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: NORFLURAZON

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYNORFLURAZON 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-Pituitary-Thyroidal (HPT) Axis...........................18

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

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................21

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.......................................................................................27

LIST OF TABLES: Table 1. Tier 1 Assay for Norflurazon...............................................................8
Table 2. Estrogenic/Anti-Estroge nic Pathway for Norflurazon.................................11
Table 3. Androgenic/Anti-Androgenic Pathway for Norflurazon...............................15
Table 4. Thyroid Pathway for Norflurazon........................................................19
APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................30
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................43

APPENDIX 3. References Not Utilized in the Norflurazon WoE Analysis..........................53NORFLURAZON 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 enerationNORFLURAZON FINAL

iv Abbreviation Terminology

F2 Second filial g eneration
Fcd Fecundit y 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/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Intera g enc y Coordinatin g Committee on the Validation of Alternative

K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

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

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level

LOQ Limit of Quantitation

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

MROD Methoxyresorufin-O-dealkylaseNORFLURAZON FINAL

v Abbreviation Terminology

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 Atlas
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 TriiodothyronineNORFLURAZON FINAL

vi Abbreviation Terminology

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 Opening

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

Page 1 of 54 Executive Summary

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

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

On December 11, 2013, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of evidence (WoE) analysis of the potential interaction of norflurazon 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).

For the estrogen pathway, results of the estrogen receptor binding and aromatase assays were negative, however, a positive response in the in vitro steroidogenesis assay was observed (1.1- to 1.3-fold increases in estradiol production). Th e positive ERTA assay result was difficult to interpret, given the effect wa s not inhibited when the cells were treated with a known ER antagonist, suggesting a false positiv e response. In the Tier 1 pube rtal assay, decreased pituitary weight and a 2-day delay in vagi nal opening were seen only at th e high dose (500 mg/kg/day) in the presence of liver toxicity (enzyme and weight changes). Additionally, the only estrogenrelated effect observed in the ma mmalian Part 158 studies was an increase in ovarian cysts in the mouse carcinogenicity study that occurred at a dose that also resulted in increased absolute and relative liver weights.

Given that the liver is a target organ for norflurazon and estrogen-re lated effects in the Tier 1 and Part 158 mammalian studies were only seen at dose s that resulted in liver weight changes and/or hepatotoxicity, the observed effects are likely secondary resulting from enhanced liver
metabolism and hormone clearance.NORFLURAZON FINAL

Page 2 of 54 In the FSTRA, effects were only seen in the pr esence of overt toxicity (decreased feeding/body weight). Decreases in eggs laid in the mallard duck study were obs erved at a concentration that decreased female (and male) body weight gain/body weight. Decreases in hatchling weight or survival were also observed in the avian repr oduction studies (the e ffects on avian offspring survival/weight are limited in th e ability to inform about pot ential estrogen (and androgen)- related effects). Therefore, ove rall, effects on the estrogen-pathwa y appeared to only occur in the presence of effects on body we ight and/or decreased feeding.

For the androgen pathway, norflurazon was negative in the Tier 1 androgen receptor binding and steroidogenesis assays. Androgen-re lated effects in the Hershberge r assay occurred only in the presence of overt toxicity. In the male pubertal assay, decreases in sex accessory organ weights were observed at the high dose in the absence of overt toxicity; however increased liver weights were also seen at this dose. No androgen -related effects were observed in the Part 158
mammalian studies. The available evidence from th e Tier 1 assays and OSRI suggests that the decreases in AST weights, are secondary to he patic enzyme induction and subsequent enhanced
testosterone metabolism and clea rance in peripubescent mammals. In the FSTRA, effects were only observed at a concentration that showed overt toxicity (decreased male body weight and length and decreased feeding). There were no an drogen-related effects obs erved in the Part 158
wildlife studies.

For the thyroid pathway, in the pubertal female assay, decreased serum T 4 levels and decreased thyroid gland colloid area were seen at the high dose (500 mg/kg/day) in the presence of increased liver weights. No thyr oid-related effects were observed in the Tier 1 male rat pubertal assay. In the Part 158 chronic rat study, thyroid weights were in creased in females at 52 weeks and in males at 104 weeks at the high dose. In the chronic dog study, thyroid weights were increased in males at the high dos e. Thyroid weight changes in these studies occurred without concomitant changes in thyroid histopathol ogy and in the presence of liver function perturbations.

Although findings of thyroid gla nd atrophy are identified in th e AMA guideline as diagnostic criteria, no effects were seen on the incidence of follicular cell hypertrophy or hyperplasia, which are expected to be more commonly affected in the presence of a thyroid-related developmental delay. Overall, the thyroid and developmenta l findings in the AMA with norflurazon are
inconsistent with a direct , thyroid-related delay.

Based on a WoE of the available data, there is no convincing evidence for a potential interaction with the estrogen or androgen pathways. While th yroid relate effects we re observed in mammals, the effects are likely due to perturbation of liv er function. The developmental effects observed inNORFLURAZON FINAL

Page 3 of 54 the AMA do not appear to be thyroid-related.

In the EDSP Tier 1 in vivo male and female pubertal assays and OSRI, there was evidence for potential interaction with the thyroid pathway in st udies conducted with adult animals but no such data exists for potential thyr oid toxicity in the young animals.

For norflurazon, the current point of departure (POD) of 1.5 mg/kg/day used for human health risk assessment is based on hepatotoxicity obs erved in dogs at 5.02 mg/kg/day (LOAEL) in a chronic toxicity study. This POD is used for deri ving the chronic Reference Dose (RfD) used for chronic dietary risk assessment. A second POD of 30 mg/kg/day is used for calculating the Margins of Exposure (MOEs) for non- dietary risk assessment is base d on maternal toxicity seen at 60 mg/kg/day (LOAEL) in a developmental toxicity study in rabbits. These PODs are approximately 16-300-fold lower than the dose (500 mg/kg/day) that caused thyroid-related effects in the female pubertal assay.

In general, since the POD and the toxicity endpo ints of concern are base d on data obtained from adult animals, there usually would be a concern that the POD may not be protective of potential thyroid toxicity in the young. However in the ca se of norflurazon, although no data exists for potential thyroid toxicity in the young, a comp arative thyroid assay (C TA) is not recommended since: 1) the PODs are markedly lower than th e dose that caused alterations in T4 level and histopathological lesions in the female pubertal assay; 2) the thyroid-related effects in the femal pubertal assay were seen in the presence of hepatotoxicity (increases in enzymes and liver weights); 3) no thyroid-rela ted effects were seen in the male pube rtal assay; 4) all thyroid effects seen in the Part 158 studies were accompanied with systemic liver effects; 5) totality of the data
shows that the critical toxic effects of norflur azon are on liver and kidney and that the effects on the E, A, and T pathways occur only when the li ver is compromised; and 6) the chronic RfD and MOEs are protective of the thyroid-related e ffects seen in the female pubertal assay. Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is NOT
recommended for norflurazon since additional testing is not exp ected to impact EPA’s current regulatory point of departur es and endpoints for human h ealth risk assessments.

No fish or amphibian EDSP Tier 2 testing is recommended since there is no convincing evidence for an interaction with estrogen, androgen or thyroid pathways in fish or amphibians.

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

Page 4 of 54 I. Introduction

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

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

On December 11, 2013, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of eviden ce analysis of the potential interaction of norflurazon with the E, A or T hormone path ways. 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).

Norflurazon has a vapor pressure of 2.89 × 10 -8 mm Hg at 25°C, and vol atilization is not expected to contribute significan tly to the dissipation of norfluraz on in the environment. It has a water solubility of 33.7 mg/L at 25°C and an octanol/water partition co efficient of 2.3 (log P ow ). It is stable to hydrolysis, but does degrade rapidly in water due to photolysis. Norflurazon has a whole body bioconcentration factor va lue in fish of approximately 12.

The available information considered in determin ing the potential interact ion of norflurazon with the E, A, T pathways include submitted EDSP Tier 1 assays and other scientifically relevant information (OSRI) such as general toxicity stud ies 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 (U SEPA, 2011), is presented in Section IV. The
EDSP Tier 2 studies r ecommendations are presen ted in Section V.NORFLURAZON FINAL

Page 5 of 54 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 for norflurazon are s hown below in Table 1. Executive Summaries are presented in Appendix 1

B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 Screening assays were submitted as OSRI by the Test Order recipients and/or the public. This included studies published in the open literatu re and/or data submitted to support pesticide registration ( e.g., Part 158 guideline studies). The Agen cy’s review of the initial OSRI is provided in the Endocrine Disr uptor Review Team on norflur azon (USEPA, 2010). Since then, the Agency has also conducted a more recent sear ch of available scientif ic literature for any
additional relevant information. Su mmaries of the Tier 1 assays and the studies cited as other scientifically relevant information (OSRI) ar e presented in Appendix 1 and 2, respectively. Additionally, literature/studies c onsidered but not util ized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

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

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

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

Page 6 of 54 EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

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

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

Page 7 of 54 endocrine-related effect from a non-endocrine toxic response.

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

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

Page 8 of 54 The WoE approach involved consideration of data ( i.e ., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

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

A. EDSP Tier 1 Screening Assays

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

.

Table 1: Tier 1 Screening Assays for Norflurazon.

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48672503)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48672504)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48672501)

Steroidogenesis Assay (Human cell line 295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48672505)

Aromatase Assay (Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48672502)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48618206)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48618203)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID No. 48618204)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID No. 48618205)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48686502)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 48686501)NORFLURAZON FINAL

Page 9 of 54 B. Effects on Hypothalamic-Pit uitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for norflurazon to interact with the estrogen pathway is summarized in Table 2

. The various targets of the estroge n pathway across the rele vant Tier 1 assays are delineated so as to facilitate determination of potential for estrogen ic, anti-estrogenic, or HPG axis effects. This
table also includes HPG-re levant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment

Norflurazon was classified as negative (or not interactive) in the Tier 1 in vitro ER binding assay and the aromatase assay. In c ontrast, a response in the ER α transcriptional activation assay was

observed at norflurazon concentrations of 10 − 5 and 10 − 4 M. The relevance of the ERTA finding is questionable given that s upplemental data provided with the ERTA showed the hER α antagonist ICI 182,780 failed to inhib it the agonist response in the cells treated with norflurazon suggesting a false positive respons e in this assay and the fact that the ER binding assay was negative. A weak response also was observed in the steroidogenesi s assay with slight increases in estradiol production (1.1- to 1.3- fold) at the two highest concen trations tested, 1 and 10 μM.

In the uterotrophic assay, norflurazon did not indu ce uterine weight increases. In the female pubertal assay, norflurazon also showed no consis tent evidence of an estrogenic response. Absolute (unadjusted and adjusted) mean pituitary weight was decreased 17 to 18% at the highdose. Additionally, vaginal openi ng was delayed 2.1 days at the high dose. The only potential estrogen-related effect observed in the Part 158 toxicity studies were the observation of cystic ovaries in the mouse carcinogenicity study at the high dose (218 mg /kg/day) and a nonstatistically significant increases in ovarian we ight (17 and 30%) seen at the mid and high dose in the 9-month chronic toxicity study.

