high dose; the decreases were 14%, 22% and 37%, at the low, mid and high dose, respectively. The absolute weights of the testes were increased (33%) at the high dose; whereas, the relative testes weight was significantly increased at both the mid (18%) and high (56%) doses. None of these weight increases were corroborated with histopathological lesions in these organs.

In the females, absolute (50%; p<0.01) and relative (46%) ovarian weights and absolute (78%; p <0.05) and relative (76%; p<0.05) uterine weights were decrea sed at the high dose. No histopathological lesi ons were seen in the ovaries, but “early” maturity of the uterus was reported in all females (5/5) at the high dose.

No treatment-related histopa thological lesions were seen in the testes, epididymides, prostate, ovaries, mammary glands, thyroid or pituitary glands. Cortical vacuolation of the adrenal glands were seen in 5/5 males and compared to 0/15 control males; incide nces in females were comp arable to the controls.

The LOAEL is 6 mg/kg/day, based on increased liver weights and increa sed serum cholesterol, triglycerides, phospholipids, and alkaline phosphata se in males and females and increased serum γ

GT and periportal hypertrophy of the hepatocytes in males. The NOAEL is 1 mg/kg/day.

Combined Chronic Toxicity/Carcinogenicity (Rat)

In a combined chronic toxicity/carcinoge nicity study (non-guideline; MRIDs 00147438 and 40401101), dichlobenil (96.7% a.i.; lot# FUN80C25A) was administered in the diet for 110 weeks to Fisher 344 rats (50/sex/dose) at daily dose levels of 0, 50, 400, or 3200 ppm (equal to 0, 2.3, 18.9, or 173.1 mg/kg/day). Interim sacrifices of 10 additio nal animals/sex/dose took place at Weeks 52 and 78. Mortality was 100% in males by Week 98 at 3200 ppm. Survival in males was similar to
controls at the mid and low doses and in females at all doses tested. No treatment-related clinical signs of toxicity were observed upon re-examination of the data (MRID 40823801).

At 3200 ppm, body weight was decreased by 33 % (p<0.001) by Week 88 in males and 34% (p<0.001) by Week 104 in females. Decrements we re proportional to time on st udy in both sexes. Similarly, mean body weight gain in males was decreased at 3200 ppm by 19% from Week 1-13 and by 160% from Week 52-66. In females, mean body weight gain was decreased at 3200 ppm by 24% from Week 1-13 and by 40% from Week 52-66. No differences were observed in food consumption when corrected for body weight, whereas food effici ency was decreased by 35% in males and 29% in females at 3200 ppm. Wa ter consumption (corrected for body weight) in high-dose animals was increased by 30% and 55% in male s and 22% and 39% in females at Weeks 5 and 15, respectively. Urinary volume was also over 2-fold higher in high-dose males at Week 78 (p<0.01).

No toxicologically significant ef fects were observed on hematological parameters. Mean blood urea nitrogen (BUN) was 3-fold higher (p<0.01) at W eek 78 in males treated with 3200 ppm; in males treated with 400 ppm, mean BUN wa s increased by 75% (p<0.05) at termination. Mean serum uricDICHLOBENIL FINAL

Page 44 of 52 acid levels were also increased by 35% (p<0.01) at 3200 ppm in male s. Mean cholesterol in males treated with 3200 ppm was increase d by 91% (p<0.05) at Week 52 and by 176% (p<0.01) at Week 78; in males treated with 400 ppm, mean cholesterol was increased by 136% (p<0.01) at termination. In females treated with 3200 ppm, mean BUN was over 3-fold high er (p<0.05) at Week 110. Mean
cholesterol in females treated w ith 3200 ppm was increased by 168% , 157%, and 83% (all p<0.01) at Weeks 52, 78, and 110, respectively. No treatment-rel ated changes in mean serum uric acid levels
were observed in females.