In the FSTRA, fertility was not affected by nor flurazon treatment, but there was a 95% decrease in fecundity (p ≤ 0.05) in the high-dose group relative to the negative control; additionally this treatment group was significantly decreased compared to the solvent contro l (the solvent control had a significant 34% decrease in fecundity compar ed to the negative cont rol). The difference in fecundity between the control group s was attributed to an unusually high output of eggs in two replicates in the negative contro l during the last week of the st udy, although a possible solvent
effect could not be definitivel y discounted. Plasma VTG and GSI concentrations in females
were not affected but increased incidences of minimal to severe oocyte atresia, decreased postovulatory follicles, and decreased staining of yolk proteins were observed in high-dose females. In this study at the high treatment level, signs of overt toxicity were observed including reduced feeding and significant decr eases in male body weight (28%) and length (6%).NORFLURAZON FINAL

Page 10 of 54 In Part 158 avian reproduction studies, decreased hatchling weights (15%) were observed at 200 mg/kg-diet in the mallard duck study and reduc tion egg production (11%) and female and male body weight gain were observed at 1000 mg/kg-di et. In the bobwhite qu ail study, decreases in hatchling survival (although not concentration-res ponsive) at 200 mg/kg-diet was observed.
There were no effects on mort ality, behavior, food consump tion, or other reproductive parameters in either study. In Part 158 early life cycle studies with fish, effects on survival and/or growth were observed at ≥ 1.5 mg/L.NORFLURAZONFINALPage 11 of 54Table 2: Estrogenic/Anti-Estrogenic Pathway for NorflurazonLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Norflurazon1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histo p atholo gy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility/Fecundity Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48672503)NERTA(MRID 48672504)P4Aromatase(MRID48672502)NSteroidogenesis(MRID 48672505)PUterotrophic(MRID 48618206)NX(H)5Female Pubertal Rat(MRID 48618204)NNN↓17-18%(H)N↑2.1 d(H)↑LW14-21%;CC6(H)NFSTRA(MRID 48686502)NENNNNNX(H)7NORFLURAZONFINALPage 12 of 54Table 2: Estrogenic/Anti-Estrogenic Pathway for NorflurazonLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Norflurazon1Study Type / LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histo p atholo gy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility/Fecundity Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRIDevelopmental toxicity(Rat; 00063621)NNXX(H)Developmental toxicity(Rabbit; MRIDs00131151, 00131152)NRNXX(H)Three-generationreproduction (Rat;MRID 00080750)NENENENE NENXNRTwo-generationreproduction (Rat;MRID 43522301)NENENNE NENXNOne-generationreproduction (Mouse;MRID 00080751 )NRNRNRNR NRNX N9-mo Chronic toxicity(Rat; MRID 00091056)NENNNEX N2-yr Chronic toxicity(Rat; MRID 00082019)NENNNX NCarcinogenicity(Mouse; MRID00111649)NENP8(H)NX NChronic toxicity (Dog;MRID 00111618)NNNNX NAvian Reproduction(Duck; MRID00052172; 42615302)P9N N10Avian Reproduction(Quail; MRID00039222; 42615301)P11N NNORFLURAZONFINALPage 13 of 541. Key to responses:L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows (↓or↑)indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight areabsolute unless otherwise indicated. LW=Liver weight, CC= Clinical chemistry2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in thiscolumn. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For details seeSection IV. A4. In the ERTA assay, norflurazon had a calculated PC50s of 10–5.3and 10–5.2M in the Runs 1 and 2; however, failure of the hERαantagonist ICI 182,780 to inhibitthe response of the cells to norflurazon suggests that the response of norflurazon in the assay may be a false positive.5. Body weight was decreased by 20% in the high dose group on Day 4.6. Clinical chemistry changes in females atthe high dose included a 100% increasein blood urea nitrogen (BUN) and 65% decrease in bile acid.7. At the high treatment level signs of overt toxicity were observed including reduced feeding and significant decreases in male body weight (28%) and length (6%).8. Observations of cystic ovaries at high dose (218 mg/kg/day).9. Decreased hatchling weights relative to the control (15%) at 200 mg/kg-diet. At 1000 mg/kg, there was an 11% reduction in eggs laid and decreased hatchlingweight.10. Adult female and male body weight gains were reduced compared to control at 1000 mg/kg-diet (male: +6g vs. -53g at 1000 ppm; female: +140g vs. +51g (64%decrease) at 1000 ppm). Female body weight was also significantly reduced 7.5%. There were no other clinical signs of toxicity.11. Decreases in hatchling survival (although not concentration-responsive) at 200 mg/kg-dietP Positive findings (in vitro)/No effect (in vivo)N Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedNORFLURAZON FINAL

Page 14 of 54 2. Effects on Androgen Pathway

The potential for norflurazon to interact w ith the androgen pathwa y are summarized in Table 3. The various targets of th e androgen pathway across the relevant Tier 1 as says are delineated so as to facilitate determination of potential for a ndrogenic, 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 toxicity are di scussed in the text but are not reported in the table and not considered furt her in the WOE assessment

Norflurazon was negative (non -binder) in the Tier 1 in vitro androgen receptor (AR) binding assay, and the steroidogenesis as say. Norflurazon was also classi fied as negative for androgenic activity in the Tier 1 in vivo Hershberger. In the anti-androge nic portion of the Hershberger assay, high-dose animals had signific ant decreases in four AST weights (i.e., seminal vesicles with coagulating glands, vent ral prostate, LABC, and Cowpe r’s gland) accompanied by a
decrease in serum testosterone concentration of 31% (NS) in the hi gh-dose animals in the presence of overt toxicity (12- 24% decrease in body weight). In the male pubertal assay, body weights at preputial separation (PPS) were not affected, but complete PPS was non-significantly delayed in the high-dose group ( 375 mg/kg/day) by 1.8 days. Seru m testosterone concentration was decreased (NS) by 16% in the high- dose anim als relative to control. Adjusted weights of AST (ventral and dorsolateral prostate and semi nal vesicle without flui d) were significantly decreased at the high dose. No histopathologi cal lesions were observed in the testes or epididymides. Significant increases in relative liver weights were seen at the low and high doses of norflurazon (14 and 23%, re spectively). There was no ev idence of effects on male reproductive parameters in the existi ng norflurazon Part 158 mammalian studies.

In the FSTRA, at the high dose, male VTG concentrations were significantly decreased by 89%, male nuptial tubercles (as a scor e) were decreased (11 vs. 29 fo r negative control) and male gonadal development was arrested in three of ei ght fish. However, these effects occurred in the presence of overt toxicity in cluding decreased feeding, decrea sed body length and decreased male body weights (10 and 28%, mi d and high). In the Part 158 wildlife studies; decreases in hatchling weight/survival were observe d in the avian reproduction studies.NORFLURAZONFINALPage 15 of 54Table 3: Androgenic/Anti-Androgenic Pathway for NorflurazonLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Norflurazon1Study Type/ LiteratureCitation AR Binding Steroidogenesis Sex Steroid hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weigh t Epididymides Histopathology Pituitary Weigh t Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48672501)NSteroidogenesis(MRID 48672505)NHershberger(MRID 48618203)NN↑LW28%(L)↑39%(H)↓BW11-24%(H)Male Pubertal Rat(MRID 48618205)NNNNN↓14%(H)↓22%,DP;↓15%,VP;↓15%,SV (H)N↑LW15-23%(H)NFSTRA(MRID 48686502)NENNNNNX(H)4OSRIDevelopmentaltoxicity (Rat;00063621)NNEX(H)NORFLURAZONFINALPage 16 of 54Table 3: Androgenic/Anti-Androgenic Pathway for NorflurazonLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Norflurazon1Study Type/ LiteratureCitation AR Binding Steroidogenesis Sex Steroid hormones Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weigh t Epididymides Histopathology Pituitary Weigh t Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) / Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Developmentaltoxicity (Rabbit;MRIDs 00131151,00131152)NXX(H)Three-generationreproduction (Rat;MRID 00080750)NE NE NE NE NE NENX NRTwo-generationreproduction (Rat;MRID 43522301)NE N NE N NE NENX NOne-generationreproduction (Mouse;MRID 00080751 )NRNRNRNRNRNRNX N9-mo Chronic toxicity(Rat; MRID00091056)NNNENENENEX N2-yr Chronic toxicity(Rat; MRID 00082019)NNNENENNX NCarcinogenicity(Mouse; MRID00111649)NNNENENNEX NChronic toxicity (Dog;MRID 00111618)NN NE N NNX NAvian Reproduction(Duck; MRID00052172; 42615302)P5N N6Avian Reproduction(Quail; MRID00039222; 42615301)P7N NNORFLURAZONFINALPage 17 of 541. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.. Arrows (↓or↑)indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight areabsolute unless otherwise indicated. Abbreviations for androgensensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Levator anibulbocavernosus (LABC), Epididymides (EP); LW=Liver weight2.The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in thiscolumn. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For details seeSection IV. A4. Decreased feeding, decreased body length, and decreased male body weights (10 and 28%, mid and high).5. Decreased hatchling weights relative to the control (15%) at 200 mg/kg-diet and at 1000 mg/kg-diet. .6. Adult female and male body weight gains were reduced compared to control at 1000 mg/kg-diet (male: +6g vs. -53g at 1000 ppm;female: +140g vs. +51g (64%decrease) at 1000 ppm). Female body weight was also significantly reduced 7.5%. There were no other clinical signs of toxicity.7. Decreases in hatchling survival (although not concentration-responsive) at 200 mg/kg-dietP Positive findings assay responseN Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedNORFLURAZON FINAL

Page 18 of 54 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 pubertal female and male (ra t) assays, and the AMA (frog).

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

The AMA demonstrated delayed development at high concentration on Day 7 and Day 21 compared to solvent control; the delays at Day 21 were not significant when compared to the negative control. An increase in mild t hyroid gland atrophy was observed at the high
concentration.

In the Tier 1 female pubertal rat assay with norflurazon, eviden ce of altered thyroid function included decreased total thyroxine (T 4 ) at both doses tested ( 250 and 500 mg/kg/day) and

decreased colloidal area at the high dose. Serum TSH levels we re unchanged in females. No thyroid-related effects were observed in the Ti er 1 male pubertal assay. Pituitary weights were decreased at the high dose in both sexes in the pubertal assays.

In the Part 158 mammalian toxicity studies, effe cts on thyroid weights were observed in a ninemonth chronic oral toxicity study but the effect s were not statistically significant in either sexes with decreased thyr oid weights in males ( ↓ 30%, NS) and increased weights in females ( ↑ 11 to 47%, NS) at the high dose (25.1 mg/kg/ day) and similar weight changes at the intermediate dose (12.5 mg/kg/day). No microsc opic alterations in the thyroid glands were reported for this study. Increases in thyroi d weights (with no repor ted histopathological effects) were reported for the two year chro nic oral toxicity/carcinogenicity study with
norflurazon in rats in the hi gh-dose group (51.25 mg/kg/day). Thyroid weights in females at 52 weeks were increased 30 to 31% and weights in males at 104 weeks were increased 30 to 32%. Finally, thyroid weights were increased in a six month chronic oral toxicity study with norflurazon in dogs. At the intermediate dose (5.02/4.77 mg/kg/day; M/F), thyroid weights were increased by 33% (p<0.05) in male dogs a nd by 37% (NS) in female dogs, and thyroid weights were increased by 43% (p<0.01) in male dogs and by 30% (NS) in female dogs at the high dose (14.27/17.75 mg/kg/day; M/F).NORFLURAZON FINAL

Page 19 of 54 Table 4. Thyroid Pathway for Norflurazon.

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

Study Type / Literature CitationThyroid WeightThyroid: Grossand HistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmentalstage (± orasynchronous) orHLLGrowth (BW,SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assays

Male Pubertal Rat (MRID 48618205) N N N N ↓ 14% (H) ↑ LW 15-23%

(H) N

Female Pubertal Rat (MRID 48618204) N ↓ colloidal

area (H) ↓ 13%, ↓ 23% (L, H) N ↓ 17- 18% (H) ↑ LW 14-21%;

CC 4 (H) N

AMA (Frog; MRID 48686501) P 5 P 6 N N

OSRI Three-generation reproduction (Rat; MRID 00080750) NE NE NE X NR

Two-generation reproduction (Rat; MRID 43522301 ) NE NE NE X N

One-generation reproduction (Mouse; MRID 00080751 ) NR NR NR X N

9-mo Chronic toxicity (Rat; MRID 00091056) N N NE X N

2-yr Chronic toxicity (Rat; MRID 00082019) Wk 52: ↑ 30-31% (Female; H);

Wk 104: ↑ 30-32% (Male; H) N N X N

Carcinogenicity (Mouse; MRID 00111649) N N N X N

Chronic toxicity (Dog; MRID 00111618) Male: ↑ 33%, ↑ 43%

(M, H) N N X N

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not appli cable in this assay. LW=liver weight; CC= clinical chemistry 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 OSRINORFLURAZON FINAL

Page 20 of 54 is indicated by an X in this column. For details see Section IV. A 4. Clinical chemistry changes in females at the high dose included a 100% increase in blood urea nitrogen (BUN) and 65% decrease in bile acid. 5. Slight increase in mild thyroid gland atrophy noted at the high dose. 6. Delayed development at high concentration on Day 7 and Day 21 compared to solvent control; the delays at Day 21 were not significant when compared to the negative control. P Positive findings N Negative findings NE Not examined NR Not reportedNORFLURAZON FINAL

Page 21 of 54 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of norflurazon 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 modes of action may elicit a number of phenotypic consequences other than those evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses 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 Tire 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues shou ld be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l disruptions in homeostasis” (USEPA, 2013).

A. Systemic/Overt to xicity in the in vivo Tier 1 Assay and OSRI

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

Page 22 of 54 1. Tier 1 in vivo Assay s

Overt toxicity at the high dos e (1000 mg/kg/day) in the anti-a ndrogen phase of the Hershberger assay manifested as increased in cidence of clinical signs (hair loss, dry red material around nose, decreased defecation), decrease s in body weight (11–24%), body weight gain (90%), and food consumption (31%) and increa sed liver weight (39%).

In the FSTRA, decreased male body weight and le ngth, and reduced feeding were seen at the high dose; additionally decreased male body weight was observed at the mid dose compared to the negative control. No overt toxicity was seen at othe r doses in these studies.

Systemic toxicity at the high doses in the pubertal assays manifested as hepatotoxicity in both sexes. In the male pubertal assay at the high dose (375 mg/k g/day), there were significant (p<0.05) increases in absolute ( ↑ 15%) and relative ( ↑ 23%) liver weights and ALP activity ( ↑ 18%) levels. In the female pubertal assay atthe high dose (500 mg/kg/day), there were significant (p<0.05) increases in absolute ( ↑ 14%) and relative ( ↑ 21%) liver weights and total BUN ( ↑ 100%) and a decrease in total bile acid ( ↓ 65%).