Mean absolute and relative li ver weights were increased in males by 48% (p<0.01) and 118% (p<0.01), respectively, after 78 weeks at 3200 ppm (cha nges at 78 weeks were si milar to those at 52 weeks and termination); however, th e increased relative liver weight was likely due to a combination
of increased absolute liver and decreased body weight. In female s at week 78, mean absolute and relative liver weights were incr eased (p<0.01) by 75% and 139%, re spectively, at 3200 ppm, and by 16% and 23%, respectively, at 400 ppm. Absolute kidney weights were increased by 16% (p<0.05) in both males and females at 3200 ppm, as was the ab solute weight of the ad renals in males (22%, p<0.01) at the same dose.

At terminal necropsy, dose-dependent increases in the incidences of enlarged liver and kidneys were
observed in males and females. The incidences of enlarged liver in males were 3/50, 9/50, 15/50, and 45/50 at 0, 50, 400, and 3200 ppm, respectively, wh ile enlarged liver was observed in 4/50, 3/50, 11/50, and 44/50 females, respectively. The inci dence of enlarged kidney was 2/50, 5/50, 8/50, and 40/50 in males and 0/50, 0/50, 2/50, and 13/50 in fe males at 0, 50, 400, and 3200 ppm, respectively. Incidences of fatty metamorphosis of th e liver was increased above controls at ≥ 50 ppm at Week 52 in males and females; however, dose-dependence was lacking for the effect in both sexes at Week 78. At terminal sacrifice, increased incidences of fa tty metamorphosis (with increased severity) of the liver were clearly observed in ma les (45/50) and females (39/40) at 3200 ppm. Liver necrosis was increased in males at 3200 ppm at Week 52 only and slightly increased in females at Week 78. Cytologic alterations (polyploidy with hepatocy tic swelling) were increased in females at ≥ 400 ppm at terminal sacrifice, whereas increased incidences of “hepatic nodules” (h yperplasia) were observed in males and females at 3200 ppm at terminal sacrific e. Clear increases in ca lcification of the kidney were observed at 3200 ppm in both males (24/50) and female s (46/47) at terminal sacrifice, whereas nephrosis (kidney damage) wa s observed in 100% males at ≥ 400 ppm (increased severity at high dose) and 98% females at 3200 ppm at terminal sacrifice. Nephrosis was also increased in both sexes
at both interim sacrifice times at ≥ 400 ppm. Last, parathyroid hyperp lasia was increased at terminal sacrifice only at ≥ 50 ppm in males and at 3200 ppm in female s. Parathyroid hyperplasia was likely a response to the reported kidney da mage (nephrosis), in order to maintain normal serum calcium
concentrations.

The LOAEL is 400 ppm (18.9 mg/kg/day), based on ch anges in clinical chemistry (increased blood urea nitrogen, cholesterol), gro ss pa thology (enlarged liver, enla rged kidney), and histopathology (nephrosis, parathyroid hyperplasi a) in males; and increased li ver weight, enlarged liver, andDICHLOBENIL FINAL

Page 45 of 52 cytologic alterations (polyploidy with hepatocytic sw elling) in the liver in females. The NOAEL is 50 ppm (2.3 mg/kg/day).

According to the Health Effects Division 2nd Ca rcinogenicity Peer Revi ew (1995), a treatmentrelated increase in the incidence of hepatocellul ar adenomas and combined adenomas/carcinomas was observed in females only at 3200 ppm. Dosing in females was considered adequate at 3200 ppm, based on decreased body weight and body weight ga in, changes in clinical chemistry and organ weights, gross pathology, and histopathology. Do sing in males was consid ered excessive at 3200 ppm, but adequate at 400 ppm, based on changes in clinical chemistry, gross pathology, and histopathology.