2. OSRI

In the two generation reproduction study, significant (p<0.05) increases in absolute and relative liver weights were seen at 102 mg/kg/day in ma les and 130 mg/kg/day in females of F0 and F1 generations. Also increased inci dence of hepatocellular hypertrophy was seen in both generations of parental rats at the high dose.

In the chronic study with dogs, absolute liver weights were increased in males by 30%, 38% and 41% at the low (1.5 mg/kg/day), mid (5 mg/ kg/day) and high (14 mg/kg/day) dose groups, respectively. In females, liver weights were increased by 23% and 44% at the mid and high dose groups. There was an increase in cholesterol leve ls in males (23-40%) and females (6-34%) at the mid and high dose levels. The weight of the thyroid gland was increased in a dose-related manner in males (16, 33% and 43% at the low, mid and high dose, respectively). In females, there was a non-dose dependent incr ease in thyroid weights (12%, 37% and 30%, at the low, mid and high dose, respectively). There was no corroborative thyroid histopathology.

In the chronic toxicity/carcinogenic ity with rats, at the interim sacr ifice (52 weeks), the weight of the liver was increased in males (17%) and in females (13%) at the mid dose (19 mg/kg/day); and in males (21%) and females (27% ) at the high dose (52 mg/kg/day). At termination, liver weight was increased in both sexes (14% in male s and 12% in females) only at the high dose. Histopathology showed increased incidence of he mosiderin pigment deposition in the liver of both sexes at 52 mg/kg/day. The weight of the le ft thyroid gland was increased by 30% in males at the high dose, while the weight of the ri ght thyroid gland was increased by 12%, 19% and 32% at the low, mid and high dose groups, respectively.NORFLURAZON FINAL

Page 23 of 54 In the carcinogenicity study with mice, absolute liver weight wa s increased by 9% in males and 15% in females at the mid dose (59 mg/kg/day), and by 27% in males and 21% in females at the high dose (219 mg/kg/day). Relati ve liver weight was increase d 43% in males and 19% in females at 219 mg/kg/day. Carc inogenic potential was evidenced by an increased incidence of hepatocellular adenomas and combined adenomas/carcinomas at the high dose.

Thus, the liver effects seen in the in vivo Tier 1 assays showed that liver is the target organ for norflurazon. This was corroborated by the liver ef fects (weights and hist opathology) observed in
the Part 158 studies at doses lower than the dose levels used in the in vivo Tier 1 rat assays.

In the Part 158 avian reproduction studies, no eff ect on mortality, behavior or food consumption were observed in the bobwhite quail study. In a mallard duck study, adult female and male body weight gains were reduced compared to contro l at 1000 mg/kg-diet (mal e: +6g vs. -53g at 1000 ppm; female: +140g vs. +51g at 1000 ppm). Female body weight was also significantly reduced 7.5%. There were no effects on mortality, behavior or food consumption.

In Part 158 early life cycle studies with fish, effects on survival and/or growth were observed at

≥ 1.5 mg/L.

B. Estrogen pathway

Results of the estrogen recepto r binding and aromatase assays we re negative, however, a positive response in the in vitro steroidogenesis assay was observed (1.1 - to 1.3-fold increases in estradiol production). The positive ERTA assay result was di fficult to interpret, given the effect was not
inhibited when the cells were treated with a known ER antagoni st, suggesting a false positive response. In the Tier 1 pubert al assay, decreased pituitary wei ght and a 2-day delay in vaginal opening were seen only at the high dose (500 mg /kg/day) in the presence of liver toxicity (enzyme and weight changes). Additionally, the only estrogen-related effect observed in the
mammalian Part 158 studies was an increase in ov arian cysts in the mouse carcinogenicity study
that occurred at a dose that also resulted in increased absolute and relative liver weights.

Given that the liver is a target organ for norflurazon and estrogen-re lated effects in the Tier 1 and Part 158 mammalian studies were only seen at dose s that resulted in liver weight changes and/or hepatotoxicity, the observed effects are likely secondary resulting from enhanced liver
metabolism and hormone clearance.

In the FSTRA, effects were only seen in the pr esence of overt toxicity (decreased feeding/body weight). Decreases in eggs laid in the mallard duck study were obs erved at a concentration that decreased female (and male) body weight gain/body weight. Decreases in hatchling weight or survival were also observed in the avian repr oduction studies (the e ffects on avian offspring survival/weight are limited in th e ability to inform about pot ential estrogen (and androgen)-NORFLURAZON FINAL

Page 24 of 54 related effects). Therefore, ove rall, effects on the estrogen-pathwa y appeared to only occur in the presence of effects on body we ight and/or decreased feeding.

C. Androgen pathway

Norflurazon was negative in the Tier 1 androgen receptor binding and steroidogenesis assays. Androgen-related effects in the Hershberger a ssay occurred only in the presence of overt toxicity. In the male pubertal assay, decreases in sex accessory organ weights were observed at the high dose in the absence of overt toxici ty; however increased liver weights were also
seen at this dose. No andr ogen-related effects were observe d in the Part 158 mammalian
studies. The available evidence from the Tier 1 assays and OSRI suggests that the decreases in AST weights, are secondary to hepatic enzyme induction and subsequent enhanced
testosterone metabolism and clea rance in peripubescent mammals.

In the FSTRA, effects were only observed at a concentration that s howed overt toxicity (decreased male body weight and length and de creased feeding). There were no androgenrelated effects observed in the Part 158 wildlife studies.

D. Thyroid pathway

In the pubertal female assay, decreased serum T 4 levels and decreased thyroid gland colloid area were seen at the high dose (500 mg/kg/day) in the presence of increased liver weights. No thyroid-related effects were observed in the Tier 1 male rat pubertal assay. In the Part 158 chronic rat study, thyroid weights were increased in females at 52 weeks and in males at 104 weeks at the high dose. In the chronic dog study, thyroid weights were increased in males at the high dose. Thyroid weight changes in these studies occurred without concomitant changes in thyroid histopathology and in the pres ence of liver function perturbations.

Although findings of thyroid gla nd atrophy are identified in th e AMA guideline as diagnostic criteria, no effects were seen on the incidence of follicular cell hypertrophy or hyperplasia, which are expected to be more commonly affected in the presence of a thyroid-related developmental delay. Overall, the thyroid and developmenta l findings in the AMA with norflurazon are
inconsistent with a direct , thyroid-related delay.NORFLURAZON FINAL

Page 25 of 54 E. Conclusions

Based on a WoE of the available data, there is no convincing evidence for a potential interaction with the estrogen or androgen pathways. While th yroid relate effects we re observed in mammals, the effects are likely due to perturbation of liv er function. The developmental effects observed in the AMA do not appear to be thyroid-related.

V. EDSP Tier 2 Testing Recommendations

In the EDSP Tier 1 in vivo male and female pubertal assays and OSRI, there was evidence for potential interaction with the thyroid pathway in st udies conducted with adult animals but no such data exists for potential thyr oid toxicity in the young animals.

For norflurazon, the current point of departure (POD) of 1.5 mg/kg/day used for human health risk assessment is based on hepatotoxicity obs erved in dogs at 5.02 mg/kg/day (LOAEL) in a chronic toxicity study. This POD is used for deri ving the chronic Reference Dose (RfD) used for chronic dietary risk assessment. A second POD of 30 mg/kg/day is us ed for calculating the Margins of Exposure (MOEs) for non- dietary risk assessment is base d on maternal toxicity seen at 60 mg/kg/day (LOAEL) in a developmental toxicity study in rabbits. These PODs are approximately 16-300-fold lower than the dose (500 mg/kg/day) that caused thyroid-related effects in the female pubertal assay.

In general, since the POD and the toxicity endpo ints of concern are base d on data obtained from adult animals, there usually would be a concern that the POD may not be protective of potential thyroid toxicity in the young. However in the ca se of norflurazon, although no data exists for potential thyroid toxicity in the young, a comp arative thyroid assay (C TA) is not recommended since: 1) the PODs are markedly lower than th e dose that caused alterations in T4 level and histopathological lesions in the female pubertal assay; 2) the thyroid-related effects in the femal pubertal assay were seen in the presence of hepatotoxicity (increases in enzymes and liver weights); 3) no thyroid-rela ted effects were seen in the male pube rtal assay; 4) all thyroid effects seen in the Part 158 studies were accompanied with systemic liver effects; 5) totality of the data
shows that the critical toxic effects of norflur azon are on liver and kidney and that the effects on the E, A, and T pathways occur only when the li ver is compromised; and 6) the chronic RfD and MOEs are protective of the thyroid-related e ffects seen in the female pubertal assay.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is NOT
recommended for norflurazon since additional testing is not exp ected to impact EPA’s current regulatory point of departur es and endpoints for human h ealth risk assessments.

No fish or amphibian EDSP Tier 2 testing is recommended since there is no convincing evidence for an interaction with estrogen, androgen or thyroid pathways in fish or amphibians.NORFLURAZON FINAL

Page 26 of 54 For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard
duck are available for norflur azon. In both species, effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from norflurazon exposure. Additional testing is not recommended.NORFLURAZON FINAL

Page 27 of 54 VI. References

Eschbach, B., Aerni, R., Hertl, P., et al . (1994) Two Generation Re production Study in Rats: Norflurazon: Revised Final Report: Lab Project Numbers: 440 R: BS4809: 94/123 I. Unpublished study prepared by Sandoz Agro, Ltd. (MRID 43522301)

Hrab, R., De Raismes, M., Ya o, C. (1983a) Dose Range-finding Study of Norflurazon in Pregnant Rabbits: Sandoz Pr oject No. T-1793. (Unpublished study submitted by Sandoz, Inc., Crop Protection). (MRID 00131151)

Hrab, R., Halbert, F., Quagliotti, M., et al . (1983b) Investigation of Teratogenic Potential of Norflurazon in the Rabbit—Se gment II: Sandoz Project T -1794. (Unpublished study received Sep 16, 1983 under 11273-13; subm itted by Sandoz, Inc., Crop Protection, San Diego, CA; CDL:251279-A). (MRID 00131152)

Hartman, H.A., Hrab, R. (1972) Compound 9789: I nvestigation of Terato genic Potential in the Rat: Report No. T-I-817172. (Unpublished study received Nov 14, 1972 under 3G 1310; submitted by Sandoz, Inc., Homestead, Fla.; CDL:092234-E). (MRID 00063621)

Klotzsche, C., Carp y, S., De Rijk, et al . (1973) San 9789 (Zorial), 6 Months Feeding Study in Dogs: Bericht Nr. 56173. (Unpublished study received May 13, 1974 under 4F1506; prepared by Sandoz-Wander, Inc., Homestea d, FL; CDL: 093995-A). (MRID 00111618)

Kuhl, A. (2011) Norflurazon — A Dose Range-F inding Oral (Gavage) Toxicity Study in Young Adult Rats. Unpublished study by WIL Research Laboratories, LLC, Ashland, OH.Laboratory Project ID: WIL639115, (MRID 48618202)

LeBlanc, G. (1982) Early life stage toxicity st udy in the fathead minnow on norflurazon: Report #BW-82-5-1166: Sandoz Project T-1767. Unpub lished study received Nov. 15, 1982, under 11273-13; prepared by EG&G Binomics, submitte d by Sandoz, Inc., Crop Protection, San Diego, CA; CDL: 248828-A. (MRID 00118047)

LeBlanc, G. (1982) Early life stage toxicity st udy in the rainbow trout on norflurazon: Report #BW-82-5-1165: Sandoz Project T -1733. U npublished study received Nov. 15,1982, under 11273-13; prepared by EG&G Binomics, submitte d by Sandoz, Inc., Crop Protection, San Diego, CA; CDL: 248829-A. (MRID 00118048)

Sawhney Coder, P. (2012). Norflu razon — Hershberger Bioassay in Orchidoepidiymectomized Rats. Unpublished st udy by WIL Research Laboratories, LLC, Ashland, OH. WIL Research Laboratories, LLC, Ashland, OH. Study No. WIL-639118. (MRID 48618203)NORFLURAZON FINAL

Page 28 of 54 Sawhney Coder, P. (2012) No rflurazon — Pubertal Development and Thyroid Function in Intact Juvenile / Peri pubertal Male Rats. Unpubl ished study by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Report No.: WIL-639117. (MRID 48618205)

Sawhney Coder, P. (2012) No rflurazon — A 14-Day Dose Ra nge-Finding Oral (Gavage) Toxicity Study in Juvenile Rats. Unpublishe d study by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Report No.: WIL-639114. (MRID 48618201)

Sawhney Coder, P. (2012) Norf lurazon — Uterotrophic Assay in Ovariectomized Rats Final Report. Unpublished study by WIL Research Laboratories, LLC, Ashland, Ohio. Laboratory Project ID: WIL-639119 (MRID 48618206)

Sawhney Coder, P. (2012) Norfluraz on — Pubertal Development and Thyroid Function in Intact Juvenile / Peripubertal Female Rats . Unpublished study by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Report No.: WIL-639116. (MRID 48618204)

Schneider, S., Martin, K., Mar tin, Kendall, T., and Krueger, H. (2012) Norflurazon — Fish Short-term Reproduc tion Assay with the Fathead Minnow ( Pimephales promelas ). Unpublished study performed by Wildlife In ternational, Ltd., Ea ston, Maryland, Report No.: 528A-223. Submitted by Syngenta Crop Protection, LLC, Greensboro, NC. (MRID 48686502)

Schneider, S., Kendall, T ., and Krueger, H. (2012) Norflurazon — Amphibian Metamorphosis Assay for the De tection of Thyroid Active Substances. Unpublished study performed by Wildlife International, Ltd., Easton, Maryla nd. Laboratory report number 528A-224. Study sponsored by Syngenta Cr op Protection, LLC, Greensboro, NC. (MRID 48686501)

USEPA. (2010) Norflurazon — Report of the OPP Endocrine Di sruptor Review Team; Test Order #: EDSP-105801-19. July 22, 2010. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

USEPA. (2011) Endoc rine Disruptor Screening Progr am — Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical Safety and Pollution Preventi on. U.S. Environmental Protection Agency, Washington, DC.

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

Wagner, H. (2012) Norflurazon — H295R Steroidogenesis Assay. Unpublished studyNORFLURAZON FINAL

Page 29 of 54 by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9047V-100330STER. (MRID 48672505)

WARF Institute, Inc. (1975) Compound 9789: Reproduction Studies in Rats and Mice: Volume I: Reproduction Studies on Rats. Final rept. (Unpublishe d study, including submitter summary, received May 8, 1975 under 5F1602; su bmitted by Sandoz, Inc., Homestead, Fla.; CDL:094369-A). (MIRD 00080750).

WARF Institute, Inc. (1975) Compound 9789: Reproduction Studies in Rats and Mice: Volume 2: Reproduction Studies in Mice. Final rept. (Unpublis hed study, including submitter summary, received May 8, 1975 under 5F1602; su bmitted by Sandoz, Inc., Homestead, Fla.; CDL:094368-A). (MRID 00080751)

WARF Institute, Inc. (1975) Compound 9789: Tw o Year Feeding Study in Rats. Final rept. (Unpublished study received May 8, 1975 under 5F1602; submitted by Sandoz, Inc., Homestead, Fla.; CDL:094370-A; 09 4371; 094372; 094373; 094374) (MRID 00082019)

WARF Institute, Incorporated. (1972) 9-Month Feeding Study in Rats: F0 Generation. Final rept. (Unpublished study received Nov 14,1972 unde r 3G1310; prepared by WARF Institute, Inc., submitted by Sandoz, Inc., Homest ead, Fla.; CDL: 092233-G). (MRID 00091056)

WARF Institute, Inc. (1973) Long Term F eeding Study—Rat: 1 Y ear Report: Compound 9789. (Unpublished study received May 13, 1974 under 4F1506; prepared by WARF Institute, Inc., submitted by Sandoz-Wander, Inc., Homestead, FL; CDL:093994-G; 093995). (MRID 00111617)

WARF Institute, Inc. (1975) Compound 9789: Tw o Year Feeding Study in Mice. Final rept. (Unpublished study received Aug 21, 1975 under 4F1506; submitted by SandozWander, Inc., Homestead, FL; CDL:096144-A; 096147; 096148). (MRID 00111649)

Wilga, P. (2012) Norfluraz on — Human Recombinant Aromat ase Assay. Unpublished study prepared by CeeTox, Inc., Kalamazoo, MI. Laboratory Report No.: 9047V-100330AROM. (MRID 48672502)

Willoughby, J. (2012) Norflura zon — Androgen Receptor Binding (Rat Prostate Cytosol). Unpublished study prepared by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9047V-100330ARB. (MRID 48672501)

Willoughby, J. (2012) Supplemental Inform ation — Laboratory Pr oficiency Data for ERTA Assays and Saturation Binding Data fo r AR and ER Binding Assays for Assorted Chemicals. Unpublished study prepared by CeeTox, Inc., Kalamazoo, MI. (MRID 48843501).

Willoughby, J. (2012) Norflu razon — Estrogen Receptor Binding (Rat Uterine Cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9047V-100330ERB. (MRID 48672503)NORFLURAZON FINAL

Page 30 of 54 APPENDIX 1. EDSP Ti er 1 Screening Assays

Amphibian Metamorphosis A ssay (AMA); (OCSPP 890.1100)

The 21-day assay (MRID 48686501) of nor flurazon (99.5% purity) on amphibian metamorphosis of African clawed frogs ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Fabe r (NF) stage 51 (80/control and treatment group; 20/replicate) were expos ed to negative and solvent (0.04 mL/L dimethylformamide; DMF) controls and nominal norflurazon con centrations of 0.10, 1.0, and 10 mg a.i./L. Meanmeasured concentrations were <0.05 (<LOQ; c ontrols), 0.094, 0.92, and 5.7 mg a.i./L. The test system was maintained at 21.5 to 22.6°C and a pH of 7.8 to 8.3.

Unless otherwise indicated, all eff ects are reported based on comparis on to the negative control.

No mortalities were observed in the negative cont rol or treatment groups, and a single mortality
was observed in the solvent contro l. Tadpoles with crooked tails we re observed for 36 to 54 of surviving tadpoles in the controls and treatment groups. In the so lvent control a nd low treatment group, one tadpole in each group wa s observed to be smaller th an the other tadpoles; an additional tadpole in the low treatment group was observed swimming erratically.

Day 21 wet weight and snout-vent length (SVL) we re significantly increa sed (p<0.05) in the solvent control group compared to the negative control. Similar though non-significant increases (p>0.05) in these parameters were obs erved in the low and mid treatment groups; Day 21 wet weight and SVL in the high treatment gro up were generally decrea sed (not significant; p>0.05) compared to the negative control. A marginally significan t increase (p=0.051; Dunnett’s) of 14.5% in Day 7 body weight was obs erved in the low treatment group compared to negative control.

Norflurazon did not significantly affect (p>0.05) Day 7 or Da y 21 normalized (for snout-vent length) hind-limb length (HLL) at any of the treatment levels. Furthermore, there was no significant acceleration or delay (p>0.05) of median NF developmental stage at Day 7 or Day 21 compared to the negative control, and no as ynchronous development was observed. The NF developmental stage at Day 7 was decreased at the high treatment level (media NF stage: 53) compared to the negative control (median NF st age: 54), but the differe nce was not significant (p=0.067; Jonckheere-Terpstra). There were some individuals excluded fr om the analysis of Day 21 growth endpoints because NF stage de velopment exceeded 60; there was no pattern noted among their occurrence: 0, 0, 5, 4, and 1 each from the negative control, solvent control, low, mid, and high treatment groups, respectively.NORFLURAZON FINAL

Page 31 of 54 There was a slight increase in mild thyroi d gland atrophy in the hi gh treatment group. Other observations included thyroid gland hypertrophy , follicular cell hypertrophy and hyperplasia, and changes in the shape of follicular cells; no treatment-related pattern was apparent for these diagnoses.

Since there were significant differences between the solvent control and negative control in the Day 21 wet weight and SVL, a comparison of th e treatment groups to the solvent control was conducted for all endpoints. At the high treatme nt level, there was a significant decrease (p<0.05) in NF stage at Day 7 a nd Day 21 as well as significant decreases in Day 7 and Day 21 wet weight and SVL. There we re no differences (p>0.05) in normalized HLL between the solvent control and treatment groups.

Androgen Receptor (AR) Binding Assay; (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48672501), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct satu ration and competitive binding experiments. The competitive binding a ssay 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 or 10 –11 to 10 –4 M) of norflurazon (99.5±0.5%, Ba tch #609218). Dimethyl sulfoxide (DMSO) was used as the solvent vehicle at a fi nal assay concentration of approximately 3.2%. The assay included dexamethasone as a weak positive control, and R1881 as the ligand
reference standard.

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. The mean dissociation constant (K d ) for [ 3 H]-R1881 was 1.080±0.690 nM, and the mean estimated B max was 1.071±0.167 fmol/100 μg protein for the single batch of prostate cytoso l that was prepared (July 23, 2 011). Only one of the three K d values was within the range re ported in the EPA validation program (0.685 to 1.57 nM), and all

B max values were below the recommended range 7 to 16 fmol/100 μg protein). Confidence in these numbers is low based on the large variation in K d estimates among runs.

In the competitive binding experiment, precipitat ion was observed at norfl urazon concentrations
of 10

− 3 M in the first run and at 10

− 4 M in the second run. Norflu razon did not significantly reduce specific binding of the [ 3 H]-ligand to the AR at any sol uble concentration tested. A log

IC 50 and RBA for norflurazon could not be determined. Based on the results of the three valid runs, norflurazon is classified as a “non- binder” with the androgen receptor under the conditions of this assay.NORFLURAZON FINAL

Page 32 of 54 The estimated log IC 50 s for R1881 and the weak positive co ntrol (dexamethasone) were –9.30 and –4.43 M, respectively. Compared to R1881, the mean relative binding affinity (RBA) for the weak positive control was 0.0022%. Confidence in these numbers is high due to the small variation. However, it was noted that the variat ions between Run 1 comp ared to Runs 2 and 3 were likely due to the concentr ation differences used for R1881 and dexamethasone. The curve for dexamethasone was not fully defined in the first run and therefore the calculation for the

IC 50 may be affected. The range of concentratio ns for R1881 in the first run fully defined the curve, but the concentration range was off by more than an order of magnitude. However, this is a minor deficiency, and the performance criteria were generally met.

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

Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP19) assay (MRID 48672502) , norflurazon (99.5±0.5% a.i. Lot # 609218) was incubated with human recombinant ar omatase and tritiated androstenedione ([1 β

- 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]-ASDN) in 1% dimethyl sulfoxide (DMSO) at log
concentrations from 10

− 11 to 10

− 3 M for 15 minutes to assess the potential of norflurazon to inhibit aromatase activity.

Aromatase activity was determined by measuring the amount of tritiated water produced at the
end of a 15-minute incubation for each concentra tion of test chemical. Tritiated water was quantified using liquid scintillation counting (LSC ). Three runs were conducted that included a full activity control, a bac kground activity control, a pos itive control series (10

− 10 to 10

− 5 M) with a known inhibitor (4-hydroxyandrostened ione; 4-OH ASDN) in duplicate, and the norflurazon series (10 –11 to 10 –3 M) in triplicate. Due to obs erved precipitation of norflurazon
at concentrations of ≥ 10

− 3.5 M, the highest successfully tested concentration was 10

− 4 M in all
three test runs.

Aromatase activity in the full activity controls ranged from 0.169 to 0.293 nmol·mgprotein

− 1 ·min

− 1 for the three test runs, with a mean and standard deviation of 0.220±0.048 nmol·mg-protein

− 1 ·min

− 1 . Activity in the response of the full activity controls and background controls were acceptable for each run.

For the positive control substance (4-OH ASDN), aromatase results were generally within the recommended ranges for the top of the curve, bottom curve, Hill slope, log IC 50 , and % CV for replicates of each concentration within runs. The estimated log IC 50 for 4-OH ASDN averaged

− 7.17 M and the Hill slope was

− 0.94.NORFLURAZON FINAL

Page 33 of 54 For norflurazon, aromatase activity av eraged 0.215 ± 0.48 nmol·mg-protein

− 1 ·min

− 1 at the
lowest concentration test ed in all three runs, 10

− 10 M, and 0.200 ± 0.41 nmol·mg-protein

− 1 ·min

− 1 at the highest successfully tested concentration, 10

− 4 M. The lowest portion of the response curve across all runs was greater than 75% activity at all soluble concentra tions of norflurazon. A log IC 50 and Hill slope could not be calculated.

Based on the data from the average response curve, norflurazon is classified as a Non-inhibitor of aromatase activity in this assay.

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

In an estrogen receptor (ER) binding assa y (MRID 48672503), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct bindi ng experiments. The saturation binding experiment was conducted to demonstrate that the ER in the ra t uterine cytosol was present in reasonable numbers and was func tioning with appropriate affinity for the
radiolabeled reference estr ogen prior to routinely conduc ting ER competitive binding experiments. A competitive bi nding experiment was conducted to measure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presen ce of increasing concentrations (10 –11 to 10 –4 M) of norflurazon (99.5±0.5% purity, Ba tch #609218). DMSO was used as the solvent vehicle at a final assay concentration of 2%. A total of four runs were performed (yielding three valid runs), and each run include d 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17 β -estradiol as the na tural ligand reference
material.