Carcinogenicity Study (Hamster)

In a high dose carcinogenicity study ( MRID 42221201 ), dichlobenil ( 99.4% a.i.; batch# FUN87EO8A/FUX011000) was administ ered in the diet for 91 (males ) or 78 (females) weeks to Bio F1D Alexander Syrian hamsters (50/sex/dose) at daily dose levels of 675, 1500, or 3375 ppm (equal to 51/55, 117/121, or 277/277 mg/kg/day for males/fe males). Control groups (0 ppm) consisted of 100 animals/sex. The criterion for study termination was 50% mortality in the control groups of each
sex. There was no difference in su rvival across groups in males afte r 91 weeks, while survival was higher in females at the high dose than in co ntrols after 78 weeks (78% vs. 50%). Submitted historical control data (M RID 42563601) indicated that the lifespan of untreated females of this strain
of hamster is less than that of untreated males (88-91 weeks vs. 77-78 weeks). There were no clinical signs of toxicity or ophthalmic abnorma lities after treatment with dichlobenil.

Body weight was decreased (>10%) in males at 3375 ppm from Week 2 of the study until termination, while overall body we ight gain in males was decr eased (p<0.05) by 68%, 136%, and 166% at 675, 1500, and 3375 ppm, respectively, from Week 0-91. Overall body weight gain in females was decreased (p<0.01) by 34%, 37%, a nd 30% at 675, 1500, and 3375 ppm, respectively, from Week 0- 78. Food consumption was decr eased (9-11%; p<0.01) at 3375 ppm in males throughout the treatment period. No adverse effects were observed on differenti al leukocyte counts in
males.

In males, enlarged liver (11/50 ; p<0.05) and liver masses (6/50; p<0.05) were observed at 3375 ppm (vs. 1/100 and 3/100, respectively, in controls), wher eas in females, the inci dence of enlarged liver was 3/50 and 30/50 (p<0.05) at 1500 and 3375 ppm, resp ectively (vs. 1/00 in controls). Pale liver
was also observed in males at incidences of 5/100, 7/50, 8/50 (p <0.05), and 13/50 (p<0.05) at 0, 675, 1500, and 3375 ppm, respectively. In cidences of minimal adipose ti ssue in males, which correlated with decreased body weight gains, were also decreased at 675, 1500, and 3375 ppm [19/50, 23/50 (p<0.01), and 27/50 (p<0.01), respectively vs. 18/100 in controls]. Mean rela tive liver weights in males were increased by 13% (p <0.01) and 53% (p<0.01) at 1500 and 3375 ppm. This is likely explained by the dose-dependent decreases in body we ight in males, since abso lute liver weights wereDICHLOBENIL FINAL

Page 46 of 52 not significantly different across dose. In female s, a 50% increase in absolute liver weight was observed at 3375 ppm, while relative liver weights were increased by 9% (p<0.05), 23% (p<0.01) and 59% (p<0.01) at 675, 1500, and 3375 ppm, respectively. Mean relative testes/epididymis weights were decreased by 12-13% at ≥ 1500 ppm. Decreases in mean absolu te heart weights in either males or females were also observed, but without histopathological correlates.

Dose-dependent increases in microscopic effects indicative of liver toxicity at ≥ 675 ppm in males included finely vacuolated hepatocytes, hepatitis, a nd brown pigment in the hepatocytes. In females, dose-dependent increases in hepatitis and pigmented giant cell s were observed at ≥ 1500 ppm. Histopathology of the testes (pigmented macrophages, dilated seminiferous tubu les) in ma les and of the urinary bladder (urothelial hyperplasia) and pitu itary (microcysts) in females was observed at the high dose only.

The LOAEL is 675 ppm (51/55 mg/kg/day in males/ females), based on decreased body weight gain
in males and females and liver hi stopathology (finely vacuolated he patocytes, hepatitis, and brown pigment in the hepatocytes) in ma les. The NOAEL wa s not identified.

According to the Health Effects Division 2nd Ca rcinogenicity Peer Revi ew (1995), there was a
treatment- related increase in liver adenomas an d combined adenomas/carcinomas in males only at 3375 ppm, when compared to controls. Dosing was considered excessive at ≥ 1500 ppm in both sexes, based on decreased body weight gains and severe hepatotoxicity.