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. The single ba tch of cytosol used for the saturation and competitive binding assays was prepared on Se ptember 27, 2010. In the saturation binding assay, the mean dissociation constant (K d ) for [ 3 H]-17 β -estradiol was 0.081±0.006 nM, and the
mean estimated B max was 52.70±1.05 fmol/100 μg protein. The K d was within the expected range of 0.03 to 1.5 nM. Confidence in these numb ers is high due to the goodness of fit and the small variation among runs.

In the competitive binding experiment, precipitat ion occurred at norflurazon concentrations of

10 –3 M in Runs 1 and 2; therefore, data from th is concentration were not used to evaluate competitive binding. Norflurazon had no effect on specific binding of [ 3 H]-17 β -estradiol at
concentrations of ≤ 10 –4 M in all three valid assay runs. No rflurazon displaced less than 25% ofNORFLURAZON FINAL

Page 34 of 54 the radiolabel estradiol bound to the ER at the highest concen trations successfully tested (10

− 4 M).

The performance criteria were met for the assa y, and the reference compounds performed as expected. Octyltriethoxysilane had no effect on binding; 17 β -estradiol and 19-norethindrone generally reduced labeled ligand binding at th e expected concentrations. The mean log IC 50s were –9.23 M for 17 β -estradiol and –5.57 M for 19-norethi ndrone, and the mean relative binding affinity (RBA) was 0.0326% for 19-noreth indrone, compared to the natural ligand. Although the cytosol preparation (isolated Sept ember 27, 2010) used in the competitive binding experiment was approximately 9-10 months old at the time of the assay, there was no apparent impact on the results of the assay.

Based on the results from the three runs, norflur azon is classified as Not Interactive in the Estrogen Receptor Binding Assay.

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

In an estrogen receptor transcript ional activation assay (MRID 48672504), hER α -HeLa-9903
cells cultured in vitro were exposed to norflurazon (99.5±0.5% a.i., Lot #609218) at logarithmically increasing concentrations from 10

− 11 to 10

− 4 M in cell culture media containing 0.1% DMSO for 24 hours in two independent test s. The experiments were performed using 96- well plates and each norflurazon concentration was te sted in 6 replicates/plate. In addition to the required reference com pound, ICI 182,780 was added to 2 re plicates/plate as a hER α antagonist. Cells were exposed to the test agen ts for 24±2 hours to induce reporter (luciferase) gene products. Luciferase expression in res ponse to activation of the estrogen receptor was measured using a proprietary luciferase assay. A total of nine runs we re reportedly conducted and results were reported from the tw o runs judged to be acceptable.

Cytotoxicity was observed at the hi ghest concentrations tested of 17 α -methyltestosterone and corticosterone in the first run, a nd at the highest concentration te sted of corticosterone in the
second run. In both runs the mean responsiveness of cells to 17 β -estradiol at concentrations

≥ 10 –9 M was 132-167% of the response to the positive control (PC; 17 β -estradiol at 10 –9 M). The responsiveness of the cells to 17 α -methyltestosterone was below the expected level, indicating a decreased sensitivity to very weak agonists.

Although precipitation of norfl urazon was noted at the 10

− 4 M concentration, norflurazon elicited a clear positive respons e in the assay at both 10

− 4 M (72.1 and 115%) and 10

− 5 M (52.9 and 78.5%), and the response at 10

− 6 M (8.9 and 9.6%) was just below the PC 10 threshold. A

PC 50 was calculated for norflurazon of 10 –5.3 M in the first run and 10 –5.2 M in the second run.NORFLURAZON FINAL

Page 35 of 54 However, data were also pr ovided showing that the hER α antagonist ICI 182,780 failed to inhibit the response of the cel ls to norflurazon, while fully inhibiting the response to 17 β

estradiol. Since the data indicates that the cell’s response to norflurazon may not be due to
hER α activation but other no-specific response of the assay, norflu razon cannot be evaluated in this test system.

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

The 28-day short-term reproduction assay (MRID 48686502) of norflurazon with fathead minnows ( Pimephales promelas) was conducted under flow-through conditions. Adult fish (20 spawning groups; 2 males and 4 females in each group; 5 months old) were exposed to norflurazon (99.5% purity) at nom inal concentrations of 0 (n egative and solvent [0.04 mL/L dimethylformamide; DMF] controls), 0.10, 1.0, and 6.0 mg a.i./L (Days 7 to 28; 10.0 mg a.i./L for Days 0 to 6); mean-measured concentrations were <0.05 (<LOQ), 0.097, 1.2, and 5.1 mg a.i./L (Days 7 to 28; 5.0 mg a.i./L for Days 0 to 6). The test was extended an extra week to provide a complete 21-day exposure du ration at the adjusted high treatment level because one test delivery interruption lasted ca. 30 hours. Therefore, the study was conducted for 28 days instead of 21. The te st system was maintained at 24.4 to 25.6 o C and a pH of 7.9 to 8.3.

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

Female survival was 100% for both controls and all treatment groups. Male survival was 87.5, 100, 87.5, 100 and 100% and overall survival was 95.8, 100, 95.8, 100 and 100% in the negative control, solvent cont rol, low, mid, and high treatment levels, respectively. Male fatpad scores were reduced (not statistically analyzed) at the high treatment level (median fatpad score: 1.5) compared to the negative c ontrol (median fatpad score: 3). There were no
other notable observatio ns of secondary sex characteris tics or clinical signs for any treatment group compared to the controls wi th the exception that reduced feeding was periodically observed from Da y 18 to test termination, with an increased occurrence among replicates in the high treatment group towards test termination.

There were significant reductions (p<0.05; Jonc kheere test) in male body weight of 10 and 28% at the mid and high treatment levels, respectively, compared to the negative control. There was also a significant reduction (p<0.05; Jonckheere test) in ma le body length of 6% at the high treatment level compared to the ne gative control. Female growth parameters were not significantly different (p>0.05) be tween the norflurazon treatment groups and the negative control; however, female body weight in the solvent contro l was significantlyNORFLURAZON FINAL

Page 36 of 54 increased (p<0.05) by 11% compared to the negative control.

Spawning occurred at least every 4 days (excep t for one replicate each in the negative and solvent control), and mean f ecundity in the negative and solvent controls was 28.1 and 18.5 eggs/female/day/replicate, re spectively (range: 22.6 to 31.6 fo r the negative control and 15.1 to 22.3 for the solvent control); fertilization success in the negative and solvent controls averaged 98.8 and 98.7%, respectively. There wa s a significant decreasing monotonic trend (p<0.05) of 34 to 95% compared to the negative control at all treatm ent levels. Additionally, there was a significant reduction in solvent co ntrol fecundity of 34% compared to the negative control; therefore, it is possible that there was a solvent interference that contributed to the reduced fecundity observed in the study, particularly in the low and mid treatment groups. The solvent co ncentration (0.04 mL/L) was consistent between the solvent
control and all norflura zon treatment groups. There was a ma rginally signif icant reduction (p=0.057) of 2.25% in fertility in the mid treatme nt group compared to the negative control.

There was an 89% reduction (p<0.05; Jonckheere test) in male vitellogenin (VTG) at the high treatment level compared to the negativ e control; there was no significant difference (p>0.05) in female VTG between treatment groups and the negative control. Male tubercle scores were significantly re duced (p<0.05; Jonckheere test ) in the high treatment group (median tubercle score: 29) compared to the negative control (median tubercle score: 11) and marginally significant reduction (p=0.053, Jo nckheere test) of 13% at the mid treatment level compared to the negative control. No tube rcles were observed in females. There were no significant differences in male or female GS I for any treatment. Sex steroids (testosterone
and 17 β -estradiol) were not measured.

There was a higher incidence of mild to modera te altered proportions of sperm precursors in 3 of 8 males in the high treatment group sugges ting a relative maturation arrest. In females,
there was evidence of an increase in minimal to severe oocyte atresia in 15 of 16 fish, a decrease in abundance of post-ovulatory follicles, and decrease in minimal to mild staining of yolk proteins in the high treatment group. Th erefore, there may be a trend towards an earlier developmental stage in males and females of the high treatment group.

Since there was a significant difference (p<0.05 ) between the negative and solvent control for two endpoints ( i.e. , fecundity and female body weight), data from the treatment groups were compared to the solvent control. There was a significant decrease (p<0.05) in male tubercle score in the mid and high treatmen t groups (median tubercles scores: 25 and 11, respectively) compared to the solvent contro l (median tubercle score: 28). Additionally, fecundity was also significantly decreased (p<0 .05) and male plasma VTG was marginally significantly decreased (p=0.057) in the high treatment group compared to the solvent
control.NORFLURAZON FINAL

Page 37 of 54 Hershberger Assay; (OCSPP 890.1400)

In a Hershberger assay (MRID 48618203) scr eening for androgenic activity, norflurazon (99.5% a.i., Batch# 609218) in 1% carboxymethylce llulose (CMC) was administered daily via oral gavage to groups of 55 to 56-day old, castrated male Sprague Dawley rats (6/group) at dose levels of 0 (vehicle), 300, or 1000 (limit dose) mg/kg/day. An androgenic positive control group consisting of six castrated ra ts exposed to 0.2 mg/kg/day te stosterone propionate (TP) in corn oil by subcutaneous (s.c .) injection plus 1% CMC by oral gavage was included.

To screen for potential anti-a ndrogenic activity, norflurazon in 1% CMC was administered daily via oral gavage to 55 to 56-day old, castrated male Sprague Dawley rats (6/group) at dose levels of 100, 300, and 1000 (limit dose) mg/kg/da y in conjunction with a daily dose of reference androgen TP at 0.2 mg/kg/day by s.c . injection. The positive control group for androgenic activity served as th e negative control group for the anti-androgenic activity. An anti-androgenic positive control group consisted of six castrat ed rats dosed orally with flutamide (FT) in corn oil at 3 mg /kg/day in conjunction with a daily s.c. dose of TP (0.2 mg/kg/day).

For both components of the assay, the animal s were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration to determine weights of the five androgen-dependent tissues. All animals survived until scheduled termination.

In the androgen agonist assay, cl inical signs included right and le ft forelimb hair loss at 300 mg/kg/day and left forelimb, abdominal, and th oracic hair loss, and dr ied red material around nose at 1000 mg/kg/day. Body weights and ove rall body weight gains for the 300 mg/kg/day treatment group were comparable to controls throughout the du ration of the assay. Animals dosed with 1000 mg/kg/day had d ecreased (p<0.05) body weights ( ↓ 11-15%) on Days 3-9, and decreased (p<0.05) overa ll body weight gains ( ↓ 46%) compared to vehicle controls. Food consumption in all norflurazon-treated groups was comparable to the vehicle control. Liver weights were increased (p ≤ 0.05) in the 300 mg/kg/day ( ↑ 28%) and 1000 mg/kg/day ( ↑ 39%) groups. No treatment-related effects were obser ved in any of the accessory sex organs of
norflurazon-dosed animals. Animals in the positive control group had increases (p ≤ 0.05) in accessory sex organ weights as follows: 359% in seminal vesicles; 676% in ventral prostate; 184% in levator ani-bulbocavernosus (LABC); 579% in Cowper’s gla nd; and 48% in glans penis.

In the anti-androgen assay, no clin ical signs of toxicity were observed in the 100 mg/kg/day dose group. At 300 mg/kg/day, clinical signs included left and right forelimb hair loss and
dried red material around nose. At 1000 mg/kg/day, clinical signs included hair loss of theNORFLURAZON FINAL

Page 38 of 54 forelimb, neck, abdominal, thoracic, and hindl imb areas, and decreased defecation. Body weights and overall body weight gains for the 100 and 300 mg/kg/day groups were comparable to negative controls throughout the duration of the assay. Animals dosed with 1000 mg/kg/day had decreased (p<0.05) body weights ( ↓ 12-24%) on Days 3-11, and decreased (p<0.05) overall body weight gains ( ↓ 90%) compared to negative controls. For the positive control group (TP + FT), overall body weight gains were decreased (p ≤ 0.05) by 18%. Food consumption in the 100 and 300 mg/kg/day treatment groups was compar able to the negative control. At 1000 mg/kg/day, food consumption was decreased (p <0.05) by 31% on Days 1-11 compared to the negative controls. The mean testosterone concentration of the 1000 mg/kg/day group was decreased ( ↓ 35%) compared to the negative controls. There were no treatm ent-related effects in any of the accessory sex organs of animal s dosed with 100 or 300 mg/kg/day norflurazon + TP. At 1000 mg/kg/day, accessory se x organ weights were decreased (p ≤ 0.05) as follows: 24% in seminal vesicles; 45% in ventral prostate ; 43% in LABC; and 31% in Cowper’s gland. Animals dosed with the positive control had decreased (p ≤ 0.05) accessory sex organ weights as follows: 70% in seminal vesicles; 73% in ventral prostate; 50% in LABC; and 68% in Cowper’s gland. No effects were observed fo r liver weights in the 100, 300, or 1000 mg/kg/day norflurazon + TP groups.