Carcinogenicity Study (Hamster)

In a low dose carcinogenicity st udy (MRIDs 41988301 an d 42015101), dichlobenil (99.4% a.i.; batch# FUN87EO8A/FUX011000) was administe red in the diet for 88 (males) or 80 (females) weeks to Bio F1D Alexander Syrian hamsters (50/sex/dose) at daily dose levels of 5, 26, 132 , or 675 ppm (equal to 0.34/0.35, 1.69/1.78, 9.39 /9.20, or 45.64/4 8.85 mg/kg/day for males/female s). Control groups (0 ppm)
consisted of 100 animals/sex. The criterion for st udy termination in male s was 50% mortality in controls. Mortality in control females at termin ation was 42%. There were no dose-dependent
differences in survival in males or females at termination. Submitte d historical control data (MRID 42563601) indicated that the lifespan of untreated females of this strain of hamster is less than that of untreated males (88-91 weeks vs. 77-78 weeks).

Mean body weight was decreased (10-11%) in males at 675 ppm from Weeks 80-88 only of the study. Mean overall body weight gain was decreased at 675 ppm by 96% (p <0.01) in males from week 0-88 and by 14% in females from Week 0-80. No differences in food consumption or efficiency were
observed across dose. No adverse effects were observed on differential leukocyte counts or ophthalmological endpoints or at necropsy.DICHLOBENIL FINAL

Page 47 of 52 Mean relative liver weights we re increased in females by 15% (p<0.01) at 675 ppm; however, the effect showed no relationship to dosing. Increased incidences (p< 0.05) of reduced secretion of the prostate and seminal vesicles were observed in males at ≥ 132 ppm, at which the dose-response curve plateaued for both endpoints. The do se-response curve also plateaued at ≥ 26 ppm, at which an increased incidence (p<0.05) of hy perplasia of the non-glandular re gion of the stom ach was observed in males. The toxicological releva nce of this endpoint is questionable, however, since humans lack a homologue for the rodent non-glandul ar stomach. Increased incidences (p<0.05) of hyperplasia of the non-glandular region of the stomach we re also observed in females; however, the effect was not dosedependent. Increased incide nces of hyperplasia (p<0.05) were also observed at several other sites at 675 ppm, including pancreatic islet cells and skin (acanthosis) in males and adrenal cortex, small intestine, and bone marrow (ster num) in females. Hypertrophy of the skin (hyperkeratosis) and hepatocytes was also observed at the high dose in females.

The LOAEL is 132 ppm (9.39 mg/kg/day) in males, based on reduced secreti on of the prostate and seminal vesicles; and 675 ppm (48.85 mg/kg/day) in females, based on decreased body weight gain, peritonitis, and hyperplasi a of the adrenal cortex, small intestine, and bone marrow (sternum). The NOAEL is 26 ppm (1.69 mg/kg/day) in males and 132 ppm (9.20 mg/kg/day) in females.

According to the Health Effect s Division 2nd Carcinogenicity Pe er Review (1995), there was no treatment- related increase in the incidence of any tumor type in this study. Dosing was considered adequate, based on decrease d body weight gains and hyperplasia in various tissues in both sexes.

Reproduction and Fert ility Effects (Rat)

In a 2-generation reproduction study ( MRIDs 41257303 and 42239101 ), dichlobenil technical (99.4% a.i.; lot #s 4 and 5; batch# F6 N87EO8A/FUX011000) was administered in the diet to Crl:CD(SD)BR rats (30/sex/dose in the P generatio n; 25/sex/dose in the F1 generati on) at daily dose levels of 0, 60, 350, or 2000 ppm (equivalent to 0, 3, 17.5, or 100 mg/kg/day) for 2 consecutive generations. One (of 30) high-dose P males was found dead during week 14 of the study. The cau se of death was liver necrosis and hemorrhage ( MRID 42239101 ). One (of 25) F1 high-dos e males was sacrificed in extremis during week 12 of the stud y due after an accidental injury to the snout. Both deaths were
considered incidental to treatment. There were no treatment-related clinical signs of toxicity in the study.

During the 10-week premating period for P anim als, mean cumulative body weight gains were decreased by 25-26% (p<0.05) at 2000 ppm in both males and females. Similarly, mean overall body weight gains for F1 males were decreased by 25% during the 10-week premating period and by 18% for F1 females at 2000 ppm. During gestation, m ean body weight gains were decreased by 13-14% in both P and F1 females at 2000 ppm. Decrements in body weight gains were not observed in adult females of either generation duri ng lactation. Mean food consump tion at 2000 ppm was decreased by 11-33% (p<0.001) in P males and by 17-28% (p<0.001) in P females during premating. In F1 males, mean food consumption during premating was decreased by 16-21% (p<0.001) and by 16-20%DICHLOBENIL FINAL

Page 48 of 52 (p<0.001) in F1 females at 2000 ppm. Mean food c onsumption for P and F1 females during gestation and lactation was not reported.

No treatment-related effects were observed on fertility index, fec undity index, gestation index, or mean gestation length in either P or F1 female s. The mean number of implantations/dam was unreported for P females and decreased at 2000 ppm in F1 females (12.3 treated vs. 14.6 in controls).

The parental systemic LOAEL is 2000 ppm (100 mg /kg/day), based on decreased body weight gains during premating (males and females) and gesta tion (females) in both generations, decreased food consumption during premating in both generations (males and females), and decreased number of implantations/dam in F1 females. The pare ntal systemic NOAEL is 350 ppm (17.5 mg/kg/day).

The reproductive LOAEL is 2000 ppm (100 mg /kg/day), based on decreased number of implantations/dam in F1 females. The re productive NOAEL is 350 ppm (17.5 mg/kg/day).

No treatment-related effects were ob served on live birth index, viabilit y index, lactation index, or sex ratio in either F1 or F2 offspring. In add ition, the mean number of pups born live/litter was unaffected in F1 offspring but de creased (p<0.001) at 2000 ppm in F2 offspring (11.0 vs. 13.7 in controls). This was likely due to the decreased number of implantations/F 1 dam at 2000 ppm. At

≥ 350 ppm, the mean pup body weight of F1 offspr ing was decreased by 16-23% (p<0.05) from postnatal day (PND) 4 (precull)-21. The effect was dose-dependent. In F2 offspring, mean pup body weight was also dose-dependently decreased by 19-22% (p<0.05) from PND 14-21. An increased incidence of pelvic cavitation of the kidney at 2000 ppm (3% vs . 0%) was observed during necropsy in weanling F2 offspring; however, the incidence was similar to the sporadic incidence observed in P
and F1 animals and was not consider ed toxicologically significant.

The offspring LOAEL is 350 ppm (17.5 mg/kg/day), ba sed on decreased body weight during weaning in both generations. The offspri ng NOAEL is 60 ppm (3 mg/kg/day).

Teratogenicity Study (Rabbit)

In a prenatal developmental t oxicity study (MRID 41257302), dichlobeni l technical (98.5% a.i.; lot#s 3 and 6; batch# FUX010000) was admi nistered by gavage in 1% gum tragacanth to 18 pregnant New
Zealand white rabbits/sex/dose from gestation day (GD) 7-19 inclusiv e at daily dose levels of 0, 15, 45, or 135 mg/kg/day. No treatment-related mortality, clinical signs of toxici ty (including abortions), or gross pathology were observed in the study.