Norflurazon was administered at up to the limit dose (1000 mg/kg/day); reduced body weights
were observed at this dose in a range-finding study.

Norflurazon was negative for androgenic activity in the Hershberger assay as no statistically significant increases in organ weights were seen in two or more of the five androgen sensitive tissues in the androgen agonist assay. Howeve r, norflurazon was positive for anti-androgenicity in the Hershberger assay as statistically significan t decreases in organ weights were seen in four of the five androgen sensitive tissues in the androgen antagonist assay.

Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48618204), 15 Crl:CD(SD) rats/dose group were treated daily by oral gavage with norflurazon ( 99.5% a.i., Batch # 609218) in 1% aqueous carboxymethylcellulose (CMC) at doses of 0 (vehicle contro l), 250 or 500 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 we ight at day of attainment we re recorded. After sacrifice on PND 42, total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels were analyzed using an electrochemiluminescent immunoassay for T 4 and a magnetic [ 125 I]rTSH radioimmunoassay for TSH. Adrenal, liver, ki dneys, pituitary, thyroi d, and urogenital organ weights were recorded and microscopic examinati on of the ovaries, uter us, thyroid, and kidneys
were conducted.NORFLURAZON FINAL

Page 39 of 54 All animals survived until the scheduled necr opsy. No treatment-related findings were observed during the detailed physical examinations of the animals. No changes in estrous cyclicity were observed, and no treatment-re lated gross necropsy findings were noted.

At 250 mg/kg/day and above, body weight gains were decreased (not sign ificant [NS]) by 8-9% for the overall treatment period (PND 22-42). VO was delayed (p<0.05) by 2.1 days at the high dose; this finding was attributed to reduced body weights in this group. Control body weights on PND 35 were similar to the high dose group on PND 37, supporting the c onclusion that the delay in VO was a result of delayed body weight gain. The lowest individual terminal body weights were noted in two 500 mg/kg/day female s; these two animals exhibited absent and delayed VO, respectively, and absent and re duced corpora lutea, respectively. It was
considered that the latter effects were s econdary to the body wei ght effects.

Mean adjusted and relative (to final body weight) liver weights were increased (p<0.05) by 14% and 21%, respectively, at the high dose and by 9% (NS) and 15%, respectively, at the low dose. Relative kidney weight was also slightly increase d (5%, p<0.05) at the high dose. Absolute and adjusted pituitary weights were decreased (p <0.05) by 17 to 18% in high-dose animals; however, this was not considered treatment-relate d as relative weights we re not affected. No other organ weights were affected by treatment with norflurazon.

Mean serum T 4 levels were decreased (p<0.05) by 13-23% in the low- and high-dose groups; however, serum TSH levels were unaffected by treatment. Thyroid colloid area was slightly reduced at the high dose; this effect was consid ered to be treatment-related and was presumed to be a response to the reduced T 4 levels in this dose group. T hyroid follicular cell height was unaffected by treatment.

The dose levels tested in this assay are adequa te based on the decreased body weights, reduced body weight gains, and increased liver weights seen at the high dose.

Male Pubertal Assay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48618204), 15 Cr l:CD(SD) rats/dose group were treated daily via oral gavage with norflurazon (99.5% a.i., Batch # 609218) in 1% aqueous carboxymethylcellulose (CMC) at doses of 0 (vehicle contro l), 185, or 375 mg/kg/day from post-natal day (PND) 23 to 53. Animals were ex amined for preputial separation (PPS) daily beginning on PND 30 and age and weight at da y of attainment was recorded. Following sacrifice on PND 53, total seru m testosterone, thyroxine (T 4 ), and thyroid stimulating hormoneNORFLURAZON FINAL

Page 40 of 54 (TSH) levels were analyzed using a chemilu minescent immunoassay for testosterone and T 4 , and radioimmunoassay for TSH. Ad renal, liver, kidney, pituitary, thyroid, and urogenital organ weights were recorded and microscopic examina tions of the testes, ep ididymides, thyroid, and kidneys were performed.

All animals survived until the scheduled necr opsy. No treatment-related findings were observed during the detailed physical examinations of the animals. There were no treatmentrelated changes in serum T 4 , TSH, or testosterone concentrations. Thyroid follicular cell height
and colloid area were similar betw een treated and control groups. No test material-related gross or microscopic pathologi cal findings were noted.

No treatment-related effects on body weights or food consumption were observed. Final body weights were decreased (not sign ificant [NS]) by 6-7% in trea ted animals, and overall (PND 23- 53) body weight gains were decreased (NS) by 7-8%. At 375 mg/kg/day, food consumption (g/cage/day) was decreased (NS) by 11-21% dur ing Days 23-26; decreases (NS) of 10% or greater were occasionally observed during the remainder of the treatment period. Complete PPS was delayed in the high-dose group by 1.8 da ys (unadjusted; PND 47.5 treated vs. PND 45.7 controls).

Increased mean organ weights in the high-dose animals were observed for unadjusted, adjusted and relative liver weights (15-23%, p<0.05) a nd relative kidney we ight (7%, p<0.05). Decreased mean organ weights in the high-dose animals were observed for unadjusted, adjusted and relative adrenal weights (18- 23%, p<0.05); adjusted and unadj usted pituitary weights (14%, p<0.05) and dorsolateral prostate weights (22%, p<0.05); and adju sted ventral prostate (15%, p<0.05) and seminal vesicle without fluid (15% , p<0.05) weights. Increased mean organ weights were observed in the low-dose group for relative liver wei ght (14%, p<0.05) and relative kidney weight (7%, p<0.05).

The study report provided normal (historical) ra nges for the clinical chemistry (but not hormone) measures. It must be noted that many of the control means fall outside of the range of the historical control data from the performi ng laboratory. The paramete rs reported that were outside the laboratory normal range are albumin, ALT, AST, bile acids, cholesterol, potassium, total protein, triglyceride a nd urea nitrogen. Mean bile acids were decreased compared to concurrent control values (p<0.05) by 54-58% at 185 mg/kg/day and greater.

The doses tested were determined to be ade quate based on the decreased final body weights, reduced overall body weight gains, and in creased liver weights in this study.

Steroidogenesis Ass ay; (OCSPP 890.1550)NORFLURAZON FINAL

Page 41 of 54 In a steroidogenesis assay (M RID 48672505), H295R cells cultured in vitro in 24-well plates
were incubated with norflurazon

, (99.5±0.5% purity, Batch #609218) at log concentrations from 0.0001 to 100 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as a vehicle, at a final assa y concentration of 0.05%.

Three independent, valid experiments were perf ormed; an initial run was rejected and the samples were not analyzed. Testosterone a nd estradiol concentrations were measured by HPLC/MS-MS with positive ion multiple reaction monitoring using internal standards. A Quality Control (QC) plate was run concurrently with each independent r un of a test chemical plate to demonstrate that the assay responded properly to pos itive control agents at two concentration levels; positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol a nd testosterone production.

Guideline acceptability recommendations and requirements were met, including lack of cytotoxicity, adequate production of testosterone and estradiol, accepta ble reproducibility (low %CV), and appropriate induction and inhibition with pos itive controls.

No evidence of cytotoxicity was observed at a ny tested concentration of norflurazon. However, precipitation was observed at the 100 μ M concentration in all three runs and at 10 μ M in one run; therefore, 10 μ M was the highest concentration evaluated. There were no statistically significant changes in testoster one production following incubation with norflurazon in all three runs, although there was a trend toward in creased testosterone levels at 1 and 10 μ M (1.15- and 1.26-fold, respectively). Estrad iol production was significantly increased in Run 3 at 10 μM (1.2-fold) and in Run 4 at 1 μ M (1.1-fold) and 10 μM (1.3-fold).

Based on the hormone responses in each of the three independent runs, norflurazon treatment resulted in a statistically si gnificant and reproducible increas es in estradiol production, but
norflurazon treatment did not result in a statistically significant and reproducible alteration in testosterone production.

Uterotrophic Assay; (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48618206) conducte d to screen for potential estrogenic activity, norflurazon (99.5% a.i., batch # 609218) in aqueous 1% carboxymethylcellulose was administered daily via oral gavage to groups of six ovariectomized Sprague Dawley rats at dose levels of 0 (vehicle), 300 or 1000 (limit dose) mg/kg/day on post-natal days (PND) 56-58. A positive control group was treated with a daily dose of 17 α -ethynyl estradiol (EE) in corn oil at 0.3 mg/kg/day by oral gavage . Body weights were determin ed daily. All animals wereNORFLURAZON FINAL

Page 42 of 54 terminated and necropsied on PND 59 appr oximately 24 hours after the final dose
administration to determine wet and blotted uterine weights.

All animals survived until sche duled termination. No significa nt clinical observations were noted in any of the animals during the study. Body weights were decreased (p<0.05) by 14% on Day 3 and 20% on Day 4 (termination) in th e 1000 mg/kg dose group comp ared to controls, resulting in a decrease (p<0.05) of 257% in ove rall (Day 1-4) body weight gains (BWG). Body weights in the low-dose (300 mg/kg) group were also decr eased (p<0.05) on Days 2 ( ↓ 8%), 3 ( ↓ 14%), and 4 ( ↓ 16%), and the overall BWG (Day 1- 4) was decreased (p<0.05) by 202% compared to controls. Body weights for th e positive control (EE) group were decreased (p<0.05) by 9% and 14% on Days 3 and 4, respec tively, and overall BWG for the EE animals was decreased (p<0.05) by 183% in comp arison with the vehicle controls.

Uterine weights in the norflur azon treated groups were comparab le to the vehicle controls. Absolute wet and blotted uterus weights for the positive control (EE) group were increased (p<0.05) as expected.

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

Page 43 of 54 APPENDIX 2. Other Scientifica lly Relevant Info rmation (OSRI)

Prenatal Developmen t in Rodents (Rat)

In a developmental toxicity study ( MRID 00063621 ), SD female rats (20/dose; Charles River) were administered 0, 100, 200, or 400 mg/kg/day norflurazon technical (purity not reported; Lot/Batch #004) by oral gavage on gestation days (GD) 6 thro ugh 15 inclusive. Insemination was by natural means with stock males. Matern al effects were noted on body weight gain during the dosing period ( ↓ 28%, ↓ 23%, and ↓ 37% in the low-, mid- , and high-dose groups), for the dosing plus post-dosing period ( ↓ 13% at the low and in termediate dose and ↓ 17% at the high dose), and for the entire study period ( ↓ 12% at the low and in termediate dose and ↓ 16% at the high dose). Food consumption also was decreased during the period of dosing, but not during the post-treatment period (GD 15- 20), and food efficiency was unaffected over the course of the study. The NOAEL for ma ternal toxicity was not determined (<100 mg/kg/day, the low dose), and the LOAEL is 100 mg/kg/day, based on decreased body weight gain. No treatment-related change s were seen in gravid uterine weight, total implantation, fetal viability, or sex ratio. Cesarean data we re unremarkable. Developmental toxicity was suggested at the high dose in the form of increases (NS) in bipartite thoracic vertebrae (10 th

– 13 th ) and rudimentary 14 th ribs. However, these increase s were considered minor and secondary to maternal effects in the hi gh-dose group. Therefore, the NOAEL for developmental toxicity is 400 mg/kg/da y and the LOAEL is not determined.

Prenatal Development in Non-Rodents (Rabbit)

In a developmental toxicity (teratology) study ( MRID 00131152 ), New Zealand White female rabbits (15/dose; Buckshire Co rporation, Perkasie, PA) were administered 0, 10, 30, or 60 mg/kg/day norflurazon technical (98.4%, Lot/B atch #80561) by oral gavage on GD 7 through 19 inclusive. Insemination was by natural means. Clinical toxicity (abortion in 2 rabbits) was observed at the high dose. Treat ment-related decreases in body weight gain were observed at
all doses in maternal rabbits. Body weight gain was decrease d 32%, 50%, and 156% during the dosing period, and 17%, 39%, a nd 57% overall in the low-, mid-, and high-dose animals. Gravid uterine weight data were not reported. The NOAEL fo r maternal toxicity is 30 mg/kg/day, and the LOAEL is 60 mg/kg/day, ba sed on clinical toxicity and decreased body weight gain.

No treatment-related changes were seen in total implantation, fetal viability, or sex ratio. Cesarean data were unremarkable, except for a decrease of 12% in mean fetal weight observed at the high dose. Developmenta l toxicity was suggested by in creases (p<0.05) in the fetal incidence of incompletely ossified frontal bones, 16 th caudal vertebrae, unossified first metacarpal, unossified middle phalanx of the fifth digit of the forelimb in the mid- and highNORFLURAZON FINAL

Page 44 of 54 dose groups, and unossified proximal epiphysis of the tibia in the low- and high-dose groups.
Litter incidence was increased fo r incompletely ossified frontal bones and unossified middle phalanx of the fifth digit of th e forelimb in the mid- and hi gh-dose groups. Therefore, the NOAEL for developmental toxicity is 10 mg/kg/ day, and the LOAEL is 30 mg/kg/day, based
on the increase in skelet al variations observed.

Three-Generation Reproduction in Rodents (Rat)

In a multi-generation reproduction study ( MRID 00080750 ), male and female SD rats (40/sex/dose and 80/se x/control; Sprague-Dawley, Ma dison, WI) were administered norflurazon technical (98.8% purit y, Lot/Batch #004) in the diet. The F0 generation of rats was
administered norflurazon at nominal dos es of 0, 125, 250, and 500 ppm (0, 6.25, 12.5, and 25 mg/kg/day} for 18 weeks prior to mating to pr oduce the F1a and F1b generation. Subsequent to this, the F1a generation wa s used in a two-year feeding study; the F1b animals were administered norflurazon at 0, 125, 375, and 1025 ppm (0, 6.25, 18.75, and 51.25 mg/kg/day) for 13 weeks prior to mating to produce th e F2a and F2b generations. The F2a animals continued to receive norflurazon at these doses, a nd were sacrificed just prior to delivery of a second litter (F3b), which was examined for developmental effects. Organ weight and histopathology data were not determined in this study.

There were no data regarding surv ival and clinical signs in any generation of pare ntal rats in this study. Body weights on LDs 4 and 21 in da ms of F1a, F1b, and F2 a litters were reported,
but no consistent treatment-relat ed effects were observed. No ot her body weight data were reported. However, subchronic a nd chronic toxicity studies co nducted with norflurazon have indicated an effective dose of 25 mg/kg/day, based on increased liver weight, microscopic changes in the kidney, and thyroid effects. Thus, the NOAEL for systemic toxicity is considered to be 18.75 mg/kg, and the LOAEL is considered to be 51.25 mg/kg for male and
female rats.

There were no significant effects of norfluraz on on reproductive performance in any of the generations in this study, although a decrease in live pups born was noted in the F3a litters. Pup weights were not affected by treatment. Th e developmental data determined with the F3b
litters was considered unreliable due to the possibility of stress-related influences on study endpoints. The NOAEL for reproductive toxici ty is 51.25 mg/kg, and the LOAEL was not
determined.

This study is classified as unacceptable/guid eline due to the lack of parental systemic toxicity at the doses tested, incl uding the high dose (51.25mg/kg/day).

Two-Generation Reproduction in Rodents (Rat)NORFLURAZON FINAL

Page 45 of 54 In a two-generation reproduction study ( MRID 43522301 ), male and female Wistar Crl:(WI) br rats (25/sex/dose; Charles River WIGA GmbH, Sulzfeld, Switz erland) received norflurazon technical (98.1% purity, Lot/Bat ch #P.148/86/31) in the diet at doses of 0, 150, 750, or 1500 ppm over 2 generations ( 0, 10.2, 50.8, and 102.5 mg/kg/day for F0 males; 12.1, 62.0, and 129.7 mg/kg/day for F0 females; 13.2, 67.8, a nd 138.6 mg/kg/day for F1 males; and 17.1, 81.7, and 173.0 mg/kg/day for F1 females). The F0 anim als were pretreated with norflurazon for 70 days prior to mating followed by treatment durin g the mating, gestation, and lactation periods for F1a and F1b generations. Animals were selected to be the F1 parents from the F1b litters at weaning (LD 21) and were pretreated for 84 days prior to the mating, gestation, and lactation periods for F2a and F2b generations. Parents were re-mated 10 days after weaning the first generation to produce the second generation. All parental animals were examined macroscopically (especially the reproductive organs), and the liv er and kidneys were weighed. Organs/tissues collected for microscopic evaluation at stud y termination were the liver, kidneys, pituitary, testes with epididymides, prostate , seminal vesicles w ith coagulating gland, uterus with cervix, vagina, and ovaries.

No treatment-related effects on survival were observed; several animals in each parental generation (three F0 and four F1 animals) from the control and all treatment groups were found dead or sacrificed in extremis prior to study termination. Mild clinical signs were limited to mid- or high-dose F0 parental males; no clinical signs were observed with F1 males or female rats on study. At the high dose, body weight gain in F0 female parental rats was decreased by
32% for Weeks 5–10 of the stud y, and body weight gain for F1 male parental rats was decreased by 31% for Weeks 0–31 of the study. Significant decrea ses in F1 male body weights were <10% (6 to 9%). No effects on food c onsumption were noted. During gestation and lactation, body weight gain in F0 female rats at the second mating was de creased by 17% at the high dose; significant decreases in body weight at various periods were <10% (6 to 8%). No weight-related effects during gestation and lact ation were observed for the F1 females. The absolute and relative weights of the liver a nd kidneys were generally increased (p<0.01 or p<0.05) in both F0 and F1 high-dose male and fe male parental rats, and were accompanied by an increased incidence of hepatocellular hypertr ophy in both sexes, and an increased incidence of single cell necrosis in female rats. At th e intermediate dose, liver and kidney weights were also increased (p<0.01 or p<0.05) in parental males (and F1 fema le kidney weights), with an increase in the incidence of hepatocellular hypert rophy similar to that obs erved in the high-dose
animals.

Based upon the significant increases in liv er and kidney weights observed in both generations of parental rats and the increased incidence of hepatocellular hypertrophy in these animals, the LOAEL for systemic toxici ty is 750 ppm (50.8 mg/k g/day for F0 males; 62.0 mg/kg/day for F0 females; 67.8 mg/kg/day for F1 males; and 81.7 mg/kg/day for F1NORFLURAZON FINAL

Page 46 of 54 females). The NOAEL is 150 ppm (10.2 mg/kg/day for F0 males; 12.1 mg/kg/day for F0 females; 13.2 mg/kg/day for F1 males; and 17.1 mg/kg/day for F1 females) for both males
and females.

Measurement of reproductive pe rformance showed decreased (p <0.05) mean pup weight in the high dose, F2b litter pups on LD 21 for male s and for females on LDs 4 and 21. Lactation
index was observed to be decr eased (NS) at the high dose (5 9% versus 80%) and at the intermediate dose (64% versus 80%) for the F2b litter. Mating index at the high dose was consistently increased (NS) over controls for both generati ons. Mean pre-coital interval was decreased (NS) at the high dose for the first li tter of the F0 parental generation, and for both litters of the F1 parental gene ration. Weight gain wa s affected in the F2b pups, with an 18% decrease (significance not re ported) observed in the high-d ose pups on LDs 4–21; the number of live pups also was decreased (p<0.01) in this group. Liver a nd kidney relative weights were increased (p<0.01 or p<0.05) at the high dose for male and female pups in all litters except the F1b and F2b males (relative li ver weights only). Occasional in creases in relative liver and kidney weights were noted in the mid-dos e pups (all female gr oups and F2b males).

In the F1 and F2 litters, reproductive toxicity wa s demonstrated at the high dose by decreased weight gain in F2b litter pups ( 18%) for LD 4–21, and by signific ant increases in absolute and relative weights of the liver and kidneys for male and female pups. There were no apparent
detrimental effects of norflura zon on reproductive perf ormance in this study. Therefore, the reproductive LOAEL is 1500 ppm (102.5 mg/kg/da y for F0 males; 129.7 mg/kg/day for F0 females; 138.6 mg/kg/day for F1 males; and 17 3.0 mg/kg/day for F1 females), and the NOAEL is 750 ppm. The LOAEL for offspring toxicity is 750 ppm based on decreased weight gain in F2b litter pups (18%) versus control for LDs 4–21 post-partum and statistically significant increases in relative weight of the liver fo r male and female pups, and the NOAEL is 150 ppm.

This reproductive toxicity study is classified acceptable and does satisfy the Guideline requirements for a two-generation reproduct ive toxicity study in the rat [870.3800; OECD

416 (§83-4a)].

One-Generation Reproduction in Rodents (Mouse)

In a single-generation reproduction study ( MRID 00080751 ), male and female CD-1 (HaM/ICR Swiss) mice (50/sex/dose and 100/ sex/control; Charles River, Wilmington, MA) were administered norflur azon technical (98.8% purity; Lot/Batch #004) in the diet at nominal doses of 0, 85, 170, and 340 ppm (0, 12.1, 24.3, and 48.5 mg/kg/day; intake data were not provided). The F0 generati on of mice received norflurazon for 18 weeks prior to mating to produce th e F1a litter. The parent anim als were sacrificed after weaning the F1a litter. Animals from the F1a li tter were subsequently used in a two-year chronic feeding study (MRID 00111649).NORFLURAZON FINAL

Page 47 of 54 There were no significant treatment-related syst emic or reproductive effects reported in parental mice or in the F1a o ffspring at any dose tested in th is study. Therefore, the NOAEL for systemic toxicity is 48.5 mg/kg, a nd the systemic LOAEL was not determined. Additionally, the NOAEL for reproductive toxi city is 48.5 mg/kg and the LOAEL was not
determined. It was concluded th at the doses used in the pres ent study were inadequate for assessment of the reproductive to xicity of norflurazon in mice.

This study is classified as unacceptable/guid eline since only a F1a litter was produced for assessment of reproductive toxicity, and the parental data presented were limited to weight gain on days 4–21 of lactati on, and the lack of any effe ct in the F1a offspring.

9-Month (Chronic) Oral Toxicity in Rodents (Rat)

This report ( MRID 00091056 ) constitutes data obtained from the F0 generation of rats used initially in a two-year chronic/carcinogenicity study ( MRID 00082019 ). SD rats (SpragueDawley, Madison, WI) were given norflurazon t echnical (98.8% purity, Lo t/Batch #004) in the diet at nominal doses of 0, 125, 250, and 500 ppm (0, 6.25, 12.5, and 25.1 mg/kg/day) for 39 weeks. Organs/tissues collected for macrosco pic and/or microscopi c evaluation at study termination included the liver, pituitary, adrenals , thyroids, kidneys, testes, uterus, and ovaries. The livers, kidneys, gonads, and thyroids were weighed at study termination.

There were no significant effects of norfluraz on on survival, body weight, body weight gain, food consumption, or food efficiency, in male or female rats at any dos e tested. In the highdose animals, liver weight was increased ( ↑ 14% and ↑ 23%, NS) in male and female rats, respectively, at 39 weeks. Kidney weight was affected (NS), but the incidence of hyaline pigment deposition in male and fe male rats (many tubules in male s and few tubules in females) was increased, as was the incidence of medulla ry congestion in high-dose female rats. The incidence of tubular degeneration was increased in both sexes at the high dose. Thyroid weight was decreased in males ( ↓ 30%, NS), but thyroid weight was increased in females ( ↑ 11 to 47%, NS). Gonadal weight (left gonad) also wa s increased in female rats by 30% (NS).

At the intermediate dose, thyroid weight was decreased by in male rats ( ↓ 30%, NS), but was increased by in female rats ( ↑ 11 to 29%, NS). However, ther e were no reported microscopic alterations in the thyroid at an y dose. Gonadal weight in fema le rats also was increased ( ↑ 17%, NS).

Based on the dose-related increase in liver weight in male and female rats at 39 weeks, the increase in gonad weight of fe males, and the microscopic change s observed in kidneys of both sexes, the LOAEL is considered to be 500 ppm (25 mg/kg/day), and the NOAEL is 250 ppm (12.5 mg/kg/day). Although thyroid weight was incr eased at the intermediate and high doses in both sexes, there were no correl ated histopathological effects. Therefore, effects on thyroidNORFLURAZON FINAL

Page 48 of 54 weight, although related to treatmen t, do not indicate a toxic effect.

This study is classified as co re supplementary, based on the fact that this study satisfies no specific Guideline requirement u nder Subdivision F guidelines. However, this study provides useful toxicity data on norflurazon in rats after 39 weeks of exposure.

Chronic Oral Toxicity/Carcin ogenicity in Rodents (Rat)

In a two-year carcinogenicity study ( MRID 00082019 , interim report MRID 00111617 ) male and female SD rats (85 rats/sex/dos e and 170 rats/sex/control; Sprague-Dawley, Madison, WI) were administered norflurazon technical (98.8% purit y, Lot/Batch #004) in the diet at doses of 0, 125, 375, and 1025 ppm (0, 6.25, 18.75, and 51.25 mg/kg/day) for 104 weeks. Organs/tissues collected for macrosco pic and/or microscopi c evaluation at study termination included the liver, pituitary, adrenals, mammary gl and, thyroids, kidneys, testes, prostate, uterus, and ovaries. The liver, kidneys, gonads, pitu itary, adrenals, and thyroids were weighed at Week 52 (n=10/sex/dose) and at study termination (Week 104).