A decrease (129%; p<0.05) in body weight gain was observed during the dosing period only (GD 7- 19) in does treated at 135 mg/kg/day. This effe ct was accompanied by a 30% (p<0.01) decrease in food consumption at the high dos e during the dosing period only. Body weight gain and food consumption in high-dose animals rebounded during the post-dosi ng period (GD 19-29).DICHLOBENIL FINAL

Page 49 of 52 The maternal LOAEL is 135 mg/kg/day, based on d ecreased body weight gain and food consumption during dosing. The maternal NOAEL is 45 mg/kg/day.

Increases in total resorptions/d am (1.3) and post-implantation loss (17.9%) were observed at 135 mg/kg/day. Although the effects we re not dose-dependent, th e incidences were outside the historical control range and considered treatme nt-related. Although generally o ccurring at very low incidences (1-3 fetuses), several external , visceral, and skeletal defect s anomalies were reported at 135 mg/kg/day. These effects were not observed in ei ther concurrent or histor ical controls or were
observed at incidences outside hist orical control ranges and were th erefore considered toxicologically significant. External anomalies included bilateral open eye (3/115 fe tuses; 3/14 litters), cleft palate, and adactyly. High-dose visceral anomalies incl uded abnormal cystic ga llbladder and distended ureter with bilateral severe hy dronephrosis. Skeletal defects at 135 mg/kg/day were composed of malformed and malpositioned right scapula, right radius absent with malpositioned ulna and humerus, fused cervical vertebral arches, asymmetrically ossified and fused cervical vertebra centra, abnormally shaped cranium with enlarged and mi sshapen fontanelle, enlarged fontanelle (19/115 fetuses; 13/14 litters), misshapen frontals (2/115 fetu ses; 2/14 litters), skull and frontals foreshortened and nasal malpositioned, and major fusion of sternebrae (3 /115 fetuses; 3/14 litters).

The developmental LOAEL is 135 mg/kg/day, base d on increased number of total resorptions/dam and post-implantation loss; and increased incidences of external (cleft palate, adactyly, bilateral open eye), visceral (abnormal cystic gallb ladder, distended ureter with bi lateral severe hydronephrosis), and skeletal (malformed and malpositioned right sca pula, right radius absent with malpositioned ulna and humerus, fused cervical vertebral arches, asy mmetrically ossified and fused cervical vertebra centra, abnormally shaped craniu m with enlarged and misshapen fontanelle, enlarged fontanelle, misshapen frontals, skull and frontals foreshorte ned and nasal malpositione d, and major fusion of sternebrae) anomalies. The developmental NOAEL is 45 mg/kg/day.

Teratogenicity Study (Rat)

In a prenatal developmental toxicity study ( MRID 00147437 ), dichlobenil technical (purity not reported; batch# FUN82B07A/FUX003000) was administered by gavage in 1% gum tragacanth to 25 pregnant Wistar Cpb:WU ra ts/sex/dose from gestation day (GD) 6- 15 inclusive at daily dose levels of 0, 20, 60, or 180 mg/kg/day. No treatment-related mo rtality or clinical signs of toxicity were observed in pregnant does in the study.

A 29% (p<0.01) and 36% (p<0.01) d ecrease in mean body weight gain was observed during the dosing period in does treated at 60 and 180 mg/kg/day, respectively. This effect was accompanied by a 15% (p<0.01) and 21% (p<0.01) decrease in food consumption at 60 and 180 mg/kg/day, respectively, as well as a respec tive 19% (p<0.05) and 24% (p<0.01) decrease in food efficiency. During the post-dosing period (GD 16-21), body weight gain, food consumption, and food efficiency values in mid- and high-dose animal s rebounded to levels that were similar to or greater than those of
controls.DICHLOBENIL FINAL

Page 50 of 52 The maternal LOAEL is 60 mg/kg/day, based on d ecreased body weight gain, food consumption, and food efficiency during dosing. The maternal NOAEL is 20 mg/kg/day.

No treatment-related changes were observed in ce sarean section parameters (death, altered growth) for either embryos or fetuses. It is unclear fr om the study report whether statistical analysis was performed for litter incidences of ex ternal, skeletal, or visceral observ ations. With regard to external anomalies, a shallow dose- dependent increase in the incidence of small subcutaneous hemorrhage or petechia was observed (0/23, 1/22, 2/22, and 3/21 litters at 0, 20, 60, and 180 mg/kg/day); however, the increase in the number of fetuses affected (0-3 ) was only slightly increa sed across do se and not statistically significant. Historical control incide nces for this specific obser vation were not reported. However, the percentage of litters affected at all doses was within the historical control range (0- 20%) of litter incidences of externally visible alterations.

An increased litter (13.6-14.3%) and fetal (3.1-3.5%) incide nce of unilateral supernumerary rib (14th)
was observed at ≥ 60 mg/kg/day. The fetal incidences were not statistically significantly different
from concurrent controls. The li tter incidence (19%) of bilateral s upernumerary rib (14th) was also increased at 180 mg/kg/day, as wa s the fetal incidence (4.7%; p<0.05). Howeve r, both the litter and fetal incidences of supernumerary (14th) rib were below the historic al control incide nces (32.26% and 10.86%, respectively). An increase (p<0.01) in the “degree” of absence of ossification of the sternebrae (expressed as transf ormed ossification values per lit ter) was also observed at 180 mg/kg/day (7.03 vs. 0.59 in controls). The increase was outside the hi storical control range (0-0.31). However, given that the concurre nt control value (0.59) as above the upper limit of the historical control range and dose-response was lacking in the effect, the calcu lated value is not considered toxicologically significant.

With respect to visceral anomalies, a very slight increase in the malformations, unilateral microphthalmia and intestinal altera tion of the situs viscerum (combi ned with focal fi brosis of the peritoneum and mesentery) was obs erved at the high dose in one an imal in 1/21 litters only. The malformation, soft consistency of th e lens/unilateral folded retina, wa s observed in a different animal in 1/21 litters only. The incidenc e of each effect was above that of the historical controls (1/2691 fetuses; 1/401 litters); ho wever, fetal incidences were not statis tically significant and a dose-response was lacking at the doses tested.

The developmental LOAEL was not identified. The developmental NOAEL is 180 mg/kg/day.

Avian Reproduction Study (Bobwhite Quail)

In a one-generation reproductive study ( MRID 45280301 ) the toxicity of dich lobenil to groups (16 pens/treatment level) of 1 male and 1 female, 20-week-old Northe rn Bobwhite quail was assessed over approximately 21weeks. Dich lobenil was administered to th e birds in the diet nominalDICHLOBENIL FINAL

Page 51 of 52 concentrations of 0 (negative control), 150,300, and 600 mg/kg-diet. Mean-measured concentrations were -30.8 (<LOD, control) , 146, 289, and 600 ppm a.i.

The most sensitive parameters was 14-day old survi vors to number of hatch lings and adult male body weight, with the reviewer's analysis detecting significant reductions at all treatment levels. Treatmentrelated effects at the 289 ppm treat ment level included reductions in eggs laid (effects on eggs set, viable embryos, live 3-week embryos, number of hatc hlings, and hatchling survival were also reduced compared to per pen, but not when compared to e ggs set). There were adve rse effects at the 600 ppm treatment level on the number of eg gs laid, ratios of number of hatc hlings to eggs set and to live 3- week embryos, as well as on the ratio of hatchling survival to eggs set (effects on eggs set, viable embryos, live 3-week embryos, num ber of hatchlings, an d hatchling survival were also reduced compared to per pen, but not when compared to eggs set)). Furthermore, eggshell thickness ( ↓ 4.2%), hatchling ( ↓ 18%) and survivor weight ( ↓ 26%), were all adversely aff ected at the 600 ppm treatment level. Adult male weight gain was significantly re duced 42, 25 and 50% compared to control at 146, 300 and 600 ppm; no effect on female body weight gai n. There were no significant treatment-related effects on adult mortality, clinical effects, food c onsumption, or necropsy findings at any test level.