No effects of norflurazon were ev ident for survival (except for an increase in mortality in the mid- and high- dose males at Week 104), body we ight, body weight gain, or food consumption in male or female rats at any dose tested. Liver weight was increased ( ↑ 17% and ↑ 13%; intermediate dose) and ( ↑ 24% and ↑ 27%; high dose) in male and fe male rats, respectively, at 52 weeks. Other organ-weight effects after 52 weeks included increased kidney weight (16 to 39%) in high-dose animals, non-dose-dependent increases in adrenal weights at all treated females, and increased thyroid weight in the high-dose females ( ↑ 30%). At 104 weeks, liver weight was increased ( ↑ 12 to ↑ 14%) in male and female rats, respectively, and kidney weight was increased 16 to 39% in high-dose animals. Th yroid weight also was increased in the highdose males at 104 weeks ( ↑ 32%). An increased incidence of hydronephrosis was observed in high-dose male rats at 52 weeks, and the incidence of nephritis al so was increased in male rats (terminal sacrifice + dying on test ) at this dose. The in cidence of tubular ca sts was increased in high-dose females that survived to study term ination. Other microscopi c alterations observed in high-dose animals included an increased inci dence of parathyroid hyperplasia (both sexes), hemosiderin pigment deposition in the spleen (males only) and liver (both sexes), and endrometritis and squam ous metaplasia of the uterus (females).

Based on the increased kidney weight and acco mpanying histopathologic changes, as well as increases in liver weight (males and female s) and thyroid weight (males), the LOAEL is considered to be 1025 ppm (51.25 mg/k g/day) and the NOAEL is 375 ppm (18.75 mg/kg/day). There was no evidence of carcinoge nicity in response to dietary administration of norflurazon in this study.NORFLURAZON FINAL

Page 49 of 54 This chronic toxicity study is classified acceptable (core minimu m) and does satisfy the Guideline requirements for a chronic/ carcinogenicity study in the rat [870.4300 (§83-5a)].

Carcinogenicity in Rodents (Mouse)

In a two-year carcinogenicity study ( MRID 00111649 ) male and female CD-1 HaM/ICR Swiss mice (125 mice/sex/dose and 250 mice/sex/ control; Charles River, Wilmington, MA) were administered norflurazon technical (98.8% purity, Lot/Batch #004) in the diet at dose levels of 0, 85, 340, and 1360 ppm (0, 12.8, 58.7, a nd 218.8 mg/kg/day; intake data were not provided) for 100–104 weeks. Organs/tissues co llected for macroscopic and/or microscopic evaluation at study termination included th e liver, pituitary, adrenals, mammary gland, thyroids, kidneys, testes, prosta te, uterus, and ovaries. The li ver, kidneys, gonads, pituitary, adrenals, and thyroids were weighe d at study termination (Week 104).

No significant effects were observed on surviv al, body weight, body weight gain, clinical toxicity, or food consumption at any dos e tested. Liver weight was increased ( ↑ 9% and ↑ 15%; intermediate dose) and ( ↑ 27% and ↑ 21%; high dose) in male and female mice, respectively. Liver to body weight ratio was increased ( ↑ 19% and ↑ 4%; intermediate dose) and ( ↑ 43% and ↑ 19%; high dose) in male and fema le mice, respectively. No statistical significance of liver
effects was noted. Increased incidences of enla rged spleen, nephritis, swollen/enlarged liver, and nodular enlargement of the liver were obs erved in high-dose ma les, and increased incidences of pyelonephritis, enlarged liver, and cystic ovaries were observed in high-dose females. The LOAEL is 340 ppm (58.7 mg/kg/day) for male mice, based on the increased incidence of enlarged spleen, increased absolute and relative liver weights, and the increased incidence of nephritis. The LOAEL is 1360 ppm (218.8 mg/kg/day) for female mice, based on the increased incidences of enlarged liver, cyst ic ovaries, and pyelonephritis, as well as the increased absolute and relative liver weights. The NOAEL estimates are 85 pm for male mice and 340 ppm for female mice (12.8 and 58.7 mg/k g/day, respectively). Dosing was considered adequate in this study due to the increased liver /body weight ratio in male and female mice at the high dose, as well as the increased incide nce of non-neoplastic path ology in the liver, spleen, and kidney in male mice. Carcinogenic potential was evidenced by an increased incidence of hepatic adenoma and combined adenoma/carcinom a in high-dose male mice.

The submitted study is classified as acceptable (core minimum) and does satisfy the guideline requirements for a carcinogenicity study in mice [870.4200 (§83-2b)].

Chronic Oral Toxicity in Non-Rodents (Dog)

. Norflurazon technical (99.2% purity, Lot/Batch not reported) was admini stered to male and female beagle dogs (4/sex/group; Buckshire Corporation, Perkasie, PA) in a six-month chronic oral toxicity study ( MRID 00111618) at doses of 0, 50, 150, and 450 ppm (1.53,NORFLURAZON FINAL

Page 50 of 54 5.02, and 14.27 mg/kg for males; 1.58, 4.77, and 17.75 mg/kg for females). Organs/tissues that were weighed at study termination, and prepared for macroscopi c and/or microscopic evaluation, included the liver, p ituitary, adrenals, thyroids, ki dneys, testes, prostate, uterus, and ovaries; the epididymides and vagina were also evaluated (but not weighed).

No significant treatment-related effects were observed on body weight, body weight gain, food consumption, or daily intake, except in high-dose females w ith decreased food consumption ( ↓ 41–47%) and increased food intake ( ↑ 58%). At the intermediate dose, liver weight was increased by 38% (p<0.01) in male dogs and by 23% (p<0.05) in female dogs, and thyroid weights were increased by 33% (p<0.05) in male dogs and by 37% (NS) in female dogs. Liver weight was increased by 41% (p< 0.01) in male dogs and by 44% (NS) in female dogs at the high dose, and thyroid weights were increased by 43% (p<0.01) in male dogs and by 30% (NS) in female dogs. The LOAEL of 150 ppm (5.02 mg/kg/day in males; 4.77 mg/kg/day in females) is based on increased absolute and relative liver we ight in both sexes, and the NOAEL is 50 ppm (1.53 mg/kg/day in ma les; 1.58 mg/kg/day in females).

This chronic toxicity study is classified acceptable (core minimu m) and does satisfy the Guideline requirements for a chronic/ carcinogenicity study in the rat [870.4100 (§83-1b)].

Avian Reproduction Toxicity (OCSPP: 850.2300)

Effects to avian species from chronic exposure are assessed with one-generation reproduction studies. Studies, two with mallard duck (Anas platyrhynchos) and two with bobwhite quail (Colinus virginianus) are used to evaluate the chronic toxicity of norflurazon to avian species. Both malla rd studies determined an NOAEC of 40 mg ai/kg-diet. In the first mallard st udy (MRID 00052172) the highest dose tested was 40 mg ai/kg-diet and no effects were reported. The second mallard study (MRID 42615302) tested norflurazon (99.4%) at co ncentrations of 40, 200 and 1000 mg ai/kg-diet. The NOAEC was the lowest c oncentration tested, based on decreased hatchling weights relative to the contro l (15%) at 200 mg/kg-di et. At 1000 mg/kg,
there was an 11% reduction in eggs laid. Adult female and male body weight gains were reduced compared to control at 1000 mg/kg-diet (male: +6g vs. -53g at 1000 ppm; female: +140g vs. +51g at 1000 ppm). Female body weight was also significantly reduced 7.5%. There were no effects on mo rtality, behavior or food consumption.

In the first quail study (MRID 00039222), th ere were no effects at the highest concentration tested (40 mg/kg-diet ). The second quail study (MRID 42615301) determined an NOAEC of 40 mg ai/kg-diet, based on decreased hatchling survival at the second highest dose (200 mg ai/kg-diet) relative to the cont rol; however, thisNORFLURAZON FINAL

Page 51 of 54 effect was not concentration-responsive. Nu mber of eggs laid were greater at 200 and 1000 mg/kg-diet compared to control (19 and 13%, respectively), however, the total number of 14-day old survivors was approximately the same in all groups.
There were no effects on mo rtality, behavior or f ood consumption, or other reproductive parameters. The LOAEC for both mallard duck and bobwhite quail is 200 mg ai/kg-diet.

Fish Early Life-Stage Toxicity (OPPTS: 850.1400)

Fathead minnows ( Pimephales promelas ) were exposed to norflurazon (96.6% purity; Batch/Lot not reported) at c oncentrations of 0.53, 1.1, 2.1, 4.2, and 8.9 mg/L in a fish early life-stage toxicity study ( MRID 00118047 ). The nominal test levels were present at or above
the stated concentration as de termined by GLC-ECD. The em bryo and larvae exposure was
initiated within 48 hours after egg fertilization, and conti nued through 30 days post-hatch. Sixty embryos were used in each test chambe r and two test chambers per control, solvent control, and nominal test concentration were utilized. Fort y larvae were utilized in replicate tanks (2) for each test concentration.

Percent hatch was not significantly differen t between treatment a nd control groups. No toxicological signs were obs erved at concentrations ≤ 1.1 mg/L. Survival of larvae exposed to
mean concentrations ≥ 4.2 mg/L norflurazon was significan tly (p=0.05) reduced compared to
the survival of control and solvent control larvae. Mean total length of larvae exposed to mean concentrations as low as 2.1 mg/L norflurazon was significantly reduced as compared to the controls. Average wet we ight of larvae exposed to 4.2 mg /L norflurazon was significantly reduced, compared to control and solvent contro l larvae, and average wet weight of larvae exposed to 2.1 mg/L was significantly reduced co mpared to control larvae only. The MATC of fathead minnows exposed to norflurazon during em bryo and larval development is >1.1 mg/L and <2.1 mg/L.

Fish Early Life-Stage Toxicity (OPPTS: 850.1400)

Rainbow trout ( Oncorhynchus mykiss ) were exposed to norfluraz on (96.6% purity; Batch/Lot not reported) at concentrati ons of 0.77, 1.5, 3.3, 5.5, and 11.2 mg/L in a fish early life-stage toxicity study ( MRID 00118048). The nominal test leve ls were present at or above the stated concentration as determined by GLC-ECD. Th e embryo and larvae exposure was initiated within 48 hours after egg fertilization, and continued through 30 days post-hatch. Fifty embryos
were used in each test chamber and two test chambers per contro l, solvent control, and nominal
test concentration were utilize d. An unknown number of larvae were utilized in replicate tanks (2) for each test concentration.

The percentage viability of rainbow trout embryos was unaff ected by exposure to mean norflurazon concentrations up to 11.2 mg/L. The percentage hatch of viable embryosNORFLURAZON FINAL

Page 52 of 54 exposed to mean concentrations ≥ 5.5 mg/L norflurazon was significantly reduced compared
to control and solven t control embryos.

Embryos began hatching on Day 23 of exposure. None of the embryos exposed to 11.2 mg/L
norflurazon hatched successful ly, and larvae exposed to 5.5 mg/L norflurazon began dying after hatching with no larvae su rviving after Day 9 pos t-hatch. Mortality and lethargic behavior were observed among larvae exposed to 3.3 mg /L norflurazon by Day 2 post-hatch, with no surviving larvae after Day 17 post-hatch. Lethargi c behavior and mortalit y also were observed among larvae exposed to 1.5 mg/L norfluraz on through Day 15 post-hatch, after which surviving larvae no longer appear ed lethargic and none died. Larv ae survival was unaffected by exposure to a mean norflurazon concentration of 0.77 mg/L. Mean total length and average wet weight of larvae exposed to 1.5 mg/L norflur azon were significantly reduced, and also appeared to have light pigmentation as compared to larvae in both c ontrol groups. Mean total length, average wet weight and pigmentation of larvae exposed to 0.77 mg/L norflurazon was comparable to growth and pigmentation of c ontrol and solvent contro l larvae. Based on the reduced percentage survival and reduced grow th of larvae exposed to a mean norflurazon concentration of 1.5 mg/L, the MATC of this compound for the rainbow trout was estimated to be >0. 77 and <1.5 mg/L.NORFLURAZON FINAL

Page 53 of 54 APPENDIX 3: References Not Utilized in the Norflurazon WoE Analysis

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

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

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

Fogleman, R.W. (1971) Evaluation of the Subchronic (90-day) T oxicity Effects of SAN-9789 in Rats: Contract No. 120-546-9-70. Final repo rt. (Unpublished study received Nov 14, 1972 under 3G1310; prepared by Affiliated Medical Enterprises, Inc., submitted by Sandoz, Inc., Homestead, Fla.; CDL: 092233-F). (MRID 00091055)

WARP Institute, Incorporated (1975) Compound 9789: Re production Studies in Rats and Mice: Volume I: Reproduction Studies on Rats. Final report. (Unpublished study, including submitter summary, received May 8, 1975 under 5F1602; su bmitted by Sandoz, Inc., Homestead, Fla.; CDL: 094369-A). (MRID 00080750)NORFLURAZON FINAL

Page 54 of 54 WARP Institute, Incorporated (1975) Compound 9789: Re production Studies in Rats and Mice: Volume 2: Reproduction Studies in Mice. Fina l report. (Unpublished study, including submitter summary, received May 8, 1975 under 5F1602; su bmitted by Sandoz, Inc., Homestead, Fla.; CDL: 094368-A). (MRID 00080751)