Avian Reproduction Study (Mallard Duck)

In a one-generation reproduction study ( MRID 45280302 ), Mallard ducks were fed diets containing nominal concentrations of 0, 150, 300, and 600 ppm a.i. Dichlobenil technical for 21 weeks. Mean measured dietary concentrations of the te st substance were 0,146, 289, and 600 ppm a.i.

There were no clear toxic-induced mortalities, pathological res ponses, or adverse effects on food consumption by adults or adult bodyweight. Signific ant reductions were detect ed for number of eggs laid and ratios involving egg production, number of hatchlings, a nd 14 day old survivors. As a result, the NOAEC was 289 ppm and th e LOAEC was 600 ppm a.i.

Early-Life Stage - Fish (Rainbow Trout)

An early-life stage study with dichlobenil on rainbow trout demonstrated a statistically significant effect on fry length at 34 days post-hatch at all concentrations tested (mean measured values of 0.33, 0.66, 1.2, 2.4, and 5.1 mg a.i./L) ( MRID 42224202 ). Therefore, the NOAEC is <0.33 mg/L and the LOEC is 0.33 mg/L.DICHLOBENIL FINAL

Page 52 of 52 APPENDIX 3: References Not Utilized in the Dichlobenil WoE Analysis

In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing Test Orders for the EDSP Tier 1 screening battery was ma de available in a Federal Register Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted as “other scientifically relevant information (OSRI) was pr ovided in the Report of the Endocrine Disruptor Review Team (USEPA, 2010).

Beginning in 2011, the agency has reviewed data cited as “OSRI which in cluded Part 158 studies previously submitted to the agency for registratio n/reregistration, published lite rature articles and/or Tier 1 assays. The agency also conducted a more r ecent search (2009 to 2014) of available scientific literature for any additional relevant information for their weight of evidence (WoE) evaluations.
These articles were evaluated in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species_rereg istration_workgroup/PDF_rot/esa_e valuation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature Toxicity Studies to Support Human Health Risk Assessment ( http://www.epa.gov/pesticides /science/lit- studies.pdf).

The following published and unpublished references were considered for use in the WoE analysis for
Dichlobenil but were not utilized due to one or mo re of the following reasons : 1) the article was not available in English; 2) the compound of interest was not used in the st udy; 3) the test material was not adequately described; 4) a formulat ed end-use product or mixture of ch emicals was utilized as the test material; 5) only acute mortality to xicity data were provided; 6) th e experimental conditions were not adequately described; 7) only an ab stract of the study was available; 8) the reference is a review article or book chapter and does not contain primary study da ta; 9) insufficient information was available to adequately assess the validity of the study results; 10) the 40 CFR Part 158 guideline study was classified as unacceptable/inadequate; 11) the stu dy dealt only with non-EDSP assay development; 12) no specific endocrine-related endpoints were assesse d in the study; and 13) the study contained only
data on invertebrates.

Barker, L. (1989) Dichlobenil: Or al (Gavage) Teratology Study in the Rabbits (Experiment II): Duphar Report No. 56645/85/87: Labor atory Project ID 562665/17B

. Unpublished study prepared by Hazleton UK. 139 p. (MRID 41257301)

Cooke, A.S. (1977) Effects of Fi eld Applications of the Herbic ides Diquat and Dichlobenil on Amphibians. Published in Environ. Pollut . (12) 1977, p. 43- 50.

Til, H.P., Feron, V.J., and de Groot, A.P. (1968) S hort-term (90-day) Toxicity Study with Dichlobenil (H133) in Beagle Dogs. Study conducted by Centra l lnstituut Voor Voedingsonderzoek for PhilipsDuphar N.V. Weesp, The Netherlands, Report No. R2362. 35 p. (MRID 48043301)